InteractiveFly: GeneBrief

piwi : Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | Evolutionary Homologs F | References

BIOLOGICAL OVERVIEW

A central enigma in epigenetics is how epigenetic factors are guided to specific genomic sites for their function. It has been reported that a Piwi-piRNA complex associates with piRNA-complementary transposon targets in the Drosophila genome and regulates their epigenetic state. This study reports that Piwi-piRNA complexes bind to numerous piRNA-complementary sequences throughout the genome, implicating piRNAs as a major mechanism that guides Piwi and Piwi-associated epigenetic factors to program the genome. To test this hypothesis, it was demonstrated that inserting piRNA-complementary sequences to an ectopic site leads to Piwi, HP1a, and Su(var)3-9 recruitment to the site as well as H3K9me2/3 enrichment and reduced RNA polymerase II association, indicating that piRNA is both necessary and sufficient to recruit Piwi and epigenetic factors to specific genomic sites. Piwi deficiency drastically changed the epigenetic landscape and polymerase II profile throughout the genome, revealing the Piwi-piRNA mechanism as a major epigenetic programming mechanism in Drosophila (Huang, 2013).

This study has systematically demonstrated the existence of the Piwi-piRNA epigenetic guidance mechanism and its function as a major mechanism of guiding epigenetic factors to their target sites in Drosophila. This mechanism provides a clear and effective answer to the long-standing question on how epigenetic factors are recruited to their specific target sites to achieve epigenetic programming throughout the genome. Given that some other Piwi proteins and piRNAs also exist in the nucleus of other organisms including mammals, this mechanism might have profound significance in diverse organisms (Huang, 2013).

Whole-genome mapping produced the high-resolution map of Piwi and piRNA binding to the genome. The perfect colocalization of Piwi and piRNA binding sites is expected given their association as molecular complexes. These Piwi-piRNA complexes directly bind to many regions in the genome, exerting epigenetic repression at most of the target sites. This may account for the diverse biological functions of Piwi in different cell types during development. In particular, many Piwi-piRNA complexes bind to transposon sequences; this may be a major mechanism that is responsible for transposon silencing by Piwi as reported in many studies (Huang, 2013).

Furthermore, this analysis has revealed, at the whole-genome scale, the dependence of HP1a and H3K9 methylation on Piwi, which suggests that HP1a and histone methylases are recruited by Piwi-piRNA complexes as a major mechanism to many sites in the genome. It is important to note that Lei and colleagues reported that, in several piRNA clusters, HP1 binding is apparently unaffected by Argonaute proteins, including Piwi (Moshkovich and Lei, 2010). This result was fully anticipated because Piwi colocalizes with HP1a at many, but not all, HP1a-containing bands on polytene chromosomes. The binding of HP1a to chromatin at Piwi-free sites must be via a different mechanism, possibly via the canonical H3K9me2/3- mediated mechanism. These data, combined with the current findings, indicate that there are at least two different ways for recruiting HP1a to the chromatin, with Piwi-piRNA mechanism as a main way of recruitment, as demonstrated in this study and suggested by previous polytene staining data (Huang, 2013).

The results demonstrate that piRNA is both necessary and sufficient to bring Piwi to specific genomic sites in a sequence-specific manner and reveal a crucial role of piRNA in guiding epigenetic factors to specific sites in the genome. This epigenetic guidance mechanism is similar to the RNAi-mediated heterochromatin formation in the fission yeast in that both are mediated by small RNAs and Piwi/Ago. However, it distinctly differs from the yeast pathway in three major aspects. First, it recruits HP1a without H3K9 methylation, which then leads to recruitment of HMT and H3K9 methylation. This is in stark contrast to the yeast RNAi pathway in which the RITS complex first recruits HMT, which then leads to the methylation of H3K9 and eventual recruitment of HP1. In addition, this is also in sharp contrast to the known H3K9 methylation-dependent mechanism of HP1a recruitment in higher eukaryotes and represents a novel H3K9 methylation-independent mechanism. The recruitment of HMT by HP1a would lead to H3K9 methylation, which would result in further recruitment of HP1a molecules to the site, thereby stabilizing the repressive state of the chromatin. Second, the Piwi-piRNA-mediated epigenetic guidance mechanism can lead to transcriptional repression or activation, depending on the genomic context Last, the Piwi-piRNA mechanism involves single-stranded piRNAs and Piwi proteins rather than double- stranded siRNAs and Ago proteins. Given the genomic complexity of the higher eukaryotes, piRNAs, mostly 24-32 nt in length, are ideal candidate molecules for conferring sequence specificity in a genome-wide context. Indeed, the extreme complexity of the identified piRNAs, with more than 20,000 piRNAs associated with Piwi alone in Drosophila and more than 58,000 piRNAs in mammals, renders the Piwi-piRNA pathway a likely major epigenetic factor guidance mechanism in Drosophila, and possibly even in mammals (Huang, 2013).

High-resolution mapping analysis suggests that piRNAs might associate with euchromatin by binding to nascent RNA transcripts of 100-800 bp yet with heterochromatin by directly binding to DNA. This is in perfect agreement with previous observation that Piwi binding to euchromatin and heterochromatin is sensitive to RNaseIII and RNaseH that selectively digest double-stranded RNA and RNA-DNA hybrid, respectively. Given the complexity of the heterochromatic context, it is not clear so far exactly how piRNA binds to heterochromatic DNA. However, it is conceivable that such direct binding might occur between piRNA and single-stranded DNA (e.g., during DNA replication or transcription) or between piRNA and DNA duplex. Future studies will resolve these hypotheses (Huang, 2013).

It is also worthy noting that the sequence specificity of piRNA binding to its targets is additive with respect to individual base pairs. Each mismatch compromises the piRNA binding efficacy by 40%, so that a piRNA carrying three point mutations retains only
10% its binding ability to target sequences. This is in contrast to siRNA targeting that requires perfect complementarity and miRNA targeting that requires a mismatch in the middle position yet requires perfect match in the base 2-7 'seed sequences'. Indeed, the graded sequence specificity of piRNA binding to its target sequences might create a mechanism of quantitative regulation that allows piRNAs to guide Piwi and epigenetic factors to even more genomic sites with graded effects as well as bestows tolerance to point mutations that frequently occur in heterochromatic and repetitive sequences (Huang, 2013).

Maternal depletion of Piwi, a component of the RNAi system, impacts heterochromatin formation in Drosophila

A persistent question in epigenetics is how heterochromatin is targeted for assembly at specific domains, and how that chromatin state is faithfully transmitted. Stable heterochromatin is necessary to silence transposable elements (TEs) and maintain genome integrity. Using reporters subject to Position Effect Variegation (PEV), this study found that depletion of key proteins in the early embryo can lead to loss of silencing assayed at adult stages. The piRNA component Piwi is required in the early embryo for reporter silencing in non-gonadal somatic cells, but knock-down during larval stages has no impact. This implies that Piwi is involved in targeting HP1a when heterochromatin is established at the late blastoderm stage and possibly also during embryogenesis, but that the silent chromatin state created is transmitted through cell division independent of the piRNA system. In contrast, heterochromatin structural protein HP1a is required for both initial heterochromatin assembly and the following mitotic inheritance. Piwi depletion leads to decreased HP1a levels in pericentric heterochromatin, particularly in TEs. The results suggest that the major role of the piRNA system in assembly of heterochromatin in non-gonadal somatic cells occurs in the early embryo during heterochromatin formation, and further demonstrate that failure of heterochromatin formation in the early embryo impacts the phenotype of the adult (Gu, 2013).

These results, coupled with earlier findings, support a model for heterochromatin targeting that utilizes Piwi in the early zygote: it is suggested that Piwi and the associated piRNA system are required (directly or indirectly) to guide HP1a to a subset of TEs, and that the deposition of HP1a further recruits other components to establish H3K9me2-enriched heterochromatin status in those TE regions. Specificity could be achieved via a base-pairing mechanism utilizing piRNAs. Subsequent mitotic transmission of this HP1a/H3K9me2 enriched heterochromatic state during development does not appear to depend on the piRNA system. This targeting mechanism may be of primary importance for TEs in border regions between heterochromatin masses and adjacent euchromatin, the situation for PEV reporters utilized in this study. When Piwi is depleted, the HP1a level is significantly decreased at these sites. Some loss of HP1a is seen in general in heterochromatic regions, presumably because heterochromatin is enriched in TEs and other repetitious elements. Thus the silencing of PEV reporters, which are dependent on the spreading of the local heterochromatin, can be released. The silent chromatin state is apparently transmitted by the heterochromatin system during development, when the piRNA system is largely absent in non-gonadal somatic cells (Gu, 2013).

Using mutant alleles, this study assayed the effect of maternal depletion (which results in depletion in early embryos) and zygotic depletion of HP1a or Piwi on the expression of PEV reporters. Functional HP1a depletion in either the early zygote or developing animals leads to suppression of variegation of the PEV reporters, coupled with decreased levels of HP1a itself as well as the silencing mark H3K9me2 in the reporter regions. This suggests a critical role for HP1a in both early establishment and subsequent maintenance of heterochromatin, and demonstrates that the impact of early depletion can be seen using an adult phenotype, even when wild type alleles of HP1a are present in the developing zygote. In the case of Piwi, only maternal or early zygotic depletion has a significant effect on the reporters in non-gonadal somatic cells. Surprisingly, zygotic Piwi depletion in embryos from wild type mothers does cause a small decrease in PEV silencing of the BL1 reporter in carcasses, and of 118E10 and w^m4 reporters in eyes. However, this effect is not as significant as that caused by maternal depletion. A small zygotic effect of Piwi depletion is consistent with prior observations. At the same time, the results of Piwi knock down in the eye lineage argue that Piwi is dispensable for the maintenance of heterochromatin silencing after embryogenesis. Note that the ey-GAL4 driver becomes active in late embryogenesis, much later than the onset of zygotic expression in the 2-hour embryo (Gu, 2013).

Overall, the data demonstrate that Piwi's role in recruiting HP1a and other components to some TE regions happens early in development, while HP1a is essential for heterochromatin formation during every cell cycle. This is in congruence with their expression patterns. Piwi mRNA is present in gonadal cells and early embryos, with little detectable expression in non-gonadal somatic cells, with some exceptions (e.g., larval fat body, possibly nerve cells), while HP1a is expressed in all cells/tissues during development. Thus any HP1a-Piwi interaction likely occurs in gonadal cells and in early embryos, where they are both highly enriched and observed to be nuclear proteins. These stages are also enriched in small RNAs and piRNA pathway components, supporting a model of piRNA-mediated heterochromatin assembly. As it is a structural protein of heterochromatin, one would anticipate that HP1a would be essential for heterochromatin formation in any dividing cell (such as those in the eye imaginal disc) when heterochromatin is re-established after DNA replication as is observed. A second protein found to be important for silencing state maintenance for some reporters is the histone methyltransferase Eggless. Egg has been suggested to be essential for heterochromatin formation in specific regions, including chromosome 4 (in somatic cells) and piRNA clusters (gonads;) (Gu, 2013).

It is of interest that cells in the mature organism 'remember' the loss of HP1a in the early zygote, exhibiting HP1a and H3K9me2 reduction in the reporter promoter region in the adult. The depletion of HP1a at the critical stage of heterochromatin establishment during early development, even when the overall HP1a level is presumably recovered soon after the onset of zygotic transcription, results in diminished heterochromatic regions that apparently cannot be fully re-established, and only partially recover. This implies that both genetic and environmental insults sustained at the critical embryonic stage can have a long-lasting impact on the individual (Gu, 2013).

The reporters exhibiting PEV used in this study either lie near the break point between heterochromatin and euchromatin caused by inversion or translocation (e.g. BL1, BL2 and w^m4), or have been inserted into heterochromatic domains by P element transposition (e.g., 118E10). Their silencing is dependent on the spreading of the adjacent heterochromatin structure, making them sensitive to even small changes in the heterochromatin environment and chromatin assembly systems. For example, when Piwi is knocked down in the early embryo, suppression of variegation of the BL2 reporter was observed coupled with significant HP1a loss at the promoter of the reporter. However, no dramatic change of HP1a enrichment was observed in most other heterochromatic regions. The sensitivity of the BL2 reporter to Piwi depletion might be explained by its position at the edge of a heterochromatic mass, and the requirement for spreading of the heterochromatic assembly. The HP1a ChIP-array data in piwi mutant larvae further confirms that depletion of Piwi will lead to a small decrease in the HP1a level at some TE classes, coupled with an overall small decrease of HP1a levels in heterochromatic sequences. However, the data obtained from the ChIP-array includes only the unique probes in the assembled genome sequence, so only a small portion of the TEs have been analyzed. It is possible that the actual overall decrease of HP1a enrichment is greater, as most of the TE sequences are not included in this analysis. Nonetheless, the PEV reporters may be particularly sensitive to Piwi manipulation, either because of their dependency on spreading of heterochromatin, or because the Piwi-dependent response itself is triggered by transcription, more likely to occur in these flanking regions (Gu, 2013).

While these studies have focused on the role of Piwi, the resulting model is consistent with earlier work examining several components of the piRNA pathway (Piwi, Aubergine, Armitage, Spn-E). Mutations in these components are reported to have an impact on the repression of transcription and maintenance of a closed chromatin structure for several TE classes when assayed in the female germ line. This study has demonstrate two additional features: first, that maternal depletion of Piwi has an impact on silencing PEV reporters that can be seen in somatic cells of larvae and adults, and second, that depleting Piwi in early zygotic cells (but not maternally) also impacts PEV assayed in later stages (Gu, 2013).

The results further suggest that the piRNA system observed in this study most likely acts in the context of multiple mechanisms for heterochromatin formation. In the yeast S. pombe the RNAi system is redundant with other heterochromatin protein interaction systems in heterochromatin establishment; such DNA-protein interaction systems have also been inferred in Drosophila. The interplay among these systems remains to be investigated. The system of selective depletion developed in this study should allow further investigation of the role of various components in targeting and maintaining heterochromatin at different heterochromatin domains (Gu, 2013).

Silencio/CG9754 connects the Piwi-piRNA complex to the cellular heterochromatin machinery

The repression of transposable elements in eukaryotes often involves their transcriptional silencing via targeted chromatin modifications. In animal gonads, nuclear Argonaute proteins of the PIWI clade complexed with small guide RNAs (piRNAs) serve as sequence specificity determinants in this process. How binding of nuclear PIWI-piRNA complexes to nascent transcripts orchestrates heterochromatin formation and transcriptional silencing is unknown. This study characterize CG9754/Silencio as an essential piRNA pathway factor that is required for Piwi-mediated transcriptional silencing in Drosophila. Ectopic targeting of Silencio to RNA or DNA is sufficient to elicit silencing independently of Piwi and known piRNA pathway factors. Instead, Silencio requires the H3K9 methyltransferase Eggless/SetDB1 for its silencing ability. In agreement with this, SetDB1, but not Su(var)3-9, is required for Piwi-mediated transcriptional silencing genome-wide. Due to its interaction with the target-engaged Piwi-piRNA complex, it is suggested that Silencio acts as linker between the sequence specificity factor Piwi and the cellular heterochromatin machinery (Sienski, 2015).

A somatic piRNA pathway in the Drosophila fat body ensures metabolic homeostasis and normal lifespan

In gonadal tissues, the Piwi-interacting (piRNA) pathway preserves genomic integrity by employing 23-29 nucleotide (nt) small RNAs complexed with argonaute proteins to suppress parasitic mobile sequences of DNA called transposable elements (TEs). Although recent evidence suggests that the piRNA pathway may be present in select somatic cells outside the gonads, the role of a non-gonadal somatic piRNA pathway is not well characterized. This study reports a functional somatic piRNA pathway in the adult Drosophila fat body including the presence of the piRNA effector protein Piwi and canonical 23-29 nt long TE-mapping piRNAs. The piwi mutants exhibit depletion of fat body piRNAs, increased TE mobilization, increased levels of DNA damage and reduced lipid stores. These mutants are starvation sensitive, immunologically compromised and short-lived, all phenotypes associated with compromised fat body function. These findings demonstrate the presence of a functional non-gonadal somatic piRNA pathway in the adult fat body that affects normal metabolism and overall organismal health (Jones, 2016).

This study has shown evidence for a fully functional piRNA pathway in a non-gonadal somatic tissue, the adult fly fat body, that is likely to be necessary for proper tissue function and overall organismal health. These results demonstrate that the adult fat body piRNA pathway exhibits canonical characteristics found in gonadal somatic cells, and its activity likely positively affects the function of a tissue important to metabolic homeostasis and physiological health. Although it has not been possible to entirely rule out a contribution of the gonadal piRNA pathway to fat body function, many of the phenotypes observed are opposite to those typically seen in animals with compromised gonadal tissue function and therefore likely represent the effect of a loss of the fat body piRNA pathway. For example, the shortened lifespan and reduced lipid stores in piRNA pathway mutants demonstrate that the piRNA pathway is essential in the health and functioning of non-gonadal somatic tissues, as reduction or ablation of gonadal function in flies often extends lifespan and increases lipid stores rather than decreasing lifespan and fat storage. Recent studies in wild-type flies have also demonstrated an important link between TE activity and longevity, and these studies demonstrating partial rescue of the shortened lifespan in flamenco mutants upon administration of a reverse transcription inhibitor further support this association (Jones, 2016).

Interest in a function for the piRNA pathway in the soma has increased recently as new roles for this pathway are being illuminated. The piRNA pathway's association with tissues that maintain a degree of immortalization similar to that exhibited in the germline is of particular interest. For example, the somatic stem cell niches of Hydra maintain an active piRNA pathway that represses TEs, possibly contributing to this organism's remarkably long lifespan. These studies, together with the current findings, suggest that the presence of a piRNA pathway in normal somatic tissues may offer an additional cellular defence against TE reactivation and possible somatic genomic damage. The finding of a role for the piRNA pathway in preserving metabolic homeostasis and the overall health of the fly suggests the potential importance of the piRNA pathway in other somatic tissues. Finally, interventions specifically augmenting the piRNA pathway may provide significant benefits to maintaining genomic integrity, tissue function and healthy lifespan (Jones, 2016).

Nuclear RNA export factor variant initiates piRNA-guided co-transcriptional silencing

The PIWI-interacting RNA (piRNA) pathway preserves genomic integrity by repressing transposable elements (TEs) in animal germ cells. Among PIWI-clade proteins in Drosophila, Piwi transcriptionally silences its targets through interactions with cofactors, including Panoramix (Panx) and forms heterochromatin characterized by H3K9me3 and H1. This study identified Nxf2, a nuclear RNA export factor (NXF) variant, as a protein that forms complexes with Piwi, Panx, and p15. Panx-Nxf2-P15 complex formation is necessary in the silencing by stabilizing protein levels of Nxf2 and Panx. Notably, ectopic targeting of Nxf2 initiates co-transcriptional repression of the target reporter in a manner independent of H3K9me3 marks or H1. However, continuous silencing requires HP1a and H1. In addition, Nxf2 directly interacts with target TE transcripts in a Piwi-dependent manner. These findings suggest a model in which the Panx-Nxf2-P15 complex enforces the association of Piwi with target transcripts to trigger co-transcriptional repression, prior to heterochromatin formation in the nuclear piRNA pathway. These results provide an unexpected connection between an NXF variant and small RNA-mediated co-transcriptional silencing (Murano, 2019).

Transposable elements (TEs) act as endogenous mobile mutagens to alter the sequence and structure of the genome, thereby changing the transcriptome and chromatin structure. TEs are often deleterious to the host by, for example, disrupting a gene, but may also be adaptive and drive genome evolution. TEs comprise nearly half of the human genome and approximately 30% of the genome of Drosophila melanogaster; thus, the successively arising families of TEs are the main drivers of genome expansion. In metazoans, TEs are silenced by the piRNA pathway, in which Piwi proteins are guided by Piwi-interacting RNAs (piRNAs) to their targets. The piRNA pathway is also essential for germline development and fertility in animals (Murano, 2019).

In the Drosophila ovary, two cytoplasmic PIWI paralogs, AGO3 and Aubergine (Aub), engage an amplification loop termed the 'ping-pong cycle' to cleave both TE transcripts and long piRNA precursor transcripts arising from piRNA clusters, which comprise a large number as well as various types of fragmented TEs, leading to the post-transcriptional silencing of TEs and production of piRNAs. These piRNAs can in turn trigger the production of phased piRNAs from piRNA precursors, which generates a dazzling variety of piRNAs and is coupled to the activity of a third Piwi protein. Phased piRNAs are also produced in ovarian somatic cells by a ping-suggests the independent mechanism, which are in turn also loaded onto Piwi. This loading initiates the transport of Piwi into the nucleus where it drives the transcriptional silencing of target TEs, by inducing specific histone modification and/or facilitating the folding of chromatin into a higher order structure (Murano, 2019).

Lack of the nuclear Piwi activity results in de-repression of TEs, which is concomitant 6 with decreases in both H3K9me3 repressive epigenetic marks and RNA polymerase II 7 (Pol II) occupancy on target TE loci This suggests a scenario of how the nuclear piRNA pathway works: Piwi and its bound piRNAs scan for target TEs by complementary base-pairing with their nascent transcripts. Upon targeting, Piwi recruits chromatin factors including H3K9me3 methyltransferases such as Eggless (also called dSetDB1) to initiate heterochromatin formation. The H3K9me3 modification is then bound by HP1a to maintain and propagate epigenetic silencing. However, thus far, direct association of the Piwi-piRNA complex with target transcripts and/or H3K9me3 methyltransferases has not been demonstrated (Murano, 2019).

Furthermore, depletion of Maelstrom (Mael), a Piwi cofactor in the nuclear piRNA pathway, does not decrease H3K9me3 levels at Piwi-targeted TE loci, suggesting that the repressive histone mark per se is not the final silencing mark for transcriptional gene silencing mediated by Piwi-piRNA complexes. Recently, linker histone H1 was identified as a component of a nuclear Piwi complex; depletion of H1 de-represses Piwi-targeted TEs and their surrounding genes without affecting the density of H3K9me3 marks and HP1a at target TE loci at target TE loci. Instead, it was demonstrated that the chromatin accessibility at Piwi-targeted TE loci is modulated by H1. These findings suggested a model in which Piwi recruits H1 and forces it to stay on target TE loci to induce chromatin compaction, thereby repressing target TE transcription and that the nuclear piRNA pathway adopts collaborated actions of H1 and H3K9me3 mark to maintain silencing of the TE state by modulating the chromatin state (Murano, 2019).

In addition to Mael and H1, biochemical and genetic analyses have also identified a number of putative Piwi cofactors including DmGTSF1/Asterix (Arx) and Panoramix (Panx)/Silencio. These findings together suggest that multiple pathways leading to Piwi-mediated TE silencing may exist, and raise the question of whether these pathways are independently initiated or have a common initial event (Murano, 2019).

To investigate this issue, Panx, which interacts with Piwi and promotes the deposition of H3K9me3 marks on target TE chromatin by H3K9me3 histone methyltransferase, Eggless, was reinvestigated. This study biochemically isolated Nxf2, nuclear RNA export factor (NXF) variant, as a component of Panx-associated complexes. Nxf2 further associates with p15 (also called Nxt1), a co-adaptor for nuclear RNA export. The NXF family comprises four members in Drosophila, among which Nxf1 is an essential mRNA nucleocytoplasmic export factor. However, other members (Nxf2-4) are gonad-specific and their functions are not known, although they share common domain structures with Nxf1. Detailed analysis of Nxf2 function in the nuclear piRNA pathway revealed that the interactions between Panx, Nxf2, and p15 are necessary to maintain the protein stability of Nxf2 and Panx. Moreover, ectopic targeting of Nxf2 initiates co-transcriptional repression of the target reporter gene prior to heterochromatin formation, and H3K9me3 marks and H1 are required at later time points. Notably, the RNA binding domain of Nxf2 is essential for recruitment of the complex to target TEs. CLIP experiments demonstrated that both Piwi and Nxf2 directly interact with Piwi-targeted TE transcripts and that the association of Nxf2 with target TE transcripts is Piwi-dependent. These results suggest that Nxf2 enforces the association of Piwi-Panx-Nxf2-p15 (PPNP) complexes with the nascent transcripts of target TEs and triggers co-transcriptional repression in the nuclear silencing pathway (Murano, 2019).

A model is proposed in which the engagement of the PPNP complex with nascent TE transcripts initiates co-transcriptional silencing prior to heterochromatin formation. Although the heterochromatin factors are not required for the initial step of the silencing, they play a significant role in maintenance of the repressed status in the later stages of TE silencing. Because the depletion of Piwi activity rapidly results in the de-repression of TEs, this also suggests that silencing initiation and heterochromatin formation undergo continuous cycling to enforce a silenced state of TEs (Murano, 2019).

Four groups have obtained the similar findings regarding the essential role of Nxf2 in the Piwi-piRNA pathway rather than in nuclear RNA export. This studies have identified a new complex of Panx-Nxf2-P15, which associates with Piwi. Although two studies did not observe significant amount of Piwi peptides in their shotgun MS analysis of Panx immunoprecipitants, they showed the interaction between Panx-Piwi in immunoprecipitation followed by Western blot analysis. The inconsistency could be due to the immunoprecipitation conditions and/or antibodies used. Additionally, the UBA domain of Nxf2 associates with Panx, and the NTF2 domain associates with p15. The first LRR domain with RNA binding activity is needed for the regulation of TEs, and direct association of TE transcripts with Nxf2. Among these four studies, this is the only one which was able to observe the initial steps of the silencing. This is because this analysis was carried out using cultured cell line OSCs, and different timepoints were taken after expression of Nxf2. The present study is the only one to consider time points as early as 48 h. The regulation observed at 96 h after transfection, representing later stages of TE silencing, was consistent with the observation by one of the four studies. An important issue to be clarified is the mechanism by which the PPNP complex initiates the silencing of TEs (Murano, 2019 and references therein).

In C. elegans, NRDE-2 (nuclear RNAi defective-2) associates with the Argonaute protein NRDE-3 in the nucleus and is recruited by the NRDE-3/siRNA complex to nascent transcripts. This complex inhibits Pol II during the elongation and directs the accumulation of Pol II at genomic loci targeted by RNAi. This study has investigated whether λN-Nxf2 directs the accumulation of Pol II at boxB sites in the co-transcriptional silencing. Although the time course of Pol II occupancy upon λN-Nxf2 was examined by ChIP-qPCR at the boxB sites, no accumulation of Pol II was seen along the reporter gene (Murano, 2019).

Recently, it was reported that Pol II-associated proteins, PAF1 and RTF1, antagonize Piwi-directed silencing, which are factors involved in the modulation of promoter release, elongation, and termination of Pol II. The Panx-Nxf2-P15 complex might interfere with Pol II via the PAF1 complex, considering that fission yeast PAF1 represses AGO1/siRNA-directed silencing. Although this study examined the effect of PAF1 and RTF1 on the co-transcriptional silencing, PAF1 and RTF1 do not appear to be involved in the co-transcriptional silencing mediated by λN-Nxf2. These results suggest that Pol II regulation by the PPNP complex differs from that of the small RNA-mediated Pol II regulation model proposed previously. It may be necessary to identify key factors involved in this regulation, possibly by performing proteome analysis of the unknown factors tethered to the reporter recruitment system (Murano, 2019).

A decrease was found in the active histone H3K4me3 marks on the reporter gene even at an earlier time point (48 hpt) at which the level of H3K9me3 marks remained low. Two reports about H3K4 methylation in Piwi-mediated silencing have been published. The level of H3K4 methylation on TEs is increased upon Piwi-KD. The expression of artificial piRNAs that target a reporter locus induced transcriptional silencing associated with a decrease in the active H3K4-methylation marks. In addition, the depletion of LSD1, which removes H3K4-methylation marks from promoters, had significant effects on the ability of Panx to repress the reporter gene. Therefore, this study performed LSD1-KD and examined the effect of LSD1 on co-transcriptional silencing by the enforced tethering of Nxf2. However, the depletion of LSD1 had a limited impact on the silencing, whereas the level of mdg1 was increased 27-fold upon LSD1-KD. These findings together suggest that the decrease observed in H3K4me3 levels at 48 hpt in the tethering assay may not have been due to LSD1 (Murano, 2019).

Nxf2 is the core factor of the PPNP complex, and the current results suggest that the nuclear export factor variant has been co-opted to repress TEs in the Piwi-piRNA pathway. It is speculated that the co-option of Nxf2 into TE silencing is not coincidental but may reflect previous TE adaptation to exploit cellular mRNA transport pathways promoting the export of TE transcripts and replication/transposition. Why is it necessary for Piwi to use the RNA binding activity of Nxf2 to silence their targets? Piwi, as a member of the Argonaute family, is thought to search for targets through random interactions between Piwi-piRNA and transcripts in the ocean of the transcriptome, many of which likely show partial complementarity with Piwi-loaded piRNAs. To distinguish 'scanning Piwi' and 'target-engaged Piwi', the silencing pathway may need an engaged-in system to repress only targets and avoid unnecessary silencing of other cellular transcripts. It is possible that Nxf2 plays a role in supporting the association of Piwi that has found its bona fide targets. Panx preferentially associates with Piwi, which loads piRNAs targeting TEs, suggesting that there may be a system for Panx-Nxf2 to form the PPNP complex, specifically with Piwi that is engaged with its target TEs. CLIP analysis shows that Nxf2 cannot associate with target TEs without the activity of Piwi, indicating that the Panx-Nxf2-P15 complex itself does not recognize target transcripts, but rather recognizes target-engaged Piwi. In line with this, while piRNA-targeted Piwi can repress the reporter gene, λN-tethered Piwi cannot. This may be due to the Panx-Nxf2-p15 complex recognizing only piRNA-directed target-engaged Piwi, but not scanning or λN-tethered Piwi. Further studies will be necessary to confirm this speculation and elucidate the precise mechanism by which the Panx-Nxf2-P15 complex selectively recognizes only the 'target-engaged Piwi' and forms a PPNP complex to induce silencing of its targets (Murano, 2019).

The nascent RNA binding complex SFiNX licenses piRNA-guided heterochromatin formation

The PIWI-interacting RNA (piRNA) pathway protects genome integrity in part through establishing repressive heterochromatin at transposon loci. Silencing requires piRNA-guided targeting of nuclear PIWI proteins to nascent transposon transcripts, yet the subsequent molecular events are not understood. This study has identified SFiNX (silencing factor interacting nuclear export variant), an interdependent protein complex required for Piwi-mediated cotranscriptional silencing in Drosophila. SFiNX consists of Nxf2-Nxt1, a gonad-specific variant of the heterodimeric messenger RNA export receptor Nxf1-Nxt1 and the Piwi-associated protein Panoramix. SFiNX mutant flies are sterile and exhibit transposon derepression because piRNA-loaded Piwi is unable to establish heterochromatin. Within SFiNX, Panoramix recruits heterochromatin effectors, while the RNA binding protein Nxf2 licenses cotranscriptional silencing. These data reveal how Nxf2 might have evolved from an RNA transport receptor into a cotranscriptional silencing factor. Thus, NXF variants, which are abundant in metazoans, can have diverse molecular functions and might have been coopted for host genome defense more broadly (Batki, 2019).

The discovery of SFiNX, a nuclear protein complex consisting of Panoramix, Nxf2, and Nxt1/p15, provides the key molecular connection between Piwi, the nascent target RNA, and the cellular heterochromatin machinery. In the absence of SFiNX, piRNA-loaded Piwi is incapable of inducing co-transcriptional silencing. Conversely, experimental recruitment of SFiNX to a nascent RNA independently of Piwi results in potent silencing and local heterochromatin formation. The data indicate that within SFiNX, Panoramix, but not Nxf2, provides the molecular link to the downstream cellular heterochromatin effectors. Based on genetic experiments, the histone methyl-transferase SetDB1/Eggless, the histone-demethylase Lsd1/Su(var)3-3, and the heterochromatin binding protein HP1/Su(var)205 are required for piRNA-guided co-transcriptional silencing (Sienski, 2015; Yu, 2015). None of these factors were found enriched in a SFiNX co-IP mass-spectrometry experiments. This suggests a transient or regulated molecular interaction between Panoramix and the cellular heterochromatin machinery. In this respect, the recent finding that the SUMOylation machinery is critically involved in piRNA-guided co-transcriptional silencing (Ninova, 2019) is of considerable interest (Batki, 2019).

The involvement of Nxf2, a nuclear RNA export variant, in cotranscriptional silencing was surprising. Based on biochemical and structural data, it is proposed that two molecular features of the ancestral NXF protein, the principal mRNA export receptor Nxf1/Tap, facilitated the evolutionary exaptation of Nxf2 into piRNA-guided silencing. First, Nxf2 retained its ability to bind RNA, thereby providing SFiNX a molecular link to the nascent target RNA. Second, crosslink-mass spectrometry data indicate that, similar to Nxf1/Tap, the RNA binding activity of Nxf2 might be gated. It is proposed that this could provide a critical regulatory switch to ensure that SFiNX only associates with transcripts that are specified as targets via the Piwi-piRNA complex. In the case of Nxf1/Tap, various proteins (e.g. SRproteins, THO-complex, UAP56, Aly/Ref) that are recruited during co-transcriptional mRNA maturation, are required to restrict Nxf1/Tap deposition onto export competent mRNAs only. Whether any of these factors is also required for loading Nxf2 onto RNA is currently unclear. It is, however, likely that target-engaged Piwi contributes a critical role in the deposition of SFiNX onto the target RNA, potentially by licensing Nxf2’s RNA binding activity. It is noted, that despite considerable experimental efforts it was not possible to establish a direct molecular link between Piwi and either Nxf2 or Panoramix. Considering that Piwi represses transcription of its targets, the steady state level of a Piwi-SFiNX complex is expected to be small. In support of this, piRNA-independent recruitment of Piwi to a nascent RNA is incapable of target silencing, indicating that the Piwi-SFiNX interaction occurs only once Piwi is bound to a target RNA via a complementary piRNA. An intriguing result from the Nxf2 CLIP experiments is that Nxf2 binds nascent, Piwi-repressed RNAs also far away from piRNA complementary sequences. One possible explanation for this is that Nxf2 or SFiNX, after initial Piwi-mediated recruitment, spread without Piwi along the target RNA. Alternatively, SFiNX might form oligomers in vivo, resulting in multiple RNA binding domains per complex that could tether SFiNX to distant parts of the target transcript. Such an ‘entangling’ of the target RNA at the transcription locus might serve an additional critical role, namely to keep the target RNA at chromatin, thereby providing sufficient time for the recruited effectors to modify chromatin at the locus (Batki, 2019).

Although no direct ortholog of Nxf2 is identifiable in vertebrates, the finding that an NXF variant is involved in transposon silencing in Drosophila could point to a more general scheme. The Nxf1/Tap ancestor diversified through several independent evolutionary radiations into numerous NXF variants in different animal lineages. In flies, the three NXF variants exhibit gonad-specific expression with Nxf2 and Nxf3 being expressed predominantly in ovaries, and Nxf4 being testis-specific. Besides Nxf2 (this study), Drosophila Nxf3 is also an essential piRNA pathway component as it is required for the nuclear export of un-processed piRNA cluster transcripts in the germline (ElMaghraby, 2019). In mice and humans, several NXF variants are preferentially expressed in testes. Intriguingly, Nxf2 mutant mice are male sterile, a phenotype shared with many piRNA pathway mutants. Considering this, it is speculated that also in vertebrates the host-transposon conflict has been a key driver of NXF protein family evolution through frequent duplication and exaptation events. This study highlights that some of these variants might have evolved novel functions, not directly related to RNA export biology (Batki, 2019).

piRNA-guided co-transcriptional silencing coopts nuclear export factors

The PIWI-interacting RNA (piRNA) pathway is a small RNA-based immune system that controls the expression of transposons and maintains genome integrity in animal gonads. In Drosophila, piRNA-guided silencing is achieved, in part, via co-transcriptional repression of transposons by Piwi. This depends on Panoramix (Panx); however, precisely how an RNA binding event silences transcription remains to be determined. This study shows that Nuclear Export Factor 2 (Nxf2) and its co-factor, Nxt1, form a complex with Panx and are required for co-transcriptional silencing of transposons in somatic and germline cells of the ovary. Tethering of Nxf2 or Nxt1 to RNA results in silencing of target loci and the concomitant accumulation of repressive chromatin marks. Nxf2 and Panx proteins are mutually required for proper localization and stability. This study mapped the protein domains crucial for the Nxf2/Panx complex formation and showed that the amino-terminal portion of Panx is sufficient to induce transcriptional silencing (Fabry, 2019).

The data identify Nxf2 and Nxt1 as critical mediators of co-transcriptional gene silencing, acting in concert with Panx to repress loci in response to Piwi-piRNA target engagement. The emerging model for piRNA-dependent silencing implies that target recognition by Piwi is necessary to recruit the PICTS complex onto the appropriate nascent RNA targets. Difficulties in detecting stable interactions between Piwi and PICTS components in vivo may arise from a requirement for Piwi target engagement to licence it for recruitment of silencing complexes, as has been suggested previously. The same mechanism may underlie the difficulties experienced in observing Piwi on its target loci by ChIP (Fabry, 2019).

Panx and Nxf2 are interdependent for their protein stability and proper subcellular localization, underscoring the fact that correct assembly of the PICTS complex is essential for TGS, while the silencing capacity, per se, resides in Panx. Of note, previous work reported a partial destabilization of Nxf2 in cells depleted of Nxt1, potentially extending the interdependency to all three proteins. RIP-seq experiments from unperturbed cells found transposon RNAs enriched only with Panx, as reported, but not with Nxf2, possibly due to low substrate availability combined with an insensitive assay. These results are consistent with another recent report that did not detect transposon enrichment in Nxf2 CLIP-seq from wild-type cells. However, two other studies identified transposon mRNA association with Nxf2 in CLIP-seq experiments upon depletion of the previously described TGS factor, Mael, or by using a stable cell line and depletion of endogenous Nxf2 . Considered together, these data suggest that Nxf2 might be important for stabilizing the binding of Panx to nascent RNAs. However, precisely how Nxf2 executes this function remains to be fully elucidated. Of note, Murano found that Panx interacts with Nxf2, Piwi, Mael and Arx, which could imply that other TGS factors come into contact with the PICTS complex, although the relationship between these factors and PICTS requires further investigation (Fabry, 2019).

Mutational analyses suggest that Panx and Nxf2 must normally bind Nxt1 to carry out transposon repression. Direct recruitment of any of the PICTS complex components to RNA reporters results in robust chromatin silencing. Upon tethering to DNA, however, Panx induces potent TGS, whereas Nxf2 leads to less prominent effects and Nxt1 shows no silencing capability in these assays. Interestingly, recruitment of the amino-terminal part of Panx alone is necessary and sufficient to induce reporter repression, pinpointing this domain of Panx as harboring the silencing effector function. Future investigations will be crucial to uncover the molecular mechanism by which the Panx amino terminus instructs the downstream chromatin silencing machinery (Fabry, 2019).

This work, and that of others indicates that piRNA-guided co-transcriptional silencing of transposons has coopted several components of the RNA export machinery, namely Nxf2 and Nxt1. Of the four NXF proteins present in flies, only two have thus far been characterized. Interestingly, while Nxf1, acting along with Nxt1, is crucial for canonical mRNA export, Nxf2 has been coopted by the piRNA pathway and functions in co-transcriptional gene silencing. Nxf3, which also is required for transposon repression in germ cells, is emerging as being critical for the export of piRNA precursors generated from dual-strand clusters in the germline. The role of Nxf4, whose expression is testis-specific, is yet to be established. This remarkable functional diversity of NXF family members correlates with tissue-specific expression patterns, and seems conserved in mammals. However, deciphering how each achieves substrate specificity will be critical to understanding how these homologs can be exclusively dedicated to different targets and confer different fates upon the RNAs that they bind (Fabry, 2019).

Importantly, the fate of the nascent transcript that is detected by Piwi and instructed by PICTS for silencing remains unclear. One hypothesis is that instead of being exported, these targets undergo degradation by the nuclear exosome. Such mechanism would be contrary to yeast, where the TREX complex subunit Mlo3 was shown to oppose siRNA-mediated heterochromatin formation at gene loci, and suggests that different lineages have evolved different silencing mechanisms. In any case, it is possible that a single transcript from a locus that is marked for silencing might pose a lesser threat than an unsilenced locus and, therefore, not be capable of exerting evolutionary pressure for the detemination of its fate (Fabry, 2019).

Piwi-piRNA complexes induce stepwise changes in nuclear architecture at target loci. EMBO J: e108345

PIWI-interacting RNAs (piRNAs) are germline-specific small RNAs that form effector complexes with PIWI proteins (Piwi-piRNA complexes) and play critical roles for preserving genomic integrity by repressing transposable elements (TEs). Drosophila Piwi transcriptionally silences specific targets through heterochromatin formation and increases histone H3K9 methylation (H3K9me3) and histone H1 deposition at these loci, with nuclear RNA export factor variant Nxf2 serving as a co-factor. Using ChEP and DamID-seq, this study has uncovered a Piwi/Nxf2-dependent target association with nuclear lamins. Hi-C analysis of Piwi or Nxf2-depleted cells reveals decreased intra-TAD and increased inter-TAD interactions in regions harboring Piwi-piRNA target TEs. Using a forced tethering system, the functional effects were analyzed of Piwi-piRNA/Nxf2-mediated recruitment of piRNA target regions to the nuclear periphery. Removal of active histone marks is followed by transcriptional silencing, chromatin conformational changes, and H3K9me3 and H1 association. These data show that the Piwi-piRNA pathway can induce stepwise changes in nuclear architecture and chromatin state at target loci for transcriptional silencing (Iwasaki, 2021).

A model is proposed where Piwi-piRNA-mediated TE regulation of targets is linked to changes in nuclear localization, chromatin conformation, and histone modifications. Interestingly, this occurs in a stepwise manner, where localization and removal of active histone marks is followed by repressive histone marks and chromatin conformational change. These results suggest that nuclear small RNA-mediated regulation modifies nuclear architecture at the genomic level (Iwasaki, 2021).

A general conundrum in this field is whether nuclear positioning controls gene expression or vice versa. There is some evidence that transcription can counteract lamina interactions and modulate Hi-C contacts. The relocation of piRNA target TEs after Piwi- or Nxf2-KD could be a secondary effect of transcriptional activation. Meanwhile, slight de-repression of piRNA target TEs upon the depletion of lamin proteins may suggest a possible defect in transcription upon disruption of lamina association. Defects in transcriptional regulation and nuclear localization upon Piwi-KD occur prior to changes in H3K9me3 histone marks and chromatin conformation. In C. elegans, heterochromatin regions with H3K9me3 histone marks are anchored to perinuclear regions. This suggests that in the Piwi-piRNA pathway, deposition of H3K9me3 at a later step reinforces the association of chromatin regions with perinuclear regions. Indeed, Piwi or Nxf2 knockdown, which decreases the Lamin DamID-seq signal at Piwi-piRNA target regions, also results in a major decrease in H3K9me3 levels. These results suggest that lamina localization may both be the cause and result of TE repression. Nuclear peripheral localization may affect gene expression to some extent, and the regulation of gene expression may be enforced due to its localization to the nuclear periphery (Iwasaki, 2021).

It is proposed that the Piwi-piRNA pathway triggers spatial regulation of genomic regions in order to repress transposable elements. In other words, Piwi-piRNA system may be able to send target regions to specific positions within the nucleus in order to efficiently regulate their expression. In OSCs, piRNAs are generated and loaded onto Piwi protein at the cytoplasmic Yb body. Therefore, target gene localization to the nuclear periphery may facilitate regulation by Piwi-piRNAs imported from the cytoplasm. Within the nucleus, Piwi-piRNA functions together with Panx-Nxf2-p15 at the nascent transcript, in order to silence target TEs. Recent studies have suggested that Panx-Nxf2-p15 complex forms condensates upon Piwi-piRNA-mediated silencing (Eastwood, 2021; Schnabl, 2021). Condensate formation is coupled to dimerization of Panx-Nxf2-p15 complex, and piRNA target regions may therefore be spatially regulated upon silencing. Moreover, it has been proposed that condensate formation is stimulated by nascent RNA produced during early steps in transcription initiation. By analogy with these observations, nuclear localization and chromatin conformational changes observed in this study may also be linked to condensate formation and possibly form a spatial structure for Piwi-piRNA regulation (Iwasaki, 2021).

Although Piwi-piRNA regulation affects nuclear localization of target chromatin regions, the effect on TAD boundaries was limited, and the effect mainly observed as changes in intra- and inter-TAD interactions. In line with this observation, it has been reported that even depletion of nuclear lamins does not result in modulation of TAD boundaries in Drosophila or mouse ES cells. This suggests that changes in nuclear localization of target chromatin regions are not directly linked to modulation of TAD boundaries, but Piwi-piRNA regulation affects expression levels of genes that are localized within the same TAD as piRNA target TEs. This is consistent with the fact that Piwi-piRNA regulation regulates not only expression, histone modifications, and chromatin accessibility of its target TEs, but also their neighboring regions (Iwasaki, 2021).

Recent studies have revealed that heterochromatin structure is dynamic and relatively large molecules can move between both heterochromatin and euchromatin regions. In addition, some essential genes have been reported to be encoded and transcribed in heterochromatin. The Piwi-piRNA system, which is reversible and can be reversed upon loss of Piwi proteins or specific piRNAs, may contribute to this dynamic regulation of heterochromatic regions. Since Piwi proteins and piRNAs are specifically expressed in the germline, it may be the case that the Piwi-piRNA system has come to regulate nuclear architecture in these inheritable populations. It is tempting to speculate that gene expression and nuclear localization of chromatin is designed to account for TE regulation by Piwi-piRNAs. Inversely, TEs may even function as 'functional motifs' in order to rewire nuclear architecture. The dynamics of heterochromatin regulation by the Piwi-piRNA pathway therefore provides insight into complex gene regulation controlled by TEs and even impacts the genome at the level of nuclear architecture (Iwasaki, 2021).

A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal

The Drosophila piwi gene is required for the asymmetric division of germ-line stem cells (GSCs) that takes place during the process of gametogenesis. GSCs are the source of gametes during gametogenesis. The process may be likened to an assembly line one can follow, from the raw material of the GSCs to the egg and sperm 'finished product'. Before a consideration of piwi, a brief review of gametogenesis is in order.

In Drosophila, germ line stem cells are present at the apical tip of the ovariole, the functional unit of the ovary. In the ovariole, GSCs are located in a specialized structure called the germarium, the structure in which the egg develops from germ line stem cells. In each germarium, two to three GSCs contact the somatic basal terminal filament cells. GSCs undergo oriented asymmetric divisions producing two daughter cells -- one is a daughter stem cell, which remains associated with the terminal filament; the other is a differentiated daughter, the cystoblast, which is displaced one cell away from the terminal filament. The germ-line cyst, derived from the cystoblast, is then enveloped by follicle cells that are produced by somatic stem cells to form an egg chamber, which buds off the germarium, joins pre-existing egg chambers in a linear array to constitute the ovariole, and will eventual develop into a mature egg. This assembly line organization, with each egg chamber representing a differentiated stem cell product whose position along the ovariole corresponds to its birth order, provides a unique opportunity for the study of stem cell division (Cox, 1998 and references).

Although piwi is expressed in the germ line, this expression is not involved in regulation of stem cell division. Instead, piwi expression in somatic cells is involved in this essential function. piwi expression in the germ line provides a maternal component for embryogenesis. Although Drosophila piwi is required for the asymmetric division of GSCs into daughter GSCs and daughter cystoblasts, piwi is not essential for the further differentiation of the cystoblast. The self-renewing ability of GSCs is controlled both by extrinsic signaling and by cell-autonomous mechanisms. piwi is not required cell autonomously in the germline, but piwi expression in adjacent somatic cells regulates GSC division (Cox, 1998).

piwi mutant ovaries contain a normal number of GSCs at the onset of oogenesis (late third instar larval stage); however, the mutation produces an equal or somewhat smaller number of gametes in the adult gonads, and they no longer contain GSCs (Lin, 1997). This failure of germ-line maintenance could be due to the following: (1) the differentiation of GSCs without self-renewing divisions; (2) a defect in the asymmetry of GSC division, producing aberrant germ cells that eventually degenerate; and/or (3) a secondary defect influenced by abnormal ovary differentiation. To examine whether the failure of germ-line maintenance is a secondary defect due to abnormal ovary development, the ovarian morphology of piwi mutants was examined. Mutant ovaries show normal morphology at the third instar larval stage. Their germ-line cells are normal in number and are correctly positioned along the medial plane of the ovary. Moreover, the expected number of terminal filaments are forming, so that at the pupal stage, the ovary differentiates normally, partitioning GSCs and their daughter cells correctly into individual germaria and ovarioles. GSCs are able to divide several times to provide a normal complement of germ cells to the germarium. Yet, mutant GSCs subsequently fail to continue self-renewing divisions, and the existing germ-line cysts often degenerate during the late pupal stage so that the adult ovarioles contain germaria lacking germ lines and fewer egg chambers than expected. These observations suggest that the failure of GSC maintenance in piwi mutants is not a secondary defect due to abnormal ovary development (Cox, 1998).

The main oogenic defect in piwi mutants is the differentiation of GSCs without self-renewing divisions immediately following the initiation of oogenesis. At this stage in wild-type ovaries, GSCs in 16-23 newly formed germaria have initiated asymmetric divisions to generate multiple developing germ-line cysts. However, in mutant ovaries, GSCs and their immediate differentiated daughters, cystoblasts, are undetectable, as indicated by the absence of spectrosome-containing germ cells (see Spectrin for the definitions of spectrosome and fusome). Instead, most ovaries contain differentiated germ-line cysts whose number approximately equals that of GSCs. These cysts are much larger in size and contain two- to multicell stage fusomes, indicating their differentiating state. By the adult stage, most ovarioles contain only two egg chambers, either normal or abnormal, derived from these cysts, but no other germ-line cells. This defect contrasts with the development of wild-type ovaries, in which ovarioles contain a fully developed germarium and a stage 1 egg chamber by the 48-hr pupal stage and have produced multiple egg chambers by the adult stage. This observation indicates that the mutant GSCs have differentiated into germ-line cysts without self-renewing divisions (Cox, 1998).

piwi is expressed in the somatic germarial terminal filament cells apical to the GSCs, in somatic follicle cells that surround the ovary, and in the germ-line cells that constitute the ovary. Theoretically, piwi could be required in either the germ-line cells or somatic cells for asymmetic cell division of the germ-line stem cells. To examine the roles of somatic and germ-line piwi in GSC asymmetric division, genetic clonal analyses were conducted on piwi¹ and piwi² mutations. Removing the piwi⁺ chromosome from the germ line allows oogenesis, including GSC division, to occur normally. Thus, the requirement of piwi for GSC division does not reside in the germ line but in somatic cells. To determine whether piwi is required in somatic cells outside the ovary for GSC maintenance, wild-type germaria were transplanted into the abdominal cavity of homozygous piwi mutant females. Transplanted germaria produce a normal number egg chambers after 7 days of incubation in mutant hosts. Thus wild-type germaria continue oogenesis at a normal rate in the piwi mutant females. Hence, piwi is not required in extra-ovarian cells for GSC maintenance. By inducing mitotic recombination at the second and third instar larval stages, piwi minus somatic clones were generated throughout ovarioles, with some egg chambers completely covered by piwi mutant follicle cells. These egg chambers developed normally, indicating that piwi function is not required in follicle cells for egg chamber development. Because follicle cells are derived from their precursor cells in region II of the germarium, this suggests that piwi is not required in somatic cells from germarial region II for GSC division and ovarian development. These results, together with the fact that piwi is expressed in the terminal filament cells at the tip of the germarium, suggest that piwi is required in these somatic cells in the anterior-most tip of the germarium to regulate GSC division (Cox, 1998).

Findings of piwi homologs in plants, C. elegans and vertebrates, and studies of their functions in various organisms suggest that piwi is involved in an evolutionarily conserved stem cell self-renewing mechanism. Among this class of genes, the significantly higher homology between piwi and human hiwi as compared with that between piwi and C. elegans prg-1/prg-2 (prg stands for piwi-related gene) suggests that hiwi function is closer to piwi. Consistent with this, GSC division and gametogenesis in humans are much more similar to the same processes in Drosophila than those in C. elegans, whose gonads contain syncytial mitotic germ-line nuclei that divide symmetrically and are capable of self-renewing only as a population. RNA interference (RNAi) experiments, which are known to mimic the effects of gene mutation, were carried out in C. elegans. RNAi by prg-1 double stranded RNA causes germ-line depletion similar to that in piwi mutants. This suggests that the piwi-mediated mechanism in germ-line self-renewal is conserved even in this evolutionarily distant organism without stereotypic GSCs. The conservation of the piwi-mediated mechanism appears to extend to the plant kingdom as well. The overall homology between Piwi and ZWILLE (ZLL) and AGO of Arabidopsis thaliana is worth noting. Intriguingly, ZLL is essential for maintaining stem cells of the shoot meristem in an undifferentiated state during the transition from embryo-specific development to repetitive organ formation through the self-perpetuating shoot meristem divisions (Moussian, 1998). AGO also plays an important role in maintaining normal apical shoot meristem function (Bohmert, 1998). Thus, the homology between piwi and ZLL and AGO further suggests the existence of a family of novel genes essential for stem cell division in diverse organisms (Cox, 1998).

Given the functional conservation of the piwi family genes between distant species such as Drosophila, C. elegans, and Arabidopsis, it is tempting to speculate that the piwi-mediated mechanism is also conserved in mammals and humans less distant from Drosophila. Given that C. elegans and Drosophila are separated by >1000 million years of evolution, and human and Arabidopsis are even further apart, this functional conservation could reflect the existence of a very ancient mechanism for stem cell maintenance and proliferation in a multicellular ancestor. The conserved piwi mechanism may mediate cell-cell interactions. In Drosophila, genetic mosaic and piwi expression analyses together suggest that piwi function is required in the apical nonmitotic somatic cells to control GSC division. Similarly, in A. thaliana, it is thought that ZLL is required to maintain the undifferentiated state of shoot meristem stem cells by relaying positional information, possibly by mediating cell-cell interactions within the center of the shoot meristem (Moussian, 1998). Further analysis of Piwi should shed light on this evolutionarily conserved cell-cell interaction mechanism (Cox, 1998).

Specific association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome

In Drosophila, Piwi (P-element-induced wimpy testis), which encodes a protein of the Argonaute family, is essential for germ stem cell self-renewal. Piwi has recently been shown to be a nuclear protein involved in gene silencing of retrotransposons and controlling their mobilization in the male germline. However, little is known about the molecular mechanisms of Piwi-dependent gene silencing. This study shows that endogenous Piwi immunopurified from ovary specifically associates with small RNAs of 25-29 nucleotides in length. Piwi-associated small RNAs were identified by cloning and sequencing as repeat-associated small interfering RNAs (rasiRNAs) derived from repetitive regions, such as retrotransposon and heterochromatic regions, in the Drosophila genome. Northern blot analyses revealed that in vivo Piwi does not associate with microRNAs (miRNAs) and that guide siRNA was not loaded onto Piwi when siRNA duplex was added to ovary lysate. In vitro, recombinant Piwi exhibits target RNA cleavage activity. These data together imply that Piwi functions in nuclear RNA silencing as Slicer by associating specifically with rasiRNAs originating from repetitive targets (Saito, 2006).

To investigate the molecular function of Piwi in gene silencing, monoclonal antibodies were produced against the protein. As an antigen, the N-terminal region of Piwi was used that shows much less similarity to the other members of the fly Argonaute family. Western blotting using the Piwi-specific antibody revealed that Piwi is strongly expressed in the ovary and in the early embryo, but later in development the protein levels in the embryo decrease. Piwi was not detected in Schneider 2 (S2) cell lysate. To examine the expression pattern of Piwi in fly testis, ovary, and embryo, immunofluorescent staining was carried out. In ovarioles, Piwi was found to be clearly accumulated in nuclei of both GSCs and somatic cells such as terminal filament cells (TFCs), cap cells, and follicle cells. piwi mRNA is detected in TFCs, and myc-tagged Piwi protein is found in both somatic and germline cells in adult ovary. Relatively low expression was observed in cystoblasts, as in the case of myc-Piwi. In embryos, Piwi was found accumulated in nuclei of pole cells. In testis, Piwi appeared to be expressed in the hub, a tiny cluster of post-mitotic somatic cells localized at the apical tip of the testis and in cyst progenitor cells. The hub functions in the maintenance of GSC identity and influences its behavior, while Piwi is required for the asymmetric division of GSCs to produce and maintain a daughter GSC but is not essential for further differentiation of committed daughter cells. Piwi seems not to be expressed in male germline stem cells (GSCs). This observation agrees well with the Western data showing that the relative amount of Piwi in testis is quite low (Saito, 2006).

Immunoprecipitation was performed from an ovary lysate using the anti-Piwi antibody. Silver staining of the immunoprecipitate failed to reveal any prominent bands copurifying with Piwi under physiological conditions, although some weak bands were perceived. Western blotting confirmed that the discrete band of ~92 kDa was Piwi itself. Whether immunoprecipitated Piwi associates with any small RNAs was investigated. Total RNAs were isolated from the immunopurified Piwi complex, labeled with ³²P-pCp, and separated on a denaturing acrylamide gel. As a control, the AGO1 complex—an essential factor in miRNA-mediated gene silencing—was also immunopurified from the ovary, and miRNAs associated with AGO1 were simultaneously visualized. The Piwi-associated small RNAs migrated slightly slower than miRNAs. Compared with the size markers, Piwi-associated small RNAs were estimated to be 25–29 nt in length. Northern blot analysis using probes specific for miRNAs (miR-1 and miR-310) clearly revealed that the Piwi-associated small RNAs are not miRNAs (Saito, 2006).

To identify the Piwi-associated small RNAs, they were cloned and sequenced. Of 800 clones sequenced, 392 hit to the Drosophila genomic sequences in databases. The largest class of cloned RNAs (84.1%) was rasiRNAs, originally found in a small RNA profiling study in Drosophila embryo and testis. Breakdown products of rRNA, tRNA, mRNA, and snoRNA were also obtained (6.6%, 0.2%, 4.8%, and 4.1%, respectively). It is noteworthy that no miRNAs were obtained in this experiment. Among the rasiRNAs (330 clones), ~40% (130 out of 330) matched to various kinds of transposable elements, including LTR (long terminal inverted repeat) retrotransposons and LINE (long interspersed nuclear elements)-like elements. Those LINE-like elements include Het-A and TART, both known to transpose only to the chromosome ends to maintain telomere length. Another 60% (200 out of 330) corresponded to heterochromatic regions in the genome. By looking at the nucleotide sequences of all the clones, it was noticed that the Piwi-associated rasiRNAs include both the sense and the anti-sense of a transposable transcript, although sense (+) orientation clones tended to be less abundant than those with an antisense (–) orientation, and that the first nucleotide at the 5' end was predominantly U (~78%) . In addition, the Piwi-associated rasiRNAs were cloned by the procedure originally developed for miRNA cloning, indicating that they contain 5' phosphates and 2',3'-hydroxyl termini. These characteristics suggested that the Piwi-associated rasiRNAs are produced from dsRNA precursors by an RNase III domain-containing enzyme. In some cases, such as stalker4, rasiRNAs are only found in an antisense (–) orientation. However, where the orientation bias of rasiRNA loading onto Piwi comes from is unknown and requires further investigation (Saito, 2006).

To confirm that rasiRNAs associated with Piwi indeed involve both the sense and antisense of a transcript, Northern blotting analysis was performed. roo rasiRNA was chosen for examination because it was one of most abundant clones obtained (11 out of 300) and in both sense and antisense orientations. Both sense and antisense roo rasiRNAs were detected in the immunopurified Piwi complex as expected. Neither was found in the AGO1 complex, indicating that the Piwi association with rasiRNAs is specific. roo rasiRNA that is derived from the roo antisense transcript (hereinafter, referred to it as roo anti-rasiRNA) seems more abundant in the Piwi complex than the roo rasiRNA that was derived from the roo transcript in the sense orientation. This was determined from the fact that the signal of roo anti-rasiRNA in the anti-Piwi lane is about threefold stronger than that of roo anti-oligo (10 fmol), whereas the signal of roo rasiRNA is a little less (~85%) compared with that of roo sense oligo (10 fmol). This observation correlated well with the fact that less roo rasiRNA than roo anti-rasiRNA was obtained in the cloning experiment. The Piwi–rasiRNA association was further verified by Northern blotting on total RNAs isolated from Piwi and AGO1 immunopurified complexes in ovary. rasiRNAs originating from copia and TART retrotransposon transcripts, and one of the repetitive elements located in the centromeric heterochromatin region, Responder, were detected only in Piwi but not in AGO1 complexes, demonstrating that the association with rasiRNAs is specific to Piwi. Whether Piwi was able to associate with siRNA was also investigated. luc siRNA duplex was first incubated in ovary lysate. Piwi and AGO2 complexes were then immunopurified from the lysate using specific antibodies to each protein. Northern blotting revealed that guide siRNA was specifically loaded onto AGO2 but not onto Piwi. These data strongly indicate the high specificity of Piwi association with rasiRNAs. It is suggested that a complex for loading rasiRNAs specifically onto Piwi exists; as the Dicer1/ Loquacious (R3D1) and Dicer2/R2D2 complexes load, after processing, mature miRNA and guide siRNA onto AGO1 and AGO2, respectively (Saito, 2006).

Using GST-tagged recombinant full-length AGO1 (GST-AGO1), it has been shown that AGO1 is able to cleave target RNA completely complementary to miRNA. In this study, full-length GST-Piwi was produced and the activity was examined in an in vitro target RNA cleavage assay. Interestingly, it was noticed that in the alignment of the Piwi domains of Piwi, AGO1, and AGO2, the D–D–H (Asp–Asp–His) motif, shown to be essential for Slicer activity of human Ago2, was not well conserved, and that the third residue was replaced with Lys. Thus, it was suspected that Piwi may not exhibit Slicer activity. However, it was observed that GST-Piwi was able to cleave luc target RNA when it was preincubated with luc guide siRNA (21 nt; luc 21) as well as GST-AGO1. By searching for peptide sequence similarity over species, it was noticed that the D–D–K triad is conserved in a member of the Argonautes in Drosophila pseudoobscura and one of the Piwi domain-containing proteins in Tetrahymena thermophila (Saito, 2006).

For several years, it has been thought that Piwi is involved in gene silencing of LTR retrotransposons since (1) Piwi is one of the members of the fly Argonaute family of proteins, and (2) piwi mutations cause higher expression levels of retrotransposon transcripts in vivo. Links between protein factors involved in RNAi and the silencing of endogenous transposable elements have also been made in other species like C. elegans and Chlamydomonas reinhardtii. However, evidence at molecular levels to support such a Piwi function has not hitherto been reported. This study is the first to show that Piwi is specifically associated with rasiRNAs in vivo and that Piwi has Slicer activity. The data suggest a novel, third kind of gene silencing pathway in Drosophila, following two distinct gene silencing pathways mediated by AGO1-miRNAs and AGO2-siRNAs. The existence of rasiRNAs in Drosophila has been demonstrated previously through investigation of small RNA expression profiling in testis and embryo. In that study, it was mentioned that rasiRNAs show some characteristics that suggest involvement of an RNase III domain enzyme in rasiRNA processing. In this study, it was observed that Piwi-associated rasiRNAs also show the same characteristics as those found in another study. Which protein factor(s) then produces rasiRNAs? Dicer1, Dicer2, and Drosha were not detected in Piwi complex immunopurified from ovary. Although it may be due to the epitope hindering by Piwi and processing factor association, it is possible that a yet-to-be unidentified protein(s) other than Dicer1, Dicer2, and Drosha might be the crutial factor(s) for the rasiRNA-producing process (Saito, 2006).

In fly testis, Piwi seems not to be expressed in GSCs, but its expression is clearly observed in the hub, the somatic cell cluster that functions to maintain GSC identity and influence its behavior. It is known that piwi¹ causes male infertility due to severe defects in spermatogenesis; thus, the necessity of the Piwi function in the hub for such processes is quite apparent. Although it also will be important to determine if Piwi is associated with rasiRNAs in the testis, these findings have set a new stage for understanding how Piwi functions in the formation and maturation of GSCs in both ovary and testis (Saito, 2006).

Piwi maintains germline stem cells and oogenesis in Drosophila through negative regulation of Polycomb group proteins

The Drosophila melanogaster Piwi protein regulates both niche and intrinsic mechanisms to maintain germline stem cells, but its underlying mechanism remains unclear. This study reports that Piwi interacts with Polycomb group complexes PRC1 and PRC2 in niche and germline cells to regulate ovarian germline stem cells and oogenesis. Piwi physically interacts with the PRC2 subunits Su(z)12 and Esc in the ovary and in vitro. Chromatin coimmunoprecipitation of Piwi, the PRC2 enzymatic subunit E(z), histone H3 trimethylated at lysine 27 (H3K27me3) and RNA polymerase II in wild-type and piwi mutant ovaries demonstrates that Piwi binds a conserved DNA motif at ∼72 genomic sites and inhibits PRC2 binding to many non-Piwi-binding genomic targets and H3K27 trimethylation. Moreover, Piwi influences RNA polymerase II activities in Drosophila ovaries, likely via inhibiting PRC2. The study hypothesizes that Piwi negatively regulates PRC2 binding by sequestering PRC2 in the nucleoplasm, thus reducing PRC2 binding to many targets and influencing transcription during oogenesis (Peng, 2016).

Antagonistic roles between Nibbler and Hen1 modulate piRNA 3' ends in Drosophila

In eukaryotes, aberrant expression of transposable elements is detrimental to the host genome. Piwi-interacting RNAs of approximately 23 to 30 nucleotides (nt) bound to PIWI-clade Argonaute proteins silence transposqons strictly dependent on their sequence complementarity. Hence, a key question in understanding piRNA pathways is to determine mechanisms that modulate piRNA sequences. This study identified a protein-protein interaction between Nibbler (Nbr), a 3'-to-5' exoribonuclease and Piwi, linking Nbr activity with piRNA pathways. A delicate interplay occurs between Nbr and Hen1, a methyltransferase involved in 2'-O-methylation at 3' terminal nucleotides of piRNAs, connecting two genes with opposing activities in biogenesis of piRNA 3' ends. With age, piRNAs become shorter and less, coupled with de-repression of select TEs. Activities of nbr and hen1 inherently contribute to TE silencing and age-dependent profiles of piRNAs. It is proposed that antagonistic roles between nbr and hen1 define a mechanism to modulate piRNA 3'ends (Wang, 2015).

Reassessment of Piwi binding to the genome and Piwi impact on RNA Polymerase II distribution

Drosophila Piwi was reported by Huang (2013) to be guided by piRNAs to piRNA-complementary sites in the genome, which then recruits heterochromatin protein 1a and histone methyltransferase Su(Var)3-9 to the sites. Among additional findings, Huang (2013; see below) also reported Piwi binding sites in the genome and the reduction of RNA polymerase II in euchromatin but its increase in pericentric regions in piwi mutants. Marinov (2015) disputed the validity of the Huang bioinformatic pipeline that led to the last two claims. This study reports an independent reanalysis of the data using current bioinformatic methods. This reanalysis agrees with Marinov (2015) that Piwi's genomic targets still remain to be identified but confirms the Huang's claim that Piwi influences RNA polymerase II distribution in the genome. This Matters Arising Response addresses the Marinov et al. (2015) Matters Arising, published concurrently in this issue of Developmental Cell (Lin, 2015).

REGULATION

The exon junction complex controls transposable element activity by ensuring faithful splicing of the piwi transcript

The exon junction complex (EJC) is a highly conserved ribonucleoprotein complex that binds RNAs during splicing and remains associated with them following export to the cytoplasm. While the role of this complex in mRNA localization, translation, and degradation has been well characterized, its mechanism of action in splicing a subset of Drosophila and human transcripts remains to be elucidated. This study describes a novel function for the EJC and its splicing subunit, RnpS1, in preventing transposon accumulation in both Drosophila germline and surrounding somatic follicle cells. This function is mediated specifically through the control of piwi transcript splicing, where, in the absence of RnpS1, the fourth intron of piwi is retained. This intron contains a weak polypyrimidine tract that is sufficient to confer dependence on RnpS1. Finally, this study demonstrates that RnpS1-dependent removal of this intron requires splicing of the flanking introns, suggesting a model in which the EJC facilitates the splicing of weak introns following its initial deposition at adjacent exon junctions. These data demonstrate a novel role for the EJC in regulating piwi intron excision and provide a mechanism for its function during splicing (Malone, 2014).

The exon junction complex is required for definition and excision of neighboring introns in Drosophila

Splicing of pre-mRNAs results in the deposition of the exon junction complex (EJC) upstream of exon-exon boundaries. The EJC plays crucial post-splicing roles in export, translation, localization, and nonsense-mediated decay of mRNAs. It also aids faithful splicing of pre-mRNAs containing large introns, albeit via an unknown mechanism. This study shows that the core EJC plus the accessory factors RnpS1 and Acinus aid in definition and efficient splicing of neighboring introns. This requires prior deposition of the EJC in close proximity to either an upstream or downstream splicing event. If present in isolation, EJC-dependent introns are splicing-defective also in wild-type cells. Interestingly, the most affected intron belongs to the piwi locus, which explains the reported transposon desilencing in EJC-depleted Drosophila ovaries. Based on a transcriptome-wide analysis, it is proposed that the dependency of splicing on the EJC is exploited as a means to control the temporal order of splicing events (Hayashi, 2014).

Piwi and transgene silencing

Two types of transgene silencing were found for the Alcohol dehydrogenase (Adh) transcription unit. Transcriptional gene silencing (TGS) is Polycomb dependent and occurs when Adh is driven by the white eye color gene promoter. Full-length Adh transgenes are silenced posttranscriptionally at high copy number or by a pulsed increase over a threshold. The posttranscriptional gene silencing (PTGS) exhibits molecular hallmarks typical of RNA interference (RNAi), including the production of 21-25 bp length sense and antisense RNAs homologous to the silenced RNA. Mutations in piwi, which belongs to a gene family with members required for RNAi, block PTGS and one aspect of TGS, indicating a connection between the two types of silencing (Pal-Bhadra, 2002).

Despite the fact that posttranscriptional silencing appears to be a matter of RNA metabolism, some indications of chromatin modification of the homologous endogenous gene under certain circumstances have emerged. (1) Transgene copies of viral genes present in the nucleus only become methylated upon infection of the plant by the homologous virus, which has a double-stranded RNA genome and which does not enter the plant nucleus. (2) In plants and rodent cells, the introduction of DNA constructs into cells can trigger the RNA degradation reaction. (3) Mutations in Arabidopsis selected for reduced DNA methylation, ddm1, a SWI2/SNF chromatin component, and met1, the major DNA methyltransferase, will relieve gene silencing, including a stochastic reversal of posttranscriptional silencing. (4) Transgene arrays in the C. elegans germline are desilenced and appear less condensed in mutant backgrounds for mut-7 and rde-2, which are both required for RNAi. (5) The ectopic transcription of promoter sequences or their introduction to the plant cell in a virus will trigger transcriptional silencing of another reporter construct in the same cell with a homologous promoter, which also becomes hypermethylated. Evidence is presented for a link between posttranscriptional and transcriptional modes of gene silencing in Drosophila (Pal-Bhadra, 2002 and references therein).

The silencing of the promoter-reporter construct white-Alcohol dehydrogenase (w-Adh) and full-length Adh transgenes occurs in Drosophila. The w-Adh effect is modulated by mutations in the Polycomb group (Pc-G) of chromatin-repressive proteins, and the silenced transgenes are associated with the Pc-G complex, whereas single highly expressed copies are not. The endogenous Adh gene is drawn into the silencing pool and is capable of further extending the effect to an Adh-w construct, which has no portion in common with w-Adh but which does share homology with the endogenous Adh 5' sequences. The silenced Adh-w construct also accumulates the Pc-G complex. Deletion of the endogenous Adh gene eliminates the silencing interaction between the two nonhomologous, reciprocally constructed transgenes, w-Adh and Adh-w (Pal-Bhadra, 2002 and references therein).

The full-length Adh transgene silencing operates posttranscriptionally as determined by nuclear run-on transcription assays that directly assess the distribution of transcriptionally-engaged Pol II. In contrast, the w-Adh and Adh-w silencing is transcriptional. The posttranscriptional silencing of Adh correlates with the appearance of 21-25 bp sense and antisense RNAs as occurs with virus and PTGS silencing in plants and RNAi in flies (Pal-Bhadra, 2002).

Mutations have been recovered in C. elegans that are defective for RNAi. Homologs to these genes exist throughout the plant and animal kingdoms. One of these mutations, rde1, has several related gene family members in flies. A homozygous viable member of this group was tested for its impact on transgene silencing. The piwi mutation drastically reduces the magnitude of posttranscriptional silencing. Surprisingly, piwi also had a strong impact on the transcriptional silencing of Adh-w by w-Adh. This result indicates that under certain circumstances the two types of silencing can be mechanistically related (Pal-Bhadra, 2002).

When one to five copies of full-length Adh transgenes are introduced in the genome, the steady-state RNAs accumulate in direct correlation with copy number. However, at higher dosage (six to ten), Adh RNA levels depart dramatically from a linear relationship with transgene copy number. The transgene analyzed contains all the Adh sequences required for normal function. Five single insert strains showing minimal positional effects were selected. By combining these insertions via genetic crosses, a series of Adh stocks was generated that carry one to ten copies. Each stock is also homozygous for the endogenous transformation recipient allele, Adh^fn6. This allele is defective at the 3' splicing acceptor of the first intron, resulting in a longer transcript. The endogenous allele produces only 5%-10% of the steady-state level of a normal Adh gene (Pal-Bhadra, 2002).

To determine whether the endogenous gene was silenced in concert with the transgenes, an RNase protection assay was used that distinguishes the two RNAs. The normal full-length Adh RNA protects two fragments, 142 and 160 bp, while the Adh^fn6 RNA protects a 355 bp fragment due to defective splicing. To correct for loading differences, a ß-tubulin probe was included, which protects a 70 bp fragment. The level of each protected fragment (142/70 or 160/70 ratio) was reduced at higher dosage. The amount of endogenous Adh^fn6 transcripts did not show any significant difference in one to five copies, suggesting an equal expression. However, these transcripts followed a similar trend as those from the transgenes at higher dosage (Pal-Bhadra, 2002).

To test for any positional influence on Adh transgene silencing, separate sets of one to nine copy stocks were examined. A similar level of Adh expression at any one dosage suggests that the silencing is not significantly affected by the insertion sites. RNA in situ analyses in embryos the Adh^fn6 strain, as well as with one, five, and seven Adh transgene copies, indicates a linear increase of Adh expression to five copies. Silencing of the seven copy stock was already evident during blastoderm, the stage where Adh RNA is accumulated initially (Pal-Bhadra, 2002).

To determine whether the Adh transgene silencing is transcriptional or posttranscriptional, nuclear run-on assays were performed. ß-tubulin was included as an internal control in order to determine the relative amount of Adh transcription, while lacZ acted as a negative control (Pal-Bhadra, 2002).

Transcription levels were estimated in adult flies carrying zero to ten copies of full-length Adh transgenes. The results reveal that the endogenous Adh^fn6copies, present in each stock, are transcribed similarly to two normal copies. In the one to ten copy series, the amount of transcription increases proportionally to the transgene copy number relative to ß-tubulin (Pal-Bhadra, 2002).

A similar experiment was performed using flies that contain selected genotypes from zero to six copies of the w-Adh hybrid constuct. In the absence of transgenes, the Adh^fn6 allele is transcribed at the normal level. When one copy of the w-Adh transgene is present, the transcript level is increased as expected. In contrast, two copies of w-Adh exhibit a plateau of transcript accumulation. In the presence of more w-Adh copies (four to six), total Adh transcription is reduced progressively. The transcribed RNA in four to six copy stocks is below the level produced by the Adh^fn6 alleles alone. The amount of Adh transcription in males is always greater than in females with equal dosage (Pal-Bhadra, 2002).

Adh transcriptional level (as detected using run-on assays), produced by multiple full-length Adh or w-Adh transgenes, was compared to steady-state mRNA levels produced by the same genotypes, as determined in Northern analyses. In the Adh series, the amount of mRNA is proportional to dosage from one to five, while in six to ten copies, the steady-state levels depart from linearity. This difference from the run-on assays indicates that silencing of full-length Adh transgenes is posttranscriptional. On the contrary, with the w-Adh dosage series, a similar curve was found when the transcription level and steady-state RNA were compared. A parallel pattern of reduction of both nascent and mature RNA suggests that w-Adh silencing, which includes an effect upon the endogenous Adh gene, is transcriptional. The reason for the different modes of silencing of the two types of transgenes is not known (Pal-Bhadra, 2002).

The silencing of w-Adh and the endogenous Adh locus can also be extended to an Adh-white (Adh-w) transgene that is the reciprocal construct of w-Adh. Run-on transcription analysis of the Adh-w transgene in a w deficiency background with varying numbers of w-Adh indicates that this type of silencing also occurs on the transcriptional level. A comparison of the run-on data with a previous Northern analysis of Adh-w with increasing w-Adh copies shows a similar relationship. The Adh-w + 1 w-Adh and Adh-w + 4 w-Adh genotypes have nearly identical levels of reduction in gene expression in the two assays. There is a residual amount of transcription in the Adh-w + 2 w-Adh genotype but no detectable RNA in the Northern analysis, which might suggest that a combination of PTGS and TGS is operating. However, these data points lie near the limit of detection for the two techniques and likely differ by chance due to exposure time because the same genotype assayed subsequently by both methods shows a similar low level of expression. Thus, at the developmental stage examined (adults), the silencing is predominantly, if not exclusively, transcriptional (Pal-Bhadra, 2002).

Biochemical experiments on RNA interference (RNAi) in Drosophila have shown that double-stranded RNAs in the form of 21-25 nt fragments are generated and these fragments are used in the sequence-specific degradation of mRNA. Therefore, the in vivo existence of such RNAs was tested in the case of Adh silencing. The 21-25 nt antisense Adh RNAs were strongly accumulated in the stocks that contain six to ten copies of the Adh transgene. At the lower doses, the same RNA was not detected or was present at very low amounts in the four and five copy stocks. Using an antisense probe for the detection of sense fragments, a similar-sized RNA was found in the silencing doses (Pal-Bhadra, 2002).

Whether transcriptional silencing induced by w-Adh transgenes was associated with small RNAs was tested by analyzing stocks containing zero to six copies of w-Adh. Hybridization with sense or antisense Adh probes revealed that low molecular weight RNA was not found in abundance at any w-Adh dosage (Pal-Bhadra, 2002).

To investigate the nature of PTGS, it was reasoned that an induced hsp70-Adh construct with four Adh transgenes would bring the Adh expression over the silencing threshold. The hsp70-Adh construct contains a heat shock promoter and the Adh reporter gene. Adh RNA from adult flies was measured relative to ß-tubulin controls in three different circumstances: no heat, heat shock (37°C for 45 min), and 20 hr after heat shock. In flies with five full-length Adh copies, the amount of RNA in heat shock and 20 hr after heat shock did not change relative to that of the untreated flies (Pal-Bhadra, 2002).

The heat treatment of adult flies carrying two copies of the hsp70-Adh gene increased their RNAs as expected. In contrast, the heat treatment of a stock that carries four copies of the Adh transgene and in addition an hsp70-Adh gene causes a rapid loss of Adh mRNA. A similar response was found in embryos. The heat shock induced increase in Adh RNA appears to surpass the threshold limit that triggers silencing. A time course sampling of RNA after heat shock showed that Adh mRNA levels were partially recovered 20 hr subsequent to heat treatment. Interestingly, the transient silencing in this case is in contrast to the systemic spread and prolonged silencing that occurs in C. elegans and plants following a localized induction of posttranscriptional silencing (Pal-Bhadra, 2002).

Whether pulsed threshold induced RNA degradation is correlated with the small species of Adh RNA was tested. The small RNAs were found at a high level in flies with the combination genotype after heat shock. Treatment of the control stock composed only of two hsp70-Adh copies does not produce the small RNAs. This result suggests a threshold level must be exceeded to initiate the synthesis of the small RNAs, which in turn participate in the destruction of the homologous mRNA (Pal-Bhadra, 2002).

Because Polycomb and Polycomb-like (Pcl) mutations have a significant effect on w-Adh transcriptional silencing and Pc-G proteins are bound at the sites of the transgenes under silencing conditions, binding of Pc-G proteins at the Adh transgene insertion sites was tested cytologically. Those sites that do not overlap (1CD and 53B) normal positions of Pc labeling were examined for evidence of binding in the single insert stocks and for the same insert in the ten copy larvae. The Pc proteins were not observed at these sites in either case. Therefore, Pc protein recruitment is not a consequence of posttranscriptional silencing of Adh (Pal-Bhadra, 2002).

A group of genes has been characterized that affects PTGS or RNAi in fungi, plants, and animals. Some of the mutations show sequence similarity to members of the piwi/sting/eIF2C/argonaute gene family conserved from plants to vertebrates. Members of this family, including piwi, have been characterized in Drosophila . To determine whether this mutation has any effect on PTGS and TGS of Adh, the role of this mutation on posttranscriptional silencing was tested. Two or four copies of fully functional Adh genes (two normal endogenous copies ± two transgenes) plus a hsp70-Adh transgene in combination with either heterozygotes or homozygotes of piwi were examined. In the stocks analyzed, a normal Adh allele, rather than Adh^fn6, resides on both the balancer chromosome present in heterozygotes and on the chromosome carrying the piwi mutations. These endogenous Adh copies contribute greater amounts of mRNA to the total pool than Adh^fn6 (Pal-Bhadra, 2002).

The heat shock and nonheat shock RNAs of the respective stocks were compared in quantitative Northern blots. The stocks that are heterozygous for the piwi ¹ or piwi ² alleles with two copies of the normal Adh gene plus hsp70-Adh exhibit an increase of RNA after heat incubation. In contrast, a sharp reduction of the Adh RNA was found in the five copy (four Adh + hsp70-Adh) stock following heat shock. In piwi homozygotes, the presence of two endogenous genes + hsp70-Adh has a similar pattern of expression as in heterozygotes following heat shock. However, the Adh mRNA level in the four Adh + hsp70-Adh stocks that are homozygous for piwi alleles is restored to nearly normal after heat shock. An almost equal level of Adh expression in nonheat shock and heat shock flies suggests that the piwi mutation disrupts the threshold-based posttranscriptional Adh silencing. The two separate piwi alleles examined as well as their heteroallelic combination, which was used to minimize any influence of linked modifiers, produced the same results (Pal-Bhadra, 2002).

Examination of 21-25 nt RNAs in the piwi-segregating classes shows that the small RNAs are present in the heat shocked class with four Adh genes plus a hsp70-Adh construct when the flies are heterozygous for piwi. In contrast, these RNAs are diminished in the piwi homozygotes that carry the same constellation of Adh genes and that were subjected to heat shock. This result suggests that piwi acts before or during the formation of these small RNAs (Pal-Bhadra, 2002).

As a first measure of the effect on transcriptional silencing, the interaction between the reciprocal w-Adh and Adh-w transgenes was examined because the expression of the latter can be observed phenotypically. One copy of w-Adh reduces and two copies nearly eliminate the expression of Adh-w. The w-Adh transgenes among themselves participate in silencing and include endogenous Adh. The w-Adh/Adh-w silencing interaction is eliminated when the endogenous Adh is deleted from the genome, suggesting that it mediates the reaction via mutually homologous sequences. The expression of the Adh-w transgene can be assayed on the RNA level by probing for white RNA because the transgene is present in a stock with the normal white gene deleted. In the same RNA populations, the silencing of w-Adh and its effect on endogenous Adh can be assayed by probing for total Adh messenger RNA (Pal-Bhadra, 2002).

piwi mutations were introduced into a background with an Adh-w and a w-Adh transgene. The eye color of Adh-w flies, which is reduced in the interaction with one w-Adh copy, is restored to a normal level in piwi¹ or piwi² homozygotes. The heteroallelic combination piwi¹/piwi² shows a similar level of restoration. These combinations of piwi alleles did not have any effect on the eye color of Adh-w in the absence of w-Adh, indicating that their effect is due to a relief of silencing (Pal-Bhadra, 2002).

w mRNA was measured in genotypes with Adh-w and zero to two copies of w-Adh segregating for the piwi alleles. The accumulation of white transcripts in the Adh-w/Y flies without a w-Adh transgene was not significantly different in the presence of the mutations. As expected, the w transcripts from Adh-w were reduced from the normal level in the presence of one copy of w-Adh. However, the presence of the homozygous mutations restored the levels significantly but not completely to the normal amount. Moreover, the w transcripts from Adh-w were significantly restored in mutant homozygotes carrying two w-Adh copies (Pal-Bhadra, 2002).

The same RNA blots were hybridized using an Adh probe to estimate the effect on w-Adh/endogenous Adh silencing. The data revealed that the chromosomes carrying either allele of piwi slightly increased the Adh mRNA in the Adh-w flies without w-Adh copies compared to the balancer chromosomes. The effect of the mutant-bearing chromosome may reflect variation at linked modifiers or at Adh itself. In the Adh-w/Y;w-Adh/+ and Adh-w/Y;w-Adh/w-Adh flies, the total Adh expression is significantly reduced as expected when piwi is heterozygous. This Adh expression is minimally increased in piwi homozygotes, although the increase in these classes is likely due to the variation on chromosome 2, rather than a release from silencing. The magnitude of this slight increase is similar with Adh-w alone and in the presence of one or two w-Adh copies, suggesting that the mutations do not interfere with silencing at this step. These data suggest that piwi has no effect on w-Adh/Adh silencing in contrast to the effect on Adh-w (Pal-Bhadra, 2002).

Run-on analysis was conducted on heterozygous and homozygous genotypes to determine the influence of piwi on the transcriptional silencing of Adh-w. The heteroallelic mutant combination has no impact on Adh-w expression in the absence of w-Adh transgenes. In the heterozygous genotypes, introduction of one or two w-Adh copies causes a progressive reduction of Adh-w expression. The magnitude of reduction in expression in the transcriptional assay is quite similar to the Northern analysis, again indicating that the silencing of Adh-w at the adult stage does not appear to have a posttranscriptional component. In the heteroallelic piwi mutant flies, Adh-w transcription is only slightly diminished by the addition of one or two copies of w-Adh. These results indicate that piwi mutations interfere with the transcriptional silencing of Adh-w (Pal-Bhadra, 2002).

Thus, a single transcription unit, namely Alcohol dehydrogenase, can experience two types of transgene silencing: transcriptional and posttranscriptional. The w-Adh/Adh/Adh-w silencing is transcriptional as might have been anticipated from the involvement of the Polycomb chromatin complex. In contrast, the full-length Adh transgene silencing is posttranscriptional. The molecular features of this silencing follow those established from biochemical studies of RNAi. In other words, the specific loss of Adh messenger RNA is accompanied by the appearance of small sense and antisense 21-25 nt length RNAs (referred to as small interfering RNAs, siRNAs). The appearance of these RNAs and the degradation of Adh messenger RNA require a certain threshold over which the silencing is triggered. When these in vivo data are considered together with in vitro analyses of RNAi, it is reasonable to suggest that at a certain concentration of Adh messenger RNA, a double-stranded RNA moiety is transiently formed, presumably by a form of RNA-dependent, RNA polymerase activity. These molecules would then be cleaved to form the siRNAs by an RNase type III nuclease and subsequently incorporated into a larger RNase complex (RISC) to specifically target the homologous mRNA for enzymatic destruction. It is noted, however, that until further study is performed, it remains a formal possibility that the siRNAs form by an alternative pathway that does not involve double-stranded RNA (Pal-Bhadra, 2002).

The piwi gene is a member of a family including the RNAi defective 1 (rde1) gene of C. elegans, which was isolated for its failure to support RNAi. Family members contain a PAZ domain (Cerutti, 2000) that is characteristic of several gene products involved with RNAi. This study demonstrates that piwi mutation blocks the posttranscriptional silencing of Adh, including the production of siRNAs. In contrast, rde1 does not inhibit siRNA formation during RNAi. Another member of this gene family, the aubergine locus, interferes with the germline silencing of the Stellate genes present on the X chromosome (Aravin, 2001). This silencing must occur for male fertility and is accomplished by repeated genes on the Y chromosome that generate siRNAs in conjunction with Stellate. The product of another family member, the argonaute2 gene, is associated with the RISC complex. Thus, several members of this gene family have been implicated in PTGS at various steps (Pal-Bhadra, 2002).

It was of interest to determine whether genes involved with posttranscriptional silencing might also have an impact on transcriptional silencing. Several lines of evidence from plant research have suggested a connection between silencing via RNA in the cytoplasm and changes in the nucleus, although no previous data in animal species have indicated such a relationship. For example, cDNAs of plant viruses (or other sequences incorporated into the virus) transformed into the nucleus are undermethylated until infection by the corresponding virus. The presence of the virus in the cytoplasm undergoing silencing causes a hypermethylation of the sequences in the nucleus. These DNA modifications are coincident in length with the homologous portion carried in the virus. While the mechanism of this RNA-directed DNA methylation is unknown, it is presumed to involve an RNA-DNA interaction. In addition, ectopic transcription of promoter sequences will cause transcriptional silencing of transgenes with a homologous promoter driving another reporter gene (Pal-Bhadra, 2002).

The piwi mutations inhibit the transcriptional silencing of Adh-w. Two aspects of the Adh-w silencing can be assayed by probing for either white or Adh messenger RNA. The silencing of w-Adh and its effect on endogenous Adh can be monitored by examining the amount of Adh RNA. With increasing dosage of w-Adh, the total Adh RNA declines. This trend is not affected by the mutations. The silencing of Adh-w itself can be determined phenotypically and by measuring the amount of w RNA. Typically, the expression of Adh-w declines with increasing dosage of w-Adh, but this response is strongly diminished in homozygotes of piwi (Pal-Bhadra, 2002).

While the evidence presented here suggests a relationship between posttranscriptional and transcriptional silencing, the basis for this connection is unknown. The step in the silencing of Adh-w that is affected by piwi requires the presence in the nucleus of the 5' regulatory sequences of Adh. These sequences are not known to be transcribed under normal circumstances (Pal-Bhadra, 2002).

There are several possibilities to draw a connection between an RNAi-like mechanism and this transcriptional silencing. First, there may be undetected, transient transcription of the Adh regulatory sequences, which in turn form homologous siRNAs. These may act in the nucleus to trigger a chromatin change at the complementary regulatory regions in an analogous fashion to experimental transcription of promoters in tobacco. The piwi mutations might block such a mechanism by inhibiting the formation of the siRNAs homologous to the regulatory sequences. Considering this scenario, in the case of the w-Adh/Adh-w interaction, one must postulate that increased dosage of w-Adh would increase the amount of ectopic transcription of the endogenous Adh regulatory sequences and that the Polycomb complex becomes targeted to Adh-w in conjunction with a homologous interaction between the siRNAs and the regulatory region of Adh-w (Pal-Bhadra, 2002).

If small RNAs trigger transcriptional silencing, it is of interest why w-Adh/Adh silencing is unaffected by piwi. One potential explanation is that the RNA involvement is needed only for the establishment of transcriptional silencing but not its maintenance through to the adult stage. The initiation of w-Adh/Adh silencing in early embryogenesis precedes that of Adh-w. It is possible that maternal contributions of piwi product in early embryos are sufficient for initiation of w-Adh/Adh silencing but are depleted to effective levels by the time of the establishment of Adh-w silencing (Pal-Bhadra, 2002).

Another explanation suggests that the piwi gene product may play dual roles in an RNAi-like mechanism and transcriptional silencing. One possibility is that it could affect some aspect of gene-to-gene association that might trigger transcriptional silencing. Pairing of transgenes or other intranuclear interactions appears to have an impact on transcriptional gene silencing. Indeed, the w-Adh transgene is silenced much more effectively when paired between homologs than when two dispersed copies are present in the nucleus. If the piwi product participates in some aspect of sequence recognition, nucleic acid associations or protein-nucleic acid interactions, its elimination might block certain steps of both posttranscriptional and transcriptional silencing. It remains a possibility that gene-to-gene associations might trigger silencing as well as dsRNA-to-gene interactions with the two acting independently but by a related mechanism. Finally, the connection between the two types of silencing could be indirect, with piwi blocking a function secondarily removed from the transcriptional silencing but that is required nevertheless (Pal-Bhadra, 2002).

Certainly, members of this gene family have diverse roles in the cell. A previously identified function of the nucleoplasmic product of piwi indicates its requirement for germline stem cell renewal. The aubergine product is required for dorsoventral patterning of the early embryo, which is mediated by an enhancement of the translation of the oskar mRNA. The aubergine product is also implicated with the expression of the Stellate (Ste) repeats and Suppressor of Stellate [Su(Ste)] genes on the X and Y chromosomes. The argonaute1 gene is required early in Drosophila embryogenesis for proper development. In mammals, a member of this gene family, eIF2C, functions in translation initiation via tRNA association with messenger RNA, while in Arabidopsis the argonaute mutation affects the functions of the meristem. In C. elegans, Drosophila, and human cells, related genes play a role in the maturation of small regulatory RNAs involved in the temporal control of development. The data presented here suggest that they can also play a direct or indirect role in transcriptional regulation. Clearly, many aspects of development and cellular metabolism use these gene products, raising the possibility that the transposon and virus defense functions may have been co-opted early in evolution from genes involved with other regulatory processes (Pal-Bhadra, 2002).

Regulatory relationship among piwi, pumilio, and bag-of-marbles in Drosophila germline stem cell self-renewal and differentiation

The transition from a Drosophila ovarian germline stem cell (GSC) to its differentiated daughter cell, the cystoblast, is controlled by both niche signals and intrinsic factors. piwi and pumilio (pum) are essential for GSC self-renewal, whereas bag-of-marbles (bam) is required for cystoblast differentiation. This study demonstrate that Piwi and Bam proteins are expressed independently of one another in reciprocal patterns in GSCs and cystoblasts. However, overexpression of either one antagonizes the other in these cells. Furthermore, piwi;bam double mutants phenocopy the bam mutant. This epistasis reflects the niche signaling function of piwi because depleting piwi from niche cells in bam mutant ovaries also phenocopies bam mutants. Thus, bam is epistatic to niche Piwi, but not germline Piwi function. Despite this, bam⁻ ovaries lacking germline Piwi contain approximately 4-fold fewer germ cells than bam⁻ ovaries, consistent with the role of germline Piwi in promoting GSC mitosis by 4-fold. Finally, pum is epistatic to bam, indicating that niche Piwi does not regulate Bam-C through Pum. It is proposed that niche Piwi maintains GSCs by repressing bam expression in GSCs, which consequently prevents Bam from downregulating Pum/Nos function in repressing the translation of differentiation genes and germline Piwi function in promoting germ cell division (Szakmary, 2005).

This study investigates the regulatory relationships between Piwi, Bam, and Pum, three key regulators of GSC versus cystoblast fates. Among them, Pum and Bam are intrinsic factors, whereas Piwi is expressed both in niche cells as an essential component of niche signaling and in GSCs to promote its division. Pum was originally identified as a maternal effect protein that heterodimerizes with Nanos (Nos) to bind and suppress the translation of its target hunchback mRNA in the posterior of the Drosophila embryo. In addition, Pum and Nos have important germline development zygotic roles, including their cell-autonomous function for GSC maintenance. In contrast to this function of Pum and Nos, Bam is necessary and sufficient in promoting GSC differentiation, even though its molecular activity is not known. bam encodes two protein isoforms: the cytoplasmic (Bam-C) and the fusomal (Bam-F) forms, with Bam-C specifically present in cystoblasts and differentiating cysts but absent in GSCs. Finally, Piwi is the founding member of the evolutionarily conserved Piwi protein family (a.k.a. Argonaute family) involved in stem cell division, RNA interference, transcriptional gene silencing, and other developmental processes. In the Drosophila ovarian germline, Piwi is a nuclear protein that is preferentially expressed in GSCs but is only weakly expressed in cystoblasts and mitotic cysts, consistent with its germline function (Szakmary, 2005).

To investigate the regulatory relationship between piwi and bam, the reciprocal expression pattern was confirmed by double immunofluorescence microscopy of wild-type germaria for Piwi and Bam-C. A fully functional myc-tagged Piwi is expressed at high levels in GSCs and is downregulated in cystoblasts and early mitotic cysts. In contrast, Bam-C is absent from GSCs but accumulates in most cystoblasts and mitotic cystocytes in germarial region 1. The downregulation of Piwi coincides with the zone of Bam-C expression. In a few cases, germ cells were observed expressing both Piwi and Bam-C in cystoblast positions. These cells might represent the transitional stage from GSCs to cystoblasts. At a very low frequency, cystoblast-like cells express low levels of Piwi, but no detectable Bam-C. On the basis of piwi;bam double mutant analysis, these cystoblast-like cells are likely to be undifferentiated or potentially apoptotic. Overall, the reciprocal expression pattern of Piwi and Bam-C proteins supports the opposing functions of piwi and bam genes (Szakmary, 2005).

To determine whether this reciprocal expression pattern is a result of mutually negative regulation toward each other's expression, Bam expression was analyzed in piwi mutants and vice versa. Because piwi mutant ovarioles typically contain germaria that are depleted of germline cells, it is difficult to assay Bam-C expression in them. For this reason, piwi¹ GSC clones were generated with the FLP-DFS (flipase-mediated dominant female sterile) technique. Bam-C is expressed normally in cystoblasts and early mitotic cysts in germaria that contain only piwi¹ germline cells. Moreover, no ectopic Bam expression was detected in GSCs. Therefore, proper Bam-C expression in the adult germline during oogenesis does not require piwi⁺ function in the germline. To address whether piwi expression in apical somatic cells affects bam expression in GSCs, Piwi was eliminated in somatic niche cells. This was achieved by using Yb mutations that eliminate Piwi expression specifically in niche cells. Yb mutants are phenotypically very similar to piwi mutants. However, if examined within the first day of eclosion, Yb mutant germaria still contain germ cells. Bam-C expression is unaffected in adult Yb mutant ovaries, suggesting that there is no specific requirement for Yb or Piwi in niche cells for proper Bam-C expression or localization in the germline. Taken together, the above analyses indicate that neither niche nor germline piwi is required for Bam-C expression (Szakmary, 2005).

Whether the absence of Bam affects Piwi expression was examined. A P[myc-piwi] transgene was introduced into a bam null mutant background to monitor Piwi expression. Ovaries were dissected from these females and stained with Myc antibody to monitor the Piwi expression and with Vasa antibodies to label germ cells. Piwi is present in all bam null germline cells. In addition, Piwi is also strongly expressed in apical somatic cells that correspond to terminal filament, cap cells, and inner sheath cells in the wild-type germarium. Therefore, bam⁺ function is dispensable for Piwi expression in the germline and the apical somatic cells (Szakmary, 2005).

Whether piwi or bam negatively regulates the expression of the other was tested. Overexpression of Piwi in apical somatic cells increases the number of GSC-like cells. These ectopic stem cell-like cells fill regions 1 and 2a of the germarium and, thus, displace Bam-C-expressing cells to region 2b. If Piwi and Bam expression are mutually antagonistic, the prediction would be that expanding Bam expression to GSCs would downregulate Piwi expression there during oogenesis. To express Bam-C protein ectopically in GSCs, a heat shock-inducible bam transgene was used that places the bam cDNA under the control of the hsp70 promoter. Flies carrying a P[myc-piwi] and a hs-bam transgene were subjected to heat shock twice daily for 3 days after eclosion. Ovaries were subsequently dissected and stained for Bam-C and Myc to monitor ectopic Bam-C expression and its effects on Piwi expression. As predicted, ectopic expression of Bam in GSCs diminishes Piwi expression specifically in these cells. Interestingly, ectopic Bam expression in both somatic cells and other germline cells within and beyond the germarium has no effect on Piwi expression in these cells. Particularly, Piwi expression in apical somatic cells (i.e., cap cells and inner sheath cells) of the germarium is unaffected by ectopic Bam expression. Thus ectopic Bam expression may specifically downregulate the germline Piwi expression (Szakmary, 2005).

The mutually independent expression of Piwi and Bam does not rule out their regulatory relationship in GSC cell fate, whereas the suppression of piwi in GSCs by ectopic bam expression suggests that these two genes interact antagonistically. To further define the interaction between piwi and bam, females were constructed lacking both piwi and bam function and the double mutants' ovaries were examined. In contrast to the piwi mutant phenotype, in which ovarioles typically contain a germlineless germarium and 2-3 egg chambers, the double mutant ovaries are characterized by 'tumorous' germaria filled with hundreds of undifferentiated germ cells. Moreover, there is no apparent egg chamber development in the double mutant ovary. This phenotype is qualitatively similar to the tumorous phenotype observed in bam mutant ovaries, which can contain up to thousands of undifferentiated germ cells. The piwi;bam double mutant phenotype therefore indicates that bam is epistatic to piwi. Given the opposing functions of piwi and bam, these results suggest that piwi acts upstream of bam to repress its function in promoting GSC differentiation (Szakmary, 2005).

Although the piwi;bam double mutant shows a bam-like phenotype, there is a difference between the defect of the double mutant and that of bam alone. The bam mutant typically contains 300-1000 undifferentiated germ cells, whereas the piwi;bam double mutants contain only 50-300 germ cells. One possible explanation for this difference is the absence of the mitosis-promoting, germline cell autonomous piwi function in the double mutant. The cell autonomous function of piwi in GSCs is to promote mitosis, resulting in a 4-fold increase of mitotic rates. In bam mutants, 'tumorous'germ cells are more mitotic because of the presence of piwi⁺ function, whereas in piwi;bam double mutants, 'tumorous' germ cells are less mitotic because of the absence of piwi⁺ function. Therefore, these analyses suggest that, whereas bam is epistatic to the niche function of piwi, the cell autonomous function of piwi is epistatic to bam (Szakmary, 2005).

To verify the complex epistasis between bam and distinct somatic versus germline functions of Piwi, the effect of specifically removing Piwi protein from either the germline or the somatic niche cells of bam mutants was investigated. The piwi (somatic);bam double mutant was achieved by generating Yb;bam double mutants because Yb specifically eliminates piwi expression in niche cells. The piwi (germline);bam double mutant was achieved by driving transgenic piwi expression specifically in the niche cells of a piwi;bam double mutant background (Szakmary, 2005).

Yb;bam double mutant ovaries display a clear bam phenotype. This phenotype, however, is not as attenuated as in piwi;bam double mutants, but rather appears to be as pronounced as in bam single mutants. This result supports the assumption that the epistasis of bam over piwi reflects the somatic piwi function, and the attenuated bam phenotype of the double mutant reflects the germline cell autonomous piwi function (Szakmary, 2005).

To further verify this hypothesis, the phenotype of piwi (germline);bam double mutants was analyzed. The piwi (germline) mutant was generated with an en-gal4 transgene to drive the expression of piwi^EP to produce specific expression of fully functional Piwi in niche cells. Because piwi^EP is inserted into the piwi locus, it is therefore a piwi mutant allele in the absence of gal4 expression. The en-gal4 piwi¹/piwi^EP transheterozygotes were generated in bam mutant and wild-type backgrounds. The piwi/piwi^EP;bam⁺ ovaries display the expected piwi mutant phenotype. The en-gal4 piwi¹/piwi^EP;bam^Δ86~/TM3 Sb ovaries appear wild-type, aside from a mild reduction in size, and give rise to females capable of laying eggs. This finding directly confirms that Piwi expression in niche cells is sufficient for GSC maintenance, whereas the observed reduction in ovary size may reflect the absence of germline piwi function in promoting GSC mitoses. As expected, the en-gal4 piwi¹/CyO;bam^Δ86/bam^Δ86 flies display typical bam mutant ovarioles. Also as expected, piwi¹/piwi^EP;bam^Δ86/bam^Δ86 and en-gal4 piwi¹/piwi^EP;bam^Δ86/bam^Δ86 ovaries display the phenotypes of piwi (somatic);bam double mutants and the piwi (germline);bam double mutants, respectively. These analyses further verified that bam is epistatic to somatic niche piwi function, yet germline piwi is epistatic to bam function (Szakmary, 2005).

The fact that Piwi expression in somatic cells has a downregulating effect on Bam-C expression in GSCs raises the question of how this signal may be relayed. The reciprocal expression patterns of Piwi and Bam-C in the germline closely resemble those of Pum and Bam-C. Pum maintains GSC self-renewal during oogenesis, whereas Bam promotes GSC differentiation. GSCs are depleted in pum mutants but overproliferate in bam mutants. This raised the possibility that Piwi may exert its functions by acting on Pum. Pum expression was therefore examined in piwi¹ mutants and Piwi expression in pum¹⁶⁸⁸ and pum²⁰⁰³ mutants. The expression of one gene was not detectably altered in the mutant background of the other, suggesting that neither gene regulates the other's expression. However, pum encodes two distinct protein isoforms (156 kDa and 130 kDa). Either isoform is sufficient for maternal function, but both are required for zygotic function, including GSC maintenance. Because pum¹⁶⁸⁸ and pum²⁰⁰³ eliminate the expression of the 156 kDa and 130 kDa Pum isoforms, respectively, these results could suggest that either the 156 kDa or the 130 kDa isoform of Pum alone is sufficient for proper germline Piwi expression. Even if this is the case, the niche expression of piwi is independent of pum because pum is not required somatically to maintain GSCs (Szakmary, 2005).

To more definitively determine the regulatory relationship between Pum and the Piwi-Bam-C pathway, pum,bam double mutants were constructed and analyzed. If somatic Piwi acts through Pum to regulate Bam-C, then pum,bam double mutants should resemble piwi;bam double mutants. This, however, was not the case. In pum^ET1,bam^Δ86/pum^ET9,bam^Δ86 double mutant flies, in which both pum^ET1 and pum^ET9 are null alleles, the majority of germaria were devoid of germ cells. Only a minority of germaria (<10%) contained a number of undifferentiated germ cells with restricted proliferation. This range of defects is indistinguishable from that of the phenotype of typical pum mutant ovaries. These results suggest that pum is epistatic to bam and that the proliferation of germ cells in bam mutants requires Pum function (Szakmary, 2005).

In summary, these results show that somatic niche Piwi function antagonizes Bam-C, which in turn antagonizes Pum and germline Piwi. The niche function of Piwi in downregulating BAM function appears to converge with the Dpp signaling pathway that is also required for GSC maintenance. This is based on three observations. (1) the expression of dpp does not require piwi. Therefore, Dpp is not a downstream signal of piwi. (2) dpp overexpression does not rescue piwi mutant defects. Therefore, Dpp and niche Piwi are functionally parallel. (3) The dpp signaling pathway directly represses bam transcription. Likewise, piwi niche signaling also downregulates bam expression because bam is epistatic over piwi and because overexpression of Piwi in germarial somatic cells causes overproliferation of GSCs and displaces Bam-C expression beyond region 1 and 2a of the germarium. Taken together, these results indicate that these two signaling pathways must converge at some point to regulate Bam function. The convergence point could be in niche cells, where piwi directly affects Dpp signal production by aiding in its modifications, stability, and/or secretion. Alternatively, it could be in GSCs, where Piwi suppresses a Dpp agonist(s) or perhaps even the Bam/Bgcn complex. This scenario would require that Piwi produce an intercellular signal independent of Dpp. At present, these two possibilities cannot be distinguished (Szakmary, 2005).

How does Bam function as a converging target in promoting GSC differentiation? It has been suggested that benign gonial cell neoplasm (Bgcn) is an obligatory partner for Bam-C as a differentiation factor. Bgcn is expressed in GSCs, but not in somatic cells. This may explain why ectopic bam expression only downregulates Piwi in GSCs, but not in somatic cells (Szakmary, 2005).

How is Pum involved in the Piwi-Bam pathway? Piwi and Pum do not affect one another's expression, yet pum is clearly epistatic to bam. This precludes the possibility that Piwi exerts its effect on Bam-C via Pum. The epistasis of pum to bam is best explained by ascribing a translational repressing function of Pum/Nos in the germline toward mRNAs that promote differentiation. This repression is released by Bam/Bgcn. In GSCs, Bam-C is itself transcriptionally silenced; therefore, Pum and Nos are active in suppressing differentiation. In cystoblasts, Bam/Bgcn are expressed, thereby antagonizing Pum/Nos function. This allows differentiation-promoting mRNAs to be translated. Bgcn is related to the DexH-box family of RNA-dependent helicases. Recently, it has been suggested that the majority of RNA helicases function by displacing proteins from RNA strands rather than by unwinding RNA. It is therefore conceivable that the Bam/Bgcn complex displaces Pum/Nos from their target RNAs (Szakmary, 2005).

A model is proposed for switching between self-renewal and differentiation of GSCs in the Drosophila germarium. The niche cells signal to GSCs by secreting Dpp/Bmp and possibly other proteins. The Dpp signal is received by GSCs through its receptors Punt and Thick Veins (TKV), and it is transduced by pMad to silence bam transcription in these cells. This is achieved via the direct binding of Smads to a discrete silencing element in the bam gene. Piwi in niche cells has an essential and cooperative functional involvement in this signal. Piwi and Dpp signaling pathways converge at some point upstream of bam, in either niche cells or GSCs. The absence of Bam allows Pum and Nos to be active, which suppresses the translation of differentiation genes, thus maintaining the stem cell fate. In the cystoblast and differentiating germline cysts, the Dpp signal is no longer effective, thereby relieving the transcriptional repression of bam. The Bam/Bgcn complexes then repress Pum/Nos function, allowing these cells to differentiate. Therefore, Pum/Nos can be considered a switch between self-renewal and differentiation, whereas niche signaling through Bam/Bgcn regulates this switch at a single cell level (Szakmary, 2005).

Gene circuitry controlling a stem cell niche

Many stem cell populations interact with stromal cells via signaling pathways, and understanding these interactions is key for understanding stem cell biology. In Drosophila, germline stem cell (GSC) maintenance requires regulation of several genes, including dpp, piwi, pumilio, and bam. GSCs also maintain continuous contact with cap cells that probably secrete the signaling ligands necessary for controlling expression of these genes. For example, dpp signaling acts by silencing transcription of the differentiation factor, bam, in GSCs. Despite numerous studies, it is not clear what roles piwi, primarily a cap cell factor, and pumilio, a germ cell factor, play in maintaining GSC function. With molecular and genetic experiments, it is shown that piwi maintains GSCs by silencing bam. In contrast, pumilio is not required for bam silencing, indicating that pumilio maintains GSC fate by a mechanism not dependent on bam transcription. Surprisingly, it was found that germ cells can differentiate without bam if they also lack pumilio. These findings suggest a molecular pathway for GSC maintenance. dpp- and piwi-dependent signaling act synergistically in GSCs to silence bam, whereas pumilio represses translation of differentiation-promoting mRNAs. In cystoblasts, accumulating Bam protein antagonizes pumilio, permitting the translation of cystoblast-promoting transcripts (Chen, 2005).

dpp-dependent silencing of bam transcription defines a key -- probably the primary -- mechanism for maintaining GSCs. By repressing bam transcription in the germ cells attached to cap cells, dpp signaling prevents these cells from forming cystoblasts and assigns them as GSCs. It is speculated that all GSC maintenance genes might act by repressing bam transcription and this prediction was tested for piwi and pumilio (Chen, 2005).

Two genetic observations suggested that piwi might negatively regulate bam expression: (1) bam was epistatic to piwi in double mutants, indicating that the piwi GSC-loss phenotype required an active bam gene; (2) Bam coexpression suppressed the formation of extra GSCs induced when piwi was overexpressed. Thus, piwi-dependent GSC formation depends on maintaining low levels of bam expression (Chen, 2005).

Both overexpression and loss-of-function phenotypes could be explained if piwi, like dpp signaling, were necessary to silence bam transcription in GSCs. This possibility was tested by scoring the expression of bam transcriptional reporters in piwi mutant GSCs. Because piwi inactivation causes GSC loss, P{bamP-GFP} reporters were assayed in piwi bgcn (benign gonial cell neoplasm) double mutant flies that preserve GSCs. GSCs lacking bgcn were GFP negative, but GSCs that lacked both piwi and bgcn were GFP positive. Thus, like dpp signaling, piwi⁺ was necessary to silence bam transcription in GSCs (Chen, 2005).

piwi⁺ action in somatic, but not germline, cells is critical for GSC maintenance, and, therefore, piwi must act indirectly to repress bam transcription. A putative piwi target (or targets) in GSCs must integrate with dpp signaling because previous work has established that the Mad:Medea binding site in bam is a sufficient silencer element. Two recent findings drew attention to the E3-ligase Dsmurf as a candidate for a germ cell piwi target: (1) Dsmurf inactivation produces extra GSCs, just as does ectopic piwi expression and (2) Dsmurf suppresses dpp signaling by targeting phosphorylated Mad for degradation (Chen, 2005).

If piwi silences bam transcription by repressing Dsmurf in GSCs, then GSCs might be restored in piwi mutants if Dsmurf were simultaneously removed. Therefore ovaries of piwi Dsmurf double mutant females were examined and it was found that most germaria contained supernumerary GSCs and a continuous supply of egg chambers. It was verified that piwi Dsmurf GSC-like cells behaved as GSCs by noting that they did not express BamC protein. In 62/80 double mutant germaria, no cells expressing BamC were detected, whereas BamC-positive germ cells were detected in 18/80 germaria. In those cases, the most apical cells, in the GSC position, were BamC negative (Chen, 2005).

Dpp signaling and piwi act as GSC maintenance factors by repressing bam transcription. pumilio (pum) is a component of an evolutionarily conserved mechanism of translational control and is also essential for ovarian GSCs. The expression of P{bamP-GFP} reporter was examined in pum mutant germ cells to determine if bam transcriptional silencing also depends on pum⁺. In contrast to piwi, the reporter was properly silenced in pum bam GSCs. For example, GSCs in 84.6% of pum^MSC bam^BG/pum²⁰⁰³ bam^Δ86 germaria were GFP negative. Because pum mutant germ cells are unstable, it was suspected that the few GFP-positive cells in the GSC position had either differentiated or were dying (Chen, 2005).

GSCs required (1) dpp⁺ and piwi⁺ signaling to repress cystoblast (CB) differentiation by silencing bam transcription and (2) pum⁺ to repress CB differentiation by a mechanism that is independent of bam silencing. Because previous work has shown that Pum forms a translational repressor complex with Nanos, it was reasoned that pum⁺ might maintain GSCs by repressing translation of CB-promoting mRNAs. One candidate target mRNA is bam itself, but, because dpp-dependent transcriptional silencing of bam fully accounts for the absence of bam from GSCs, it is unlikely that Pum sustains GSCs by repressing bam translation (Chen, 2005).

The Pum:Nos repressor complex probably blocks translation of other unidentified target mRNAs that are essential for CB differentiation. Cystoblast formation would then depend on relieving this block, and, because bam is both necessary and sufficient to induce CB differentiation, bam might antagonize or bypass translational repression. The phenotypes of double mutants can distinguish between these possibilities. If bam bypasses translational repression, pum bam germ cells would not form CBs and would resemble bam mutant gonads. If, however, bam antagonizes Pum/Nos-mediated translational repression, pum bam germ cells might differentiate (Chen, 2005).

Ovaries formed in various pum and bam genotypes were compared with several alleles of each gene. Double mutant ovaries produced a complex phenotype that was distinct from either single mutant. Staining nuclei with DNA dyes revealed a mixture of apparently undifferentiated cells and overtly polyploid cells. Indeed, in many cases the polyploid chromosomes were also thick and expanded like nurse cell chromosomes. Most remarkably, these pseudo-nurse cells were occasionally organized within an epithelial layer of follicle cells, like a cyst, although these cysts never contained a full complement of 16 cystocytes. Cells with hallmarks of post-CB differentiation occurred only in the pum bam double mutant ovaries, where they were seen in over half the ovarioles scored (see Table S2) (Chen, 2005).

The appearance of pseudo-nurse cells and even cysts suggested that double mutant germ cells had formed functional cystoblasts, remarkably bypassing the requirement for bam⁺ expression. To verify that pum bam germ cells were undergoing differentiation, the double mutant cells were examined with several additional markers of differentiation (Chen, 2005).

Orb is expressed in all germ cells, but its levels increase dramatically in the cystocytes of developing cysts. Orb protein levels remain at very low levels in bam mutant cells. Double mutant cells, however, expressed Orb at levels seen in differentiating cysts and well above the levels in bam cells. Orb accumulation revealed that many of the pum bam germ cells that did not yet have obvious pseudo-nurse cell chromosomes had, in fact, progressed well beyond the "pre-CB" stage of bam cells. Pseudo-nurse cells also had high levels of Orb expression, similar to accumulation seen in developing nurse cells (Chen, 2005).

Ring canal formation is a distinctive feature of germ cell cysts, and the multiple cell pum bam cysts contained ring canals. The incidence of these pum bam cysts was modest but reproducible in all double-mutant combinations, including those containing null alleles of bam and very strong or null alleles of pum. It is suspected that the infrequent appearance of multi-nurse cell cysts is due to a second requirement for bam⁺ to drive cystocyte divisions during cyst formation. This requirement would not have been recognized previously because bam mutations arrested cells as 'pre-CBs' (Chen, 2005).

Although they are not normal, the appearance of these cysts is a striking manifestation that CBs lacking bam could differentiate as long as they also lacked pum. Combined with previous studies showing that ectopic bam expression is sufficient to direct GSC differentiation, the pum bam phenotype strongly suggests that bam acts as a CB-promoting factor by antagonizing, rather than bypassing, pum action. The data suggests further that dpp signaling, which directly regulates bam expression, does not control pum⁺ expression. A similar conclusion has been reached about the relationship between dpp signaling and nanos expression on the basis of studies of primordial germ cell differentiation (Chen, 2005).

A unifying model is proposed to explain the gene circuitry of GSC and CB fate within the GSC niche. The results suggest that Drosophila ovarian GSCs are retained as stem cells because Pum:Nos complexes repress translation of a pool of mRNAs that induce CB differentiation (Chen, 2005).

In wild-type GSCs that contact cap cells, Pum:Nos translational repression remains active because dpp signaling from stromal cells silences bam transcription and thus blocks the formation of Bam:Bgcn complexes that would antagonize Pum:Nos translational repression. Expression of piwi in stromal cells contributes a key, but unknown, signal that stabilizes or strengthens the Dpp response and bam transcriptional silencing (Chen, 2005).

After the GSC divides, the strength of Dpp signaling falls to levels that can no longer efficiently silence bam transcription in the cell displaced to the posterior and away from cap cells. This could be due to declining Dpp levels or diminished piwi-dependent signals that lead to reduced phospho-Mad levels. As bam transcription increases, Bam:Bgcn complexes antagonize Pum:Nos action and cause derepression of CB-promoting mRNAs, initiating the events of CB differentiation. Because pumilio and nanos are evolutionarily conserved proteins, it will be important to determine if a similar 'multiple-negative' circuitry is at work in mammalian stem cell niches (Chen, 2005).

RNAi components are required for nuclear clustering of Polycomb group response elements

Drosophila Polycomb group (PcG) proteins silence homeotic genes through binding to Polycomb group response elements (PREs). Fab-7 is a PRE-containing regulatory element from the homeotic gene Abdominal-B. When present in multiple copies in the genome, Fab-7 can induce long-distance gene contacts that enhance PcG-dependent silencing. Components of the RNA interference (RNAi) machinery are involved in PcG-mediated silencing at Fab-7 and in the production of small RNAs at transgenic Fab-7 copies. In general, these mutations do not affect the recruitment of PcG components, but they are specifically required for the maintenance of long-range contacts between Fab-7 copies. Dicer-2, PIWI, and Argonaute1, three RNAi components, frequently colocalize with PcG bodies, and their mutation significantly reduces the frequency of PcG-dependent chromosomal associations of endogenous homeotic genes. This suggests a novel role for the RNAi machinery in regulating the nuclear organization of PcG chromatin targets (Grimaud, 2006).

The RNAi machinery has been implicated in a wide variety of biological processes. One of these processes is the formation of heterochromatin. In S. pombe, this involves bidirectional transcription of RNA molecules from repetitive sequences and their cleavage into short interfering RNAs (siRNAs) of 21–23 nt by an RNase III enzyme called Dicer-1. siRNAs guide the RNA-induced initiation of transcriptional gene silencing (RITS) complex to homologous sequences in the nucleus (Noma, 2004; Verdel, 2004). Clr4, the homolog of the histone methyltransferase Su(Var) 3-9, is recruited along with the RITS complex to chromatin, where it methylates lysine 9 of histone H3 (H3K9). This epigenetic mark promotes the formation of heterochromatin by recruiting the heterochromatin protein Swi6, the homolog of HP1, via its chromodomain (Grewal, 2004). Consistent with these data, a redistribution of H3K9 methylation has been observed in Drosophila chromosomes in flies mutant for components of the RNAi machinery (Pal-Bhadra, 2004; Grimaud, 2006).

The RNAi machinery is also required for cosuppression, a phenomenon whereby the introduction of multiple transgenic copies of a gene phenocopies its loss of function instead of increasing its expression. In Drosophila, cosuppression can act at either the transcriptional or posttranscriptional level and involves PcG proteins as well as the RNAi machinery (Pal-Bhadra, 1997, Pal-Bhadra, 1999 and Pal-Bhadra, 2002; Grimaud, 2006 and references therein).

The Drosophila RNAi machinery includes two Dicer proteins encoded by the dicer-1 (dcr-1) and dicer-2 (dcr-2) genes. Dcr-2 is specifically required to process double-stranded RNAs into siRNAs and mediates the assembly of siRNAs into the RNA-induced silencing complex (RISC). Dcr-1 is involved in the metabolism of siRNAs as well as the processing of pre-microRNAs into microRNAs. RNA silencing also involves several highly conserved genes coding for PAZ-domain proteins. Argonaute1 (AGO1) and Argonaute2 (AGO2) are involved in microRNA biogenesis and RNA interference (RNAi). piwi is involved in cosuppression, silencing of retrotransposons, and heterochromatin formation. aubergine (aub) was first isolated based on its role in germline development but is also responsible for maintaining the silenced state of an X-linked male fertility gene locus (Stellate) via RNAi. The Aub protein is required for RNAi and RISC assembly in ovaries. In addition, homeless/spindle-E (hls) is involved in silencing of Stellate and in heterochromatin formation. This study tested whether RNAi components are involved in the PcG pathway. The results show that the RNAi machinery affects the PcG response via a novel regulatory function in nuclear organization (Grimaud, 2006).

The role of a variety of RNAi components in a specific transgenic line called Fab-X was tested. This line contains a construct carrying a 3.6 kb fragment from the Fab-7 region, cloned upstream of a mini-white reporter and inserted into the X chromosome. In the Fab-X line, the presence of the Fab-7 sequence is sufficient to induce PcG-dependent silencing, both of the mini-white eye-color reporter gene and of the endogenous scalloped (sd) gene, which is required for wing-blade morphogenesis and is located 18.4 kb downstream of Fab-7. These two repressed phenotypes are abolished in the presence of mutations in PcG genes and are not present in heterozygous females and hemizygous males, indicating that both mini-white and sd expression are subject to PSS (Grimaud, 2006).

The eye-color and wing phenotypes were used as a basis to analyze the effect of the RNAi machinery on PcG-dependent repression. Mutations in RNAi components were introduced into the Fab-X line and placed over a balancer chromosome containing a GFP marker. As the AGO1 mutant alleles involve P element insertions containing the mini-white reporter gene, they could not be tested using the eye phenotype. A null mutation in dcr-2 (dcr-2^L811fsX) decreased silencing of the mini-white reporter gene relative to the Fab-X line when in the homozygous state. Likewise, two different mutant alleles of piwi (piwi¹ and piwi²) decreased mini-white silencing, with the effect being more pronounced in piwi² mutant flies. This effect was not restricted to the Fab-X line since it was also observed when piwi² was recombined into another Fab-7-containing line. In contrast to the effects seen for dcr-2 and piwi alleles, Fab-X females homozygous mutant for hls^E1 or that carried the heteroallelic hls^E1/hls^E616 combination silenced mini-white like wt Fab-X females (Grimaud, 2006).

The sd phenotype was then analyzed in all mutant backgrounds at 28.5°C, a temperature inducing a strong wing phenotype in Fab-X. A preselection of non-GFP female larvae was carried out in order to selectively analyze homozygous or trans-heterozygous mutant adults. This analysis revealed that mutating any of the components of the RNAi machinery, except for hls and the heterozygous dcr-1 mutation, leads to a strong decrease in the sd phenotype. These data show that the RNAi machinery can affect PcG-mediated silencing. The fact that hls mutants had no effect suggests that this process might be mechanistically distinct from the role of RNAi components in heterochromatin formation (Grimaud, 2006).

While most RNAi components are not required for binding of PcG proteins to PREs, they are required to mediate long-range contacts between multiple copies of the Fab-7 element. Moreover, Dcr-2, PIWI, and AGO1 colocalize with PH in the cell nucleus, and their effect correlates with the presence of small RNAs homologous to Fab-7 sequences. Finally, in addition to their effects on transgenic Fab-7 copies, mutations in these genes also reduce the frequency of long-distance contacts between endogenous PcG target genes. Taken together, these results reveal a novel and unexpected role for the RNAi machinery in the regulation of euchromatic genes in the nuclear space (Grimaud, 2006).

The effects caused by mutations in different RNAi components suggest the existence of distinct molecular roles for these proteins in the regulation of PcG function. First, the hls gene does not seem to play a major role in silencing at the Fab-7 PRE or maintaining long-distance Fab-7 contacts. Since Hls has been shown to play a central role in heterochromatin formation (Pal-Bhadra, 2004), there may be different subtypes of nuclear RNAi machineries for heterochromatin formation and for regulating PcG function. No effect was found when a mutation in the dcr-1 gene was analyzed at the heterozygous state, but the elucidation of the function of dcr-1 in PcG-mediated silencing awaits further analysis in a homozygous mutant background. A second class of RNAi components that participate in PcG-mediated repression contains dcr-2, AGO1, and the aub gene. Loss of any of these RNAi gene products affects PcG-dependent silencing at Fab-7, although it does not impact the binding of PcG proteins to Fab-7. Only mutations in piwi affected the binding of PcG proteins to Fab-7, at least in polytene chromosomes, but even PIWI did not affect recruitment of PcG factors at endogenous genes. In S. pombe, both RNAi components as well as DNA binding proteins are involved in recruiting heterochromatin proteins to the mating-type region (Jia, 2004). At PREs, multiple DNA binding factors and chromatin-associated proteins are known to contribute to PcG protein recruitment, such as PHO, the GAGA factor, DSP1, and the CtBP proteins. Their combinatorial action might play a key role in the robust and specific chromatin tethering of PcG proteins, while, in contrast to the situation in S. pombe, the RNAi machinery might play a relatively minor role (Grimaud, 2006).

It is interesting to note that piwi mutations affected recruitment of E(Z) and PC to Fab-7 in polytene chromosomes but had no effects on PH, another PRC1 component. In the current model for recruitment of PcG proteins to PREs, histone H3 methylation by the E(Z) protein recruits PRC1 via the chromodomain of PC. The current results indicate that multiple mechanisms might be used to anchor different PRC1 components to PREs and that the loss of PC does not necessarily lead to the disintegration of the entire PRC1 complex at PREs (Grimaud, 2006).

To date, all transcriptional gene-silencing phenomena that depend on the RNAi machinery involve the production of small-RNA molecules. RNAi components were also shown to affect telomere clustering in S. pombe, although binding of Swi6 and H3K9me to individual telomeres is not affected. The production of siRNAs is believed to be essential for the nuclear clustering of telomeres since cells carrying a catalytically dead RNA-dependent RNA polymerase (which abolishes siRNA production) are defective in telomere clustering. Consistent with a role for small RNAs in mediating gene contacts, sense and antisense transcription of Fab-7 as well as small-RNA species were found in Fab-7 transgenic lines. Moreover, a mutant allele of dcr-2 producing a truncated polypeptide lacking the RNase III domain, which is required for dsRNA processing, is defective in long-range interactions of PcG target sequences as well as in accumulation of Fab-7 small RNAs. These data suggest that small-RNA species could be involved in these gene contacts. However, no small Fab-7 RNA species was detected in the wt situation, although RNAi mutants affect the contact of the endogenous Fab-7 locus with the Antp gene. This might indicate that other RNA species produced in the endogenous Hox genes could contribute to gene clustering. However, the possibility remains that RNA-independent functions of RNAi proteins contribute to the maintenance of gene contacts, in particular in the case of endogenous PcG target genes (Grimaud, 2006).

Interestingly, none of the RNAi mutants tested are defective in the establishment of long-distance chromosomal interactions. Fab-7 contacts are correctly established during embryogenesis but decay during later stages of development. This suggests that the RNAi machinery is not required to initiate contacts but rather to maintain them via the stabilization of gene clustering at specific nuclear bodies. This clustering could be important in cosuppression, where transgene silencing can occur at the transcriptional and posttranscriptional levels, both requiring the RNAi machinery (Pal-Bhadra, 2002). It is difficult, however, to understand how a relatively modest increase in transcript levels caused by an increase in the copy number of a gene could trigger a robust silencing of all copies. This is particularly puzzling considering that the transcript levels of endogenous single-copy genes can vary, e.g., during normal physiological gene regulatory processes, without triggering gene silencing. One explanation might be that cosuppressed genes are clustered in the cell nucleus. Indeed, clustering of multiple gene copies has been reported in plant cells (Grimaud, 2006).

It is proposed that the RNAi machinery, perhaps in conjunction with PcG proteins, might stabilize this gene-clustering phenomenon. Specifically, the colocalization of multiple gene copies with components of the RNAi machinery might increase the local concentration of RNA species. Once this concentration overcomes a critical threshold, double-stranded RNAs might assemble and be cleaved in situ by the enzymatic activity of the RNAi machinery. RNA molecules might contribute to hold together loci containing PcG proteins that produce noncoding transcripts encompassing PREs. This gene clustering might involve contacts with components of the RNAi machinery as well as PcG proteins assembled in the same nuclear compartments (Grimaud, 2006).

One important question is, what is the role of the RNAi machinery in the regulation of endogenous PcG target genes? The data indicate that RNAi components affect only a subset of these genes since the colocalization of PcG bodies with RNAi bodies is limited. Hox loci are characterized by extensive noncoding RNA transcription, and, recently, other PcG target genes have been shown to be associated to intergenic transcription. RNAi components might be targeted to this subset of PcG target genes, while other PcG target genes that are characterized by the absence of noncoding transcripts might be independent on RNAi factors (Grimaud, 2006).

The fact that no homeotic phenotypes are visible in RNAi mutant backgrounds suggests that the function of RNAi components can be rescued by other chromatin factors. Indeed, the decrease in the level of nuclear interaction between the homeotic complexes was incomplete in RNAi mutant backgrounds. The data suggest that, while the RNAi machinery does not act in the establishment of PcG-dependent gene silencing, RNAi factors might help stabilize silencing during development by clustering PcG target genes at RNAi nuclear bodies. Thus, in addition to its role in defending the genome against viruses, transposons, and gene duplications, the RNAi machinery might participate in fine tuning the expression of PcG target genes through the regulation of nuclear organization. Finally, it must be noted that the developmental expression profile of the components of the RNAi machinery is highly specific. The function of specific RNAi components is therefore likely to be highly variable in different cell types and as a function of time. It will be of great interest to explore this issue in the developmental context of the whole organism, in Drosophila as well as in other species (Grimaud, 2006).

Piwi, Aubergine and Ago3 bind and cleave piwi-interacting RNAs to regulate of transposon activity in Drosophila

Drosophila Piwi-family proteins have been implicated in transposon control. This study examined piwi-interacting RNAs (piRNAs) associated with each Drosophila Piwi protein; Piwi and Aubergine were found to bind RNAs that are predominantly antisense to transposons, whereas Ago3 complexes contain predominantly sense piRNAs. As in mammals, the majority of Drosophila piRNAs are derived from discrete genomic loci. These loci comprise mainly defective transposon sequences, and some have previously been identified as master regulators of transposon activity. These data suggest that heterochromatic piRNA loci interact with potentially active, euchromatic transposons to form an adaptive system for transposon control. Complementary relationships between sense and antisense piRNA populations suggest an amplification loop wherein each piRNA-directed cleavage event generates the 5' end of a new piRNA. Thus, sense piRNAs, formed following cleavage of transposon mRNAs may enhance production of antisense piRNAs, complementary to active elements, by directing cleavage of transcripts from master control loci (Brennecke, 2007).

Mobile genetic elements, or their remnants, populate the genomes of nearly every living organism. Potential negative effects of mobile elements on the fitness of their hosts necessitate the development of strategies for transposon control. This is critical in the germline, where transposon activity can create a substantial mutational burden that would accumulate with each passing generation. Hybrid dysgenesis exemplifies the deleterious effects of colonization of a host by an uncontrolled mobile element. The progeny of intercrosses between certain Drosophila strains reproducibly show high germline mutation rates with elevated frequencies of chromosomal abnormalities and partial or complete sterility (Bucheton, 1990; Castro, 2004; Kidwell, 1977). Studies of the molecular basis of this phenomenon (Pelisson, 1981; Rubin, 1982) linked the phenotype to transposon mobilization (Brennecke, 2007).

Hybrid dysgenesis occurs when a transposon, carried by a male that has established control over that element, is introduced into a naive female that does not carry the element. The transposon becomes active in the progeny of the naive female, causing a variety of abnormalities in reproductive tissues that ultimately result in sterility. Since the dysgenic phenotype is often not completely penetrant, a fraction of the progeny from affected females may survive to adulthood. Such animals can develop resistance to the mobilized element, although in many cases, several generations are required for resistance to become fully established (Pelisson, 1987). Immunity to transposons can only be passed through the female germline (Bregliano, 1980), indicating that there are both cytoplasmic and genetic components to inherited resistance (Brennecke, 2007).

Studies of hybrid dysgenesis have served a critical role in revealing mechanisms of transposon control. In general, two seemingly contradictory models have emerged. The first model correlates resistance with an increasing copy number of the mobile element. A second model suggests that discrete genomic loci encode transposon resistance. The first model is supported by studies of the I element. Crossing a male carrying full-length copies of the I element to a naive female leads to I mobilization and hybrid dysgenesis. The number of I copies builds during subsequent crosses of surviving female progeny until it reaches an average of 1015 per genome. At this point, I mobility is suppressed, as the initially nave strain gains control over this element. Thus, a gradual increase in I element copy number over multiple generations was implicated in the development of transposon resistance (Brennecke, 2007).

The second model, which attributes transposon resistance to specific genetic loci, is illustrated by studies of gypsy transposon control (Bucheton, 1995). Genetic mapping of gypsy resistance determinants led to a discrete locus in the pericentric b-heterochromatin of the X chromosome that was named flamenco (Pelisson, 1994). Females carrying a permissive flamenco allele (one that allows gypsy activity) showed a dysgenic phenotype when crossed to males carrying functional gypsy elements. Permissive flamenco alleles exist in natural Drosophila populations but can also be produced by insertional mutagenesis of animals carrying a restrictive flamenco allele. Despite extensive deletion mapping over the flamenco locus, no transposon repressor from flamenco has been identified. For P elements, a repressor of transposition has been identified as a 66 kDa version of the P element transposase. Expression of the repressor was proposed to correlate with increasing P element copy number, leading to a self-imposed limitation on P element mobility. However, studies of resistance determinants indicated that control over P elements could also be established by insertion of P elements into specific genomic loci, arguing for an alternative, copy number-independent control pathway. Studies of inbred lines or of wild isolates with natural P element resistance indicated that P insertions near the telomere of X (cytological position 1A) were sufficient to confer resistance if maternally inherited. Additionally, several groups isolated insertions of incomplete P elements in this same cytological location that acted as dominant transposition suppressors. Importantly, these defective P elements lacked sequences encoding the repressor fragment of transposase (Brennecke, 2007 and references therein).

Both models of transposon resistance, those determined by specific genomic loci and those caused by copy number-dependent responses might be linked to small RNA-based regulatory pathways. Copy number-dependent silencing of mobile elements is reminiscent of copy number-dependent transgene silencing in plants (cosuppression) and Drosophila. In both cases, silencing occurs through an RNAi-like response where high-copy transgenes provoke the generation of small RNAs, presumably through a double-stranded RNA intermediate. Moreover, mutations in RNAi pathway genes impact transposon mobility in flies and C.elegans. Finally, small RNAs (rasiRNAs) corresponding to transposons and repeats have been isolated from flies and zebrafish (Brennecke, 2007 and references therein).

At the core of the RNAi machinery are the Argonaute proteins, which directly bind to small RNAs and use these as guides for the identification and cleavage of their targets. In animals, Argonautes can be divided into two clades (Carmell, 2002). One contains the Argonautes, which act with microRNAs and siRNAs to mediate gene silencing. The second contains the Piwi proteins. Genetic studies have implicated Piwi proteins in germline integrity. For example, piwi mutations cause sterility and loss of germline stem cells. aubergine is a spindle-class gene that is required in the germline for the production of functional oocytes. The third Drosophila Piwi gene, Ago3, has yet to be studied. Mutation of Piwi-family genes also affects mobile elements. For example, piwi mutations mobilize gypsy, and aubergine mutations impact TART and P elements. Finally, both Piwi and Aubergine bind rasiRNAs targeting a number of mobile and repetitive elements. These complexes are enriched for antisense small RNAs, as might be expected if they were actively involved in silencing transposons by recognition of their RNA products (Brennecke, 2007 and references therein).

Recently, a new class of small RNAs, the piRNAs, was identified through association with Piwi proteins in mammalian testes. These 26-30 nt RNAs are produced from discrete loci, generally spanning 50-100 kb. Interestingly, mammalian piRNAs are relatively depleted of transposon sequences. Despite apparent differences in the content of Piwi-associated RNA populations in mammals and Drosophila, Piwi-family proteins share essential roles in gametogenesis, with all three murine family members, Miwi2 , being required for male fertility. In order to probe mechanisms of transposon control in Drosophila and to understand the relationship between Piwi protein function in flies and mammals, a detailed analysis was undertaken of small RNAs associated with Piwi proteins in the Drosophila female germline. These studies indicate that Drosophila Piwi-family members function in a transposon surveillance pathway that not only preserves a genetic memory of transposon exposure but also has the potential to adapt its response upon contact with active transposons (Brennecke, 2007).

In C. elegans, effective RNAi depends upon an amplification mechanism. Small RNAs from the primary dsRNA trigger are largely dedicated to promoting the use of complementary targets as templates for RNA-dependent RNA polymerases (RdRPs) in the generation of secondary siRNAs. In Drosophila, no RdRPs have been identified. However, the ability of Piwi-mediated cleavage to prompt the production of new piRNAs could create an amplification cycle that serves the same purpose as the RdRP-driven secondary siRNA generation systems in worms (Brennecke, 2007).

The cycle, termed Ping-Pong amplification (Aravin, 2007b; see Properties and biogenesis of piRNA) is initiated by generating primary piRNAs, sampled from the piRNA clusters that have been identified in this study. As these are composed mainly of defective transposon copies, they serve as a genetic memory of transposons to which the population has been exposed. piRNAs that are antisense to expressed, dispersed transposons would identify and cleave their targets, resulting in the genesis of a new, sense piRNA in an Ago3 complex. The Ago3 bound sense piRNA would then seek a target, probably a precursor transcript from a master control locus that contains antisense transposon sequences. Ago3-directed cleavage would then generate additional antisense piRNAs capable both of actively silencing their target element and reinforcing the cycle through the creation of additional sense piRNAs (Brennecke, 2007).

The existence of such an amplification cycle essentially permits sense and antisense piRNAs act in concert to increase production of silencing-competent RNAs in response to the activity of individual transposons. Since Argonautes act catalytically, a significant amplification of the response could be achieved by even a relatively low level of sense piRNAs in Ago3 complexes. This model predicts that piRNAs participating in this process, namely those with complementary partners, should be more abundant than piRNAs without detectable partners. In accord with this hypothesis, the most frequently cloned Aub and Ago3-associated piRNAs show an increased probability of having partners within the data set. Interestingly, Piwi-associated RNAs do not follow this pattern. Since the amplification cycle consumes target transposon transcripts as part of its mechanism, posttranscriptional gene silencing may be sufficient to explain transposon repression. However, the possibility that transcriptional silencing may also be triggered by Piwi-family RNPs cannot be ruled out (Brennecke, 2007).

The amplification cycle may not be absolutely essential for silencing of all elements, since loci such as flamenco may operate in somatic follicle cells where the absence of Aub and Ago3 forces it to act in a stoichiometric fashion. In this regard, flamenco is unusual in that the vast majority of transposon fragments within this locus exist in a common orientation, which can lead to the production of antisense primary piRNAs given a long, unidirectional, precursor transcript (Brennecke, 2007).

In contrast to flamenco, most piRNA loci appear to be both bidirectionally transcribed and contain transposon sequences in random orientation. Nevertheless, the marked asymmetry of Piwi/Aub and Ago3 complexes is conserved in piRNAs that uniquely map to clusters. Among piRNAs that match transposons, 77% and 79% of unambiguously cluster-derived Piwi- and Aub-associated RNAs are antisense, while 73% of those in Ago3 are sense. These observations strongly suggest that piRNA clusters themselves participate in choice. According to this model, the remarkable strand asymmetry in piRNA populations hinges on informative interactions between master control loci and active transposons, which by their nature produce sense RNAs. These observations identify Ago3 as the principal recipient of piRNAs derived from transposon mRNAs. Thus, as long as there is an input to the system from active transposon transcripts via Ago3 and a preferential relationship between Ago3 and Aub for generating secondary piRNAs in their reciprocal complexes, a strand bias can be maintained even if primary and secondary piRNAs can both be derived from master control loci (Brennecke, 2007).

The amplification cycle must be initiated by primary piRNAs. Presently, the biogenesis pathway that generates primary piRNAs from piRNA clusters remains obscure. The data suggest that the piRNA precursor is a long, single-stranded transcript that is cleaved, preferentially at U residues. Transcripts have been detected from piRNA loci by RT-PCR that encompass multiple transposon fragments and find numerous small RNAs that cross junctions between adjacent transposons. In the case of flamenco, P element insertions near the proximal end of the locus have a polar effect both on these long RNA transcripts and on flamenco piRNAs (Brennecke, 2007).

Equally mysterious is the generation of piRNA 3' ends. Mature piRNAs could arise by two cleavage events and subsequent loading into Piwi complexes. Alternatively, piRNAs could be created following 5' end formation and incorporation of a long RNA into Piwi by resection of their 3' ends. The latter model is attractive, since it could provide an explanation for observed size differences between RNAs bound to individual Piwi proteins, since piRNA size would simply reflect the footprint of each Piwi protein. Although de novo biogenesis mechanisms must exist, maternally inherited piRNA complexes could also serve to initiate the amplification cycle. All three Piwi proteins are loaded into the developing oocyte, and Piwi and Aub are concentrated in the pole plasm, which will give rise to the germline of the next generation. Inherited piRNAs could enhance resistance to transposons that are an ongoing challenge to the organism, augmenting zygotic production of primary piRNAs. Indeed, maternally loaded rasiRNAs were detected in early embryos, and their presence was correlated with suppression of hybrid dysgenesis in D. virilis (Brennecke, 2007).

These data point to a comprehensive strategy for transposon repression in Drosophila that incorporates both a long-term genetic memory and an acute response to the presence of potentially active elements in the genome. The model that emerges from these studies shows many parallels to adaptive immune systems. The piRNA loci themselves encode a diversity of small RNA fragments that have the potential to recognize invading parasitic genetic elements. Throughout the evolution of Drosophila species, a record of transposon exposure may have been preserved by selection for transposition events into these master control loci, since this is one key mechanism through which control over a specific element can be achieved. Evidence has already emerged that X-TAS can act as a transposition hotspot for P elements, raising the possibility the piRNA clusters in general may attract transposons. Once an element enters a piRNA locus, it can act, in trans, to silencing remaining elements in the genome, either directly through primary piRNAs or by engaging in the amplification model described above. A comparison of D. melanogaster piRNAs to transposons present in related Drosophilids shows a lack of complementarity when comparisons are made at high stringency. However, when even a few mismatches are permitted, it is clear that piRNA loci might have some limited potential to protect against horizontal transmission of these heterologous elements. The existence of a feed-forward amplification loop can be compared to clonal expansion of immune cells with the appropriate specificity following antigen stimulation, leading to a robust and adaptable response (Brennecke, 2007).

Small interfering RNAs function through the PIWI subfamily of Argonautes to ensure silencing of retrotransposons

In Drosophila, repeat-associated small interfering RNAs (rasiRNAs) are produced in the germ line by a Dicer-independent pathway and function through the PIWI subfamily of Argonautes to ensure silencing of retrotransposons. Small RNAs were sequenced associated with the PIWI subfamily member AGO3. Although other members of PIWI, Aubergine (Aub) and Piwi, associated with rasiRNAs derived mainly from the antisense strand of retrotransposons, AGO3-associated rasiRNAs arose mainly from the sense strand. Aub- and Piwi-associated rasiRNAs showed a strong preference for uracil at their 5' ends, and AGO3-associated rasiRNAs showed a strong preference for adenine at nucleotide. Comparisons between AGO3- and Aub-associated rasiRNAs revealed pairs of rasiRNAs showing complementarities in their first 10 nucleotides. Aub and AGO3 exhibited Slicer activity in vitro. These data support a model in which formation of a 5' terminus within rasiRNA precursors is guided by rasiRNAs originating from transcripts of the other strand in concert with the Slicer activity of PIWI (Gunawardane, 2007).

Small noncoding RNAs trigger various forms of sequence specific gene silencing, including RNA interference (RNAi), translational repression, and heterochromatin formation in a variety of eukaryotic organisms, commonly referred to as RNA silencing. Members of the Argonaute family of proteins are essential components of RNA silencing. In Drosophila, five genes encode distinct members of the Argonaute family: AGO1, AGO2, Aubergine (Aub), Piwi, and AGO3. AGO1 and AGO2 constitute the Argonaute (AGO) subfamily and bind microRNA (miRNA) and small interfering RNA (siRNA), respectively. Aub, Piwi, and AGO3 belong to the PIWI subfamily of the Argonaute family and are enriched in germline cells (Williams, 2002), and Aub and Piwi have been shown to play important roles in germline cell formation. They are involved in silencing retrotransposons and other repetitive elements and exhibit target RNA cleavage (slicing) activity in vitro . Both Aub and Piwi associate with repeat-associated siRNAs (rasiRNAs) (Vagin, 2006; Saito, 2006). Aub- and Piwi-associated rasiRNAs are derived mainly from the antisense strand of retrotransposons, with little or no phasing, and have a strong preference for uracil (U) at the 5' end. Small RNA processing factors such as Dicer and Drosha are known to cleave preferentially at the 5' side of U; however, rasiRNAs are thought to be produced by a Dicer-independent pathway (Vagin, 2006). The mechanisms governing rasiRNA production remain to be elucidated (Gunawardane, 2007).

Very little is known about the function of AGO3, the third member of the Drosophila PIWI subfamily. Full-length cDNA of AGO3 revealed that the AGO3 gene is 83 kb in length. Peptide sequence alignments among Drosophila Argonaute proteins revealed that AGO3 is most similar to Piwi. The Asp-Asp-His motif in the PIWI domain, originally identified as the catalytic center for Slicer activity in human AGO2, is conserved in AGO3 (Gunawardane, 2007).

Embryonic RNA expression patterns of AGO3 are very similar to those of Piwi and Aub; they are expressed maternally, but their expression disappears by embryonic stages 10 to 12. To confirm these results, a monoclonal antibody (mAb) to AGO3 was produced, that revealed that AGO3 is strongly expressed in earlier embryonic stages but decreases as development proceeds. AGO3 accumulates in the cytoplasm of germline cells including germline stem cells (GSCs), germline cyst cells, nurse cells, and oocytes at earlier stages. In testes, AGO3 is expressed in GSC, primary gonial cells, and early spermatocytes. Unlike Piwi, AGO3 expression was undetected in the hub, a tiny cluster of postmitotic somatic cells localized at the apical tip of the testis that functions as a niche for GSC). Thus, with respect to expression in germline cells, AGO3 is more similar to Aub than to Piwi (Gunawardane, 2007).

All of the other members of the fly Argonautes are specifically associated with a subset of small RNAs: siRNAs, miRNAs, or rasiRNAs. Therefore whether AGO3 also associates with small RNAs produced in the fly ovary was tested. Immunoprecipitation with AGO3 mAb from ovary lysate revealed small RNAs 23 to 26 nucleotides (nt) long. The size distribution of AGO3-associated small RNAs is similar to that of Aub-associated small RNAs; in both cases, the peak is 24 nt and the longest is 27 nt. Small RNAs associated with AGO3 are likely to lack either a 2' or 3' hydroxyl group, because they do not migrate faster after beta-elimination as opposed to a synthetic siRNA that has 2' and 3' hydroxyl groups at the 3' end, the latter being the hallmarks of Dicer cleavage. These results suggest that AGO3-associated small RNAs in the ovary are produced by a pathway similar to those involved in production of rasiRNAs that associate with Aub and Piwi (Gunawardane, 2007).

A cDNA library was constructed of small RNAs associated with AGO3 in the ovary. Of 420 clones sequenced, 410 matched Drosophila genomic sequences in a database search, and most were rasiRNAs (86%; 353 of 410), as in the case of Aub and Piwi. Like rasiRNAs associated with Aub or Piwi, rasiRNAs associated with AGO3 included various kinds of transposable elements, both LTR (long terminal repeat) retrotransposons and LINE (long interspersed nuclear element)like elements. rasiRNAs associated with Aub or Piwi in ovaries are derived mainly from the antisense strand of retrotransposons, and the 5' end is predominantly U. These characteristics were not found for rasiRNAs associated with AGO3. However, AGO3-associated rasiRNAs were derived mainly from the sense strand of retrotransposons (82%), and they showed a strong preference for adenine (A) at nucleotide 10, but no preference for U at the 5' end. These results suggest that AGO3-associated rasiRNAs belong to a subset of rasiRNAs that are distinct from Aub- and Piwi-associated rasiRNAs (Gunawardane, 2007).

Some Argonaute proteins exhibit Slicer activity that directs cleavage of its cognate mRNA target across from nucleotides 10 and 11, measured from the 5' end of the small RNA guide strand. Thus, these findings suggest a model for rasiRNA biogenesis, in which the 5' end of Aub- and Piwi-associated rasiRNAs is determined and cleaved by AGO3-rasiRNA complexes, and the 5' end of AGO3-associated rasiRNAs is determined by Aub- and Piwi-rasiRNA complexes through a similar rasiRNA-guided cleavage event. For instance, AGO3 associated with a rasiRNA with A at nucleotide 10 can target a long RNA molecule by Watson-Crick base pairing and cleave the target RNA, resulting in sliced RNAs with U at the 5' end. Similarly, when Aub or Piwi associated with rasiRNAs with U at the 5' end slices its cognate RNA target, the resulting cleaved RNA will have A at nucleotide 10 (Gunawardane, 2007).

To test this model, AGO3 was examined for Slicer activity by performing in vitro target RNA cleavage assays with glutathione S-transferase (GST)AGO3 fusions. The target RNA, luc passenger siRNA (21 nt long, 5' end labeled with 32P), was efficiently cleaved by GST-AGO3, as was the case for GST-AGO1 and GST-Aub. The size of the cleaved products (9 nt) indicated that they direct cleavage of target RNA across from nucleotides 10 and 11 as measured from the 5' end of the small RNA guide strand. Both GST-Aub and GST-AGO3 with a longer guide RNA (26 nt) were also able to cleave a long transcript (180 nt). Long precursors of rasiRNAs both in sense and antisense orientations appear to exist in fly ovaries. These results corroborate the model in which the 5' end of rasiRNAs within the precursors is determined by rasiRNAs and cleaved by members of PIWI that associate with these rasiRNAs (Gunawardane, 2007).

The model predicts that some AGO3-associated rasiRNAs should be complementary to the first 10 nt of Aub- and Piwi-associated rasiRNAs. Sequence comparison between AGO3- and Aub-associated rasiRNAs indeed revealed pairs of rasiRNAs that show complementarities at their first 10 nt. Sixteen of 353 AGO3-associated rasiRNAs had such pairs with 11 of 676 Aub-associated rasiRNAs. However, such pairings were only found between AGO3- and Aub-associated rasiRNAs, and no pairs were observed between AGO3- and Piwi-associated rasiRNAs (353 versus 330). Like Aub-associated rasiRNAs, Piwi-associated rasiRNAs arise mainly from the antisense strand and their 5' ends show a strong preference for U; thus, it is difficult to argue that Piwi is not involved in this type of rasiRNA biogenesis. One possible reason is that Piwi is nuclear, whereas AGO3 and Aub are cytoplasmic. This type of rasiRNA biogenesis may operate in the cytoplasm. Alternatively, formation of 5' ends of Piwi-associated rasiRNAs may occur only at an earlier time during germline development (Gunawardane, 2007).

RasiRNAs are involved in genome surveillance by silencing repetitive elements and controlling their mobilization in the Drosophila germ line. It was recently shown that rasiRNAs are produced by a mechanism that requires neither Dicer-1 nor Dicer-2 in flies. These data suggest that rasiRNAs in a sense orientation guide formation of the 5' end of rasiRNAs in an antisense orientation, and vice versa; as well, this cycle of mutual dependency elaborates optimal rasiRNA production. In this model, proteins of the PIWI subfamily function as Slicer for formation of the 5' end during rasiRNA biogenesis. This model requires that sliced rasiRNA precursors then be cleaved again at the 3' end by an as yet unidentified endonuclease (or nibbled by exonuclease) to produce mature rasiRNAs before or after loading of the resulting cleavage products onto another member of the PIWI. Once 'primary' complexes of rasiRNAs with proteins of PIWI are produced, these complexes will in turn function as the 'initiator' of secondary rasiRNA biogenesis, and so nascent rasiRNAs should be continuously supplied in the ovary and testis. Such a process may occur through rasiRNA germline transmission. Of the PIWI members, at least Aub is accumulated to the posterior pole in oocytes and remains in polar granules in early embryos. It is then incorporated in pole cells, the progenitor of the Drosophila germ line (10) (Gunawardane, 2007).

An epigenetic activation role of Piwi and a Piwi-associated piRNA in Drosophila

Heterochromatin, representing the silenced state of transcription, consists largely of transposon-enriched and highly repetitive sequences. Implicated in heterochromatin formation and transcriptional silencing in Drosophila are Piwi (P-element induced wimpy testis) and repeat-associated small interfering RNAs (rasiRNAs). Despite this, the role of Piwi in rasiRNA expression and heterochromatic silencing remains unknown. This study reports the identification and characterization of 12,903 Piwi-interacting RNAs (piRNAs) in Drosophila, showing that rasiRNAs represent a subset of piRNAs. Piwi promotes euchromatic histone modifications and piRNA transcription in subtelomeric heterochromatin (also known as telomere-associated sequence, or TAS), on the right arm of chromosome 3 (3R-TAS). Piwi binds to 3R-TAS and a piRNA uniquely maps to 3R-TAS (3R-TAS1 piRNA). In piwi mutants, 3R-TAS loses euchromatic histone modifications yet accumulates heterochromatic histone modifications and Heterochromatin Protein 1a (HP1a). Furthermore, the expression of both the 3R-TAS1 piRNA and a white reporter gene in 3R-TAS becomes suppressed. A P element inserted 128 base pairs downstream of the 3R-TAS1 piRNA coding sequence restores the euchromatic histone modifications of 3R-TAS and the expression of 3R-TAS1 piRNA in piwi mutants, as well as partly rescuing their defects in germline stem-cell maintenance. These observations suggest that Piwi promotes the euchromatic character of 3R-TAS heterochromatin and its transcriptional activity, opposite to the known roles of Piwi and the RNA-mediated interference pathway in epigenetic silencing. This activating function is probably achieved through interaction with at least 3R-TAS1 piRNA and is essential for germline stem-cell maintenance (Yin, 2007).

The above findings reveal the complexity of small RNA-mediated epigenetic regulation, namely that Piwi can exert opposite effects on different genomic regions. piRNAs may have a function in guiding Piwi to the target sites, yet the opposite effects of Piwi at different target sites might be mediated by the local chromatin context, which would render the selective binding of the Piwi-piRNA complex to different partners such as HP1a or JmJC domain-containing histone demethylases. The activating effect of Piwi, 3R-TAS1 piRNA and possibly other piRNAs in 3R-TAS can be explained by a heterochromatin/euchromatin counterbalance model in which the repetitive nature of 3R-TAS by default is a substrate for heterochromatization. The heterochromatic state could be established and maintained by the Polycomb group proteins or an RNAi pathway mediated by Ago proteins, or yet another mechanism. However, the association of Piwi with 3R-TAS by means of the 3R-TAS1 piRNA or P{w+,ry+}A4-4 insertion as a roughly 20-kb unique sequence counteracts the heterochromatization. The finding that Piwi is required for the epigenetic activation of the subtelomeric region starts to reveal a mechanism underlying epigenetic regulation and stem-cell maintenance (Yin, 2007).

Pimet, the Drosophila homolog of HEN1, mediates 2'-O-methylation of Piwi- interacting RNAs at their 3' ends

Piwi-interacting RNAs (piRNAs) consist of a germline-specific group of small RNAs derived from distinct intergenic loci in the genome. piRNAs function in silencing selfish transposable elements through binding with the PIWI subfamily proteins of Argonautes. This study shows that piRNAs in Drosophila are 2'-O-methylated at their 3' ends. Loss of Pimet/Hen1 (piRNA methyltransferase), the Drosophila homolog of Arabidopsis HEN1 methyltransferase for microRNAs (miRNAs), results in loss of 2'-O-methylation of fly piRNAs. Recombinant Pimet shows single-stranded small RNA methylation activity in vitro and interacts with the PIWI proteins within Pimet mutant ovary. These results show that Pimet mediates piRNA 2'-O-methylation in Drosophila (Saito, 2007; full text of article).

In Pimet mutant ovary, piRNAs associated with Aub and Piwi were not methylated at the 3' ends, most likely due to loss of Pimet expression. Whether GST-Pimet is able to methylate these piRNAs associated with the PIWI proteins from Pimet mutant ovary was investigated. Aub-piRNA complexes were immunopurified with a specific antibody against Aub and subjected to in vitro methylation assays. As a control, miRNAs associated with AGO1 were also obtained through immunoprecipitation using anti-AGO1 from ovary lysate. It was found that piRNAs were methylated even in a complex form with Aub. piRNA methylated in the assay showed resistance to oxidation and β-elimination treatment. Interestingly, miRNAs associated with AGO1 were not methylated, although these miRNAs are single-stranded in a complex form with AGO1. Confirmation that the miRNA levels were several-fold higher than those of piRNAs was provided by phosphorylation of these small RNAs. It seems that small RNA methylation by Pimet is largely influenced by the accessibility of the 3' ends of the substrates to Pimet itself. Structural analysis of Argonaute proteins suggests that the 5' end of the small guide RNA is anchored in a highly conserved pocket in the PIWI domain, whereas the 3' end of the small RNA is embedded in the PAZ domain. Taken together, these results suggest that the 3' ends of Aub-associated piRNAs are not tightly bound to the PAZ domain, but are exposed to the surface of the protein. In contrast, the 3' ends of AGO1-associated miRNAs are likely to be embedded in the PAZ domain and therefore are not exposed to the surface of the protein. Alternatively, but not mutually exclusively, it is conceivable that Pimet may interact only with PIWI proteins and not with AGO proteins, thereby methylating only small RNAs associated with PIWI proteins. To test this, whether Pimet associates with PIWI proteins was investigated. A GST pull-down assay was performed; GST-Pimet was first incubated with Pimet mutant ovary lysate, and after extensive washing the eluates were probed with PIWI protein antibodies. Aub, Piwi, and AGO3 were clearly detected in the bound fraction with GST-Pimet but not with GST itself. By contrast, AGO1 was not observed. These results indicated that Pimet is capable of physically interacting with PIWI proteins containing piRNAs that can serve as substrates for Pimet methylation. Addition of RNaseA did not affect the interaction of Pimet with Aub, suggesting that Pimet is able to associate directly with the PIWI proteins. In Drosophila, piRNA methylation may occur after matured piRNAs are loaded onto PIWI proteins. If so, it clearly differs from the case of miRNA methylation in plants, which likely occurs prior to miRNA loading onto the AGO proteins when miRNAs are still in a duplex form with the complementary miRNA* molecules (Saito, 2007).

Mutations in Arabidopsis hen1 cause reduced fertility. Thus, is the piRNA methylation by Pimet crucial in Drosophila? piRNAs function in genome surveillance in germlines in concert with PIWI proteins. Mutations in aub, piwi, and others like spindle-E (homeless) cause piRNAs not to be accumulated in gonads, and lead to germ cell malformation and sterility. This clearly indicates that piRNAs are necessary for perpetuation of organisms. However, the Pimet mutant fly seems to be viable and fertile. Steady-state levels of piRNAs in the methylation-defective mutant are also similar to those in wild type. Expression levels of retrotransposons do not seem to be changed by loss of Pimet expression. Thus, the function of 3' end methylation is currently unknown. Further investigation such as by immunohistochemistry may be required to obtain a more detailed morphology of the mutant. Extensive analyses of the mechanisms underlying piRNA methylation may also provide important clues to more fully elucidating piRNA biogenesis. Aub and AGO3, which determine and form the 5' end of piRNAs in piRNA biogenesis, were shown to be in the protein fraction associated with Pimet. Identifying more Pimet-associated proteins may reveal the factors required for formation of the 3' end of piRNAs (Saito, 2007).

Telomeric trans-silencing: an epigenetic repression combining RNA silencing and heterochromatin formation

The study of P-element repression in Drosophila led to the discovery of the telomeric Trans-Silencing Effect (TSE), a repression mechanism by which a transposon or a transgene inserted in subtelomeric heterochromatin (Telomeric Associated Sequence or TAS) has the capacity to repress in trans in the female germline, a homologous transposon, or transgene located in euchromatin. TSE shows variegation among egg chambers in ovaries when silencing is incomplete. This study reports that TSE displays an epigenetic transmission through meiosis, which involves an extrachromosomal maternally transmitted factor. This silencing is highly sensitive to mutations affecting both heterochromatin formation (Su(var)205 encoding Heterochromatin Protein 1 and Su(var)3-7) and the repeat-associated small interfering RNA (or rasiRNA) silencing pathway (aubergine, homeless, armitage, and piwi). In contrast, TSE is not sensitive to mutations affecting r2d2, which is involved in the small interfering RNA (or siRNA) silencing pathway, nor is it sensitive to a mutation in loquacious, which is involved in the micro RNA (or miRNA) silencing pathway. These results, taken together with the recent discovery of TAS homologous small RNAs associated to PIWI proteins, support the proposition that TSE involves a repeat-associated small interfering RNA pathway linked to heterochromatin formation, which was co-opted by the P element to establish repression of its own transposition after its recent invasion of the D. melanogaster genome. Therefore, the study of TSE provides insight into the genetic properties of a germline-specific small RNA silencing pathway (Josse, 2007).

Repression of transposable elements (TEs) involves complex mechanisms that can be linked to either small RNA silencing pathways or chromatin structure modifications depending on the species and/or the TE family. Drosophila species are particularly relevant to the study of these repression mechanisms since some families of TEs are recent invaders, allowing genetic analysis to be carried out on strains with or without these TEs. In some cases, crossing these two types of strains induces hybrid dysgenesis, a syndrome of genetic abnormalities resulting from TE mobility. In D. virilis, repression of hybrid dysgenesis has been correlated to RNA silencing since small RNAs of the retroelement Penelope, responsible for dysgenesis, were detected in nondysgenic embryos but not in dysgenic embryos. In D. melanogaster, repression of retrotransposons can be established by noncoding fragments of the corresponding element (I factor, ZAM, and Idefix) and can be in some cases (gypsy, mdg1, copia, Het-A, TART, and ZAM, Idefix) sensitive to mutations in genes from the Argonaute family involved in small RNA silencing pathways. In the same species, strong repression of the DNA P TE, by a cellular state that has been called 'P cytotype', can be established by one or two telomeric P elements inserted in heterochromatic 'Telomeric Associated Sequences' (TAS) at the 1A cytological site corresponding to the left end of the X chromosome. This includes repression of dysgenic sterility resulting from P transposition. This P cytotype is sensitive to mutations affecting both Heterochromatin Protein 1 (HP1) (Ronsseray, 1996) and the Argonaute family member AUBERGINE (Reiss, 2004). P repression corresponds to a new picture of TE repression shown, using an assay directly linked to transposition, to be affected by heterochromatin and small RNA silencing mutants (Josse, 2007).

In the course of the study of P cytotype, a new silencing phenomenon has been discovered. Indeed, a P-lacZ transgene or a single defective P element inserted in TAS can repress expression of euchromatic P-lacZ insertions in the female germline in trans, if a certain length of homology exists between telomeric and euchromatic insertions. This homology-dependent silencing phenomenon has been termed Trans-Silencing Effect (TSE) (Roche, 1998). Telomeric transgenes, but not centromeric transgenes, can be silencers and all euchromatic P-lacZ insertions tested can be targets. TSE is restricted to the female germline and has a maternal effect since repression occurs only when the telomeric transgene is maternally inherited (Ronsseray, 2001). Further, when TSE is not complete, variegating germline lacZ repression is observed from one egg chamber to another, suggesting a chromatin-based mechanism of repression. Recently, an extensive analysis of small RNAs complexed with PIWI family proteins (AUBERGINE, PIWI, and AGO3) was performed in the Drosophila female germline. The latter study showed that most of the RNA sequences associated to these proteins derive from TEs. TSE corresponds likely to such a situation (Josse, 2007).

This study analyzed the genetic properties of TSE and shows that it has an epigenetic transmission through meiosis, which involves an extrachromosomal maternally transmitted stimulating component. Further, in order to investigate the mechanism behind TSE, a candidate gene analysis was performed to identify genes whose mutations impair TSE. It was found that TSE is strongly affected both by mutations in genes involved in heterochromatin formation and in the recently discovered small RNA silencing pathway called 'repeat-associated small interfering RNAs' (rasiRNA) pathway. In contrast, this study shows that TSE is not sensitive to genes specific to the classical RNA interference pathway linked to small interfering RNAs (siRNA) or to the micro RNA (miRNA) pathway. This suggests thus that TSE involves a rasiRNA pathway linked to heterochromatin formation and that such a mechanism, working in the germline, may underlie epigenetic transmission of repression through meiosis (Josse, 2007).

Probing the initiation and effector phases of the somatic piRNA pathway in Drosophila

Combining RNAi in cultured cells and analysis of mutant animals, this study probed the roles of known Piwi-interacting RNA (piRNA) pathway components in the initiation and effector phases of transposon silencing. Squash associates physically with Piwi, and reductions in its expression led to modest transposon derepression without effects on piRNAs, consistent with an effector role. Alterations in Zucchini or Armitage reduced both Piwi protein and piRNAs, indicating functions in the formation of a stable Piwi RISC (RNA-induced silencing complex). Notably, loss of Zucchini or mutations within its catalytic domain led to accumulation of unprocessed precursor transcripts from flamenco, consistent with a role for this putative nuclease in piRNA biogenesis (Haase, 2010).

Eukaryotic small RNAs regulate gene expression through various mechanisms, intervening at both transcriptional and post-transcriptional levels. Small RNAs are divided into classes according to their mechanism of biogenesis and their particular Argonaute protein partner. Piwi-interacting RNAs (piRNAs) bind Piwi-clade Argonaute proteins and act mainly in gonadal tissues to guard genome integrity by silencing mobile genetic elements (Haase, 2010).

Conceptually, the piRNA pathway can be divided into several different phases. During the initiation phase, small RNAs, called primary piRNAs, are produced from their generative loci, so-called piRNA clusters. These give rise to long, presumably single-stranded precursor transcripts, which are processed via an unknown biogenesis mechanism into small RNAs that are larger than canonical microRNAs (~24-30 nucleotides [nt]). Primary piRNAs become stably associated with Piwi proteins to form Piwi RISCs (RNA-induced silencing complexes), which also contain additional proteins that facilitate target recognition and silencing. During the effector phase, Piwi RISCs identify targets via complementary base-pairing. In some cases, for example, with Aubergine as a piRNA partner, there is strong evidence for target cleavage in vivo. This nucleolytic destruction of transposon mRNAs is probably the main Aubergine effector mechanism, although this has not been rigorously demonstrated. Piwi also conserves the Argonaute catalytic triad; however, in this case, both its nuclear localization and its association with certain chromatin proteins suggest the possibility of transcriptional and post-transcriptional effector pathways. An additional phase, adaptation, is restricted to germ cells and constitutes the ping-pong cycle. During this phase, transposon mRNA cleavage directed by primary piRNAs triggers the production of secondary piRNAs, whose 5' ends correspond to cleavage sites. These generally join Ago3 and enable it to recognize and cleave RNAs with antisense transposon content, perhaps piRNA cluster transcripts. Cleavage by Ago3 RISC again triggers piRNA production from the target, closing a loop that enables the overall small RNA population to adjust to challenge by a particular transposon. Finally, piRNA populations present in germ cells can be transmitted to the next generation to prime piRNA responses in progeny (Haase, 2010).

In Drosophila follicle cells, only the initiation and effector phases appear relevant. Here, the piRNA pathway relies on the coupling between a single Piwi protein (Piwi itself) and a principal piRNA cluster (flamenco) to silence mainly gypsy family retrotransposons. Drosophila ovarian somatic sheet cells (OSS) display many of the properties of follicle cells, and represent a convenient system to study the initiation and effector phases of the piRNA pathway without the complications inherent in the study of complex tissues in vivo. This study therefore sought to leverage information derived from the use of RNAi in OSS cells with the analysis of ovaries derived from mutant animals to probe the roles of known piRNA pathway components in the initiation and effector phases of transposon silencing (Haase, 2010).

Several prior studies have proposed models in which Piwi proteins silence targets by interfering with their transcription. Since piRNAs are largely absent from somatic tissues, impacts underlying these changes are presumed to have occurred during development and to have been epigenetically maintained in the adult. Drosophila Piwi protein is mainly localized to the nucleus and has been shown to interact with HP1, a core component of heterochromatin. Considered together, this body of evidence pointed strongly to an effector mechanism in which Piwi-associated small RNAs direct heterochromatin formation and silencing of targets (Haase, 2010).

Loss of piwi has dramatic effects on transposon expression in somatic follicle cells. Genetic mutants result in an absence of Piwi protein throughout development. This could lead to a failure to create heterochromatic marks that could have otherwise maintained epigenetic silencing of transposons in the absence of continuous Piwi expression. Alternatively, there could be an ongoing requirement for Piwi to maintain silencing, irrespective of whether it acted via transcriptional or post-transcriptional mechanisms (Haase, 2010).

To discriminate between these possibilities, OSS cells were transfected with dsRNAs corresponding to piwi, and followed impacts on Piwi mRNA and protein levels. Maximal suppression was reached by 3 d, and silencing persisted through day 6. At 6 d post-transfection, impacts on two elements known to be derepressed in the follicle cells of piwi mutant ovaries were probed: gypsy and idefix. Both showed derepression (up to 10-fold) upon piwi silencing. Additional elements were also tested, with blood being impacted strongly. Previous studies have also implicated zucchini (zuc) in the function of the somatic piRNA pathway. RNAi against this gene also increased gypsy, blood, and idefix expression. Considered together, these results demonstrate that the integrity of the piRNA pathway is essential for the ongoing repression of mobile elements and argue against a model in which silent epigenetic states, once set by the action of piwi proteins on chromatin, can autonomously maintain transposon silencing (Haase, 2010).

Nearly a dozen proteins have been linked to the fully elaborated piRNA pathway that operates in germ cells. Many of these show germ cell-specific expression patterns consistent with their selective biological effects. Mutations in armitage (armi) result in coincident loss of the characteristic nuclear accumulation of Piwi protein and a reduction in Piwi-associated piRNAs. Unlike most germline-specific pathway components, an examination of RNA-seq data from OSS cells indicated substantial armi expression. Therefore armi was suppressed by RNAi and effects on transposon expression were examined. Notably, gypsy, blood, and idefix were strongly derepressed, implying a role for armi in both the somatic and germline compartments (Haase, 2010).

The Drosophila mutant armi¹ represents a P-element insertion in the 5' untranslated region (UTR) of armitage. A second allele, armi^72.1, was derived from armi¹ by imprecise excision. RNA-seq data covered the armi ORF in OSS, but no reads were detected corresponding to the germ cell 5' UTR. This raises the possibility that armi expression might be driven by an alternative promoter in somatic cells, and that the armi alleles examined thus far may have spared the activity of that promoter (Haase, 2010).

To investigate whether Armi and Zuc act at the initiation or effector phase of the piRNA pathway, piRNAs were examined. Silencing of piwi reduced levels of two abundant piRNAs, corresponding to gypsy, or idefix. Similar effects were noted upon silencing of armi or zuc. Aggregate OSS piRNA levels can be measured qualitatively by radioactive phosphate exchange of small RNAs in Piwi immunoprecipitates. As expected, RNAi against piwi virtually eliminated piRNAs in immunoprecipitates. Silencing of armi or zuc produced indistinguishable effects (Haase, 2010).

In germ cells, armi mutation causes loss of the prominent nuclear localization of Piwi. A similar phenotype was observed upon knockdown of armi in somatic OSS cells. Because of the mixed cell types present in ovaries, previous studies had not been able to distinguish whether Armi loss simply caused Piwi mislocalization or whether Armi influenced Piwi expression or stability. In OSS cells, knockdown of armi reduced Piwi protein levels by approximately fivefold, equivalent to a targeted knockdown of Piwi itself without affecting piwi mRNA. A similar loss of Piwi protein from the nuclei in cells exposed to zuc-dsRNAs was noted. In this case, Piwi protein but not mRNA levels also fell (Haase, 2010).

Considered together, these data strongly suggest roles of Armi and Zuc in the initiation phase of the piRNA pathway. A role for Armi, along with a previously unrecognized component, Yb, in the somatic pathway, is also supported by a recent report. Either protein could play a role in primary piRNA biogenesis, aiding piRNA production or loading, with this model resting on the presumption that association with mature piRNAs influences Piwi protein stability. Alternatively, Armi or Zuc could be core components of mature Piwi RISC, with loss of either subunit destabilizing associated components of the complex (Haase, 2010).

To investigate these alternative models, proteomic analysis of Piwi RNPs was performed. Piwi immunoprecipitates contained a number of peptides from Armi, suggesting that this protein is present in Piwi RISC. Of note, association of both Piwi and Armi with Squash (Squ), another previously identified piRNA pathway component, was also detected. Piwi could be also detected in Squ immunoprecipitates by Western blotting. Although no Zuc peptides were seen in multidimensional protein identification technology (MudPIT), Piwi could be detected to a low extent in Zuc immunoprecipitates. Overall, the emerging picture suggests that both Armi and Squ are components of Piwi RISC. Lower levels of Piwi associated with Zuc might indicate a weaker or more transient association of Zuc with Piwi RISC (Haase, 2010).

Mutations in squash (squ) show little impact on piRNA populations in mutant ovaries. Similarly, upon sequencing of small RNAs in Piwi immunoprecipitates, no differences were detected in associated piRNA populations upon comparison of squ homozygous mutant animals to heterozygous siblings. Animals harboring two squ alleles interrupted by early stop codons did, however, display an effect on transposon silencing (Haase, 2010).

As compared with heterozygous siblings, squ mutants showed significant derepression of gypsy. This occurred without any detectable change in an abundant gypsy piRNA or overall Piwi levels. In contrast, no substantial changes were detected in idefix or ZAM; however, I-element and blood were strongly derepressed (Haase, 2010).

Considered together, these results point to a role of squash in the effector phase of the piRNA pathway. A slight but reproducible reduction was noted in Piwi protein levels in homozygous squ mutants. However, this was well within the range observed in Piwi heterozygotes, where the piRNA pathway functions completely normally (Haase, 2010).

In the initial screen that placed zuc within the piRNA pathway, two alleles were identified. zuc^HM27 represents an early stop mutation resulting in a putative null allele (referred to as zuc mut). This mutant strongly affects piRNA silencing in both germline and somatic cells of the ovary. While somatic piRNAs are depleted in this mutant, ping-pong signatures remain intact. This places Zuc outside of the adaptive phase, consistent with accumulating evidence for a role in the initiation phase (Haase, 2010).

While the biochemical properties of Zuc have yet to be analyzed, its protein sequence places it as a member of the phospholipase D (PLD) family of phosphodiesterases. These share a HxK(x)₄D motif, whose integrity is essential for catalytic activity. The second zuc mutation that emerged in the original screen, zuc^SG63, contains a H --> Y mutation within the phosphodiesterase motif that is predicted to render it catalytically inactive. To probe a role for Zuc catalytic activity in the piRNA pathway, the presumed null (zuc mut) and catalytically dead (zuc H --> Y) alleles were compared for their effects on piRNAs and transposon silencing (Haase, 2010).

Total ovarian small RNAs were analyzed from animals that were heterozygous or homozygous for the zuc H --> Y allele and the resulting profiles were compared to previously published analyses of the presumed zuc mut allele. In both cases, strong reductions were seen in total piRNAs and in populations that mapped uniquely to the flamenco locus, regardless of the normalization method used to compare libraries. Slightly stronger impacts were apparent when profiles of Piwi immunoprecipitates were compared. Here, piRNA populations corresponding to flamenco were almost completely lost. An accumulation was noted of 21-nt species in Piwi immunoprecipitates from both zuc mutant lines. These were enriched for a 5' U, although not to the extent for longer piRNA species. The nature of these shorter, apparently Piwi-associated RNAs remains mysterious (Haase, 2010).

Both the presumed null and H --> Y zuc alleles impacted transposon silencing. Between fivefold and 20-fold increases in gypsy, ZAM, and idefix were noted in comparison with heterozygous controls. Even stronger derepression could be observed for I-element, HeT-A, 1731, and blood. The zuc H --> Y and zuc mut alleles also showed similar impacts on piRNA populations and the overall levels of Piwi protein (Haase, 2010).

Considered together, these data point to a requirement for the presumed catalytic center of Zuc in the initiation phase of the piRNA pathway. Other PLD family nucleases that have been characterized to date cleave nucleic acids leaving 5' phosphate and 3' hydroxyl termini. These are the characteristics one might expect for a processing enzyme that catalyzed primary piRNA biogenesis. Previous studies have posited the requirement for several nucleolytic activities in the piRNA pathway. One is thought to form the 5' ends of primary piRNAs. The 3' ends of these species could be formed prior to Piwi loading or could be coupled to protein binding, as is posited for the ping-pong cycle. The nucleolytic center of Piwi proteins themselves form the 5' ends of secondary piRNAs, with their 3' ends proposed to be created by a separate enzyme. Based on its impacts in the soma on Piwi complexes, it is imagined that the Zuc catalytic center might form either the 5' or 3' ends of primary piRNAs (Haase, 2010).

To evaluate this hypothesis, RNAs derived from the flamenco locus were examined in control ovaries or in tissues from animals homozygous for either of the two zuc mutant alleles. The prevailing model holds that the flamenco locus is transcribed as a continuous, single-stranded precursor spanning >150 kb. It was reasoned that a defect in primary processing might result in an accumulation of long RNAs from this locus, since they would not be effectively metabolized into piRNAs. By quantitative PCR (qPCR) using primer pairs spanning three different regions of flamenco, 15-fold to 45-fold increases were seen in flamenco-derived long RNAs in zuc mutant ovaries (Haase, 2010).

Considered as a whole, these results strongly support a role for Zucchini in the primary processing of piRNAs from the flamenco locus. Given its size, it is virtually impossible to follow the fate of the intact flamenco transcript by Northern blotting. Three different segments of the locus do show an accumulation consistent with their failure to be parsed into piRNAs. However, several alternative explanations can also be envisioned. For example, if Zucchini impacts Piwi stability, feedback controls might operate to inhibit primary biogenesis. Without a direct, biochemical demonstration that Zucchini processes piRNA cluster transcripts, its assignment as a primary biogenesis enzyme must be viewed as provisional. However, any alternative model must account for the requirement for its phosphodiesterase active site, and, at present, a direct role in piRNA biogenesis seems the most parsimonious conclusion (Haase, 2010).

While these studies do not ascribe specific functions to Armitage and Squash, they do support their assignment to the initiator and effector phases, respectively. Armitage is a putative helicase, although no analyses as yet indicate whether this biochemical activity is required for its function. Placement of this protein in the initiation phase and its intimate association with Piwi perhaps suggest a role in loading or stability of Piwi RISC. Squash, of all the components examined in this study, had the most variable effects on transposon control in somatic cells of the ovary, but both its physical association with Piwi RISC and its impact on transposons without an effect on piRNAs imply a role in the effector phase. While the studies reported herein can suggest roles for known pathway components at specific points in the piRNA pathway, a definitive conclusion regarding the part played by any of these proteins will require reconstitution of the pathway in vitro (Haase, 2010).

The cochaperone shutdown defines a group of biogenesis factors essential for all piRNA populations in Drosophila

In animal gonads, PIWI proteins and their bound 23-30 nt piRNAs guard genome integrity by the sequence specific silencing of transposons. Two branches of piRNA biogenesis, namely primary processing and ping-pong amplification, have been proposed. Despite an overall conceptual understanding of piRNA biogenesis, identity and/or function of the involved players are largely unknown. This study demonstrates an essential role for the female sterility gene shutdown in piRNA biology. Shutdown, an evolutionarily conserved cochaperone collaborates with Hsp90 during piRNA biogenesis, potentially at the loading step of RNAs into PIWI proteins. Shutdown is shown to be essential for both primary and secondary piRNA populations in Drosophila. An extension of this study to previously described piRNA pathway members revealed three distinct groups of biogenesis factors. Together with data on how PIWI proteins are wired into primary and secondary processing, a unified model for piRNA biogenesis is proposed (Olivieri, 2012).

PIWI interacting RNAs (piRNAs) are a class of animal small RNAs. They are bound by PIWI family proteins and guide the sequence specific silencing of selfish genetic elements such as transposable elements (TEs). Defects in the piRNA pathway lead to TE derepression, genomic instability and ultimately sterility (Olivieri, 2012).

In Drosophila, most piRNAs are generated from two sources; on the one hand, these are piRNA cluster transcripts that originate from discrete genomic loci and serve as reservoirs of TE sequences; on the other hand, these are RNAs derived from active TEs that engage - together with cluster transcripts - in a piRNA amplification loop called the ping-pong cycle (Olivieri, 2012).

Two modes of piRNA biogenesis exist: (1) during primary piRNA biogenesis, a single stranded RNA is processed into pre-piRNAs, which are loaded onto PIWI proteins and are subsequently 3' trimmed and methylated, yielding mature piRNA induced silencing complexes (piRISCs). (2) piRISCs with active slicer activity can trigger secondary piRNA biogenesis, where a new piRNA is formed out of the sliced target RNA. In the presence of corresponding sense and antisense precursor RNAs, secondary piRNA biogenesis acts as the ping-pong amplification loop. The two piRNAs engaged in ping-pong have opposite orientation and exhibit a characteristic ten nucleotide 5' overlap (ping-pong signature) (Olivieri, 2012).

Primary and secondary piRNA biogenesis co-occur in germline cells, complicating the genetic and mechanistic dissection of these processes. However, somatic cells of the gonad also harbor a piRNA pathway and this is based exclusively on primary piRNA biogenesis. The Drosophila ovary is therefore ideally suited to identify and characterize factors required for either primary or secondary piRNA biogenesis or both (Olivieri, 2012).

Somatic support cells of the Drosophila ovary express Piwi as the only PIWI family protein. Primary piRNA biogenesis is thought to take place in peri-nuclear Yb-bodies, where the RNA helicases Armitage (Armi) and Yb as well as the TUDOR domain protein Vreteno (Vret) accumulate. In addition to these three factors, the putative mitochondria-localized nuclease Zucchini (Zuc) and the RNA helicase Sister of Yb (SoYb) are essential for piRNA biogenesis in the soma. Formation of mature Piwi-RISC triggers its nuclear import, while failure in piRNA biogenesis results in destabilization of presumably unloaded Piwi. Mature Piwi-RISC triggers TE silencing by an unknown mechanism that requires Piwi's nuclear localization but not its slicer activity (Olivieri, 2012).

With the exception of Yb, the above mentioned biogenesis factors are also essential in germline cells for the formation of Piwi-RISC. Germline cells, however, express two additional PIWI proteins, Aubergine (Aub) and Argonaute 3 (AGO3), which localize to the cytoplasm and are enriched in peri-nuclear nuage. Aub and AGO3 are the main players in the ping-pong cycle. Several factors with essential or modulatory roles in the ping-pong cycle have been identified. These are the RNA helicases Spindle-E and Vasa and the TUDOR domain proteins Krimper, Tejas (Tej), Qin and Tudor (Olivieri, 2012).

The analysis of piRNA populations from wild-type and piRNA pathway mutant ovaries indicated that Piwi is mainly a recipient of primary piRNAs, while Aub and AGO3 are predominantly or exclusively recipients of secondary piRNA biogenesis. Given this, three major questions arise: (1) Are primary and secondary piRNA biogenesis processes genetically and mechanistically separate or do common factors act in both processes? (2) In which processing step do identified piRNA biogenesis factors act? (3) How are the three PIWI family proteins wired into piRNA biogenesis? In other words, are certain PIWI proteins only receiving primary or only secondary piRNAs (Olivieri, 2012)?

This study shows that the female sterility gene shutdown encodes a piRNA biogenesis factor. Shu is required for all piRNA populations in ovaries and it acts downstream of known piRNA biogenesis factors. A comparison of Shu to several other pathway factors led to the definition of three major groups of piRNA biogenesis factors. In combination with data on how PIWI proteins are wired into piRNA biogenesis, a model is proposed that accounts for the distinct association of piRNA subpopulations with specific PIWI proteins in Drosophila (Olivieri, 2012).

The outcome of this work is threefold: (1) The cochaperone Shutdown is essential for the biogenesis of all Drosophila piRNA populations. (2) Three major groups of piRNA biogenesis factors can be distinguished. (3) Piwi and Aub but not AGO3 are loaded with primary piRNAs, explaining how the cell maintains highly specific piRNA populations in the three PIWI proteins (Olivieri, 2012).

A remarkable feature of the shu mutant phenotype is that piRNA populations for every TE collapse. This already points to a common piRNA biogenesis step downstream of the primary and secondary branches. Epistatic analysis in somatic follicle cells is consistent with Shu acting at a late step in piRNA biogenesis: Shu is not required for the localization of any known biogenesis factor to Yb-bodies. On the other hand, Shu's localization to Yb-bodies depends on all other biogenesis factors and even on Piwi, arguing that unloaded Piwi recruits Shu to the Yb-body. Similarly, Shu colocalizes with nonloadable AGO3 in OSCs as well as in ovaries defective of ping-pong in discrete foci that also contain and are dependent on Krimp. Thus, in wild-type and in biogenesis mutants, Shu appears to colocalize with unloaded PIWI proteins (Olivieri, 2012).

Shu's C-terminal TPR domain falls into the class of Hsp90 binders and Hsp90 is important for small RNA loading into Argonaute proteins (Iki, 2010; Iwasaki, 2010; Miyoshi, 2010). In addition, the plant cochaperone Cyp40 interacts with Hsp90 via its TPR domain and is a critical cofactor for small RNA loading into AGO1. The genetic and localization data support an analogous role for Shu and Hsp90 during small RNA loading into PIWI proteins. Clearly, in vitro assays will be crucial to dissect the precise order of events and the molecular role of Shu, especially its PPIase domain (Olivieri, 2012).

A major challenge in the field is to assemble piRNA biogenesis factors into pathways that explain the stereotypic populations of piRNAs in vivo. Advantage was taken of efficient germline specific knockdowns to study the impact of several factors on piRNA populations. Based on levels and localization of PIWI proteins as well as on piRNA populations obtained from several pathway factor knockdowns, three major groups of piRNA biogenesis factors are proposed (Olivieri, 2012).

Group I factors are required for primary piRNA biogenesis but dispensable for secondary biogenesis. In fact, piRNAs that initiated ping-pong in group I knockdowns were amplified and ping-pong signatures of such TEs were strongly increased, presumably as primary piRNAs that do not feed into ping-pong were absent (Olivieri, 2012).

Group II factors are specific for ping-pong, as primary piRNA biogenesis feeding into Piwi was unaffected. An alternative explanation that cannot be excluded is that some or all group II genes are required specifically for Aub biology (primary and secondary) per se. This would similarly leave Piwi bound piRNAs intact and would lead to a collapse in ping-pong. Given the data on Aub loading in OSCs, a model is favored however where the primary biogenesis machineries that feed Aub and Piwi are very similar (Olivieri, 2012).

Finally, group III factors are required for the biogenesis of Piwi/Aub/AGO3 bound piRNAs. The prototypic member of this group is Shu. Loss of Shu affects essentially all piRNA populations to the same extent. It is noted that analysis of piRNA populations from vret mutants indicated a role for this group III factor in primary biogenesis but not ping-pong (Handler, 2011; Zamparini, 2011). The distorted tissue composition of vret mutant ovaries coupled with perdurance of maternal Vret protein or RNA may underlie this discrepancy. The existence of group III factors predicts that primary and secondary piRNA biogenesis feed into a final piRISC maturation step that requires a set of common factors for all PIWI proteins. Given that piRNA biogenesis -- irrespective of the source of the precursor RNA -- requires an RNA loading step as well as a 3' trimming step, the existence of group III factors suggests itself (Olivieri, 2012).

The three proposed groups serve as a rough classification of biogenesis factors. Clearly, at a molecular level, the precise role of each factor within the biogenesis process will vary considerably. Of note, the classification of group I and group II genes extends to the mouse piRNA pathway. The Armi and Zuc orthologs MOV10L1 and PLD6 are required for primary piRNA biogenesis, whereas mouse VAS and TDRD9 (mouse Spn-E) were reported to be dispensable for primary biogenesis but are required for secondary biogenesis pathway (Olivieri, 2012).

The data indicate that Aub is not only loaded via ping-pong, but also via primary piRNA biogenesis. It is also postulate that Aub and Piwi proteins are wired into primary piRNA biogenesis processes in a very similar manner, meaning that they require the same or highly overlapping core factors (e.g., Armi or Zuc). In agreement with this, ectopically expressed Aub is loaded in OSCs that harbor a fully functional primary pathway but lack critical ping-pong factors such as Vas. The genetic requirements for Aub loading in OSCs are identical to those for Piwi. It is extrapolated from this that the core primary biogenesis machinery that loads Piwi in the soma also loads Piwi and Aub in the germline. Analyses of piRNA populations from armi versus piwi or aub-GLKDs support a model where Armi and Zuc are required for the biogenesis of both Piwi and Aub bound primary piRNAs. The possibility is not excluded that - despite a similar biogenesis machinery - populations of primary piRNAs in Aub and Piwi are different. For example, differences in subcellular localizations of PIWI proteins as well as piRNA precursor RNAs might result in such differences (Olivieri, 2012).

In contrast to Aub, AGO3 was unstable in OSCs. Coexpression of Aub or simultaneous knockdown of krimp had no impact on AGO3 stability. It is therefore concluded that primary piRNA biogenesis is incompatible with AGO3. In fact, also in the germline genetic data indicated that AGO3 depends on secondary piRNA biogenesis for being loaded. Blocking AGO3's access to the primary biogenesis machinery would allow the cell to load AGO3 with a unique class of piRNAs if it couples AGO3 loading to a precursor RNA originating from Aub-slicer mediated cleavage of an active TE. This would explain the remarkable bias of AGO3 bound piRNAs being sense and carrying an Adenosine at position ten (Olivieri, 2012).

Interestingly, on a primary sequence level Aub -- despite its significantly different biology -- is more closely related to Piwi than to AGO3. A critical question emanating from this is to which extent Piwi is participating in ping-pong, and if it does not, why. A weak, yet statistically significant, ping-pong signature has been observed between Piwi and AGO3 bound piRNAs. This could mean that there is indeed low level of Piwi-AGO3 ping-pong. An alternative explanation is that the Piwi-AGO3 signal is a misleading computational signal: If Piwi and Aub are loaded via the same primary biogenesis machinery, initiator piRNAs for ping-pong that end up in Aub also end up in Piwi. As primary piRNA biogenesis appears to be nonrandom and preferentially processed piRNAs likely trigger ping-pong more robustly, an 'artificial' AGO3/Piwi ping-pong signature might result (Olivieri, 2012).

What could be the molecular basis of why Piwi does not or only moderately participate in ping-pong? Either, specific features of Aub (e.g., symmetric Arginine methylation) are funneling this protein into ping-pong and similar features are absent on Piwi. Or, the mere sequestration of Piwi into the nucleus prevents Piwi from participating in ping-pong. Notably, N-terminally truncated Piwi that is still loaded but that cannot translocate into the nucleus is enriched in nuage the proposed site of secondary piRNA biogenesis. A simple difference in the subcellular localization of Aub and Piwi might thus contribute to the dramatic differences of piRNA populations residing in Aub or Piwi (Olivieri, 2012).

piRNA-guided slicing of transposon transcripts enforces their transcriptional silencing via specifying the nuclear piRNA repertoire

PIWI clade Argonaute proteins silence transposon expression in animal gonads. Their target specificity is defined by bound approximately 23- to 30-nucleotide (nt) PIWI-interacting RNAs (piRNAs) that are processed from single-stranded precursor transcripts via two distinct pathways. Primary piRNAs are defined by the endonuclease Zucchini, while secondary piRNA biogenesis depends on piRNA-guided transcript cleavage and results in piRNA amplification. This study analyzed the interdependencies between these piRNA biogenesis pathways in developing Drosophila ovaries. Secondary piRNA-guided target slicing is the predominant mechanism that specifies transcripts-including those from piRNA clusters-as primary piRNA precursors and defines the spectrum of Piwi-bound piRNAs in germline cells. Post-transcriptional silencing in the cytoplasm therefore enforces nuclear transcriptional target silencing, which ensures the tight suppression of transposons during oogenesis. As target slicing also defines the nuclear piRNA pool during mouse spermatogenesis, these findings uncover an unexpected conceptual similarity between the mouse and fly piRNA pathways (Senti, 2015).

PROTEIN INTERACTIONS

Drosophila PIWI associates with chromatin and interacts directly with HP1a

The interface between cellular systems involving small noncoding RNAs and epigenetic change remains largely unexplored in metazoans. RNA-induced silencing systems have the potential to target particular regions of the genome for epigenetic change by locating specific sequences and recruiting chromatin modifiers. Noting that several genes encoding RNA silencing components have been implicated in epigenetic regulation in Drosophila, a direct link was sought between the RNA silencing system and heterochromatin components. This study shows that Piwi, an Argonaute/Piwi protein family member that binds to Piwi-interacting RNAs (piRNAs), strongly and specifically interacts with heterochromatin protein 1a (HP1a), a central player in heterochromatic gene silencing. The HP1a dimer binds a PxVxL-type motif in the N-terminal domain of Piwi. This motif is required in fruit flies for normal silencing of transgenes embedded in heterochromatin. Piwi, like HP1a, is itself a chromatin-associated protein whose distribution in polytene chromosomes overlaps with HP1a and appears to be RNA dependent. These findings implicate a direct interaction between the Piwi-mediated small RNA mechanism and heterochromatin-forming pathways in determining the epigenetic state of the fly genome (Brower-Toland, 2007).

Thus, Drosophila PIWI interacts directly with HP1a and is distributed on chromosomes in a pattern overlapping HP1a. Association of Piwi with constitutive heterochromatin domains including pericentric heterochromatin, telomeres, and part of the banded portion of chromosome 4 is consistent with the profile of Piwi-associated small RNAs, which includes sequences homologous to telomeric and centromeric repetitive elements as well as some that are overrepresented in chromosome 4. The PIWI-HP1a interaction is mediated through binding of a PxVxL-type motif by dimerized chromoshadow domains of HP1a. Furthermore, the intact PxVxL motif is required in vivo for effective heterochromatic silencing. This is consistent with the wealth of data implicating Piwi at the interface of RNA silencing mechanisms with epigenetic phenomena, in particular heterochromatin-induced silencing. Piwi is a nuclear protein required for silencing of transposons and retroviruses. Piwi is required for effective silencing of multiple copies of dispersed transgenes, of tandem repeats of the white gene at euchromatic insertion sites, and of white reporter genes in the pericentric heterochromatin or fourth chromosome. Silencing in the latter two cases is dependent on HP1a. This study shows that Piwi interacts directly with HP1a. Some Piwi signal overlaps with HP1a in polytene chromosomes, suggesting that Piwi and HP1a together regulate the epigenetic state of diverse regions in the genome. This overlap is spatially complex and could therefore represent numerous varied roles for Piwi, HP1a, and the Piwi-HP1a interaction. Heterochromatin is first assembled early, while the Drosophila embryo is still a syncytium (nuclear division cycles 10-14). This is critical for the silencing observed in PEV, which may be compromised later in development. While the HP1a-PIWI association observed on polytene chromosomes might be similar to that inferred in the embryo, it might also represent a different function (Brower-Toland, 2007).

Recently, Piwi has been shown to bind in the germline to piRNAs, many of which are from heterochromatic regions. The colocalization of Piwi and HP1a in pericentric heterochromatin may indeed reflect a simple relationship between the piRNA pathway, histone modification, and heterochromatin formation similar to the S. pombe system wherein AGO1-mediated transcriptional gene silencing locally targets H3K9 methylation to create a binding site for the HP1 homolog Swi6. In Drosophila, these data on the specific interaction between PIWI and HP1a raise the possibility of an alternate pathway to HP1a-mediated heterochromatinization: A PIWI-piRNA complex might directly recruit HP1a to piRNA-corresponding genomic sequences, which could then recruit HMTs such as SU(VAR)3-9 to effect nucleation/spreading. This would represent an H3K9me-independent mode for initial HP1 localization, an alternative but potentially equally effective means for triggering local formation of heterochromatin. Conversely, if heterochromatin formation is targeted by a different mechanism, the presence of HP1a could allow stable binding of Piwi to heterochromatin for PTGS, a process that might be necessary to maintain silencing throughout the lifetime of the fly (Brower-Toland, 2007).

Arginine methylation of Piwi proteins catalysed by dPRMT5 is required for Ago3 and Aub stability

Piwi family proteins are essential for germline development and bind piwi-interacting RNAs (piRNAs). The grandchildless gene aub of Drosophila encodes the piRNA-binding protein Aubergine (Aub), which is essential for formation of primordial germ cells (PGCs). This study reports that Piwi family proteins of mouse, Xenopus laevis and Drosophila contain symmetrical dimethylarginines (sDMAs). Piwi proteins are expressed in Xenopus oocytes and numerous Xenopus piRNAs were identified. This paper reports that the Drosophila homologue of protein methyltransferase 5 (dPRMT5, capsuleen/dart5), which is also the product of a grandchildless gene, is required for arginine methylation of Drosophila Piwi, Ago3 and Aub proteins in vivo. Loss of dPRMT5 activity led to a reduction in the levels of piRNAs, Ago3 and Aub proteins, and accumulation of retrotransposons in the Drosophila ovary. These studies explain the relationship between aub and dPRMT5 (csul/dart5) genes by demonstrating that dPRMT5 is the enzyme that methylates Aub. These findings underscore the significance of sDMA modification of Piwi proteins in the germline and suggest an interacting pathway of genes that are required for piRNA function and PGC specification (Kirino, 2009).

Piwi family proteins are expressed in the germline and bind ~26 to ~30 nucleotide (nt) piRNAs. Drosophila express three Piwi proteins: Aub, Piwi and Ago3. Mice also express three Piwi proteins termed Miwi, Mili/PiwiL2 and Miwi2/PiwiL4. Tens of thousands of distinct piRNAs have been described and most of them are species-specific. In Drosophila , Piwi proteins and piRNAs (also known as rasiRNAs - repeat associated small interfering RNAs) silence transposons in the germline. A similar function has been found for a subset of mouse and zebrafish piRNAs. An amplification loop of piRNAs has been described but how primary piRNAs are generated is unknown. sDMA modifications occur in sequence motifs composed of arginines flanked by glycines (GRG) or alanines (GRA or ARG) that are often found as repeats. PRMT5 and its cofactors MEP50/WD45 and pICln form the methylosome that methylates Sm proteins. A highly specific monoclonal antibody (17.8) was produced that recognizes Mili by Western blot, immunoprecipitation and immunofluorescence microscopy. By serendipity it was discovered that the widely used Y12 monoclonal antibody recognizes mouse Mili and Miwi proteins and their bound piRNAs. The Sm proteins of spliceosomal small nuclear ribonucleoproteins (snRNPs) constitute the main antigen for Y12. piRNAs were not identified in immunoprecipitates of snRNPs, heterogeneous ribonucleoproteins (hnRNPs) or of the Survival of Motor Neurons (SMN) complex using various antibodies. By Northern blot analysis it was found that piR-1, but not miR-16, an abundant miRNA, was found in Y12 immunoprecipitates, suggesting that Y12 recognizes Piwi but not Ago proteins (Kirino, 2009).

The epitope that Y12 recognizes on Sm proteins consists of symmetrically dimethylated arginines, in the glycine-arginine rich regions of the proteins. It was reasoned that Y12 likely reacted with sDMA-containing epitopes in Mili and Miwi, and arginine residues were sought that could be symmetrically methylated. Intriguingly, it was found that most animal Piwi proteins contain sDMA motifs that are typically clustered close to the amino terminus, while no animal Ago proteins contained such motifs. However, it was found that four of ten Arabidopsis Ago proteins contained sDMA motifs (Kirino, 2009).

To test whether Miwi and Mili contain sDMAs, SYM11 and ASYM24 antibodies, which specifically recognize proteins containing sDMA-glycine or aDMA-glycine repeats, respectively, were used. SYM11, as well as Y12, reacted strongly with endogenous Miwi and Mili, while ASYM24 showed only faint reactivity towards endogenous Miwi. In contrast, recombinant Flag-Mili or Flag-Miwi purified from baculovirus-infected Sf9 cells, were not recognized by Y12 or SYM11 (or ASYM24). This is entirely consistent with the finding that recombinant human Sm proteins expressed in Sf9 cells also do not contain sDMAs because Sf9 cells do not express type II PRMTs and thus cannot produce sDMA modifications. These findings indicate that Mili and Miwi proteins contain sDMAs. The putative sDMA motifs of Miwi are concentrated very close to the amino terminus with the exception of one GRG triplet. Flag-tagged full-length Miwi and two truncated forms of Miwi (aa 68-862 or 1-212) were transfected in 293T cells, by Flag immunoprecipitation and subject to western blot with SYM11 antibody, SYM11 antibody recognizes the amino terminus of Miwi protein (Kirino, 2009).

Next it was asked whether the sDMA modification was conserved in Piwi family proteins from other species. A stumbling block in studying the molecular functions of Piwi proteins and piRNAs has been the lack of suitable cell culture systems. It was reasoned that Xenopus laevis oocytes might express Piwi proteins and piRNAs and thus prove very useful not only to confirm that sDMAs of Piwi proteins are conserved but also as a model to study the function of Piwi proteins and piRNAs. By searching the Gurdon EST database at Xenbase three Xenopus Piwi proteins were identified which were named Xili, Xiwi and Xiwi2. All three Xenopus Piwi proteins contain putative sDMA motifs. Immunoprecipitations with Y12 from X. laevis oocytes (defolliculated, mixed Dumont stages I-VI), testis and liver revealed the presence of two proteins at ~95 kDa and ~110 kDa specifically in the Y12 immunoprecipitates from oocytes and testis that were identified by mass spectrometry as Xiwi and Xili respectively. As shown in the western blots, Y12 recognizes both Xiwi and Xili, while anti-Mili (17.8) reacts only with Xili. In addition, both Xiwi and Xili are recognized by SYM11, indicating that Xiwi and Xili contain sDMAs (Kirino, 2009).

X. laevis piRNAs were isolated and analyzed from Y12 immunoprecipitates. ~26-29 nt piRNAs are present in the Y12 immunoprecipitates and their 3'-termini are not eliminated by periodate oxidation and are thus likely 2'-O-methylated, as seen in piRNAs from other species (Kirino, 2009).

Deep sequencing was performed of X. laevis piRNAs from Y12 immunoprecipitates of oocytes and testis. The nucleotide composition of X. laevis piRNAs shows enrichment of Uridine in the first nucleotide position and of Adenine in the tenth nucleotide position. There is also enrichment for piRNAs whose first 10 nucleotides are complementary to the first 10 nucleotide of other piRNAs. These features indicate that a fraction of X. laevis piRNAs target transposon transcripts and that they also participate in a piRNA amplification loop, as has been described for Drosophila and zebrafish piRNAs and prepachytene mouse piRNAs. By Northern blot XL-piR-3, a representative piRNA, is expressed specifically in oocytes and by in situ hybridization XL-piR-3 is localized predominantly in the cytoplasm of X. laevis oocytes and it is expressed in higher levels in immature oocytes (Kirino, 2009).

Genetic disruption of either Drosophila PRMT5 (dPRMT5; also know as capsuleen - csul- , and dart5) or its cofactor valois, (the Drosophila homolog of MEP50/WD45), results in complete loss of sDMA modifications of Sm proteins in ovaries. However, unlike the situation in mammals, the levels or function of Sm proteins is not affected by loss of sDMAs (Kirino, 2009).

Null or hypomorphic alleles of dPRMT5 (csul, dart5) phenocopy aub null alleles and it was reasoned that dPRMT5 might be the methyltransferase that produces sDMAs in Aub, Piwi and Ago3, in vivo. To test this, ovaries were used from csul^RM50/Df(2R)Jp7 females, which give rise to embryos that are genetic nulls for dPRMT5 and w^- was used as a wild-type control. Western blots of ovary lysates from wt and maternal null csul showed that there was near complete loss of SYM11 reactivity, indicating dramatic reduction of sDMA modified proteins in csul ovaries. There was no change in ASYM24 reactivity between wt and csul, indicating that aDMA modified proteins were not affected. These findings confirm that dPRMT5 (csul, dart5) activity is required specifically for sDMA modification. Piwi and Aub proteins were immunoprecipitated from wt and csul mutant ovaries and the immunoprecipitates were probed with SYM11 and ASYM24. SYM11 reacted very strongly with Aub and also with Piwi immunopurifed from wt but not csul ovaries; ASYM24 reacted only weakly with Aub from wt ovaries. Immunoprecipitates of Ago3 were also with SYM11 and ASYM24 and it was observed that only Ago3 from wt ovaries reacted with SYM11. These results indicate that, like the mouse and X. laevis Piwi family proteins, Drosophila Piwi, Aub and Ago3 contain sDMAs and that dPRMT5 is the methylase that produces sDMAs of these proteins (Kirino, 2009).

In Aub the four arginines that are putative substrates for symmetrical dimethylation are found in tandem very close to the amino terminus. Site-directed mutagenesis was used to change these arginines into lysines that are not subjected to methylation by PRMTs. Flag-tagged wild-type (WT) or mutant (M) Aub were stably transfected into Drosophila S2 cells (which express dPRMT5), the proteins were purified by Flag immunoprecipitation and subjected to western blot with Flag, SYM11 and ASYM24 antibodies. SYM11 antibody reacted only with wild-type Aub. Next the binding of wild-type and mutant Aub to a synthetic piRNA was assayed. Immunopurified, wild-type or mutant Flag-Aub were incubated with a 5'-end radiolabeled synthetic piRNA containing 4-thio-Uridine at the first position, followed by crosslinking with Ultraviolet light and NuPAGE analysis. There was similar binding between wild-type and mutant Aub proteins. These findings indicate that one or more of the four arginines in the amino terminus of Aub are symmetrically dimethylated and arginine methylation does not impact piRNA binding (Kirino, 2009).

Next, RNAs bound to Piwi and Aub were isolated and analyzed from wt or csul ovaries. piRNAs remain bound to Piwi and Aub proteins in the csul ovaries. There is mild reduction of Piwi-piRNAs and marked reduction of Aub-piRNAs in csul ovaries corresponding to concordant reduction of protein levels of Piwi and Aub. The Piwi associated piRNAs were gel purified and subjected to periodate oxidation followed by β-elimination and it was revealed that Piwi-associated piRNAs purified from csul ovaries retain 2'-O-methylation of their 3' termini. These findings indicate that the lack of sDMA modifications of Piwi and Aub in csul ovaries does not impair the methylation of piRNAs or their binding to Piwi and Aub (Kirino, 2009).

Next the protein levels of Piwi family proteins were compared between wt, heterozygous and homozygous csul ovaries. Western blot analysis showed that there was marked reduction of Aub and Ago3 protein levels and lesser reduction of Piwi levels in csul ovaries, whereas the levels of the miRNA binding protein Ago1 were not affected. Since mRNA levels of Aub, Piwi and Ago3 are the same between wt and csul ovaries, dPRMT5 activity might be required to stabilize the Aub, Ago3 and Piwi proteins most likely by symmetrically methylating their arginines. The level of a representative piRNA (roo-rasiRNA), was decreased in csul ovaries in accord with reduction of Piwi family proteins, while the level of a representative miRNA, miR-8, was not affected. The homozygous csul ovaries showed a 30-fold increase in the levels of the HeT-A retrotransposon transcript, whose expression is most sensitive to mutations that disrupt piRNA-directed silencing in the female germline. Collectively these results indicate that loss of dPRMT5 activity impairs the amounts of Piwi proteins and piRNAs, resulting in disruption of their function of transposon silencing (Kirino, 2009).

Next the localization of Ago3, Aub and Piwi was analyzed by confocal microscopy in wt and homozygous csul early stage egg chambers. Previous studies have shown that Piwi is localized predominantly in the nuclei of follicle and germ cells while Ago3 and Aub are localized in the cytoplasm of germ cells. In oocytes, Aub is concentrated in the germ (pole) plasm. Representative images reveal that the level of Ago3 is markedly reduced in csul early stage egg chambers, while there is only a mild reduction of Aub and Piwi protein levels (Kirino, 2009).

Germ cell (PGC) formation in Drosophila requires that cytoplasmic determinants are localized to the posterior pole of the embryo. Genetic screens have identified grandchildless maternal genes that are required for PGC specification and invariably the protein or RNA products of these genes are concentrated in the pole plasm and are incorporated into the PGCs. Among these genes are Aub, dPRMT5 (csul, dart5), and its cofactor valois and tudor, whose protein product contains eleven tudor domains. The localization of Aub was tested in csul oocytes by confocal microscopy. Representative results show that the levels of Aub in the pole plasm of stage 10 egg chambers are markedly reduced. Western blotting reveals marked reduction of Aub protein levels in csul ovaries while confocal microscopy shows that Aub levels are subtly reduced in early stage egg chambers but markedly reduced in later stage egg chambers, suggesting that lack of sDMAs affects Aub levels at later stages in oogenesis (Kirino, 2009).

These studies show that sDMA modification of Piwi family proteins is a conserved post-translational modification, and the methyltransferase PRMT5 (csul/dart5) is identified as the enzyme that catalyzes sDMAs of Piwi, Ago3 and Aub in Drosophila ovaries, in vivo. Both Aub and csul/dart5 (dPRMT5) are grandchildless genes and the finding that Aub is a substrate for dPRMT5, indicates that an important function of dPRMT5 in pole plasm function and PGC specification involves methylation of Aub. Intriguingly, tudor domains bind to sDMAs and Tudor protein is also a grandchildless gene that is required for pole plasm assembly and function. These findings suggest that pole plasm function may involve an interacting network of genes whose protein products contain sDMAs (Aub), the methylase (dPRMT5) and its cofactor (valois/dMEP50) that produce sDMAs and tudor domain (Tudor) proteins that may bind to sDMA-containing proteins. It is noted that both PRMT5 and tudor-domain-containing genes are found in all species that express Piwi family proteins and knockout of tudor domain containing 1/mouse tudor repeat 1 in mice leads to spermatogonial cell death and male sterility. Furthermore, it is noted that other Drosophila proteins whose genes are required for piRNA accumulation or function, such as Spindle-E/homeless, contain tudor domains. It will be interesting to test whether tudor domain containing proteins interact with sDMA-modified Piwi family proteins and to elucidate their function (Kirino, 2009).

Without Argonaute3, Aubergine-bound piRNAs collapse but Piwi-bound piRNAs persist

Piwi-interacting RNAs (piRNAs) silence transposons in the germ line of animals. They are thought to derive from long primary transcripts spanning transposon-rich genomic loci, 'piRNA clusters.' piRNAs are proposed to direct an auto-amplification loop in which an antisense piRNA, bound to Aubergine or Piwi protein, directs the cleavage of sense RNA, triggering production of a sense piRNA bound to the PIWI protein Argonaute3 (Ago3). In turn, the new piRNA is envisioned to direct cleavage of a cluster transcript, initiating production of a second antisense piRNA. This study describes strong loss-of-function mutations in ago3, allowing a direct genetic test of this model. It was found that Ago3 acts to amplify piRNA pools and to enforce on them an antisense bias, increasing the number of piRNAs that can act to silence transposons. A second piRNA pathway was found centered on Piwi and functioning without benefit of Ago3-catalyzed amplification. Transposons targeted by this second pathway often reside in the flamenco locus, which is expressed in somatic ovarian follicle cells, suggesting a role for piRNAs beyond the germ line (Li, 2009).

The ability to tame transposons while retaining them in the genome is a particular specialty of eukaryotes. Transposons, repetitive sequences, and other forms of 'selfish' DNA comprise as much as 42% of the human genome and nearly 30% of the genome of Drosophila melanogaster. In metazoa, transposons are silenced by the piRNA pathway, which is guided by 23-30 nt RNAs. The piRNA pathway is distinct from other RNA silencing pathways in that its small RNA guides are not produced by dicing. In contrast, both small interfering RNAs (siRNAs) and microRNAs (miRNAs) are cleaved by double-stranded RNA-specific endonucleases, Dicers, to yield double-stranded intermediates-siRNA or miRNA/miRNA* duplexes-that are loaded into members of the Argonaute family of proteins. piRNAs, too, act as guides for Argonaute proteins, but they appear not to exist as stable double-stranded intermediates at any point in their biogenesis. piRNAs bind PIWI proteins, a sub-family of Argonaute proteins that are expressed in germ-line cells. PIWI proteins were first identified by their roles in maintaining and patterning Drosophila germ cells. The defects in the organization of embryonic pattern in piRNA pathway mutations are likely an indirect consequence of their larger role in maintaining genomic stability. The three Drosophila PIWI proteins, Piwi, Aubergine (Aub), and Argonaute3 (Ago3), are expressed in the male and female germ line cells (Li, 2009).

The prevailing model for piRNA biogenesis-the 'ping-pong' model-reflects the discovery that the first 10 nt of piRNAs bound to Aub or Piwi, which are largely antisense and typically begin with uridine, are often complementary to the first 10 nt of piRNAs bound to Ago3, which are largely sense and typically bear an adenosine at position 10. Many Argonaute proteins can act as RNA-guided RNA endonucleases, and all such Argonautes cut their target RNAs 5' to the base that pairs with the tenth nucleotide of the small RNA guide; all three fly PIWI proteins retain their endonuclease activity. Thus, the observed 10 nt 5' complementarity between piRNAs suggests that the 5' ends of piRNAs bound to Aub or Piwi are defined by Ago3-catalyzed cleavage, and, reciprocally, that the 5' ends of piRNAs bound to Ago3 are defined by piRNAs bound to Aub or Piwi. The ping-pong model seeks to explain these observations, as well as the role of piRNA cluster transcripts in piRNA biogenesis, the function of piRNAs in silencing transposons, and the extraordinary antisense bias of piRNAs generally. At its core, the model proposes that piRNAs participate in an amplification loop in which transposon sense transcripts (e.g., transposon mRNAs) trigger the production of new, antisense piRNAs. Ago3, guided by sense piRNAs, lies at the heart of the amplification loop (Li, 2009).

To test for the ping-pong model, strong loss-of-function mutations were isolated in ago3. This study report the detailed analysis of ago3 and aub mutant flies. The data provides strong support for an amplification cycle in which Ago3 amplifies piRNA pools and enforces on them a strong antisense bias, increasing the number of piRNAs that can act to destroy transposon mRNAs. Moreover, a second, perhaps somatic, piRNA pathway was detected, centered on Piwi and functioning without benefit of Ago3-catalyzed amplification. Most of the transposons targeted by this second pathway reside in the flamenco piRNA cluster, which was first identified as a repressor of transposon expression in somatic follicle cells (Li, 2009).

Because ovaries contain both germ-line and somatic cells, the data conflate two distinct cell lineages. Combining the data with extensive genetic studies of gypsy and other transposon families represented in the flamenco locus, this study has attempted to disentangle germ-line and somatic piRNA function. It is proposed that the somatic piRNA pathway is the more straightforward, involving only Piwi and not Ago3 or Aub. The data suggest that Piwi cannot act alone to amplify piRNAs. It is envisioned that Piwi-bound piRNAs in the soma are produced by a ribonuclease that randomly generates single-stranded guides that are subsequently loaded into Piwi and trimmed to length. Although Piwi-bound piRNAs generally begin with U and Piwi shows in vitro a preference for binding small RNA that begins with U, current evidence cannot distinguish between a putative piRNA-generating ribonuclease cleaving mainly at U and Piwi selecting U1 piRNAs from a set of RNAs with all possible 5' nucleotides (Li, 2009).

A model is presented for piRNA biogenesis. The Aub- and Ago3-dependent piRNA amplification cycle is envisioned to operate only in the germ line, whereas a Piwi-dependent, Aub- and Ago3-independent pathway is shown for somatic cells. In the germ line, Piwi can also partner with Ago3 to amplify piRNAs. Without an amplification cycle to ensure an antisense bias, some other mechanism must operate to explain why Piwi-bound piRNAs are overwhelmingly antisense. A plausible but somewhat unsatisfying explanation comes from flamenco itself, whose constituent transposons are nearly all oriented in a single direction, so that the ∼160 kb flamenco transcript is almost entirely antisense to the transposons. How such a non-random array of transposons could arise is unknown. Other non-randomly oriented piRNA clusters may explain the smaller number of transposons in group III that are not present in flamenco (Li, 2009).

The transposons in most piRNA clusters do not show such a pronounced non-random orientation. These likely act in the germ line to produce primary piRNAs that load into Aub. The observed antisense bias of Aub-bound piRNAs arises subsequently, when Aub generates Ago3-bound secondary piRNAs and Ago3 acts, in turn, to produce Aub-bound secondary piRNAs. It is proposed that in the absence of Ago3, the sense/antisense ratio of Aub-bound piRNAs reverts to the inherent sense/antisense bias of the transposable element sequences present in the transcripts of piRNA clusters (Li, 2009).

For this cycle to skew the Aub-bound piRNA population toward antisense, the substrate for cleavage by primary piRNA-bound Aub must be largely sense RNA. The best candidate for such sense RNA is mRNA derived from actively transcribed transposon copies. If such sense mRNA were largely found in the cytoplasm, it would be spatially segregated from the cluster transcripts, which is envisioned to be retained in the nucleus. Supporting this idea, sense transcripts from the group I transposon, I element, normally accumulate only in the nuclei of germ-line nurse cells (Chambeyron, 2008), likely because they are destroyed in the cytoplasm by Aub-bound primary piRNAs and Aub-bound piRNAs produced by Ago3-dependent amplification. In the absence of Aub, these sense transposon transcripts accumulate in the cytoplasm instead, consistent with the strong desilencing of I element in aub and ago3 mutants (Li, 2009).

The piRNA ping-pong hypothesis predicts a role for Ago3 in the production of Aub-bound antisense piRNAs, but the current finding that loss of Ago3 also reduced the abundance of Piwi-bound antisense piRNAs was unexpected. The majority of Aub and Ago3 is found in nuage and in the cytoplasm, but Piwi is predominantly nuclear. How then can Ago3 direct the production of Piwi-bound piRNAs? Perhaps Piwi transits the nuage en route from its site of synthesis, the cytoplasm, to where it accumulates, the nucleus. In this view, cytoplasmic Piwi is predicted to lack a small RNA guide. Piwi would then acquire its small RNA guide in the nuage, through a process that requires Ago3. Loading a piRNA into Piwi might then license it for entry into the nucleus, where it could act post-transcriptionally or transcriptionally to silence transposon expression. In this view, mutations in genes required for nuage assembly or stability, such as vasa, as well as genes required for Piwi loading would reduce the amount of nuclear Piwi. A similar mechanism may operate in mammals, where the PIWI protein MILI is found in cytoplasmic granules, whereas MIWI2 is nuclear. In the absence of MILI, MIWI2 delocalizes from the nucleus to the cytoplasm, although MIWI2 is not required for the localization of MILI (Li, 2009).

Such a model cannot explain the loading of Piwi in the somatic follicle cells, which contain little or no Ago3 or Aub and which do not contain nuage. A simple but untested hypothesis for these cells is that in the absence of nuage, empty Piwi readily enters the nucleus, where it obtains its small RNA guide. It might reasonably be expected that in germ cells the absence of nuage would impair the loading of Piwi by eliminating the Ago3-dependent, germ-line specific Piwi-loading process, but also facilitate entry of some empty Piwi into the nucleus, where it could obtain small RNA guides. Consistent with this idea, some Piwi is detected in the nucleus in ago3 ovaries. The simplicity of this hypothesis, of course, belies the complexity of testing it (Li, 2009).

Why are there two distinct piRNA production pathways? Retrotransposons 'reproduce' by producing sense RNA encoding transposases and other proteins that allow them to jump to new locations in the germ cell genome. The conservation of the piRNA ping-pong cycle in animals suggests that it is an ancient and conserved germ-line defense against retrotransposition. In flies, the gypsy family of retroelements appears to have moved its reproductive cycle to the somatic follicle cells adjacent to the germ line, which it infects using retrovirus-like particles. gypsy thus appears to avoid germ-line piRNA surveillance by transcribing and packaging its RNA in the soma. Perhaps expression of Piwi in Drosophila follicle cells reflects an adaptive evolutionary counter move to the gypsy reproductive strategy. The simplicity of the direct loading of Piwi with antisense piRNAs derived from flamenco may have made this counter defense more evolutionarily accessible than a strategy requiring expression of all the proteins needed for the Ago3:Aub ping-pong mechanism. In the future, more extensive analysis of the cellular and genetic requirements for ping-pong-independent and ping-pong-dependent piRNA mechanisms in Drosophila melanogaster and in more ancient animal species may provide a test for these ideas (Li, 2009).

Roles for the Yb body components Armitage and Yb in primary piRNA biogenesis in Drosophila

PIWI-interacting RNAs (piRNAs) protect genome integrity from transposons. In Drosophila ovarian somas, primary piRNAs are produced and loaded onto Piwi. This study describes roles for the cytoplasmic Yb body components Armitage and Yb in somatic primary piRNA biogenesis. Armitage binds to Piwi and is required for localizing Piwi into Yb bodies. Without Armitage or Yb, Piwi is freed from the piRNAs and does not enter the nucleus. Thus, piRNA loading is required for Piwi nuclear entry. It is proposed that a functional Piwi-piRNA complex is formed and inspected in Yb bodies before its nuclear entry to exert transposon silencing (Saito, 2010).

In Drosophila, three sets of endogenous small RNAs have been identified so far: microRNAs (miRNAs), endogenous siRNAs (endo-siRNAs/esiRNAs), and PIWI-interacting RNAs (piRNAs). Of these, piRNAs are considered unique because of their germline-specific expression and specific interaction with germline-specific Argonaute proteins, PIWI proteins. The identification of the piRNAs associated with three PIWI proteins (Aubergine [Aub], Argonaute 3 [AGO3], and Piwi) has revealed distinct features of piRNAs associated with each PIWI and has led to two models for piRNA biogenesis: the primary processing pathway and the amplification loop pathway. In the amplification loop model, the Slicer (endonuclease) activity of Aub and AGO3 determines the formation of the 5' end of piRNAs. Zucchini (Zuc), a putative cytoplasmic nuclease, is involved in the primary processing pathway; however, its precise molecular function remains unclear. Furthermore, the factors other than zuc required for primary piRNA biogenesis are unknown (Saito, 2010).

The ovarian somatic cell (OSC) line consists of ovarian somas only. The expression of Aub and AGO3 is not detectable in OSCs because both proteins are germ cell-specific. This implies that the amplification loop does not operate in OSCs. However, OSCs express piRNAs and are loaded onto Piwi, indicating that the piRNAs in OSCs are generated specifically through the primary processing pathway. Thus, OSCs are an ideal tool to elucidate the molecular mechanisms of primary piRNA processing and Piwi function. Loss of zuc function drastically reduced the level of primary piRNAs in the ovaries. This was recapitulated in OSCs: Zuc depletion by RNAi caused a severe reduction in the piRNA level in OSCs. This result prompted a screen for other factors necessary for primary piRNA production using RNAi in OSCs (Saito, 2010).

To identify the genes required for somatic primary piRNA biogenesis, RNAi-based screening was performed in OSCs. The genes screened included armitage (armi), spindle-E (spn-E), and maelstrome (mael), all of which are implicated in piRNA biogenesis. However, their roles in somatic primary piRNA production remain unknown. Depletion of Armi reduced the piRNA levels to an extent very similar to that of Piwi and Zuc depletion, indicating that Armi is necessary for primary piRNA biogenesis in OSCs. Depletion of Mael and Spn-E showed little or no effect on piRNA accumulation in OSCs. Mutations in both genes have been shown to significantly reduce the piRNA levels in ovaries. Thus, spn-E and mael are factors functioning in the amplification loop. Depletion of Dicer1 and Dicer2 had little or no effect on the piRNA levels, confirming that neither protein is necessary for piRNA production (Saito, 2010).

Armi is the Drosophila ortholog of Arabidopsis Silencing-Defective 3 (SDE3) and mammalian Moloney leukemia virus 10 (MOV10). These orthologs contain a conserved ATP-dependent RNA helicase domain at their C termini and have been implicated in small RNA-mediated gene silencing. However, their precise functions remain unknown. To gain further insight into the function of Armi in somatic primary piRNA processing, a monoclonal antibody was produced against Armi. Western blotting showed a discrete band in both ovary and cultured Schneider2 (S2) cell lysates, indicating that Armi expression is not germline-specific. The ~150-kDa protein immunopurified from S2 cells with the anti-Armi antibody was confirmed to be Armi by mass spectrometry (Saito, 2010).

Immunostaining of OSCs and ovaries with the anti-Armi antibody confirmed an earlier observation that Armi is a cytoplasmic protein. The Armi signals were detected in both somatic and germ cells of ovaries. The somatic signal was considered a background signal because it did not disappear even in armi homozygous mutant egg chambers. In the present study, the cytoplasmic signal in OSCs mostly disappeared when Armi was depleted by RNAi. Thus, it is concluded that Armi is expressed in both somatic and germ cells in ovaries (Saito, 2010).

The subcellular localization of Armi in the armi trans-heterozygous mutants appeared very similar to that in the homozygous mutants. In addition, Western blotting revealed a band corresponding to Armi in the armi ovaries. By what mechanisms Armi is expressed in the mutant somas remains unclear. The simplest explanation is that the armi gene uses two distinct genomic elements as promoters in ovarian somas. In fact, armi homozygous mutants weakly express a shorter armi transcript than that expressed in the wild-type strain (Saito, 2010).

The Armi signal in germ cells was rather weak, and only a small proportion of Armi accumulated at, or near, the nuage, an electron-dense structure associated with nurse cell nuclei. Thus, Armi might not be a component of the nuage per se. This correlates well with the fact that armi mutations barely affected the ability of the ovaries to amplify endogenous piRNAs. In ovarian somas, Armi accumulated strongly at discrete cytoplasmic foci. Each somatic cell contained one or several foci. Interestingly, the Armi-positive foci were often located near the nucleus in both ovaries and OSCs (Saito, 2010)

Piwi is required for the silencing of transposons in gonads. In fact, Piwi depletion in OSCs caused derepression of transposons, as with Armi, Yb, and Zuc depletion. Under conditions where endogenous Piwi was depleted, expression of myc-Piwi-r, which was designed to be RNAi-insensitive, rescued transposon silencing. However, myc-Piwi-δN, which lacks 72 amino acids at the N terminus of Piwi and thus does not localize to the nucleus, did not rescue transposon silencing, although it does associate with piRNAs to the same extent as does the wild-type Piwi. myc-Piwi-δN13, which lacks 13 amino acids at the N terminus, behaved similarly. On the other hand, myc-Piwi-DDAA-r, a Slicer mutant of Piwi, could bind to mature piRNAs in OSCs, as does the wild-type Piwi, and rescued transposon silencing. These results might suggest that Piwi must be localized in the nucleus to silence the transposable elements, and that Piwi Slicer activity is unnecessary for its function. It is assumed that this system has evolved to prevent nascent Piwi, not loaded with piRNAs, from being imported into the nucleus. In other words, only the functional Piwi-piRNA complex (piRISC) formed at Yb bodies could be transported to the nucleus. At present, the mechanisms of this control system remain unclear. In the nongonadal somatic S2 cell line, where the expression of piRNAs is undetectable, transfected Piwi is localized to the nucleus, indicating that 'empty' Piwi can be transported to the nucleus. It seems that the machineries necessary for the nuclear transport of Piwi might recognize different features of Piwi in different cell types (Saito, 2010).

How is piRNA-free Piwi restrained in the cytoplasm in OSCs? One possibility is that some unknown protein binds the N-terminal end of Piwi, where its NLS (nuclear localization signal) resides, and interferes with the nuclear import machinery's ability to recognize Piwi as a cargo. The nuclear localization inhibitory factors may be retained on Piwi until a functional Piwi-piRNA complex is formed at Yb bodies. Once the complex is formed, a conformational change in Piwi would be induced, which would release the regulatory factors and reveal the Piwi NLS for recognition by the nuclear import machinery. It would be very interesting to determine the proteins that are associated with Piwi in OSCs under conditions of Armi or Zuc depletion, thus identifying the protein factors that restrain Piwi in the cytoplasm until it is loaded with mature piRNAs at Yb bodies (Saito, 2010).

Vreteno, a gonad-specific protein, is essential for germline development and primary piRNA biogenesis in Drosophila

In Drosophila, Piwi proteins associate with Piwi-interacting RNAs (piRNAs) and protect the germline genome by silencing mobile genetic elements. This defense system acts in germline and gonadal somatic tissue to preserve germline development. Genetic control for these silencing pathways varies greatly between tissues of the gonad. This study identified Vreteno (Vret), a novel gonad-specific protein essential for germline development. Vret is required for piRNA-based transposon regulation in both germline and somatic gonadal tissues. Vret, which contains Tudor domains, associates physically with Piwi and Aubergine (Aub), stabilizing these proteins via a gonad-specific mechanism that is absent in other fly tissues. In the absence of vret, Piwi-bound piRNAs are lost without changes in piRNA precursor transcript production, supporting a role for Vret in primary piRNA biogenesis. In the germline, piRNAs can engage in an Aub- and Argonaute 3 (AGO3)-dependent amplification in the absence of Vret, suggesting that Vret function can distinguish between primary piRNAs loaded into Piwi-Aub complexes and piRNAs engaged in the amplification cycle. It is proposed that Vret plays an essential role in transposon regulation at an early stage of primary piRNA processing (Zamparini, 2011).

Propagation of all sexually reproducing organisms depends upon the faithful development and function of reproductive organs. In Drosophila, oogenesis requires the coordinated differentiation of two distinct cell lineages, the germline and the gonadal somatic cells, to produce an egg. The germarium, where oogenesis initiates, contains both germline and somatic stem cells. Asymmetric cell division of germline stem cells (GSCs) within the germarium generates both a stem cell and a differentiated daughter cell, the cystoblast, which gives rise to a sixteen-cell interconnected cyst. One of the sixteen cells in the cyst differentiates into an egg and the remaining cells become nurse cells. Somatic cell populations are intimately associated with germ cells during oogenesis: niche cells provide GSC maintenance signals and are tightly connected to GSCs via adhesion and gap junctions; inner sheath cells (ISCs) intermingle with the differentiating cystoblast and early dividing cysts to promote formation of the sixteen-cell cyst; follicle stem cells and their progeny, the follicle cells, surround each germline cyst as it buds off from the germarium and provide the maturing egg chamber with the positional cues needed for establishment of anterior-posterior and dorsal-ventral polarity of the embryo (Zamparini, 2011).

In addition to germline development, genomic integrity must be preserved to generate viable progeny. In Drosophila, transposable elements occupy nearly one third of the genome and mobilization of even one of almost 150 transposon classes found can lead to defects in gametogenesis and sterility. Therefore, organisms have evolved small RNA-based defense systems to fight these elements (Malone, 2009). In Drosophila, both germline and somatic cells of the ovary rely on Piwi proteins and their 23-29 nt Piwi-interacting RNAs (piRNAs) to combat transposon activity. All three Drosophila Piwi proteins, Piwi, Aubergine (Aub) and Argonaute 3 (AGO3), are expressed in germline cells, whereas Piwi is also expressed in somatic gonadal cells. Interestingly, mutations in all known piRNA pathway components lead to oocyte and embryonic patterning defects and, ultimately, to sterility, believed to be an indirect consequence of transposon-induced genomic instability and activation of a DNA double-strand break checkpoint (Zamparini, 2011 and references therein).

In contrast to other small RNAs, such as microRNAs and siRNAs, which are produced from double-stranded RNA precursors, piRNAs are derived from single-stranded RNA precursors, independently of the endonuclease Dicer. piRNA precursors originate from either active transposon transcripts or discrete genomic loci known as 'piRNA clusters'. In Drosophila, piRNA clusters provide the primary source of antisense transposon transcripts, whereas active transposons predominantly provide sense transcripts. piRNAs associated with Piwi and Aub are mostly derived from piRNA clusters, mapping complementary to active transposons, whereas AGO3-bound piRNAs appear to be derived from the transposon itself. This relationship and a 10 nt overlap observed between sense and antisense piRNA pairs led to a model of piRNA amplification termed 'ping-pong', in which 5' ends of new piRNAs are generated through cleavage by the Piwi proteins themselves (Brennecke, 2007; Gunawardane, 2007). In the Drosophila ovary, piRNA 'ping-pong' is restricted to germline cells in which Piwi, Aub and AGO3 are present, although Piwi appears to be mostly dispensable for 'ping-pong' amplification (Malone, 2009). In gonadal somatic cells, in which only Piwi is expressed, an alternative pathway functions. Here, single-stranded piRNA clusters or gene transcripts are processed to produce 'primary' piRNAs that are directly loaded into Piwi, targeting active transposons or endogenous genes (Li, 2009; Malone, 2009; Saito, 2009). The overlapping genetic requirements for Piwi in the germline and ovarian somatic cells suggest that Piwi may also engage primary piRNAs in the germline. Like Piwi, the germline-specific Aub engages piRNAs complementary to transposons, but has not been directly linked to primary piRNAs. Therefore, the precise relationship between primary piRNAs and 'ping-pong' in the germline remains largely unknown (Zamparini, 2011).

The restriction of piRNA production and transposon control in gonadal tissues raises the question of how the piRNA biogenesis machinery has evolved specifically in the gonad. This study has identified Vreteno (Vret), a gonad-specific, Tudor domain-containing protein that functions specifically in the germline and somatic gonadal tissues during oogenesis. Vret broadly regulates transposon levels and has an essential role in primary piRNA biogenesis, leaving 'ping-pong' amplification intact (Zamparini, 2011).

This study identified a novel protein with critical roles in oocyte polarity, germline and soma differentiation, survival and transposon control. Vret, a Tudor-domain containing protein, associates with Piwi proteins in the cytoplasm of Drosophila ovarian cells and regulates their stability, as well as Piwi nuclear localization and localization of Aub to nuage. In the absence of Vret, piRNAs are dramatically reduced and transposons mobilized. By ordering the function of Vret within the network of the piRNA-transposon-based system, it is concluded that Vret functions in primary piRNA biogenesis at the stage of primary piRNA loading onto Piwi and Aub complexes (Zamparini, 2011).

Loss of Vret in the soma or germline has strikingly different morphological consequences. Molecular analysis, however, suggests the same underlying cause for these defects: a failure to produce biologically active piRNAs. Morphologically, the vret germline phenotype resembles that of mutants defective in germline piRNA biogenesis, such as aub, spnE and krimper. In these mutants, transposon mobilization activates a DNA damage checkpoint that leads to defects in transport and translation of maternal RNAs necessary for oocyte polarity and embryonic patterning. Interestingly, lack of vret in the soma resembles the piwi mutant phenotype, in which GSCs fail to differentiate as a consequence of somatic cell death, an event presumably associated with transposon misregulation. Thus, loss of vret in the germline and gonadal soma resembles loss of both Piwi and Aub. This, together with the findings that Vret associates with Piwi and Aub in ovarian extracts and affects the stability of both, strongly suggests that Vret regulates both proteins in a similar fashion (Zamparini, 2011).

Surprisingly, Vret is not required for piRNA 'ping-pong' amplification per se, suggesting that Vret might selectively interact with Aub and Piwi bound to primary piRNAs and not to those engaged in 'ping-pong'. In this scenario, it would be possible for maternally deposited Aub to initiate the 'ping-pong' cycle with AGO3, even in the absence of Vret (Brennecke, 2008). As some Aub protein remains in vret mutant ovaries, an active pool of Vret-independent Aub could maintain 'ping-pong' activity throughout the adult ovary. Therefore, it is proposed that a 'ping-pong'-independent pool of Aub within the cytoplasm depends upon primary piRNA loading, downstream of Vret function. It would be interesting to examine whether piRNAs associated with the Vret-dependent complex can, at any level, contribute to 'ping-pong', or whether Aub-bound primary piRNAs are functionally or enzymatically distinct from those involved in the piRNA amplification cycle (Zamparini, 2011).

In contrast to Aub, only a small subset of Piwi-bound piRNAs showed a 10 nt overlap with those bound to AGO3. Indeed, Piwi is genetically dispensible for 'ping-pong' and might be only marginally involved in 'ping-pong', if at all (Brennecke, 2007; Li, 2009). As Piwi slicer activity does not appear to be required for Piwi function (Saito, 2009), it seems most plausible that Piwi would act as a recipient, and not as an 'active' component of 'ping-pong' amplification. Regardless, the majority of Piwi-bound primary piRNAs act independently of 'ping-pong' and depend upon Vret for stability (Zamparini, 2011).

An ectopic expression experiment suggests that Piwi is not 'intrinsically unstable', but becomes unstable in the gonad in the absence of Vret. Furthermore, Vret is not required for Piwi or Aub transcription or translation. Vret, therefore, could either coordinate the process of biogenesis and loading of primary piRNAs into Piwi and Aub complexes or be involved in stabilizing the mature RISC (RNA-induced silencing complex). Armi, a putative helicase, and Zucchini (Zuc), a member of the phospholipase D (PLD) family of phosphodiesterases, act like Vret in the soma and germline; they specifically affect Piwi protein stability and primary piRNA levels leaving the 'ping-pong' cycle intact. Unlike Vret, the levels of unprocessed precursor RNA from flam are increased in zuc mutants implicating Zuc in piRNA cluster transcript processing. Therefore the hypothesis is favored that Vret, possibly together with Armi, is an essential component of Piwi and Aub RISC complexes. Vret is one of many Tudor domain proteins in Drosophila that affects piRNA biogenesis and contains conserved residues that are known to be required for binding of sDMAs found in Piwi proteins (Siomi, 2010). When mutated, each of these genes displays a rather distinct phenotype. Krimper and SpnE regulate transposon levels in the germline whereas fs(1)Yb is soma-specific. Vret is, at this point, the only Tudor domain protein known to be required in both tissues, suggesting a conserved and global role for this gene in piRNA regulation. It remains to be determined whether the mammalian Tudor homolog could fulfill a similar function (Zamparini, 2011).

PAPI, a novel TUDOR-domain protein, complexes with AGO3, ME31B and TRAL in the nuage to silence transposition

The nuage is a germline-specific perinuclear structure that remains functionally elusive. Recently, the nuage in Drosophila was shown to contain two of the three PIWI proteins - Aubergine and Argonaute 3 (AGO3) - that are essential for germline development. The PIWI proteins bind to PIWI-interacting RNAs (piRNAs) and function in epigenetic regulation and transposon control. This study reports a novel nuage component, PAPI (Partner of PIWIs), that contains a TUDOR domain and interacts with all three PIWI proteins via symmetrically dimethylated arginine residues in their N-terminal domain. In adult ovaries, PAPI is mainly cytoplasmic and enriched in the nuage, where it partially colocalizes with AGO3. The localization of PAPI to the nuage does not require the arginine methyltransferase dPRMT5 or AGO3. However, AGO3 is largely delocalized from the nuage and becomes destabilized in the absence of PAPI or dPRMT5, indicating that PAPI recruits PIWI proteins to the nuage to assemble piRNA pathway components. As expected, papi deficiency leads to transposon activation, phenocopying piRNA mutants. This further suggests that PAPI is involved in the piRNA pathway for transposon silencing. Moreover, AGO3 and PAPI associate with the P body component TRAL/ME31B complex in the nuage and transposon activation is observed in tral mutant ovaries. This suggests a physical and functional interaction in the nuage between the piRNA pathway components and the mRNA-degrading P-body components in transposon silencing. Overall, this study reveals a function of the nuage in safeguarding the germline genome against deleterious retrotransposition via the piRNA pathway (Liu, 2011).

Although the nuage has long been discovered in the germline of diverse organisms, little is known about its function. In this study identified and molecularly characterized a novel nuage component, PAPI. PAPI is a TUDOR-domain-containing protein that recruits PIWI proteins, especially AGO3, to the nuage and stabilizes them. The interaction between PAPI and AGO3 in the nuage is mediated by sDMAs in the N-terminal domain of AGO3 but is RNA independent. Previous studies have suggested the nuage as the cytoplasmic loci where post-transcriptional silencing of transposons occurs. In addition, loss of Drosophila TUDOR protein has been shown to affect the localization of AUB to the nuage and to alter the piRNA profile. The new findings of this study indicate that TUDOR-domain-containing proteins might serve as a platform for the recruitment of PIWI proteins to the nuage and for the assembly of piRNA pathway components. A subset of transposons are de-repressed in papi deficient ovaries, suggesting that PAPI is involved in transposon silencing in the nuage, just like other piRNA pathway components. This study thus reveals a function of the nuage in safeguarding the germline genome against deleterious retrotransposition via the piRNA pathway (Liu, 2011).

Furthermore, a physical association of PAPI and AGO3 with the TRAL/ME31B complex has been identifie along with their colocalization in the nuage, and the role of these P body proteins in silencing the expression of some transposons. The TRAL/ME31B complex has been shown to interact with CUP, which also associates with the nuclear pore complex component NUP154 (Grimaldi, 2007). The current findings reveal an exciting physical and functional link between the piRNA machinery and the P body components in the nuage and a mechanism for nuage localization to the nuclear periphery. The P body proteins are well known for their function in mRNA processing and degradation, yet the piRNA machinery regulates transposon silencing by reducing the level of their mRNAs. The physical interaction between these two machineries, with the functional relationship among known components of these two machineries in the nuage illustrated in a working model, raises the intriguing possibility that these two pathways work together in the nuage as a post-transcriptional mechanism to degrade transposon mRNAs, leading to transposon silencing. In addition, these data implicate the interaction of between the TRAL/ME31B complex and NUP154 via CUP as a mechanism of nuage localization to the nuclear periphery (Liu, 2011).

Tudor-SN interacts with Piwi antagonistically in regulating spermatogenesis but synergistically in silencing transposons in Drosophila

Piwi interacts withTudor-SN (Tudor staphylococcal nuclease, TSN) antagonistically in regulating spermatogenesis but synergistically in silencing transposons. However, it is not required for piRNA biogenesis. This study shows that TSN colocalizes with Piwi in primordial germ cells (PGCs) and embryonic somatic cells. In adult ovaries and testes, TSN is ubiquitously expressed and enriched in the cytoplasm of both germline and somatic cells. The tsn mutants display a higher mitotic index of spermatogonia, accumulation of spermatocytes, defects in meiotic cytokinesis, a decreased number of spermatids, and eventually reduced male fertility. Germline-specific TSN-expression analysis demonstrates that this function is germline-dependent. Different from other known Piwi interacters, TSN represses Piwi expression at both protein and mRNA levels. Furthermore, reducing piwi expression in the germline rescues tsn mutant phenotype in a dosage-dependent manner, demonstrating that Piwi and TSN interact antagonistically in germ cells to regulate spermatogenesis. However, the tsn deficiency has little, if any, impact on piRNA biogenesis but displays a synergistic effect with piwi mutants in transposon de-silencing. These results reveal the biological function of TSN and its contrasting modes of interaction with Piwi in spermatogenesis, transposon silencing, and piRNA biogenesis (Ku, 2018).

Oncogenic transformation of Drosophila somatic cells induces a functional piRNA pathway

Germline genes often become re-expressed in soma-derived human cancers as 'cancer/testis antigens' (CTAs), and piRNA (PIWI-interacting RNA) pathway proteins are found among CTAs. However, whether and how the piRNA pathway contributes to oncogenesis in human neoplasms remain poorly understood. This study found that oncogenic Ras combined with loss of the Hippo tumor suppressor pathway reactivates a primary piRNA pathway in Drosophila somatic cells coincident with oncogenic transformation. In these cells, Piwi becomes loaded with piRNAs derived from annotated generative loci, which are normally restricted to either the germline or the somatic follicle cells. Negating the pathway leads to increases in the expression of a wide variety of transposons and also altered expression of some protein-coding genes. This correlates with a reduction in the proliferation of the transformed cells in culture, suggesting that, at least in this context, the piRNA pathway may play a functional role in cancer (Fagegaltier, 2016).

c-Fos repression by Piwi regulates Drosophila ovarian germline formation and tissue morphogenesis

Drosophila melanogaster Piwi functions within the germline stem cells (GSCs) and the somatic niche to regulate GSC self-renewal and differentiation. How Piwi influences GSCs is largely unknown. This study uncovered a genetic interaction between Piwi and c-Fos in the somatic niche that influences GSCs. c-Fos is a proto-oncogene that influences many cell and developmental processes. In wild-type ovarian cells, c-Fos is post-transcriptionally repressed by Piwi, which destabilized the c-Fos mRNA by promoting the processing of its 3' untranslated region (UTR) into Piwi-interacting RNAs (piRNAs). The c-Fos 3' UTR was sufficient to trigger Piwi-dependent destabilization of a GFP reporter. Piwi represses c-Fos in the somatic niche to regulate GSC maintenance and differentiation and in the somatic follicle cells to affect somatic cell disorganization, tissue dysmorphogenesis, oocyte maturation arrest, and infertility (Klein, 2016).

Co-chaperone Hsp70/Hsp90 organizing protein (Hop) is required for transposon silencing and piRNA biogenesis

piRNAs are 26-30nt germ-line specific small non-coding RNAs that have evolutionarily conserved function in mobile genetic element (transposons) silencing and maintenance of genome integrity. Drosophila Hsp70/90 Organizing Protein Homolog (Hop), a co-chaperone, interacts with piRNA binding protein Piwi and mediates silencing of phenotypic variations. However, it is not known if Hop has a direct role in piRNA biogenesis and transposon silencing. This study shows that knockdown of Hop in the germ-line nurse cells (GLKD) of Drosophila ovaries leads to activation of transposons. Hop GLKD females can lay eggs at the same rate as wild type counterparts but the eggs do not hatch into larvae. Hop GLKD leads to the accumulation of γ-H2Av foci in the germline indicating increased DNA damage in the ovary. Hop GLKD induced transposon up-regulation is due to inefficient piRNA biogenesis. Based on these results, the study concludes that Hop is a critical component of piRNA pathway and it maintains genome integrity by silencing transposons (Karem, 2017).

Piwi interacts with chromatin at nuclear pores and promiscuously binds nuclear transcripts in Drosophila ovarian somatic cells

Piwi in a complex with Piwi-interacting RNAs (piRNAs) triggers transcriptional silencing of transposable elements (TEs) in Drosophila ovaries, thus ensuring genome stability. To do this, Piwi must scan the nascent transcripts of genes and TEs for complementarity to piRNAs. The mechanism of this scanning is currently unknown. This study reports the DamID-seq mapping of multiple Piwi-interacting chromosomal domains in somatic cells of Drosophila ovaries. These domains significantly overlap with genomic regions tethered to Nuclear Pore Complexes (NPCs). Accordingly, Piwi was coimmunoprecipitated with the component of NPCs Elys and with the Xmas-2 subunit of RNA transcription and export complex, known to interact with NPCs. However, only a small Piwi fraction has transient access to DNA at nuclear pores. Importantly, although 36% of the protein-coding genes overlap with Piwi-interacting domains and RNA-immunoprecipitation results demonstrate promiscuous Piwi binding to numerous genic and TE nuclear transcripts, according to available data Piwi does not silence these genes, likely due to the absence of perfect base-pairing between piRNAs and their transcripts (Ilyin, 2017).

Drosophila PAF1 modulates PIWI/piRNA silencing capacity

To test the directness of factors in initiating PIWI-directed gene silencing, this study employed a Piwi-interacting RNA (piRNA)-targeted reporter assay in Drosophila ovary somatic sheet (OSS) cells. This assay confirmed direct silencing roles for piRNA biogenesis factors and PIWI-associated factors but suggested that chromatin-modifying proteins may act downstream of the initial silencing event. These data also revealed that RNA-polymerase-II-associated proteins like PAF1 and RTF1 antagonize PIWI-directed silencing. PAF1 knockdown enhances PIWI silencing of reporters when piRNAs target the transcript region proximal to the promoter. Loss of PAF1 suppresses endogenous transposable element (TE) transcript maturation, whereas a subset of gene transcripts and long-non-coding RNAs adjacent to TE insertions are affected by PAF1 knockdown in a similar fashion to piRNA-targeted reporters. Additionally, transcription activation at specific TEs and TE-adjacent loci during PIWI knockdown is suppressed when PIWI and PAF1 levels are both reduced. This study suggests a mechanistic conservation between fission yeast PAF1 repressing AGO1/small interfering RNA (siRNA)-directed silencing and Drosophila PAF1 opposing PIWI/piRNA-directed silencing (Clark, 2017).

Structural insights into the sequence-specific recognition of Piwi by Drosophila Papi

The Tudor domain-containing (Tdrd) family proteins play a critical role in transposon silencing in animal gonads by recognizing the symmetrically dimethylated arginine (sDMA) on the (G/A)R motif of the N-terminal of PIWI family proteins via the eTud domains. Papi, also known as "Tdrd2," is involved in Zucchini-mediated PIWI-interacting RNA (piRNA) 3'-end maturation. Intriguingly, a recent study showed that, in papi mutant flies, only Piwi-bound piRNAs increased in length, and not Ago3-bound or Aub-bound piRNAs. However, the molecular and structural basis of the Papi-Piwi complex is still not fully understood, which limits mechanistic understanding of the function of Papi in piRNA biogenesis. This study determined the crystal structures of Papi-eTud in the apo form and in complex with a peptide containing unmethylated or dimethylated R10 residues. Structural and biochemical analysis showed that the Papi interaction region on the Drosophila Piwi contains an RGRRR motif (R7-R11) distinct from the consensus (G/A)R motif recognized by canonical eTud. Mass spectrometry results indicated that Piwi is the major binding partner of Papi in vivo. The papi mutant flies suffered from both fertility and transposon-silencing defects, supporting the important role conferred to Papi in piRNA 3' processing through direct interaction with Piwi proteins (Zhang, 2018).

A critical role for nucleoporin 358 (Nup358) in transposon silencing and piRNA biogenesis in Drosophila

Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNAs that bind Piwi proteins to silence transposons and to regulate gene expression. In Drosophila germ cells, the Aubergine (Aub)-Argonaute 3 (Ago3)-dependent ping-pong cycle generates most germline piRNAs. Loading of anti-sense piRNAs amplified by this cycle enables Piwi to enter the nucleus and silence transposons. Nuclear localization is crucial for Piwi function in transposon silencing, but how this process is regulated remains unknown. It is also not known whether any of the components of the nuclear pore complex (NPC) directly function in the piRNA pathway. This study shows that nucleoporin 358 (Nup358) and Piwi interact with each other and that a germline knockdown (GLKD) of Nup358 with short hairpin RNA prevents Piwi entry into the nucleus. The Nup358 GLKD also activated transposons, increased genomic instability, and derailed piRNA biogenesis because of a combination of decreased piRNA precursor transcription and a collapse of the ping-pong cycle. These results point to a critical role for Nup358 in the piRNA pathway, laying the foundation for future studies to fully elucidate the mechanisms by which Nup358 contributes to piRNA biogenesis and transposon silencing (Parikh, 2018).

Panoramix enforces piRNA-dependent cotranscriptional silencing

The Piwi-interacting RNA (piRNA) pathway is a small RNA-based innate immune system that defends germ cell genomes against transposons. In Drosophila ovaries, the nuclear Piwi protein is required for transcriptional silencing of transposons, though the precise mechanisms by which this occurs are unknown. This study show that the CG9754 protein is a component of Piwi complexes that functions downstream of Piwi and its binding partner, Asterix, in transcriptional silencing. Enforced tethering of CG9754 to nascent messenger RNA transcripts causes cotranscriptional silencing of the source locus and the deposition of repressive chromatin marks. CG9754 has been named "Panoramix," and it is proposed that this protein could act as an adaptor, scaffolding interactions between the piRNA pathway and the general silencing machinery that it recruits to enforce transcriptional repression (Yu, 2015).

The Piwi interacting RNA (piRNA) pathway controls transposons through a number of distinct, but likely interlinked, mechanisms. Whereas cytoplasmic Piwi proteins silence their targets posttranscriptionally through piRNA-directed cleavage and the ping-pong cycle, nuclear Piwi-piRNA complexes function at the transcriptional level. Piwi-directed repression of transcription is thought to be dependent on piRNA-guided recognition of nascent transposon transcripts. Transcriptional gene silencing (TGS) correlates with the presence of histone H3 lysine 9 trimethylation (H3K9me3) marks, yet the mechanism through which Piwi binding promotes the deposition of these marks remains enigmatic. With the exception of the zinc finger protein Asterix (also known as DmGTSF1), the components of Piwi effector complexes at target loci are largely unexplored (Yu, 2015).

This study systematically mined candidate genes from RNA interference (RNAi) screens for potential TGS effector proteins and identified CG9754 in three independently published screens as being critical in both the germ cells and follicle cells for transposon silencing. Loss of CG9754 had essentially no effect on the abundance or content of piRNA populations or on the nuclear localization of Piwi protein, suggesting that it is probably an effector component. CG9754 encodes a ~60-kD nuclear protein with no identifiable domains. The expression of CG9754 is restricted to the female gonads, as is seen for other core piRNA pathway components such as Asterix (Yu, 2015).

To examine global effects on transposon expression, RNA sequencing (RNA-seq) was used to measure steady-state RNA levels from ovaries with germline-specific knockdowns of either CG9754 or Piwi. Piwi knockdown caused a sharp rise in transposon transcripts, with minimal effects on protein-coding gene expression. Knockdown of CG9754 caused effects very similar to those of Piwi, with most transposon targets being shared. Changes in steady-state RNA levels could have resulted from alterations in either element transcription or the stability of transposon mRNAs. Global run-on sequencing (GRO-seq) was used to measure nascent RNA synthesis following gene knockdown. Loss of either CG9754 or Piwi produced very similar profiles, suggesting that CG9754 is specifically required for transcriptional silencing of transposons targeted by Piwi (Yu, 2015).

Piwi-mediated TGS correlates with the presence of H3K9me3 marks at silenced transposons. Depletion of either CG9754 or Piwi resulted in nearly identical losses of H3K9me3 over transposons. Four independent frameshift mutations of CG9754 generated via the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein system were isolated. A consistent global up-regulation of transposable elements and the corresponding loss of H3K9me3 marks in CG9754 mutant ovaries were observed without changes in piRNA levels. Similarly to other core piRNA pathway mutants, female flies lacking CG9754 were sterile. Moreover, flies double-mutant for CG9754 and Asterix showed transposon derepression comparable to that of flies with either single mutation, suggesting that both genes act in the same pathway. Thus, CG9754 functions along with Piwi and Asterix in the repression of transposon transcription (Yu, 2015).

Next it was asked whether the presence of CG9754 at a target locus might be sufficient to induce its silencing. Because piRNAs likely direct binding to nascent RNAs rather than to the DNA of their targets, CG9754 was delivered via protein-RNA interactions. A series of luciferase reporters was constructed with BoxB sites in their 3' untranslated regions (UTRs) and they were used to create transgenic reporter flies. BoxB sites are bound by the λN protein, which can also bring other components to the RNA as part of a fusion. Flies were also generated expressing λN proteins fused to CG9754, Asterix, nuclear Piwi, and, as a negative control, a cytoplasmic Piwi missing its nuclear localization signal (dN-Piwi). When coexpressed with the reporter, the dN-Piwi fusion failed to induce any change in luciferase expression. Of the remainder, only the CG9754 fusion considerably reduced luciferase activity. Silencing appeared to be dosage-dependent, as the degree of repression correlated with the number of BoxB binding sites inserted into the reporter mRNA. Consistent with a role for CG9754 in transcriptional silencing, the abundance of the reporter mRNAs was significantly reduced upon tethering (Yu, 2015).

Although CG9754-triggered repression appeared to be independent of chromatin context, integration of the reporter into genomic DNA appeared to be critical for repression. Transient cotransfection of reporter constructs into the OSS cell line, which contains an active piRNA pathway, resulted in little to no detectable silencing. In contrast, tethering Drosophila Ago1 (λN-dAgo1) to the luciferase reporter mRNA in OSS cells caused substantial repression of the same reporter. These results indicate that CG9754 can function properly only in the context of chromatin, likely acting at the transcriptional level, by interacting with nascent transcripts (Yu, 2015).

To test the hypothesis that λN-CG9754 acts on nascent transcripts, a reporter was generated for which the BoxB binding sites were located within the intron of the primary transcript. λN-CG9754 maintained the ability to repress this reporter but not a similar transcript carrying BoxB sites in the antisense orientation integrated into the same genomic locus. Because the spliced, mature reporter transcripts lack the BoxB sites, it was reason that λN-CG9754 must be able to exert its effects by binding to unspliced precursor mRNAs. Given that splicing occurs cotranscriptionally, this implies that CG9754 confers its effects by interaction with the nascent transcript (Yu, 2015).

If CG9754 mediates Piwi-dependent transcriptional silencing, delivery of CG9754 alone might recapitulate hallmarks of piRNA-directed repression. Tethering of CG9754 had a highly specific effect, changing levels of only the reporter mRNA. Repression occurred at the transcriptional level, as GRO-seq indicated a loss of nascent RNA from the integrated reporter. Repression by CG9754 also correlated with specific deposition of H3K9me3 marks over the reporter locus. Tethering of CG9754 failed to trigger piRNA production from the reporter, as has been seen previously for some loci that become targets of the piRNA pathway. Of note, spreading of H3K9me3 marks to other regions of the reporter gene was observed, as described previously for regions flanking piRNA-targeted transposon insertions. Thus, delivering CG9754 to the nascent RNA causes repression of a locus in a manner that mimics targeting by the piRNA pathway (Yu, 2015).

Whether CG9754 might be a component of Piwi complexes, as predicted by epistasis experiments, was tested. Functional GFP-Piwi fusion proteins copurified with hemagglutinin (HA)-tagged CG9754 from OSS cells, but not with a negative-control fusion (HA-mKate2). Conversely, Flag-tagged CG9754 was able to specifically precipitate endogenous Piwi proteins from OSS cell lysates, confirming the interaction between these two proteins. Given its properties, CG9754 was named 'Panoramix,' after the mentor who empowers the French comic book character Asterix to perform his feats of strength (Yu, 2015).

The identification of Panoramix as a key mediator of piRNA-directed TGS presented an opportunity to use the tethering assay to dissect the mechanism of transcriptional silencing. RNAi was used to deplete selected piRNA pathway genes in flies in which λN-Panoramix was tethered to the luciferase-BoxB reporter. Knockdown of Panoramix itself weakened the repression significantly, as compared with a control knockdown (mCherry). Silencing of factors required for piRNA biogenesis (Zuc and Armi) or those that are expected to act upstream of Panoramix (Piwi and Asterix) did not significantly affect repression. Depletion of dLSD1/Su(var)3-3 and its cofactor, CoREST, which normally form a complex that removes H3K4me2 marks from promoters, had significant effects on the ability of Panoramix to repress the reporter. Because H3K4me2 marks actively transcribed genes, it is possible that dLSD1-mediated removal of these marks is a key step in Panoramix-mediated transcriptional silencing. This raises a potential parallel with piRNA-directed silencing in mice, wherein engagement by DNMT3L, which is necessary for piRNA-induced DNA methylation, requires removal of such marks. Similarly, knockdown of HP1a caused derepression, in agreement with its role as a constitutive heterochromatin component required for transposon silencing and with the observation that the presence of Panoramix is correlated with the deposition of H3K9me3 marks at target loci. The H3K9 methyltransferase Eggless/dSETDB1 and its cofactor Windei appeared to be required specifically for Panoramix-mediated silencing, as knockdown of G9a, another H3K9 methyltransferase, showed no effect on the reporter. In eggless mutants, essentially complete derepression of the reporter was observed, despite Panoramix tethering. In contrast, the piRNA biogenesis mutant zuc showed little to no effect on the repression of the reporter, as also observed in zuc RNAi experiments. These data raise the possibility that Eggless could be one of the enzymes responsible for the deposition of H3K9me3 marks over silenced transposons in a Piwi-targeted fashion (Yu, 2015).

Panoramix functions downstream of Piwi and Asterix and is both necessary and sufficient to elicit transcriptional repression when bound to nascent transcripts. Panoramix represents an example in metazoans of a protein inducing cotranscriptional silencing when recruited to the nascent transcript from a locus. In fact, only cotranscriptional silencing can resolve the conundrum of a target being transcriptionally repressed while transcripts from that target locus are responsible for recruiting their own repressors. Orthologs of some of the general silencing factors that act with Panoramix to deposit and interpret repressive chromatin marks have also been implicated in mammalian transposon silencing, in which the pathway functions by causing heritable DNA methylation. Though one cannot identify a mammalian ortholog of Panoramix based on primary sequence alone, the overall conservation of the piRNA-mediated transcriptional machinery suggests that a protein with an equivalent function likely exists in mammals (Yu, 2015).

Piwi nuclear localization and its regulatory mechanism in Drosophila ovarian somatic cells

In Drosophila ovarian somatic cells (OSCs), Piwi represses transposons transcriptionally to maintain genome integrity. Piwi nuclear localization requires the N terminus and PIWI-interacting RNA (piRNA) loading of Piwi. However, the underlying mechanism remains unknown. This study shows that Importinalpha (Impalpha) plays a pivotal role in Piwi nuclear localization and that Piwi has a bipartite nuclear localization signal (NLS). Impalpha2 and Impalpha3 are highly expressed in OSCs, whereas Impalpha1 is the least expressed. Loss of Impalpha2 or Impalpha3 forces Piwi to be cytoplasmic, which is rectified by overexpression of any Impalpha members. Extension of Piwi-NLS with an additional Piwi-NLS leads Piwi to be imported to the nucleus in a piRNA-independent manner, whereas replacement of Piwi-NLS with SV40-NLS fails. Limited proteolysis analysis suggests that piRNA loading onto Piwi triggers conformational change, exposing the N terminus to the environment. These results suggest that Piwi autoregulates its nuclear localization by exposing the NLS to Impalpha upon piRNA loading (Yashiro, 2018).

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DEVELOPMENTAL BIOLOGY

Embryonic

Eggs produced from homozygous piwi mutant germ line stem cell (GSC) clones are arrested in embryogenesis, not rescuable by the paternal piwi⁺ gene. Approximately 30% of the arrested embryos show severe mitotic defects during cleavage stage. The remaining embryos show various morphological defects during late embryogenesis, including a high frequency of severe deformation of the head region. These defects demonstrate that piwi expression in the germ line provides an essential maternal contribution for embryogenesis (Cox, 1998).

Adult

To investigate in which cells piwi is expressed to regulate germ line stem cell (GSC) division, the expression pattern of piwi was examined during oogenesis by in situ hybridization of whole mount Drosophila ovaries with DIG-labeled DNA probes prepared from the piwi cDNA clone. PIWI mRNA is detected in the somatic terminal filament cells apical to GSCs in the germarium and anterior sheath cells as well as in the germ line. In the germ line, it is first abundantly expressed in region 2 of the germarium in which 16-cell germ-line cysts are formed, persists at a lower level through stages 1-6 of oogenesis, is at its lowest level between stage 7-9, becomes strongly expressed again at stage 10, and eventually accumulates in early embryos. Given that removing sheath cells does not affect oogenesis, and the terminal filament cells play a role in regulating GSC division, it is likely that the somatic expression in the terminal filament is responsible for piwi function in regulating GSC division (Cox, 1988).

To elucidate the function of piwi in regulating GSC division, the expression and subcellular behavior of the Piwi protein in vivo was studied. The Piwi protein was tagged by inserting a sequence encoding a myc epitope into the piwi gene, at the 5' end of the piwi open reading frame. The p[5'-myc-piwi] transgene (denoted as myc-piwi) was introduced into Drosophila via P-element mediated germline transformation. myc-piwi fully restores the fertility and gametogenesis of piwi mutant males and females. Thus, the myc-PIWI protein confers wild-type Piwi function. In contrast, a p[piwi-3'- myc] transgene (denoted as piwi-myc) with the myc sequence inserted in the highly conserved C-terminal region fails to rescue any piwi mutant phenotype (Cox, 2000).

The expression of myc-PIWI was confirmed by immunoblot analysis using a monoclonal anti-myc antibody as a probe. A single 97.5 kDa band was revealed in the ovarian extract of myc-piwi. In the third instar larval ovary, where germ-line stem cells (GSCs) begin their asymmetric divisions, myc-PIWI has been found in the nucleus of all germ cells of the ovary. In addition, myc-PIWI is also present in the nuclei of the forming terminal filament cells. In adult ovaries, myc-PIWI is present in the nuclei of both the somatic and germline, as predicted by Piwi RNA in situ analyses (Cox, 1998). Specifically, in the germarium, myc-PIWI is expressed in all the somatic cells, including the main terminal filament cells, the cap cells and the inner sheath cells, consistent with the previous genetic clonal analyses that suggest that piwi is required in these cells to maintain GSCs. Piwi is also expressed in somatic stem cells and the follicle cells in the germarium. In the germline, myc-PIWI shows a dynamic nuclear expression pattern: it is present abundantly in GSCs, but is down-regulated in cystoblasts and developing cysts. In 16-cell cysts in regions 2 and 3, the myc-PIWI regains its high level expression, and remains so in nurse cells and oocytes in post-germarial egg chambers throughout oogenesis. In all post-germarial egg chambers, myc-PIWI is also specifically expressed in the anterior polar follicle cells. The consistent nuclear localization of myc-PIWI in various types of somatic and germline cells during oogenesis indicates that Piwi is a nuclear protein (Cox, 2000).

Piwi is also expressed as a nuclear protein during spermatogenesis. In the third instar larval testes which contain mostly premeiotic germ cells, myc-PIWI is localized to the nuclei of apical somatic cells, including the hub cells, which are the testicular equivalent of terminal filament cells. myc-PIWI is also present in somatic stem cells and their progeny, the cyst progenitor. At the apical germline, Piwi is present in the nuclei of GSCs and their immediate daughter cells. In newly formed 16-cell cysts of primary spermatocytes somewhat away from the apex, the myc-PIWI staining is sharply reduced in the germline. The staining is only present in the cyst progenitor cells, which are equivalent to follicle cells in the ovary. Once the developing cyst enters the spermatocyte growth phase, myc-PIWI expression is completely undetectable. This pattern of expression is maintained in the adult testis which displays the same apical-distal organization but now contains more differentiated post-meiotic germ cells in the basal region of the testis. Given the essential role of piwi in testicular germline stem cell maintenance (Lin, 1997), the nuclear localization of Piwi in the testis should also be functionally important. In interphase somatic and germline nuclei, myc-PIWI is not associated with the chromatin or the nuclear envelope, but is localized in the nucleoplasm (Cox, 2000).

Previous clonal analyses have indicated that piwi functions in the apical somatic cells of the germarium to regulate GSC maintenance (Cox, 1998). However, because Piwi is also present in the nuclei of GSCs, attempts were made to test the potential function of Piwi in GSCs by removing Piwi from a single stem cell using the FLP/FRT-mediated clonal technique. piwi mutations were used for generating piwi minus deficient clones. Mitotic recombination was induced immediately prior to oogenesis and looked-for piwi minus GSC clones were sought 1 week, 2 weeks, and 3 weeks following oogenesis. piwi minus GSCs are present even three weeks following oogenesis, consistent with the observation that Piwi in GSCs is not required for GSC maintenance. The number of marked piwi minus and piwi plus germline cysts in tester and control germaria was examined to compare the division rate between the piwi minus and piwi plus GSCs within the germarium. The analysis reveals that piwi minus GSCs divided four-fold slower than wild-type GSCs. Thus, in addition to its somatic function, Piwi acts cell-autonomously in the stem cells to facilitate their division. The piwi minus cysts and postgermarial piwi minus egg chambers were examined by DAPI staining and by Nomarski microscopy for potential developmental defects. They usually developed normally, suggesting that piwi does not play an important cell-autonomous role in germline cyst development and subsequent stages of oogenesis (Cox, 2000).

Overexpression of Piwi increases the number of germline stem cells. Wild-type germaria typically have 2-3 spectrosome-containing cells, with one often being a cystoblast that is not associated with the terminal filament. Interestingly, the number of the spectrosome-containing germ cells increases to an average of 7.5 cells in germarium in which Piwi is overexpressed. In the most extreme case, up to 15 spectrosome-containing cells are observed in a single germarium. Thus, overexpression of Piwi in the soma leads to a 3- to 4-fold increase in the number of GSCs and/or cystoblasts. To distinguish whether the spectrosome-containing cells are stem cells or cystoblasts, the germaria were stained with anti-cytoplasmic Bag of marbles (Bam) antibody, since Bam is only expressed in cystoblasts and early mitotic cysts but not in GSCs. In piwi-overexpressed germaria, Ban staining is strongly present in early cysts. However, Bam staining is conspicuously absent from all the spectrosome-containing cells. This observation suggests that the ectopically induced spectrosome-containing cells are GSC-like cells (Cox, 2000).

The ectopic GSC-like cells appear to be functional GSCs. (1) They incorporate BrdU, an indicator of DNA replication, at a level similar to the wild-type GSCs. This suggests that they are not arrested in the cell cycle.(2) These GSC-like cells can all differentiate within 4 days following the withdrawal of heat shock, leaving the germaria with only 2-3 GSCs at their normal locale. No signs of cell death, such as pycnotic nuclei or apoptotic bodies, were detected by DAPI staining and Nomarski optics. This suggests that the GSC-like cells are capable of oogenesis. Thus, the dependence of GSC number on the Piwi level reveals that piwi-mediated somatic signaling controls the number of GSCs via a dosage dependent mechanism. Since over-expression of piwi in somatic cells increases the number of GSCs, yet loss of piwi function from the soma and germline in piwi mutants abolishes stem cell division and thus their maintenance, one might expect that over-expression of piwi in the soma would also increase the rate of GSC division. In addition to an increase in the number of stem cells, piwi overexpression also increases the rate of GSC division. Because the GSCs under examination are located in their normal niche, the increase in their mitotic frequency should be mostly, if not entirely, due to the increased Piwi expression in the terminal filament (Cox, 2000).

Since Piwi is a nucleoplasmic protein, it is not likely to be a somatic signal itself, but rather an essential component of the somatic signaling machinery responsible for producing the signal. Given its nucleoplasmic localization, Piwi may be involved in post-transcriptional mRNA processing in the nucleus. Alternatively, it may be involved in nuclear functions indirectly related to gene expression. In either case, the somatic activity of Piwi appears to act via a dosage-dependent mechanism to control the number of GSCs in the germline. Therefore, these results show that piwi may help to define a stem cell niche in the germarium for GSC maintenance, with the size of the niche corresponding to the level of Piwi activity. How can piwi acts in two distinct cell types and in two different mechanisms? This is probably because the Piwi protein directly or indirectly mediates gene expression in the nucleus either at the transcriptional or post-transcriptional level. The different piwi target genes or their products in different cell types then lead to the distinctly different cellular function of piwi essential for germline stem cell maintenance during Drosophila oogenesis (Cox, 2000).

The coordinated division of distinctive types of stem cells within an organ is crucial for organogenesis and homeostasis. Genetic interactions among fs(1)Yb (Yb), piwi, and hedgehog (hh) regulate the division of both germline stem cells (GSCs) and somatic stem cells (SSCs) -- the two constituent stem cell populations of the Drosophila ovary. Yb, coding for an ATP/GTP-binding site motif A (P-loop) domain protein, is required for both GSC and SSC divisions; loss of Yb function eliminates GSCs and reduces SSC division, while Yb overexpression increases GSC number and causes SSC overproliferation. Yb acts via the piwi- and hh-mediated signaling pathways that emanate from the same signaling cells to control GSC and SSC division, respectively. hh signaling also has a minor effect in GSC division (King, 2001).

Yb is expressed in terminal filament and cap cells to control GSC self-renewing divisions. The loss-of-function and overexpression phenotype of Yb reported suggests that Yb is also involved in regulating SSC divisions. It is possible that Yb achieves this dual function indirectly by regulating GSC division, which in turn affects SSC division via an unknown coordination mechanism, or vice versa. These possibilities seem unlikely, since all other mutations are known to only affect either GSCs or SSCs, but not both, as judged from their reported phenotype. For example, piwi and dpp mutations cause failure of GSC maintenance, while bam and bgcn mutations as well as piwi and dpp overexpression cause an accumulation of germline cells without a corresponding increase in somatic cells. Similarly, hh activity regulates SSC division without significant effect on GSC divisions. It is therefore unlikely that all these mutations, except for Yb, have a dual effect on GSC or SSC division and on the coordination mechanism between GSCs and SSCs. Thus, Yb appears to be the only known gene that plays a major role in regulating both GSC and SSC divisions. This dual role of Yb is further supported by the regulatory relationship between Yb, piwi, and hh (King, 2001).

The somatic function of Yb is very similar to that of hh. Like hh, Yb is specifically expressed in cap and terminal filament cells to regulate follicle cell division. Loss of either hh or Yb function leads to reduced follicle cell proliferation, while overexpression of either gene by heat shock leads to overproliferation of follicle cells that exceeds the need for egg chamber formation. The relationship between Yb and hh is further defined by observations that Yb is required for the expression of hh in cap cells and, to a lesser extent, terminal filament cells, and that Yb overexpression significantly elevates hh expression in cap cells and, also to a lesser extent, terminal filament cells. Yb overexpression causes less follicle cell overproliferation than hh overexpression. This can be explained by the fact that Yb overexpression only elevates HH expression in cap cells and terminal filament cells, while heat shock causes HH to be overexpressed all over the germarium. Since hh signaling is the main, if not the only, signaling pathway that controls SSC division, the similar mutant and overexpression phenotype between hh and Yb suggests that Yb is a positive regulator of hh expression in cap and terminal filament cells. In addition, these data provide strong evidence that cap cells play a central role in controlling SSC and GSC divisions, a hypothesis that has been proposed based on the expression pattern and function of Yb, hh, dpp, and piwi, as well as on the mitotic behavior of GSCs (King, 2001).

A parallel situation exists between Yb and piwi in controlling GSC division: (1) both Yb and piwi are expressed in cap and terminal filament cells, and this expression is essential for GSC maintenance; (2) Yb and piwi mutants share a very similar, if not identical, GSC phenotype; (3) overexpressing either Yb or piwi in somatic cells causes a similar increase in the number of GSC-like cells; (4) Yb is required for piwi expression in cap and terminal filament cells. These observations suggest that Yb is also a positive regulator of piwi expression in these somatic cells that controls GSC division. In addition, it suggests that cap cells may play a central role in GSC division, because these cells express higher levels of Yb and piwi and directly contact GSCs. The Yb-piwi mechanism apparently does not control the production of the DPP signal required for GSC maintenance, since overexpression of dpp does not produce similar effects as does Yb or piwi and does not rescue the piwi phenotype (King, 2001).

The hypothesis that Yb controls GSC and SSC divisions by regulating piwi and hh expression, respectively, in cap cells and terminal filament cells is favored. The Piwi protein, as a nuclear factor, in turn controls GSC division by promoting the production of a somatic signal 'S,' which is received by its receptor 'R' in GSCs. In parallel, the Hh signaling molecule suppresses the Ptc receptor activity in SSCs to promote SSC division. Meanwhile, Hh also participates in promoting GSC divisions through the Ptc receptor on the GSC surface, since either overexpressing Hh in Yb mutants or removing PTC activity from GSCs in Yb mutants has a similar effect in rescuing GSC division and maintenance. The expression of hh may also be controlled by engrailed (en), a known transcription regulator of hh that is also specifically expressed in cap cells and terminal filament cells. dpp appears to act independent of the Yb-mediated pathway in regulating GSC division. This bifurcating model with Yb as a common upstream regulator of both GSC and SSC divisions represents a working hypothesis to address how the coordinated division of two distinct types of stem cells is possibly controlled (King, 2001).

An interesting aspect of the above model is that the HH signaling pathway, in addition to its essential role in SSC division, is involved in regulating GSC division. This GSC function of hh, however, appears to be somewhat redundant, since the loss of hh function only affects the maintenance of ~20% of GSCs, while overexpression of hh only stimulates a slight increase in GSC-like cells. Despite this, hh overexpression is sufficient to restore GSC divisions in both Yb and piwi mutants. These observations suggest that the hh signaling pathway is a dispensable mechanism that safeguards the GSC maintenance. It remains to be determined whether other known regulators of hh, such as engrailed, are involved in regulating hh, piwi, or Yb expression in cap cells and the terminal filament. What is the somatic signal and what is its receptor also remains to be determined in the piwi branch of the bifurcating pathway. Finally, it awaits to be established whether or how the Yb-mediated extrinsic signaling mechanism regulates the asymmetric expression and activity of intracellular stem cell genes, such as pumilio, bam, and nanos, during GSC division. The study of these questions should significantly advance understanding of the stem cell mechanism in general (King, 2001).

Btk29A promotes Wnt4 signaling in the niche to terminate germ cell proliferation in Drosophila

Btk29A is the Drosophila ortholog of the mammalian Bruton's tyrosine kinase (Btk), mutations of which in humans cause a heritable immunodeficiency disease. Btk29A mutations stabilized the proliferating cystoblast fate, leading to an ovarian tumor. This phenotype was rescued by overexpression of wild-type Btk29A and phenocopied by the interference of Wnt4-β-catenin signaling or its putative downstream nuclear protein Piwi in somatic escort cells. Btk29A and mammalian Btk directly phosphorylate tyrosine residues of β-catenin, leading to the up-regulation of its transcriptional activity. Thus, this study identified a transcriptional switch involving the kinase Btk29A/Btk and its phosphorylation target, β-catenin, which functions downstream of Wnt4 in escort cells to terminate Drosophila germ cell proliferation through up-regulation of piwi expression. This signaling mechanism likely represents a versatile developmental switch (Hamada-Kawaguchi, 2014).

Stem cell maintenance and differentiation are not entirely autonomic, but instead are under strict control by supporting cells that form the 'niche'. Recent studies in Drosophila have shown that the dynamics of Piwi and its associated piRNAs, a protein-RNA complex for gene silencing, are required in not only germ cells but also distinct niche-forming somatic cells (escort cells for germ cell development); however, their regulatory mechanisms remain largely unknown. This study identified a transcriptional switch involving the factor Bruton's tyrosine kinase (Btk) and its phosphorylation target, β-catenin, operating downstream of Wnt4 in escort cells to terminate Drosophila germ cell proliferation through modulation of piwi expression (Hamada-Kawaguchi, 2014).

Drosophila Btk29A type 2 is the ortholog of human BTK. The type 1 isoform is present and the type 2 is absent in Btk29A^ficP mutants. Germ stem cells (GSCs) and transit amplifying cystoblasts (CBs) are localized in the germarium situated at the anterior tip of an ovariole, posteriorly flanked by region 2, in which each CB divides twice and differentiates into cystocytes. The 16 cystocytes originating from a single CB remain interconnected by the fibrous structure fusome, a derivative of the spectrosome. GSCs and CBs both carry the spectrosome, a round, tubulin-enriched structure. The Btk29A mutant germarium contains significantly more germ cells than does the wild-type germanium. Although supernumerary cells were observed with spectrosomes in the Btk29A^ficP germarium, many of the excess cells appear to be cystocytes, as they were accompanied by a branched fusome structure. A large excess of cystocytes in grossly deformed ovarioles has been observed in female Drosophila that are mutant for mei-P26, a gene encoding a TRIM-NHL (tripartite motif and Ncl-1, HT2A, and Lin-41 domain) protein that binds to the argonaute protein Ago-1 for microRNA regulation. In mei-P26 mutants, an ovarian tumor 'cystocytoma' is formed because cystocytes regain the ability to self-renew after they enter the differentiation path. This suggests that mei-P26 normally terminates CB proliferation. Intriguingly, the following phenotypes of mei-P26 were recapitulated in Btk29A^ficP. First, phospho-histone H3-positive mitotic germline cells, which were restricted to the anterior tip of the wild-type germarium, were detected throughout the ovarioles. Second, the expression of Bam, a protein that induces differentiation of GSCs into CBs in the wild type, was markedly increased in CB-like GSC daughters. Third, oo18 RNA-binding protein (Orb) remained expressed in multiple cells in a cyst, contrasting to a wild-type cyst, where Orb expression becomes restricted to an oocyte (Hamada-Kawaguchi, 2014).

The reduction in mei-P26 transcription in Btk29A^ficP places mei-P26 downstream of Btk29A. Notably, mei-P26 functions cell-autonomously in germ cells. However, the almost complete rescue of germ cell defects in Btk29A^ficP was attained by overexpression of Btk29A⁺ type 2 via bab1-Gal4, which showed high levels of expression in terminal filament cells and cap cells (TF and CPC, respectively) and lower levels of expression in escort cells (EC). bab1-Gal4 was effective in inducing germ cell overproduction when used to knockdown Btk29A. hh-Gal4 with expression in the terminal filament cells and cap cells and c587-Gal4 with expression in escort cells were also used to target UAS-Btk29ARNAi expression; c587-Gal4, but not hh-Gal4, led to the overproduction of spectrosome-bearing cells, and therefore, the escort cells were considered as likely sites of Btk29A action. These observations imply that Btk29A is required in the escort cells for soma-to-germ signaling to control the switch from proliferation to differentiation in germ cells, where mei-P26 functions as a core component of the switch (Hamada-Kawaguchi, 2014).

Bone morphogenetic protein (BMP) signaling and piwi-dependent signaling compose two different pathways in the niche to control proliferation and differentiation of GSCs and their daughters. BMPs are secreted morphogens, and Piwi is an argonaute protein regulating gene expression. The Btk29A^ficP mutation abrogated piwi expression with little effect on decapentaplegic (dpp) or glass bottom boat (gbb) expression, two BMPs operating in the germarium, and the BMP downstream component Mothers against Dpp (Mad) was normally phosphorylated in Btk29A^ficP GSCs. Furthermore, somatic piwi knockdown mimicked the Btk29A^ficP ovarian phenotypes (Hamada-Kawaguchi, 2014).

Immunohistochemistry revealed that the Btk29A^ficP mutation or somatic Btk29A knockdown abrogated Piwi expression in the niche, but not in germ cells. This reduction in Piwi expression was reversed by the somatic Btk29A⁺ overexpression. Furthermore, the loss-of-function piwi allele dominantly enhanced the Btk29A mutant phenotype. Moreover, somatic overexpression of piwi⁺ in Btk29A^ficP alleviated the germ cell hypertrophy and reduced Bam expression to the normal level. It is therefore considered that Btk29A regulates the Piwi-dependent pathway in the niche to control germ cell proliferation (Hamada-Kawaguchi, 2014).

Piwi and piRNAs constitute a major transposon-silencing pathway. Somatic knockdown of Btk29A resulted in an increase in the expression of gypsy-lacZ that monitored the activity of the gypsy transposon. Also, transcript levels of the ZAM, DM412, and mdg1 transposons were significantly increased in Btk29A^ficP. It is therefore concluded that the Piwi deficiency due to the impairment of Btk29A results in derepression of transposon activities (Hamada-Kawaguchi, 2014).

Genome instability associated with transposon mobilization may lead to the activation of a DNA double-strand break (DSB) checkpoint. A mutation in DSB signaling, mnk, did not ameliorate the germ cell phenotype induced by somatic Btk29A knockdown, indicating that the germ cell hypertrophy by the Btk29A deficiency is not a consequence of the DSB checkpoint activation (Hamada-Kawaguchi, 2014).

Next, potential substrates of Btk29A in the niche were sought. Btk29A type 2 was enriched in the interface between cells where Drosophila melanogaster epithelial (DE)-cadherin and associated Arm, the β-catenin ortholog, are the major structural components. No sign were found of tyrosine phosphorylation of DE-cadherin, whereas Arm contained a high level of phosphotyrosine, which was almost entirely absent from Btk29A^ficP ovaries. However, Arm immunoprecipitated from Btk29A^ficP was strongly phosphorylated in vitro by the exposure of Arm to active Btk29A protein that had been immunoprecipitated from wild-type ovaries. These results demonstrate that Btk29A mediates the tyrosine phosphorylation of Arm in vivo (Hamada-Kawaguchi, 2014).

The anti-Arm labeling intensity of cell adhesion sites was stronger in Btk29A^ficP than in the wild type. Immunoprecipitation assays revealed that the relative amount of Arm associated with DE-cadherin was greater in Btk29A^ficP than in the wild type , suggesting that the tyrosine phosphorylation of Arm facilitates its release from the membrane to the cytoplasm, as in mammalian cells (Hamada-Kawaguchi, 2014).

Mammalian β-catenin is tyrosine-phosphorylated at residues Y86, Y142, and Y654. When transfected into mammalian Cos7 cells, Drosophila Btk29A type 2 phosphorylated all these tyrosine residues of β-catenin. Moreover, the antibodies against phosphorylated Y142 (anti-pY142) and anti-pY654 recognized Arm phosphorylated at the conserved site Y150 and Y667, respectively, in the immunoprecipitates from ovarian lysates (Hamada-Kawaguchi, 2014).

Expression of unphosphorylatable Arm-Y150F in the escort cells via c587-GAL4 or bab1-GAL4, but not hh-Gal4, induced germ cell hypertrophy, whereas another unphosphorylatable mutant, Y667F, or wild-type Arm exerted little effect. In addition, somatic arm knockdown resulted in an increase in spectrosome-containing cells, reduced piwi expression in escort cells, and increased Bam expression in germ cells. Considering these results together, it is proposed that Btk29A acts on Arm, which in turn regulates piwi in the niche (Hamada-Kawaguchi, 2014).

Arm functions in the canonical Wnt pathway. Therefore the ovaries of wg, Wnt2, Wnt4, and Wnt5 mutants were examined; the germ cell overproduction was detected only in Wnt4. Somatic knockdown of Wnt4 aided by bab1-GAL4 resulted in a reduction in the expression of Piwi, accompanied by an accumulation of germ cells carrying spectrosomes with an increase in germline Bam expression. These findings support the hypothesis that Arm in the escort cells regulates germ cell proliferation under the control of Wnt4, which was likely derived from somatic cells other than cap cells and terminal filament cells, as hh-GAL4 selective for these cells was least effective to induce germ cell overproduction when used to drive Wnt4RNAi expression (Hamada-Kawaguchi, 2014).

To evaluate the ability of Arm to activate transcription, T cell factor (TCF) reporter assays were used with Cos7 cells transiently transfected with human Btk (hBtk). The wild-type hBtk alone was sufficient to induce phosphorylation at Y142 and Y654 of β-catenin, whereas the kinase-dead hBtk (Btk-K430E) was not. Tyrosine phosphorylation of β-catenin was completely blocked by two antagonists of hBtk. Similarly, Btk29A type 2 phosphorylated Y142 and Y654 of mammalian β-catenin. Notably, the TCF reporter activity was six times as high when hBtk was transfected into Cos7 cells compared with the mock-transfected control, indicating that hBtk modulates the TCF-dependent transcriptional activation mechanism, in which Arm-β-catenin is involved as a coactivator (Hamada-Kawaguchi, 2014).

The expression of an arm-dependent Ubx-lacZ reporter was examined in the embryonic midgut. Btk29A knockdown abrogated the expression of this reporter, demonstrating that Btk29A supports Arm-dependent transcription in vivo (Hamada-Kawaguchi, 2014).

Btk29A was shown to phosphorylate Arm-β-catenin on conserved tyrosine residues, one of which (Arm-Y150) is pivotal for the niche function to prevent GSC daughters from overproliferating. Notably, most GSCs in Btk29A mutants do not express Bam (fig. S1R). This suggests that the presumptive Btk29A-Arm-Piwi pathway selectively regulates the proliferation of differentiating GSC daughters without interfering with GSC maintenance. Without Btk29A type 2, cystoblasts fail to exit the cell cycle, leading to the overproduction of germ cells, many of which are unable to complete differentiation and contribute to the genesis of an ovarian tumor (Hamada-Kawaguchi, 2014).

β-Catenin exerts multiple functions through its promiscuous binding abilities in cell-to-cell interactions and transcription. This protein plays critical roles in stem cell biology, and β-catenin malfunction results in a variety of cancers. These findings add a new dimension to the study of β-catenin by highlighting the pivotal role of the tyrosine phosphorylation of β-catenin in the control of transcription in the nucleus, in addition to the regulated control of the stability and motility of cell adhesion (Hamada-Kawaguchi, 2014).

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EFFECTS OF MUTATION

To identify genes that control asymmetric cell divisions, a search was carried for mutations that affect germline stem cell division by screening a collection of single P element enhancer-trap female sterile mutants using anti-Vasa immunostaining (germ cells stain darkly with anti-Vasa antibody) and electron microscopic analyses. Females bearing mutations in stem cell function would be expected to be weakly fertile or sterile and contain small ovaries in which the 2-3 stem cells in each ovariole had differentiated into egg chambers. Several mutations with such effects were identified and found to fall into two complementation groups. The first locus is represented by eight female sterile mutations and was named ovarette; it maps to the pumillo locus (Lin, 1997).

The second gene identified in the screen was named piwi (for P-element induced wimpy testis). The piwi gene is defined by two independent, non-complementing P-element insertional mutations, piwi¹ and piwi⁶⁸⁴³, and a third allele piwi². piwi appears to be a new gene based on complementation tests with known mutations in the region. Among the three alleles, piwi¹ and piwi² show the strongest oogenic phenotype. Moreover, piwi¹ also causes male sterility: mutant males show severe defects in spermatogenesis. The effects of the piwi¹ mutation on germ line stem cell behavior were examined by analyzing mutant germaria labeled with various antisera and by electron microscopy. The ovarioles of piwi¹ females are most commonly devoid of germline cells, as indicated by Nomarski, DAPI, anti-Vasa and electron microscopic imaging analyses. Although individual ovarioles are present as revealed by electron microscopic analysis, they are often difficult to recognize. At low frequency, some ovarioles contain a few developing egg chambers or mature eggs. A small number of ovarioles contain 2-3 small clusters of germ cells, with each cluster composed of only a few germ cells. Given that the mutant ovaries initially contain normal number of germline stem cells, this oogenic phenotype suggests that most of these stem cells either die or differentiate into an egg chamber, and thus fail to maintain themselves. The few surviving egg chambers in the mutant ovaries often show a variety of defects, including abnormal egg chamber polarity and reduced nurse cell number. The spermatogenic defects of piwi¹ mutants parallel its oogenic defects. Wild-type testes contain germline cells at all stages of spermatogenesis, including 5-8 germline stem cells at the apical tip as well as numerous bundles of mature sperm that occupy most of the lumenal space in the testis. In contrast, the piwi¹ mutant testes only contain 1-6 bundles of mature sperm but no other germline cells. Since each sperm bundle derives from a single product of a stem cell division, this defect suggests that germline stem cells in the mutant testes either die or differentiate to found a spermatogenic cyst (Lin, 1997).

To analyze whether the mutant gonads initially contain a normal complement of germline cells, the germline in the piwi¹ mutant was examined during embryonic and larval development using the anti-Vasa antibody to label germ cells and the anti-spectrin antibody to visualize spectrosomes and fusomes. Germ cells in mutant embryos develop normally during embryogenesis. The number of germline stem cells in the third instar larval ovaries is also found to be normal, and the spectrosomes in these cells appear normal following staining with anti-spectrin antibodies. However, the stem cells often do not reside in the middle of the ovary. These analyses suggest that the piwi¹ mutation does not affect the initial proliferation of germline stem cell population but acts later to disrupt stem cell division or maintenance during gametogenesis in both sexes. To confirm this conclusion, the developing germline cysts were examined in piwi¹ mutant third instar testes. In wild-type third instar larval testes, the oldest cysts have progressed into meiosis. Premeiotic cysts contain an elaborate fusome connecting the 16 primary spermatocytes. These cysts grow in size as they leave the apical stem cell region and eventually enter meiosis. The mutant piwi¹ testes at this stage show four defects: (1) they often contain fewer growing cysts, suggesting a defect in germline stem cell division; (2) the premeiotic cysts frequently contain fewer then 16 spermatocytes, suggesting a defect in spermatocyte division; (3) the germline cysts often appear to develop aberrantly, as indicated by defective fusome morphology, suggesting a defect in spermatocyte differentiation; (4) presumably because of the above defects, the mutant testes often are smaller than the wild-type testes. As with the defects seen in females, these results suggest that piwi function is required both to maintain germline stem cells and subsequently for the division and differentiation of the stem cell progeny in both sexes (Lin, 1997).

Since the pumillio ovarette and piwi mutations were induced by enhancer trap P elements, the pattern of lacZ expression was examined in these lines in order to see if there is any overlap in the expression domains of the two genes. pum 2003, as well as four other ovt alleles, show lacZ expression specifically in the terminal filament, a group of apical somatic cells involved in regulating germline stem cell division. No expression in the germline stem cells or other germ line cells is detected. In contrast, the piwi elements cause lacZ to be expressed in the germline both in the third instar larval ovary and in the germarium in the adult ovary. These observations raise the prospect that pum ovt and piwi mutations may act in different cells (Lin, 1997).

In Drosophila, the endogenous retrovirus gypsy is repressed by the functional alleles (restrictive) of an as-yet-uncloned heterochromatic gene called flamenco. Using gypsy-lacZ transcriptional fusions, this repression is shown to take place not only in the follicle cells of restrictive ovaries, as has been previously observed, but also in restrictive larval female gonads. Analyses of the role of gypsy cis-regulatory sequences in the control of gypsy expression are also presented. They rule out the hypothesis that gypsy would contain a single binding region for a putative Flamenco repressor. Indeed, the ovarian expression of a chimeric yp3-lacZ construct was shown to become sensitive to the Flamenco regulation when any of three different 5'-UTR gypsy sequences (ranging from 59 to 647 nucleotides) was incorporated into the heterologous yp3-lacZ transcript. The piwi mutation, which is known to affect RNA-mediated homology-dependent transgene silencing, was also shown to impede the repression of gypsy in restrictive female gonads. Finally, an RNA-silencing model is also supported by the finding in ovaries of short RNAs (25-27 nucleotides long) homologous to sequences from within the gypsy 5'-UTR (Sarot, 2004).

Screens for piwi suppressors in Drosophila identify dosage-dependent regulators of germline stem cell division

The Drosophila piwi gene is the founding member of the only known family of genes whose function in stem cell maintenance is highly conserved in both animal and plant kingdoms. piwi mutants fail to maintain germline stem cells in both male and female gonads. The identification of piwi-interacting genes is essential for understanding how stem cell divisions are regulated by piwi-mediated mechanisms. To search for such genes, the Drosophila third chromosome (~36% of the euchromatic genome) was screened for suppressor mutations of piwi², and six strong and three weak piwi suppressor genes/sequences were identified. These genes/sequences interact negatively with piwi in a dosage-sensitive manner. Two of the strong suppressors represent known genes -- serendipity-delta and similar, both encoding transcription factors. These findings reveal that the genetic regulation of germline stem cell division involves dosage-sensitive mechanisms and that such mechanisms exist at the transcriptional level. In addition, three other types of piwi interactors were identified. The first type consists of deficiencies that dominantly interact with piwi² to cause male sterility, implying that dosage-sensitive regulation also exists in the male germline. The other two types are deficiencies that cause lethality and female-specific lethality in a piwi² mutant background, revealing the zygotic function of piwi in somatic development (Smulders-Srinivasan, 2003).

It is amazing that germline stem cell defects in the piwi mutants can be rescued by the removal of one copy of another gene ( i.e., by 50% reduction in the activity). The identification of six dominant suppressors of piwi at the deficiency level, as well as six strong and three weak suppressors at the individual gene/sequence level, shows that one or more dosage-sensitive mechanisms must negatively interact with piwi in regulating germline stem cell division in the Drosophila ovary. The fact that each of these suppressors can restore the self-renewing ability of germline stem cells in piwi mutants suggests the importance of the dosage-sensitive mechanisms. The existence of multiple suppressors further implies that such negatively interacting and dosage-sensitive mechanisms may be a significant component of the molecular machinery that regulates germline stem cell division. Finally, four deficiencies in the 89E-91B region dominantly interact with piwi² to cause male sterility. This implies that dosage-sensitive mechanisms may also exist in the male germline (Smulders-Srinivasan, 2003).

Such dosage-sensitive mechanisms may not be manifested as the solo act of an individual gene. The P{wA}4-4 suppressor mutation is inserted in the subtelomeric heterochromatic repeats of chromosome 3R, but does not interrupt TART or HeT-A elements in that region. This suggests that the insertion could affect either the transcriptional or the transpositional activity of the retrotransposons or the epigenetic state of that chromosomal region. The latter possibility in turn would suggest that epigenetic effect in the subtelomeric region is involved in regulating germline stem cell division via the piwi-mediated mechanism (Smulders-Srinivasan, 2003).

The discovery of sry-delta, similar, and tango as suppressors of piwi further suggests that the dosage-sensitive mechanism operates at least at the transcriptional level. Sry-delta is a Cys-2/His-2 zinc-finger DNA-binding protein that is present in both germline and somatic cells in the ovary and the testis, as well as in many other tissues and stages of development. It is known to act as a homodimer to activate the transcription of bicoid during oogenesis. Although sry-delta mutants are homozygous late embryonic lethal, hemizygous and intra-allelic escapers are sterile, indicating sry-delta's function during oogenesis. The three sry-delta alleles used in this study (sry-delta¹², sry-delta^SF1, and sry-delta^SF2) are all single-amino-acid changes in the third zinc-finger domain of the protein. However, they are not equivalent mutations. For example, sry-delta^SF2 hemizygotes show many more gonadal defects than do the other alleles, while sry-delta^SF1 shows a lower escaper rate than does sry-delta^SF2. This phenotypic difference leads to a suspicion that the sry-delta^SF2 mutation tends to perturb a subset of downstream effectors necessary for gonadal function, while the sry-delta^SF1 mutation tends to disrupt more general downstream factors, leading to higher lethality. Consistent with this speculation, sry-delta^SF2 is the strongest suppressor of the piwi² phenotype. It would be interesting to conduct genomic screens to identify the target genes of sry-delta whose transcription is selectively affected by sry-delta^SF1 but not by sry-delta^SF2 mutation. Such target genes would likely be involved in germline stem cell division and gonadogenesis (Smulders-Srinivasan, 2003).

Like Sry-delta, Similar and Tango play a key role in the dosage-sensitive regulation of germline stem cell division. Similar is homologous to a large group of heterodimerizing transcriptional activators. It shows closest homology to the human hypoxia inducible factor-1alpha (HIF-1alpha) and has been shown to function in hypoxic response in Drosophila. HIF-1alpha binds to HIF-1ß to drive transcription of downstream genes. Since Tango is the only Drosophila homolog of HIF-1ß, it is likely to be a partner of Similar. Indeed, Tango interacts with Similar in the yeast two-hybrid system. However, Tango is also known to bind to two other Drosophila bHLH-PAS family proteins, Single-minded and Trachealess, to mediate the transcription of their downstream targets. By showing that tango suppresses the germline stem cell phenotype of piwi², this study suggests that Tango heterodimerizes with Similar in the dosage-sensitive transcriptional activation of genes involved in germline stem cell division (Smulders-Srinivasan, 2003).

Although the biochemical properties of the Piwi family proteins have not been systematically characterized, this family of proteins has been extensively implicated in RNA-related processes. Piwi itself is necessary for both transcriptional and post-transcriptional gene silencing in Drosophila. The aubergine (a.k.a. sting) gene, a Drosophila homolog of piwi, functions in the regulation of the stellate transcript, as well as in the translational regulation of oskar and gurken. ago2 in Drosophila, rde-1 in C. elegans, ago1 in A. thaliana, and qde-2 in Neurospora crassa are necessary for post-transcriptional gene silencing. Most recently, the piwi family genes have also been implicated in epigenetic modification and even in genomic rearrangement via microRNA-mediated mechanisms. Then, what are the biochemical activities of Piwi that would allow it to achieve these regulatory functions? Piwi family genes contain the highly conserved PAZ domain in the central region and the Piwi domain at the C terminus of the proteins. The N-terminal portion of the Piwi domain in Miwi, a murine member, has been shown to bind selectively to poly(G) sequences in vitro. Thus, this region of the protein may represent an RNA-binding domain. Consistent with these data, Miwi complexes with its target mRNAs in vivo. All these data suggest that Piwi family proteins possess RNA-binding ability (Smulders-Srinivasan, 2003).

The results of this study help in the understanding of the potential biochemical function of piwi. Since Sry-delta and Similar/Tango are transcription factors, it is possible that they suppress the piwi² phenotype by promoting the transcription of a transcriptional repressor that represses piwi expression. In piwi²/piwi²; Sry-delta/+ or piwi²/piwi²; similar/+ flies, the reduced Sry-delta or Similar level leads to a decreased repressor level, which in turn leads to increased piwi transcription. This possibility, however, is less likely because piwi² produces a 1.65-kb truncated transcript, which suggests that piwi² is a strong or even null mutation. Upregulating such a truncated transcript is unlikely to restore piwi function (Smulders-Srinivasan, 2003).

Therefore, the following two possibilities are favored. One possibility is that the suppression of piwi mutations by Sry-delta and similar may reflect the fact that Piwi interacts with their target mRNAs . Given that Piwi is localized in the nucleoplasm in the ovary, it is possible that Piwi interacts with the pre-mRNAs or mRNAs of Sry-delta and Similar/Tango target genes in the nucleus to attenuate their processing, life span, or their transport into the cytoplasm. If this is the case, in piwi mutants, the mRNAs of Sry-delta and Similar/Tango target genes become more highly expressed, which compromises the self-renewing ability of germline stem cells. A corresponding decrease in the transcriptional activity of Sry-delta and Similar/Tango may then compensate for this effect. Finally, such regulation probably occurs in the somatic signaling cells, since piwi function is required in these cells for germline stem cell maintenance. Alternatively, it is possible that Piwi-mediated signaling in somatic cells indirectly downregulates Similar/Tango-mediated transcription in germline stem cells. If this is the case, in piwi²/piwi²; similar/+ flies, even though there is no Piwi-mediated downregulation of the Similar/Tango activity, the reduced Similar dosage may mimic the effect of the downregulation to keep the transcription at an appropriate level for the self-renewal of the stem cells. Further molecular experiments are required to distinguish between these possibilities (Smulders-Srinivasan, 2003).

It has been shown that piwi and hh represent two parallel pathways under the control of female sterile (1) Yb (Yb) in maintaining germline stem cells and that the defect of piwi mutants can be rescued by overexpressing hh. However, hh transcription is not increased in flies carrying a piwi suppressor mutation. Hence, the suppressors could either act in parallel with the Hh signaling pathway or affect the activity of hh via post-transcriptional mechanisms. Further experiments are required to distinguish between these two possibilities (Smulders-Srinivasan, 2003).

Although Piwi as a maternal component is essential for embryogenesis, it is not known whether its zygotic activity is involved in somatic development. This is partly because the existing mutations of piwi do not show lethal phenotype. The deficiencies that cause the general and female-specific lethality of the piwi² mutant suggest that piwi is involved in somatic development as a zygotic gene. Such synthetic lethality could reflect that piwi is not an essential gene for somatic development. This would not be surprising, given the existence of four piwi homologs in Drosophila. Alternatively, it remains possible that a null allele of piwi will display lethality. In either case, this study reveals the involvement of zygotic piwi activity in somatic development (Smulders-Srinivasan, 2003).

A distinct small RNA pathway silences selfish genetic elements in the germline: Requirement for Piwi

In the Drosophila germline, repeat-associated small interfering RNAs (rasiRNAs) ensure genomic stability by silencing endogenous selfish genetic elements such as retrotransposons and repetitive sequences. Whereas small interfering RNAs (siRNAs) derive from both the sense and antisense strands of their double-stranded RNA precursors, rasiRNAs arise mainly from the antisense strand. rasiRNA production appears not to require Dicer-1, which makes microRNAs (miRNAs), or Dicer-2, which makes siRNAs, and rasiRNAs lack the 2',3' hydroxy termini characteristic of animal siRNA and miRNA. Unlike siRNAs and miRNAs, rasiRNAs function through the Piwi, rather than the Ago, Argonaute protein subfamily. These data suggest that rasiRNAs protect the fly germline through a silencing mechanism distinct from both the miRNA and RNA interference pathways (Vagin, 2006).

In plants and animals, RNA silencing pathways defend against viruses, regulate endogenous gene expression, and protect the genome against selfish genetic elements such as retrotransposons and repetitive sequences. Common to all RNA silencing pathways are RNAs 19 to 30 nucleotides (nt) long that specify the target RNAs to be repressed. In RNA interference (RNAi), siRNAs are produced from long exogenous double-stranded RNA (dsRNA). In contrast, ~22-nt miRNAs are endonucleolytically processed from endogenous RNA polymerase II transcripts. Dicer ribonuclease III (RNase III) enzymes produce both siRNAs and miRNAs. In flies, Dicer-2 (Dcr-2) generates siRNAs, whereas the Dicer-1 (Dcr-1)–Loquacious (Loqs) complex produces miRNAs. After their production, small silencing RNAs bind Argonaute proteins to form the functional RNA silencing effector complexes believed to mediate all RNA silencing processes (Vagin, 2006 and references therein).

In Drosophila, processive dicing of long dsRNA and the accumulation of sense and antisense siRNAs without reference to the orientation of the target mRNA are hallmarks of RNAi in vitro. Total small RNA was prepared from the heads of adult males expressing a dsRNA hairpin that silences the white gene via the RNAi pathway. white silencing requires Dcr-2, R2D2, and Ago2. siRNAs were detected with a microarray containing T_M (melting temperature)–normalized probes, 22 nt long, for all sense and antisense siRNAs that theoretically can be produced by dicing the white exon 3 hairpin. Both sense and antisense white siRNAs were detected in wild-type flies but not in dcr-2^L811fsX homozygous mutant flies. The Dcr-2–dependent siRNAs were produced with a periodicity of ~22 nt, consistent with the phased processing of the dsRNA hairpin from the end formed by the 6-nt loop predicted to remain after splicing of its intron-containing primary transcript (Vagin, 2006).

Drosophila repeat-associated small interfering RNAs (rasiRNAs) can be distinguished from siRNAs by their longer length, 24 to 29 nt. rasiRNAs have been proposed to be diced from long dsRNA triggers, such as the ~50 copies of the bidirectionally transcribed Suppressor of Stellate [Su(Ste)] locus on the Y chromosome that in testes silence the ~200 copies of the protein-coding gene Stellate (Ste) found on the X chromosome (Vagin, 2006).

Microarray analysis of total small RNA isolated from fly testes revealed that Su(Ste) rasiRNAs detectably accumulate only from the antisense strand, with little or no phasing. As expected, Su(Ste) rasiRNAs were not detected in testes from males lacking the Su(Ste) loci (cry¹Y). Su(Ste) rasiRNAs were also absent from armitage (armi) mutant testes, which fail to silence Ste and do not support RNAi in vitro. armi encodes a non–DEAD-box helicase homologous to the Arabidopsis thaliana protein SDE3, which is required for RNA silencing triggered by transgenes and some viruses, and depletion by RNAi of the mammalian Armi homolog Mov10 blocks siRNA-directed RNAi in cultured human cells. Normal accumulation of Su(Ste) rasiRNA and robust Ste silencing also require the putative helicase Spindle-E (Spn-E), a member of the DExH family of adenosine triphosphatases (Vagin, 2006).

The accumulation in vivo of only antisense rasiRNAs from Su(Ste) implies that sense Su(Ste) rasiRNAs either are not produced or are selectively destroyed. Either process would make Ste silencing mechanistically different from RNAi. In support of this view, mutations in the central components of the Drosophila RNAi pathway—dcr-2, r2d2, and ago2—did not diminish Su(Ste) rasiRNA accumulation. Deletion of the Su(Ste) silencing trigger (cry¹Y) caused a factor of ~65 increase in Ste mRNA, but null or strong hypomorphic mutations in the three key RNAi proteins did not (Vagin, 2006).

Fly Argonaute proteins can be subdivided into the Ago (Ago1 and Ago2) and Piwi [Aubergine (Aub), Piwi, and Ago3] subfamilies. Unlike ago1 and ago2, the aub, piwi, and ago3 mRNAs are enriched in the germline. Aub is required for Ste silencing and Su(Ste) rasiRNA accumulation. In aub^HN2/aub^QC42 trans-heterozygous mutants, Su(Ste) rasiRNAs were not detected by microarray or Northern analysis, and Su(Ste)-triggered silencing of Ste mRNA was lost completely. Even aub^HN2/+ heterozygotes accumulated less of the most abundant Su(Ste) rasiRNA than did the wild type. That the Ago subfamily protein Ago2 is not required for Ste silencing, whereas the Piwi subfamily protein Aub is essential for it, supports the view that Ste is silenced by a pathway distinct from RNAi. Intriguingly, Su(Ste) rasiRNAs hyperaccumulated in piwi mutant testes, where Ste is silenced normally (Vagin, 2006).

Mutations in aub also cause an increase in sense, but not antisense, Su(Ste) RNA; these results suggest that antisense Su(Ste) rasiRNAs can silence both Ste mRNA and sense Su(Ste) RNA, but that no Su(Ste) rasiRNAs exist that can target the antisense Su(Ste) transcript. The finding that Su(Ste) rasiRNAs are predominantly or exclusively antisense is essentially in agreement with the results of small RNA cloning experiments, in which four of five Su(Ste) rasiRNAs sequenced were in the antisense orientation, but is at odds with earlier reports detecting both sense and antisense Su(Ste) rasiRNAs by non-quantitative Northern hybridization (Vagin, 2006).

Is germline RNA silencing of selfish genetic elements generally distinct from the RNAi and miRNA pathways? The expression of a panel of germline-expressed selfish genetic elementswas examined in mutants defective for eight RNA silencing proteins: three long terminal repeat (LTR)-containing retrotransposons (roo, mdg1, and gypsy); two non-LTR retrotransposons (I-element and HeT-A, a component of the Drosophila telomere), and a repetitive locus (mst40). All selfish genetic elements tested behaved like Ste: Loss of the RNAi proteins Dcr-2, R2D2, or Ago2 had little or no effect on retrotransposon or repetitive element silencing. Instead, silencing required the putative helicases Spn-E and Armi plus one or both of the Piwi subfamily Argonaute proteins, Aub and Piwi. Silencing did not require Loqs, the dsRNA-binding protein required to produce miRNAs (Vagin, 2006).

The null allele dcr-1^Q1147X is homozygous lethal, making it impossible to procure dcr-1 mutant ovaries from dcr-1^Q1147X/dcr-1^Q1147X adult females. Therefore, clones of dcr-1^Q1147X/dcr-1^Q1147X cells were generated in the ovary by mitotic recombination in flies heterozygous for the dominant female-sterile mutation ovo^D1. RNA levels, relative to rp49 mRNA, were measured for three retrotransposons (roo, HeT-A, and mdg1) and one repetitive sequence (mst40) in dcr-1/dcr-1 recombinant ovary clones and in ovo^D1/TM3 and dcr-1/ovo^D1 nonrecombinant ovaries. The ovo^D1 mutation blocks oogenesis at stage 4, after the onset of HeT-A and roo rasiRNA production. Retrotransposon or repetitive sequence transcript abundance was unaltered or decreased in dcr-1/dcr-1 relative to ovo^D1/TM3 and dcr-1/ovo^D1 controls. It is concluded that Dcr-1 is dispensable for silencing these selfish genetic elements in the Drosophila female germline (Vagin, 2006).

roo is the most abundant LTR retrotransposon in flies. roo silencing was analyzed in the female germline with the use of microarrays containing 30-nt probes, tiled at 5-nt resolution, for all ~18,000 possible roo rasiRNAs; the data was corroborated at 1-nt resolution for those rasiRNAs derived from LTR sequences. As observed for Su(Ste) but not for white RNAi, roo rasiRNAs were nonhomogeneously distributed along the roo sequence and accumulated primarily from the antisense strand. In fact, the most abundant sense rasiRNA peak corresponded to a set of probes containing 16 contiguous uracil residues, which suggests that these probes nonspecifically detected fragments of the mRNA polyadenylate [poly(A)] tail. Most of the remaining sense peaks were unaltered in armi mutant ovaries, in which roo expression is increased; this result implies that they do not contribute to roo silencing. No phasing was detected in the distribution of roo rasiRNAs (Vagin, 2006).

As for Su(Ste), wild-type accumulation of antisense roo rasiRNA required the putative helicases Armi and Spn-E and the Piwi subfamily Argonaute proteins Piwi and Aub, but not the RNAi proteins Dcr-2, R2D2, and Ago2. Moreover, accumulation of roo rasiRNA was not measurably altered in loqs ^f00791, an allele that strongly disrupts miRNA production in the female germline (Vagin, 2006).

Loss of Dcr-2 or Dcr-1 did not increase retrotransposon or repetitive element expression, which suggests that neither enzyme acts in rasiRNA-directed silencing. Moreover, loss of Dcr-2 had no detectable effect on Su(Ste) rasiRNA in testes or roo rasiRNA in ovaries. The amount of roo rasiRNA and miR-311 was measured in dcr-1/dcr-1 ovary clones generated by mitotic recombination. Comparison of recombinant (dcr-1/dcr-1) and nonrecombinant (ovo^D1/TM3 and dcr-1/ovo^D1) ovaries by Northern analysis revealed that roo rasiRNA accumulation was unperturbed by the null dcr-1^Q1147X mutation. Pre–miR-311 increased and miR-311 declined by a factor of ~3 in the dcr-1/dcr-1 clones, consistent with about two-thirds of the tissue corresponding to mitotic dcr-1/dcr-1 recombinant cells. Yet, although most of the tissue lacked dcr-1 function, improved, rather than diminished, silencing was observed for the four selfish genetic elements examined. Moreover, the dsRNA-binding protein Loqs, which acts with Dcr-1 to produce miRNAs, was also dispensable for roo rasiRNA production and selfish genetic element silencing. Although the possibility that dcr-1 and dcr-2 can fully substitute for each other in the production of rasiRNA in the ovary cannot be excluded, biochemical evidence suggests that none of the three RNase III enzymes in flies—Dcr-1, Dcr-2, and Drosha—can cleave long dsRNA into small RNAs 24 to 30 nt long (Vagin, 2006).

Animal siRNA and miRNA contain 5' phosphate and 2',3' hydroxy termini. Enzymatic and chemical probing was used to infer the terminal structure of roo and Su(Ste) rasiRNAs. RNA from ovaries or testes was treated with calf intestinal phosphatase (CIP) or CIP followed by polynucleotide kinase plus ATP. CIP treatment caused roo and Su(Ste) rasiRNA to migrate more slowly in polyacrylamide gel electrophoresis, consistent with the loss of one or more terminal phosphate groups. Subsequent incubation with polynucleotide kinase and ATP restored the original gel mobility of the rasiRNAs, indicating that they contained a single 5' or 3' phosphate before CIP treatment. The roo rasiRNA served as a substrate for ligation of a 23-nt 5' RNA adapter by T4 RNA ligase, a process that requires a 5' phosphate; pretreatment with CIP blocked ligation, thus establishing that the monophosphate lies at the 5' end. The rasiRNA must also contain at least one terminal hydroxyl group, because it could be joined by T4 RNA ligase to a preadenylated 17-nt 3' RNA adapter. Notably, the 3' ligation reaction was less efficient for the roo rasiRNA than for a miRNA in the same reaction (Vagin, 2006).

RNA from ovaries or testes was reacted with NaIO₄, then subjected to ß-elimination, to determine whether the rasiRNA had either a single 2' or 3' terminal hydroxy group or had terminal hydroxy groups at both the 2' and 3' positions, as do animal siRNA and miRNA. Only RNAs containing both 2' and 3' hydroxy groups react with NaIO₄; ß-elimination shortens NaIO₄-reacted RNA by one nucleotide, leaving a 3' monophosphate terminus, which adds one negative charge. Consequently, NaIO₄-reacted, ß-eliminated RNAs migrate faster in polyacrylamide gel electrophoresis than does the original unreacted RNA. Both roo and Su(Ste) rasiRNA lack either a 2' or a 3' hydroxyl group, because they failed to react with NaIO₄; miRNAs in the same samples reacted with NaIO₄. Together, these results show that rasiRNAs contain one modified and one unmodified hydroxyl. Because T4 RNA ligase can make both 3'-5' and 2'-5' bonds, the blocked position cannot currently be determined. Some plant small silencing RNAs contain a 2'-O-methyl modification at their 3' terminus (Vagin, 2006).

Drosophila and mammalian siRNA and miRNA function through members of the Ago subfamily of Argonaute proteins, but Su(Ste) and roo rasiRNAs require at least one member of the Piwi subfamily for their function and accumulation. To determine whether roo rasiRNAs physically associate with Piwi and Aub, ovary lysate were prepared from wildtype flies or transgenic flies expressing either myc-tagged Piwi or green fluorescent protein (GFP)–tagged Aub protein; they were immunoprecipitated with monoclonal antibodies (mAbs) to myc, GFP, or Ago1; and then the supernatant and antibody-bound small RNAs were analyzed by Northern blotting. Six different roo rasiRNAs were analyzed. All were associated with Piwi but not with Ago1, the Drosophila Argonaute protein typically associated with miRNAs; miR-8, miR-311, and bantam immunoprecipitated with Ago1 mAb. No rasiRNAs immunoprecipitated with the myc mAb when lysate was used from flies lacking the myc-Piwi transgene (Vagin, 2006).

Although aub mutant ovaries silenced roo mRNA normally, they showed reduced accumulation of roo rasiRNA relative to aub/+ heterozygotes, which suggests that roo rasiRNAs associate with both Piwi and Aub. The supernatant and antibody-bound small RNAs were analyzed after GFP mAb immunoprecipitation of ovary lysate from GFP-Aub transgenic flies and flies lacking the transgene. roo rasiRNA was recovered only when the immunoprecipitation was performed with the GFP mAb in ovary lysate from GFP-Aub transgenic flies. The simplest interpretation of these data is that roo rasiRNAs physically associate with both Piwi and Aub, although it remains possible that the roo rasiRNAs are loaded only into Piwi and that Aub associates with Piwi in a stable complex. The association of roo rasiRNA with both Piwi and Aub suggests that piwi and aub are partially redundant, as does the modest reduction in roo silencing in piwi but not in aub mutants. Alternatively, roo silencing might proceed through Piwi alone, but the two proteins could function in the same pathway to silence selfish genetic elements (Vagin, 2006).

These data suggest that in flies, rasiRNAs are produced by a mechanism that requires neither Dcr-1 nor Dcr-2, yet the patterns of rasiRNAs that direct roo and Ste silencing are as stereotyped as the distinctive siRNA population generated from the white hairpin by Dcr-2 or the unique miRNA species made from each pre-miRNA by Dcr-1. A key challenge for the future will be to determine what enzyme makes rasiRNAs and what sequence or structural features of the unknown rasiRNA precursor lead to the accumulation of a stereotyped pattern of predominantly antisense rasiRNAs (Vagin, 2006).

The role of PIWI and the miRNA machinery in Drosophila germline determination

The germ plasm has long been demonstrated to be necessary and sufficient for germline determination, with translational regulation playing a key role in the process. Beyond this, little is known about molecular activities underlying germline determination. This study reports the function of Drosophila Piwi, Dicer-1, and dFMRP (Fragile X Mental Retardation Protein) in germline determination. Piwi is a maternal component of the polar granule, a germ-plasm-specific organelle essential for germline specification. Depleting maternal PIWI does not affect Osk or Vasa expression or abdominal patterning but leads to failure in pole-plasm maintenance and primordial-germ-cell (PGC) formation, whereas doubling and tripling the maternal piwi dose increases Osk and Vasa levels correspondingly and doubles and triples the number of PGCs, respectively. Moreover, Piwi forms a complex with dFMRP and Dicer-1, but not with Dicer-2, in polar-granule-enriched fractions. Depleting Dicer-1, but not Dicer-2, also leads to a severe pole-plasm defect and a reduced PGC number. These effects are also seen, albeit to a lesser extent, for dFMRP, another component of the miRISC complex. Because Dicer-1 is required for the miRNA pathway and Dicer-2 is required for the siRNA pathway yet neither is required for the rasiRNA pathway, the data implicate a crucial role of the Piwi-mediated miRNA pathway in regulating the levels of Osk, Vasa, and possibly other genes involved in germline determination in Drosophila (Megosh, 2006).

It has been nearly a century since the discovery of germ plasm and its function in germline fate determination in diverse organisms. In recent decades, the components and assembly of the polar granule in Drosophila and its equivalent in C. elegans have been effectively explored. Translational regulation has also been implicated in pole plasm for abdominal patterning and germline determination. In addition, germ cell-less (gcl) and mitochondrial large-subunit ribosomal RNAs (mtlr RNAs) have been shown to be required for germline determination. However, the biochemical activities of these molecules remain largely unknown. This study identified Piwi and likely the miRNA machinery as a germ-plasm regulatory activity that is involved in germline fate determination (Megosh, 2006).

Germ-plasm assembly occurs in a stepwise fashion. Step 1 involves the transport of polar granule materials to the posterior end of the oocyte during oogenesis, a process that involves a microtubule-based transport system as well as genes such as cappuccino and staufen. Step 2 is the assembly of polar-granule components at the posterior end, a process that is almost concurrent with the transport and that is completed by stage 12 of oogenesis. A critical component for the assembly is Osk, which determines the pole-cell number in a dose-dependent manner and has the ability to recruit Vasa and Tud as well as to induce pole-cell formation at ectopic sites within the embryo. Three lines of data suggest that Piwi is downstream of Osk, Tud, and Vasa in the assembly process: (1) Osk, Tud, and Vasa appear to assemble normally into the pole plasm in Piwi-depleted developing oocytes; (2) Piwi cannot recruit Osk or Vasa ectopically to the anterior pole, yet Osk can recruit Piwi to the anterior pole; (3) Osk, Tud, and Vasa all have both germline determination and posterior-patterning functions, but Piwi does not appear to have a detectable function in patterning (Megosh, 2006).

Although the assembly of polar-granule components occurs in a hierarchical fashion, there is growing evidence for interactions between polar-granule components beyond what is required for assembly. For example, a regulatory relationship between nanos and tudor has been reported. In nanos mutant embryos, both Tudor levels and the number of pole cells increase. Other experiments suggest that the presence of mtlrRNA in the polar granules is required for stabilization of the polar-granule components Vasa, Gcl, nos mRNA, and pgc mRNA. The regulatory function reported in this study for Piwi toward Osk, Vasa, and Nos further supports the interplay and interdependency among pole-plasm components. A previous study implicates osk as a rate-limiting factor for all aspects of pole-plasm function. The results suggest that Piwi, likely working through the miRNA pathway, is also a limiting factor for germ-cell formation. This function of Piwi is likely achieved via regulation of the levels of Osk, Tud, and Vasa, and possibly that of other polar-granule components, in a dose-dependent fashion (Megosh, 2006).

The regulation of Piwi toward the expression of Osk, Tud, Vasa, and Nos appears to be dispensable; Piwi-deficient oocytes and early embryos do not display detectable defects in their expression of Osk, Tud, Vasa, and Nos. This redundancy is likely due to an overlapping function of Piwi with other proteins involved in the RNAi pathway and/or colocalized in nuage during oogenesis; such proteins might include Maelstrom, Armitage, and Aubergine. Among these proteins, Aubergine, a close homolog of Piwi, is a known polar-granule component in early embryos. It regulates the translation of Osk during oogenesis and is required for both pole-cell formation and posterior patterning during embryogenesis (Megosh, 2006).

It is intriguing that Piwi regulates Osk and Vasa expression yet does not display a posterior-patterning phenotype. This function is different from that of Aubergine, so it is possible that Piwi and Aubergine each have their own regulatory targets in addition to Osk and Vasa. The Piwi targets may be specifically involved in maintaining polar-granule localization and may not be subject to Aubergine regulation, whereas Aubergine targets might be involved in both germline determination and posterior patterning. In support of this possibility, it has recently been shown that the generation of certain rasiRNAs shows varying dependencies on Piwi and Aubergine. The regulation of Piwi toward its specific target genes may be activated during oocyte maturation, similar to the oocyte maturation-dependent activation of RNAi as observed for aubergine and spindle-E. Thus, Piwi is not required for Osk and Vasa localization during oogenesis but is required for maintaining their localization during embryogenesis. An alternative hypothesis is that Piwi, like Aubergine, also regulates patterning genes but that this function is redundant. This hypothesis, however, does not explain the fact that neither ectopic expression nor overexpression of Piwi causes a detectable defect in posterior patterning (Megosh, 2006).

Given the association of Piwi with Dcr-1 and dFMRP, the Piwi-mediated regulation is likely via the miRNA but not the siRNA mechanism, which is Dcr-2-dependent, or the rasiRNA mechanism, which does not depend on either Dcr-1 or Dcr-2. This hypothesis is further supported by the similar phenotypes observed in embryos depleted of Piwi, Dcr-1, and dFMRP but not Dcr-2. It is possible that Piwi might bind to novel small RNAs to achieve this function, given recent findings that mammalian Piwi subfamily proteins bind to Piwi-interacting RNAs (piRNAs). If so, these novel RNAs must function in a Dcr-1-dependent pathway in the cytoplasm given Piwi's localization to the cytoplasm in early pole cells. The function of the Piwi/DCR-1-mediated miRNA or novel small-RNA pathway in germline specification is very similar to that of other germ-cell regulators, such as gcl and mtlr RNAs, in that these genes are required for pole-cell formation but not for abdominal segmentation. However, unlike embryos from the gcl-bcd females, embryos from the piwi-bcd females exhibit no cell-cycle delays in the anterior nuclei and no significant changes in the morphology of anterior nuclei. Furthermore, GCL mediates a transcriptional repression mechanism [72]. Thus, the effect of the Piwi-miRNA mechanism on pole-cell formation may be distinct from the gcl-mediated mechanism (Megosh, 2006).

It is important to note that the Piwi-mediated miRNA pathway positively regulates the expression of Osk and Vasa, in contrast to the known translational repression role of the miRNA pathway. In support of this observation, the Piwi ortholog in the mouse, MIWI, also appears to positively regulate gene expression, likely by enhancing mRNA stability and translation. Alternatively, it is possible that Piwi regulates an unidentified intermediate protein whose function is to repress the expression of Osk and Vasa (Megosh, 2006).

piwi is essential for the self-renewal of adult germline stem cells in Drosophila. Recent studies have demonstrated that the miRNA pathway is involved in division and self-renewal of adult germline stem cells in the Drosophila ovary. This study further connects Piwi and the miRNA pathway and reveals their crucial role in germline fate determination during embryogenesis. These observations suggest that the germline and stem cells may share a common miRNA-mediated mechanism in defining their fates. Given the high degree of conservation of the miRNA machinery during evolution, this pathway may function in diverse organisms in determining the germline and stem cell fates (Megosh, 2006).

Ectopic expression of germline genes drives malignant brain tumor growth in Drosophila

Model organisms such as the fruit fly Drosophila melanogaster can help to elucidate the molecular basis of complex diseases such as cancer. Mutations in the Drosophila gene lethal (3) malignant brain tumor cause malignant growth in the larval brain. This study shows that l(3)mbt tumors exhibited a soma-to-germline transformation through the ectopic expression of genes normally required for germline stemness, fitness, or longevity. Orthologs of some of these genes were also expressed in human somatic tumors. In addition, inactivation of any of the germline genes nanos, vasa, piwi, or aubergine suppressed l(3)mbt malignant growth. These results demonstrate that germline traits are necessary for tumor growth in this Drosophila model and suggest that inactivation of germline genes might have tumor-suppressing effects in other species (Janic, 2010).

The Drosophila tumor-suppressor gene l(3)mbt was identified as a temperature-sensitive mutation that caused malignant growth in the larval brain. Other l(3)mbt mutant alleles obtained later show the same temperature-sensitive phenotype. L(3)mbt's closest homologs, Drosophila Scm (Sex comb on midleg) and Sfmbt (Scm-related gene containing four mbt domains), encode Polycomb Group proteins. L3MBTL1, the human homolog of Drosophila L(3)MBT, is a transcriptional repressor that is found in a complex with core histones, heterochromatin protein 1γ (HP1γ), and RB (Retinoblastoma protein) and can compact nucleosomes. Drosophila L(3)MBT is a substoichiometric component of the dREAM-MMB complex, which includes the two Drosophila Retinoblastoma-family proteins and the Myb-MuvB (MMB) complex. Depletion of components of the dREAM/MMB complex in Drosophila Kc cells by RNA interference results in genome-wide changes in gene expression. These data strongly suggest that l(3)mbt function might contribute to establishing and maintaining certain differentiated states through the stable silencing of specific genes (Janic, 2010).

To identify the genes whose misexpression might account for the growth of l(3)mbt tumors (henceforth referred to as mbt tumors), genome-wide gene expression profiling was carried out of l(3)mbt^E2 and l(3)mbt^ts1 homozygous and transheterozygous larval brains raised at restrictive temperature (29°C). l(3)mbt^ts1 tumors were also analyzed at the 1st, 5th, and 10th rounds of allograft culture in adult flies (T1, T5, and T10, respectively). Brains from homozygous white¹¹¹⁸ (w¹¹¹⁸), l(3)mbt^E2, or l(3)mbt^ts1 larvae raised at permissive temperature (17°C) were used as controls. For comparison, larval brain malignant neoplasms caused by mutation in brain tumor (brat) as well as allograft cultures at T1,T5, and T10 of tumors caused by mutants in brat, lethal giant larvae (lgl), miranda (mira), prospero (pros), and partner of inscuteable (pins), were also profiled (Janic, 2010).

Hierarchical clustering plots of these data reveal three distinct clusters that include control larval brains, mbt larval brain tumors, and cultured l(3)mbt^ts1 tumors, respectively. From these data, 151 genes were identified that were either overexpressed or underexpressed in all three larval mbt tumor types compared to all three controls. From this list, those genes were removed that were also up- or down-regulated) in larval brat neoplasms and, hence, likely to encode functions generally required for larval brain tumor growth. The expression levels of the remaining 102 up-regulated genes are referred to as as the mbt signature (MBTS). MBTS is notably enhanced in cultured mbt tumors and can be used unequivocally to distinguish mbt tumors from other cultured malignant brain neoplasms like lgl, mira, pros, pins, or brat. Individual MBTS genes, however, are also up-regulated in some of these tumors (Janic, 2010).

The function of most MBTS genes remains unknown. However, a quarter of them (26 of 102) are genes required in the germ line. For instance, nanos (nos), female sterile(1)Yb (fs(1)Yb), and zero population growth (zpg) function in the establishment of the pole plasm in the egg and cystoblasts differentiation. The gonad-specific thioredoxins ThioredoxinT (TrxT) and deadhead (dhd), giant nuclei (gnu), corona (cona), hold'em (hdm), matotopetli (topi), and the female germline-specific γTUB37C isoform function during oocyte differentiation, meiosis, and syncytial embryo development. Also piwi, aubergine (aub), krimper (krimp), and tejas (tej) are involved in the biogenesis of Piwi-interacting RNAs (piRNAs) that protect germline cells against transposable elements and viruses. Some of these genes also have functions that are not germline related. For instance, some piwi alleles display synthetic lethality), and nos is required during nervous system development (Janic, 2010).

Driven by the high percentage of MBTS genes that have germline functions, other germline-related genes were sought that do not meet the stringent criteria applied to select the 102 MBTS genes, but are overexpressed in mbt tumors. Among these, the genes were found that encode the synaptonemal complex protein Crossover suppressor on 3 of Gowen [C(3)G] and the cell cycle kinase Pan gu (PNG), which interact with the proteins encoded by the MBTS genes cona and gnu, respectively. The same applies to Squash (SQU), Spindle-E (SPN-E), Maelstrom (MAEL), and AGO3, components of the piRNA machinery, which colocalize with other MBTS proteins in nuage (Janic, 2010).

To determine whether the mRNAs found ectopically expressed in mbt tumors are translated, protein expression was examined with a selected number of currently available antibodies. Given the key role of VASA in the assembly of the pole plasm and germline development, it was included in this study, even though vasa mRNA levels are not significantly increased in mbt tumors. By Western blot, it was confirmed that PIWI, AUB, and VASA are ectopically expressed in mbt tumors. Immunofluorescence studies also revealed the ectopic expression in l(3)mbt^ts1 brains raised at 29°C of C(3)G, SQU, and VASA. These results show that some of the germline genes ectopically expressed in mbt tumors are translated. However, it has not been possible to confirm the expression of other proteins, including MAEL, ORB, BAM, GNU, and TOPI, which suggests that, possible technical problems aside, either the corresponding mRNAs are not translated or these proteins might be unstable in such an ectopic environment. The expression of VASA, by contrast, suggests that other mRNAs whose levels are not appreciably increased in mbt tumors might actually be ectopically translated (Janic, 2010).

Prompted by the expression in l(3)mbt^ts1 brains of several genes involved in the biogenesis and regulation of piRNAs, 23- to 30-nucleotide RNAs were sequenced from l(3)mbt^ts1 larval brain tumors and from wild-type brains and ovaries. 117 known piRNAs and microRNAs (miRNAs) were detected in l(3)mbt^ts1 larval brain tumor samples. Of these, 31 are either not expressed in wild-type brains or are expressed there at less than 10% their level in larval brain tumors. Most of them are highly expressed in wild-type ovaries, thus substantiating further the ectopic acquisition of germline traits that characterizes mbt tumors (Janic, 2010).

It is not known which, if any, of the germline genes that are up-regulated in mbt tumors are direct targets of l(3)mbt or if their ectopic expression is a downstream consequence of intermediate events. The putative direct targets of l(3)mbt are many. The dREAM-MMB complex, of which L(3)MBT is a substoichiometric component, has been found to be promoter-proximal to 32% of Drosophila genes, and MMB factors are known to regulate transcription of a wide range of genes in Drosophila Kc cells. In addition, there is no estimate for the number of proteins like VASA that, despite their low mRNA expression levels, might be up-regulated in mbt tumors. Indeed, many of these genes, as well as the piRNAs and miRNAs expressed in mbt tumors, might themselves regulate the basal transcription and translation machineries, adding a further layer of gene expression modulation (Janic, 2010).

The extent to which ectopic expression of germline genes contributes to mbt tumor growth was determined. To this end, larval brain growth was quantified in individuals that were mutant for l(3)mbt^ts1 alone, or double mutant for l(3)mbt^ts1 and one of several of the germline genes that are ectopically expressed in mbt tumors. Measured as the total amount of protein, the average brain size in l(3)mbt^ts1 is about seven times as large as that in control w¹¹¹⁸ larvae, a difference that is not significantly reduced by the additional loss of zpg, Pxt, or AGO3. However, brain overgrowth is reduced to a size similar to that of the control in l(3)mbt^ts1 larvae that are also mutant for either piwi, vasa, aub, or nos. The loss of piwi does not prevent brain overgrowth in brat ^k06028 mutant larvae. Then tumor growth was quantified after allograft in adult flies. The frequency with which l(3)mbt^ts1 homozygous larval brain tissue develops tumors in this assay is not significantly reduced by the additional loss of zpg or AGO3 and is only moderately reduced by the loss of Pxt, but it is markedly reduced by the additional loss of piwi, vasa, aub), or nos. The frequency of brat ^k06028 tumor formation is not affected by the loss of piwi or nos. These results demonstrate that the ectopic expression of germline genes, particularly piwi, vasa, nos, and aub, significantly contributes to mbt tumor growth (Janic, 2010).

A closely reminiscent soma-to-germline transformation observed in mutants in the Caenorhabditis elegans Rb homolog LIN-35, as well as in long-lived C. elegans strains, has led some to propose that the acquisition of germline characteristics by somatic cells might contribute to increased fitness and survival, a mechanism that could contribute to the transformation of mammalian cells. Also in humans, some genes that are predominantly expressed in germline cells and have little or no expression in somatic adult tissues become aberrantly activated in various malignancies, including melanoma and several types of carcinomas. These are known as cancer-testis (CT) genes or cancer-germline (CG) genes. A subset of these CG genes encode antigens that are immunogenic in cancer patients and are being pursued as biomarkers and as targets for therapeutic cancer vaccines (Janic, 2010 and references therein).

Human CG genes are suspected to contribute to oncogenesis germline traits like immortality, invasiveness, and hypomethylation, but their actual role in cancer remains unknown. The current results demonstrate that ectopic germline traits are necessary for tumor growth in Drosophila mbt tumors, suggesting that their inactivation might have tumor-suppressing effects in other species. Some germline genes up-regulated in mbt tumors are orthologs of human CG genes like PIWIL1/piwi, NANOS1/nanos, and SYCP1 /c(3)G. The list of genes up-regulated in mbt tumors includes many other germline genes that might also be relevant in human cancer (Janic, 2010).

PIWI proteins are essential for early Drosophila embryogenesis

PIWI proteins, a subfamily of the ARGONAUTE/PIWI protein family, have been implicated in transcriptional and posttranscriptional gene regulation and transposon silencing mediated by small non-coding RNAs, especially piRNAs. Although these proteins are known to be required for germline development, their somatic function remains elusive. This study examined the maternal function of all three PIWI proteins in Drosophila; Piwi, Aubergine (Aub) and Argonaute3 (Ago3) during early embryogenesis. In syncytial embryos, Piwi displays an embryonic stage-dependent localization pattern. Piwi is localized in the cytoplasm during mitotic cycles 1-10. Between cycles 11 and 14, Piwi remains in the cytoplasm during mitosis but moves into the somatic nucleus during interphase. Beyond cycle 14, it stays in the nucleus. Aub and Ago3 are diffusely cytoplasmic from cycle 1 to 14. Embryos maternally depleted of any one of the three PIWI proteins display severe mitotic defects, including abnormal chromosome and nuclear morphology, cell cycle arrest, asynchronous nuclear division and aberrant nuclear migration. Furthermore, all three PIWI proteins are required for the assembly of mitotic machinery and progression through mitosis. Embryos depleted of maternal PIWI proteins also exhibit chromatin organization abnormalities. These observations indicate that maternal Piwi, Aub and Ago3 play a critical role in the maintenance of chromatin structure and cell cycle progression during early embryogenesis, with compromised chromatin integrity as a possible cause of the observed mitotic defects. This study demonstrates the essential function of PIWI proteins in the first phase of somatic development (Mani, 2013).

Zfrp8/PDCD2 is required in ovarian stem cells and interacts with the piRNA pathway machinery

The maintenance of stem cells is central to generating diverse cell populations in many tissues throughout the life of an animal. Elucidating the mechanisms involved in how stem cells are formed and maintained is crucial to understanding both normal developmental processes and the growth of many cancers. Previously, studies have shown that Zfrp8/PDCD2 is essential for the maintenance of Drosophila hematopoietic stem cells. This study shows that Zfrp8/PDCD2 is also required in both germline and follicle stem cells in the Drosophila ovary. Expression of human PDCD2 fully rescues the Zfrp8 phenotype, underlining the functional conservation of Zfrp8/PDCD2. The piRNA pathway is essential in early oogenesis, and this study found that nuclear localization of Zfrp8 in germline stem cells and their offspring is regulated by some piRNA pathway genes. Zfrp8 forms a complex with the piRNA pathway protein Maelstrom and controls the accumulation of Maelstrom in the nuage. Furthermore, Zfrp8 regulates the activity of specific transposable elements also controlled by Maelstrom and Piwi. These results suggest that Zfrp8/PDCD2 is not an integral member of the piRNA pathway, but has an overlapping function, possibly competing with Maelstrom and Piwi (Minakhina, 2014).

These studies on Zfrp8 requirement in the Drosophila ovary show that the gene is essential in stem cells. The results suggest that Zfrp8 is not required in cells with limited developmental potential, as transient wild-type and mutant clones were similar in number and size. No difference was found in Zfrp8 and wild-type escort cell clones, indicating that Zfrp8 is not required in these cells that multiply by self-duplication. Furthermore, Zfrp8 and wild-type MARCM clones induced in third instar larvae were indistinguishable in the adult antenna and legs 20 days after induction (ACI). These results support the conclusion that Zfrp8 function is primarily required in stem cells (Minakhina, 2014).

Despite this functional requirement, Zfrp8 protein was not enriched in Drosophila GSCs and FSCs. This is surprising, because in mice Zfrp8/PDCD2 is enriched in several types of stem cell. Zfrp8/PDCD2 is also highly expressed in human bone marrow and cord blood stem and precursor cells with protein levels decreasing significantly as these cells differentiate (Minakhina, 2014).

Loss of Zfrp8 in the Drosophila germline did not affect signaling from the niche to the stem cells. But the stem cells themselves are highly sensitive to loss of Zfrp8. In both Zfrp8germline stem cell clones and Zfr8 KD germaria abnormal spectrosomes reminiscent of fusomes were observed. These phenotypes suggest that these germline stem cells are losing stem identity and show features of a stem cell and a more advanced cystocyte. Germline and somatic stem cells and their daughter cells ultimately stop dividing when depleted of Zfrp8 but continue to survive for several days, as evident from the phenotype of the persistent stem cell clones. Similarly, in leukemia and in cancer cell lines that initially have high levels of the protein, reduction of Zfrp8/PDCD2 correlates with delay or arrest of the cell cycle rather than cell death (Minakhina, 2014).

The most severe abnormalities were observed 10-20 days ACI in Zfrp8 GSC clones induced in larvae and adults. The phenotype of Zfrp8 KD ovarioles also became more pronounced with age, starting from a relatively normal-looking germarium and a few egg chambers in young flies, to ovarioles made up of disorganized cysts, and finally, to ovarioles in which germ cells were almost entirely absent. The temporal change in phenotype can be explained in two ways. First, it is possible that Zfrp8 levels are initially high enough in mutant and KD stem cells to support a few divisions and the formation of mutant cysts. However, as Zfrp8 is gradually depleted the cells stop dividing and are eventually lost. Alternatively, lack of Zfrp8 may induce changes in parental cells that affect the developmental potential of the daughter cells. For instance, chromatin modifications could be affected in the absence of Zfrp8, but it could take several cell generations for these changes to have a phenotypic effect. In both these scenarios, loss of Zfrp8 would predominantly affect cells undergoing constant or rapid divisions, such as stem cells and cancer cells (Minakhina, 2014).

The loss of asymmetry in the stem cells, the mislocalization of BicD and Orb proteins to and within the oocyte, the mislocalization of Zfrp8 protein in GSCs of several piRNA pathway mutants, and the genetic interaction of Zfrp8 with piRNA pathway genes suggested a connection between Zfrp8 and the piRNA pathway. The de-repression of the subset of transposons in Zfrp8 KD ovaries further links the gene with the piRNA pathway (Minakhina, 2014).

Several LTR and non-LTR retroelements were tested that represent three major TE classes based on their tissue-specific activity in the germline, soma or in both tissues (intermediate). When Zfrp8 is depleted in the germline, two out of seven intermediate and germline elements tested, HeT-A and TART, show significant de-repression. These elements are different from the majority of Drosophila TEs. The HeT-A, TART and the TAHRE elements are integral components of fly telomere. Their activity is tightly regulated and is required to protect chromosome ends. These elements, like other TEs, are controlled by the piRNA machinery, but their primary piRNAs are likely to be derived from the same telomeric loci that are also their targets for repression. By contrast, the majority of primary piRNAs are derived from piRNA clusters and target TEs dispersed throughout the genome. Furthermore, the repression of TART and HeT-A in the germline involves an unusual combination of piRNA factors. It was found that at early stages of oogenesis they appear to be regulated by piwi and mael, but not by the germline-specific Piwi family member Aub. This result is in agreement with recent studies on piwi function in the soma and germline that showed that HeT-A and TART elements are among the TEs most strongly regulated by Piwi in the germline. Thus, Zfrp8 may target the same TEs as Piwi and Mael but not those regulated by Aub (Minakhina, 2014).

De-repression of TEs caused by Zfrp8 KD could be responsible for the enhancement of developmental defects seen in piRNA pathway mutants. For instance, the increase of TE transcripts may enhance dorsoventral patterning defects in armi, AGO3, aub, spnE and vas because of the competition between TE transcripts and oocyte polarity factors for the same RNA transport machinery. However, the interaction of Zfrp8 with the piRNA pathway machinery seems to be more complex. Zfrp8 enhanced the egg phenotype of only three mutants, spnE, AGO3 and vas, and in these mutants the nuclear localization of Zfrp8 protein was also affected. These results suggest that Zfrp8 functions downstream of the three factors (Minakhina, 2014).

Both piwi and mael are dominantly suppressed by Zfrp8. Both these factors have important nuclear functions, regulating chromatin modifications and controlling TEs at the transcriptional level, and both are required to repress HeT-A and TART-A elements. Zfrp8 could suppress piwi or mael by inducing a competing chromatin modification at the genomic loci targeted by Piwi or Mael. Chromatin modifications are generally stable through several cell generations. Such a function would therefore be consistent with the temporal changes of phenotypes in Zfrp8 ovarian clones and KD ovarioles (Minakhina, 2014).

Although Piwi and Mael target the same genomic loci, no interaction between the two proteins have been detected. Co-immunoprecipitation experiments suggest that Zfrp8 complexes with Mael but not with Piwi, indicating that the observed genetic interaction between Zfrp8 and piwi may be mediated by mael. Mael is one of the most enigmatic proteins in the piRNA pathway. It is found in the cytoplasm, nuage and nucleus, and has been implicated in diverse cellular processes including the ping-pong piRNA amplification cycle in the germline, MTOC assembly in the oocyte and Piwi-dependent chromatin modification in somatic cells. Zfrp8/PDCD2 is also required in the soma and germline and may function both in the cytoplasm and in nuclei. However, in contrast to mael, Zfrp8 homozygous mutants are lethal and Zfrp8 ovaries show a stronger phenotype. Based on the observation that Mael and Zfrp8 are found in the same complex and that Zfrp8 dominantly suppresses Mael, it is proposed that they act in opposite fashion on a common target, whether during piRNA biogenesis or chromatin modification (Minakhina, 2014).

A role for Drosophila Cyclin J in oogenesis revealed by genetic interactions with the piRNA pathway

Cyclin J (CycJ) is a poorly characterized member of the Cyclin superfamily of cyclin-dependent kinase regulators, many of which regulate the cell cycle or transcription. Although CycJ is conserved in metazoans its cellular function has not been identified and no mutant defects have been described. In Drosophila, CycJ transcript is present primarily in ovaries and very early embryos, suggesting a role in one or both of these tissues. The CycJ gene (CycJ) lies immediately downstream of armitage (armi), a gene involved in the Piwi-associated RNA (piRNA) pathways that are required for silencing transposons in the germline and adjacent somatic cells. Mutations in armi result in oogenesis defects but a role for CycJ in oogenesis has not been defined. This study assessed oogenesis in CycJ mutants in the presence or absence of mutations in armi or other piRNA pathway genes. CycJ null ovaries appeared normal, indicating that CycJ is not essential for oogenesis under normal conditions. In contrast, armi null ovaries produced only two egg chambers per ovariole and the eggs had severe axis specification defects, as observed previously for armi and other piRNA pathway mutants. Surprisingly, the CycJ armi double mutant failed to produce any mature eggs. The double null ovaries generally had only one egg chamber per ovariole and the egg chambers frequently contained an overabundance of differentiated germline cells. Production of these compound egg chambers could be suppressed with CycJ transgenes but not with mutations in the checkpoint gene mnk, which suppress oogenesis defects in armi mutants. The CycJ null showed similar genetic interactions with the germline and somatic piRNA pathway gene piwi, and to a lesser extent with aubergine (aub), a member of the germline-specific piRNA pathway. The strong genetic interactions between CycJ and piRNA pathway genes reveal a role for CycJ in early oogenesis.These results suggest that CycJ is required to regulate egg chamber production or maturation when piRNA pathways are compromised (Atikukke, 2014).

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EVOLUTIONARY HOMOLOGS

Structure of Piwi proteins

RNA silencing regulates gene expression through mRNA degradation, translation repression and chromatin remodelling. The fundamental engines of RNA silencing are RISC and RITS complexes, whose common components are 21-25 nt RNA and an Argonaute protein containing a PIWI domain of unknown function. The crystal structure of an archaeal Piwi protein (AfPiwi) is organised into two domains, one resembling the sugar-binding portion of the lac repressor and another with similarity to RNase H. Invariant residues and a coordinated metal ion lie in a pocket that surrounds the conserved C-terminus of the protein, defining a key functional region in the PIWI domain. Furthermore, two Asp residues, conserved in the majority of Argonaute sequences, align spatially with the catalytic Asp residues of RNase H-like catalytic sites, suggesting that in eukaryotic Argonaute proteins the RNase H-like domain may possess nuclease activity. The conserved region around the C-terminus of the PIWI domain, which is required for small interfering RNA (siRNA) binding to AfPiwi, may function as the receptor site for the obligatory 5' phosphate of siRNAs, thereby specifying the cleavage position of the target mRNA (Parker, 2004).

Plant Piwi homologs

An allelic series of the novel argonaute mutant (ago1-1 to ago1-6) of the herbaceous plant Arabidopsis thaliana has been isolated. The ago1 mutation pleotropically affects general plant architecture. The apical shoot meristem generates rosette leaves and a single stem, but axillary meristems rarely develop. Rosette leaves lack a leaf blade but still show adaxial/abaxial differentiation. Instead of cauline leaves, filamentous structures without adaxial/abaxial differentiation develop along the stem and an abnormal inflorescence bearing infertile flowers with filamentous organs is produced. Two independent T-DNA insertions into the AGO1 locus led to the isolation of two corresponding genomic sequences as well as a complete cDNA. The AGO1 locus was mapped close to the marker mi291a on chromosome 1. Antisense expression of the cDNA results in a partial mutant phenotype. Sense expression causes some transgenic lines to develop goblet-like leaves and petals. The cDNA encodes a putative 115 kDa protein with sequence similarity to translation products of a novel gene family present in nematodes as well as humans. No specific function has been assigned to these genes. Similar proteins are not encoded by the genomes of yeast or bacteria, suggesting that AGO1 belongs to a novel class of genes with a function specific to multicellular organisms (Bohmert, 1998).

Postembryonic development in higher plants is marked by repetitive organ formation via a self-perpetuating stem cell system, the shoot meristem. Organs are initiated at the shoot meristem periphery, while a central zone harbors the stem cells. The ZWILLE (ZLL) gene is specifically required to establish the central-peripheral organization of the embryo apex and this step is critical for shoot meristem self-perpetuation. zll mutants correctly initiate expression of the shoot meristem-specific gene SHOOT MERISTEMLESS in early embryos, but fail to regulate its spatial expression pattern at later embryo stages and initiate differentiated structures in place of stem cells. The ZLL gene encodes a novel protein, and related sequences are highly conserved in multicellular plants and animals but are absent from bacteria and yeast. It is proposed that ZLL relays positional information required to maintain stem cells of the developing shoot meristem in an undifferentiated state during the transition from embryonic development to repetitive postembryonic organ formation (Moussian, 1998).

Several lines of evidence indicate that the adaxial leaf domain possesses a unique competence to form shoot apical meristems. Factors required for this competence are expected to cause a defect in shoot apical meristem formation when inactivated and to be expressed or active preferentially in the adaxial leaf domain. PINHEAD, a member of a family of proteins that includes the translation factor eIF2C, is required for reliable formation of primary and axillary shoot apical meristems. In addition to high-level expression in the vasculature, low-level PINHEAD expression defines a novel domain of positional identity in the plant. This domain consists of adaxial leaf primordia and the meristem. These findings suggest that the PINHEAD gene product may be a component of a hypothetical meristem forming competence factor. Defects are described in floral organ number and shape, as well as aberrant embryo and ovule development associated with pinhead mutants, thus elaborating on the role of PINHEAD in Arabidopsis development. In addition, embryos doubly mutant for PINHEAD and ARGONAUTE1, a related, ubiquitously expressed family member, fail to progress to bilateral symmetry and do not accumulate the SHOOT MERISTEMLESS protein. Therefore PINHEAD and ARGONAUTE1 together act to allow wild-type growth and gene expression patterns during embryogenesis (Lynn, 1999).

Introduction of transgene DNA may lead to specific degradation of RNAs that are homologous to the transgene transcribed sequence through phenomena named post-transcriptional gene silencing (PTGS) in plants, quelling in fungi, and RNA interference (RNAi) in animals. It was shown previously that PTGS, quelling, and RNAi require a set of related proteins (SGS2, QDE-1, and EGO-1, respectively). This study reports the isolation of Arabidopsis mutants impaired in PTGS which are affected at the Argonaute1 (AGO1) locus. AGO1 is similar to QDE-2 required for quelling and RDE-1 required for RNAi. Sequencing of ago1 mutants reveals one amino acid essential for PTGS that is also present in QDE-2 and RDE-1 in a highly conserved motif. Taken together, these results confirm the hypothesis that these processes derive from a common ancestral mechanism that controls expression of invading nucleic acid molecules at the post-transcriptional level. As opposed to rde-1 and qde-2 mutants, which are viable, ago1 mutants display several developmental abnormalities, including sterility. These results raise the possibility that PTGS, or at least some of its elements, could participate in the regulation of gene expression during development in plants (Fagard, 2000).

Piwi homologs in Tetrahymena

During development of the somatic macronucleus from the germline micronucleus in ciliates, chromosome rearrangements occur in which specific regions of DNA are eliminated and flanking regions are healed, either by religation or construction of telomeres. A gene, TWI1, has been identified in Tetrahymena thermophila that is homologous to piwi and is required for DNA elimination. Small RNAs are specifically expressed prior to chromosome rearrangement during conjugation. These RNAs are not observed in TWI1 knockout cells. The RNAs require PDD1, another gene required for rearrangement, for expression. It is proposed that these small RNAs function to specify sequences to be eliminated by a mechanism similar to RNA-mediated gene silencing (Mochizuki, 2002).

Like most ciliated protozoa, Tetrahymena exhibits nuclear dimorphism. Each cell contains a germline micronucleus and a somatic macronucleus. During the sexual process of conjugation, two cells pair and their micronuclei undergo meiosis, followed by fertilization. The zygotic nucleus divides to produce the next generation of macro- and micro-nuclei in each cell (Mochizuki, 2002).

Macro- and micro-nuclei differ in structure and function. During vegetative growth, the micronucleus is transcriptionally inert, while the macronucleus is transcriptionally active. The micronucleus is diploid and divides mitotically; the macronucleus is polyploid and divides by amitosis. Although derived from the same zygotic nucleus, they differ in the organization of their genomes. The macronucleus lacks ~15% of DNA sequences found in the zygotic nucleus and in the micronucleus. This is due to sequence elimination associated with two types of DNA rearrangements that occur in developing macronuclei during the late stages of conjugation. The first type occurs by deletion of ~6000 internal eliminated sequences (IESs), accompanied by ligation of the flanking macronucleus-destined sequences. IESs 0.5 to >20 kb in length occur in noncoding regions. Excision of IESs can occur reproducibly (within a few nucleotides) at a specific site, or (with similar fidelity) at a limited number of alternative sites. The second type of rearrangement involves chromosome breakage followed by small deletions (<50 bp) and addition of telomeres. This 'chromosome healing' occurs at precise locations, producing 2–300 macronuclear chromosomes from the 5 chromosomes in the micronuclear (haploid) genome. This type of eliminated sequence is referred to as a breakage eliminated sequence (BES). Little is known about the mechanisms that produce IESs or BESs. BESs contain a conserved, 15 bp chromosome breakage sequence (Cbs), a likely site for recognition by (unknown) proteins. The only common elements associated with IESs analyzed to date are short (<10 bp), direct repeats of varied sequences at the ends of the IESs that have no known function in IES removal. The flanking regions of some IESs have cis-acting sequences required for IES removal, but no sequence homology has been observed between different elements. Thus, how IESs are recognized and eliminated is obscure (Mochizuki, 2002).

DNA sequences in the parental macronucleus guide DNA rearrangements. In Tetrahymena, introduction of DNA containing an IES sequence into the parental macronucleus inhibits DNA elimination of that IES when the new macronucleus forms. In another ciliate, Paramecium, an IES in the G surface antigen coding region that had aberrantly been retained in the macronucleus inhibited elimination of this IES when the new macronucleus formed, and deletion of another (A type) surface antigen gene in macronuclei resulted in deletion of this locus in new macronuclei, even though the gene in the micronucleus was intact. These phenomena suggest that sequence-specific information is transferred from the parental to the new macronucleus (Mochizuki, 2002 and references therein).

Evidence is presented for involvement of small RNAs in DNA rearrangement in Tetrahymena, and it is proposed that these RNAs transfer sequence information from the old to the new macronucleus. This evidence derives from analysis of a gene related to piwi, a member of the PPD (PAZ and Piwi domains) gene family defined by conserved PAZ and Piwi domains of unknown function. Some PPD gene family members are involved in posttranscriptional (PTGS) and/or transcriptional (TGS) gene silencing evoked by introduction of transgenic DNA or RNA. For example, AGO1 is required for PTGS in Arabidopsis, qde-2 is required for PTGS (quelling) in Neurospora, and rde-1 is required for RNA interference (RNAi) in C. elegans. In Drosophila, AGO1 and AGO2 are involved in RNAi; aubergine (sting) is required for RNAi and for silencing the Stellate locus, and piwi is required for silencing induced by multiple copies of Adh. In many of these events, small (20-26 nt) RNAs, processed by a Dicer-related, double-stranded RNase, play a pivotal role, but how PPD genes are related to small RNAs is not well understood (Mochizuki, 2002).

The piwi-related gene TWI1 is required for DNA rearrangement in Tetrahymena thermophila. In TWI1 knockout cells, both IES elimination and chromosome breakage are affected. Small (~28 nt) RNAs specifically expressed during conjugation do not accumulate in the absence of TWI1 or PDD1 (another gene required for IES elimination) expression (Mochizuki, 2002).

It is hypothesized that micronuclear transcripts or the small RNAs derived from them (scan RNAs or scnRNAs) are then transferred to the new macronucleus, possibly in association with Twi1p, Pdd1p, and Pdd2p, all of which show the remarkable property of localizing to the new macronucleus after they appear in the old one. Interestingly, Pdd1p contains three chromodomains and Pdd3p (another protein associated with IESs during macronuclear development) contains one, and chromodomains can interact with RNA. In the new macronucleus, the selected scnRNAs are hypothesized to target the DNAs to which they are homologous for elimination. The scnRNAs (and proteins complexed with them) could participate in the elimination process directly, or change some state of the chromatin region with which they interact by chemical (DNA methylation or histone modifications) or structural (chromatin remodeling) modification. Enzymes required for IES excision could then recognize one of these 'marked' sequences and excise it (Mochizuki, 2002).

Early origins and evolution of microRNAs and Piwi-interacting RNAs in animals

In bilaterian animals, such as humans, flies and worms, hundreds of microRNAs (miRNAs), some conserved throughout bilaterian evolution, collectively regulate a substantial fraction of the transcriptome. In addition to miRNAs, other bilaterian small RNAs, known as Piwi-interacting RNAs (piRNAs), protect the genome from transposons. This study identifies small RNAs from animal phyla that diverged before the emergence of the Bilateria. The cnidarian Nematostella vectensis (starlet sea anemone), a close relative to the Bilateria, possesses an extensive repertoire of miRNA genes, two classes of piRNAs and a complement of proteins specific to small-RNA biology comparable to that of humans. The poriferan Amphimedon queenslandica (sponge), one of the simplest animals and a distant relative of the Bilateria, also possesses miRNAs, both classes of piRNAs and a full complement of the small-RNA machinery. Animal miRNA evolution seems to have been relatively dynamic, with precursor sizes and mature miRNA sequences differing greatly between poriferans, cnidarians and bilaterians. Nonetheless, miRNAs and piRNAs have been available as classes of riboregulators to shape gene expression throughout the evolution and radiation of animal phyla (Grimson, 2008).

These results indicate that miRNAs and piRNAs, as classes of small riboregulators, have been present since the dawn of animal life, and indeed might have helped to usher in the era of multicellular animal life. However, metazoan miRNA evolution seems to have been very dynamic: all miRNAs have been lost in Trichoplax, and the pre-miRNAs of Porifera, Cnidaria and Bilateria have assumed distinct sizes. In addition, no miRNAs have recognizable conservation between poriferans, cnidarians and bilaterians, with only one of the Nematostella miRNAs displaying recognizable homology to bilaterian miRNAs, either because it is the only homologue of extant bilaterian miRNAs or because divergence has obscured common ancestry of other miRNAs. The wholesale shifts in miRNA function implied by this plasticity are congruent with the report that, although thousands of miRNA-target interactions have been maintained within each of the nematode, fly and vertebrate lineages, very few appear to be conserved throughout all three lineages. The plasticity of miRNA sequences over long timescales helps to explain why the rich small-RNA biology in basal organisms had escaped detection for so long (Grimson, 2008).

Analysis of Hydra PIWI proteins and piRNAs uncover early evolutionary origins of the piRNA pathway

To preserve genome integrity, an evolutionarily conserved small RNA-based silencing mechanism involving PIWI proteins and PIWI-interacting RNAs (piRNAs) represses potentially deleterious transposons in animals. Although there has been extensive research into PIWI proteins in bilaterians, these proteins remain to be examined in ancient phyla. This study investigated the PIWI proteins Hywi and Hyli in the cnidarian Hydra and found that both PIWI proteins are enriched in multipotent stem cells, germline stem cells, and in the female germline. Hywi and Hyli localize to the nuage, a perinuclear organelle that has been implicated in piRNA-mediated transposon silencing, together with other conserved nuage and piRNA pathway components. These findings provide the first report of nuage protein localization patterns in a non-bilaterian. Hydra PIWI proteins possess symmetrical dimethylarginines: modified residues that are known to aid in PIWI protein localization to the nuage and proper piRNA loading. piRNA profiling suggests that transposons are the major targets of the piRNA pathway in Hydra. These data suggest that piRNA biogenesis through the ping-pong amplification cycle occurs in Hydra and that Hywi and Hyli are likely to preferentially bind primary and secondary piRNAs, respectively. Presumptive piRNA clusters are unidirectionally transcribed and primarily give rise to piRNAs that are antisense to transposons. These results indicate that various conserved features of PIWI proteins, the piRNA pathway, and their associations with the nuage were likely established before the evolution of bilaterians (Lim, 2014).

C. elegans Piwi homologs

The high degree of sequence homology between piwi and its homologs in other organisms suggests a potential functional conservation. This hypothesis was tested in C. elegans. The injection of specific antisense RNA into the germ-line syncytium of C. elegans eliminates maternal and zygotic gene activity, producing a gene-specific loss-of-function effect that may persist through several generations. This technique, as refined by Fire (1998) and termed RNA-mediated interference (RNAi), was used to assess the function of prg-1 and prg-2, two C. elegans piwi homologs. Given the extremely high homology between prg-1 and prg-2, an anti-prg-1 RNA was used for injection to interfere with the function of both genes. The F₁ progeny of the injected worms are designated prg-RNAi worms for simplicity (Cox, 1998).

In wild-type C. elegans, two germ-line precursor cells, Z2 and Z3, give rise to ~2000 germ cells in the adult hermaphrodite. Germ-line proliferation occurs throughout most of larval development (L1-L4) and continues in the adult. This proliferation and maintenance of the germ line requires signals from the somatic distal tip cell (DTC) at the tip of each gonadal arm. During gonadal development, DTC migration results in the formation of two U-shaped gonadal arms by the L4 stage. Germ-line proliferation is limited to the distal end of each arm, forming a mitotic proliferation zone (MPZ), which serves as the GSC equivalent in C. elegans. Moving proximally, near the U turn of the gonad, germ cells enter meiotic prophase and then further differentiate into gametes at the proximal half of each arm, producing sperm in L4 and then oocytes in young adults (Cox, 1998).

The phenotype of prg-RNAi worms was examined both by quantitating their fertility and by assessing their gonadal and germ-line development. The prg-RNAi worms show three major aspects of germ-line defects. (1) As indicated by the distribution of the number of progeny per F₁ animal, the fertility difference between the prg-RNAi and control worms is highly significant. On average, prg-RNAi worms produced 92 progeny, with 75% of prg-RNAi worms giving rise to less than125 progeny. In contrast, control RNAi animals on average produced 191, with 90% of these animals giving rise to greater than125 progeny. The significant reduction of the fertility, as well as other defects described below, may still only reflect a partial loss of prg-1 and prg-2 function, because the RNAi technique is known to phenocopy partial rather than complete loss-of-function mutants (Cox, 1998).

(2) DNA staining reveals a dramatic shortening of the mitotic proliferation zone (MPZ) in prg-RNAi worms. On average, adult prg-RNAi worms exhibit a 50% reduction in the number of mitotic nuclei as compared with control animals. Associated with the reduction of the mitotic zone is a gonadal shortening. Fifty-seven percent of prg-RNAi worms exhibit a moderate to severe shortening. In the most severe case, the gonadal arm never makes the U turn. The number of sperm produced in these worms is greatly reduced as well. In contrast, only 7% of control animals exhibit a mild gonadal shortening. The above observed gonadal defects in prg-RNAi worms suggest that prg-1 and prg-2 are essential for germ-line proliferation and maintenance. Because the gonadal shortening may be due to a defect in DTC migration, it is also possible that prg-1 and prg-2 may play a role in proper gonadogenesis. (3) In addition to the MPZ shortening, the mitotic index in the remaining mitotic zone is further reduced by 5.5-fold in prg-RNAi worms with mildly to moderately shortened gonads. This indicates the important role of prg-1 and prg-2 in maintaining the mitotic ability of the germ-line nuclei (Cox, 1998).

RNAi is a gene-silencing phenomenon triggered by double-stranded (ds) RNA and involves the generation of 21 to 26 nt RNA segments that guide mRNA destruction. In Caenorhabditis elegans, lin-4 and let-7 encode small temporal RNAs (stRNAs) of 22 nt that regulate stage-specific development. Inactivation of genes related to RNAi pathway genes, a homolog of Drosophila Dicer (dcr-1), and two homologs of rde-1 (alg-1 and alg-2), cause heterochronic phenotypes similar to lin-4 and let-7 mutations. Further dcr-1, alg-1, and alg-2 are necessary for the maturation and activity of the lin-4 and let-7 stRNAs. These findings suggest that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation (Grishok, 2001).

One of the remarkable aspects about RNA interference (RNAi) in C. elegans is that the trigger molecules, dsRNA, can be administered via the animal's food. Assays were performed to see whether this feature is a universal property of the species by testing numerous strains that have been isolated from different parts of the globe. Strains were assayed on bacterial clones that were genetically modified to produce dsRNA homologous to different C. elegans genes. All isolates, with one exception, were sensitive to RNAi by feeding dsRNA of a germline-expressed gene, par-1. The one exceptional isolate, from Hawaii, subsequently named PPW-1, had a defect in RNAi that was specific to the germline and was a result of multiple mutations in a PAZ/PIWI domain-containing protein. ppw-1 animals have a defect in RNAi specifically for those mRNA targets that are expressed in the germline: somatically expressed endogenous genes are fully sensitive. Also, when ppw-1 animals are compared to wild-type animals, transgenically driven GFP expression in somatic tissues is equally sensitive to RNAi. Interestingly, although ppw-1 animals are fully resistant to all clones inducing embryonic lethality, in some of these clones the hatched larvae fail to progress to adulthood, suggesting that RNAi is not completely lost. Deleting ppw-1 in the canonical C. elegans strain Bristol N2 makes it resistant to feeding of dsRNA directed against germline-expressed genes. PPW-1 is a member of the Argonaute family of proteins; this protein family acts in posttranscriptional gene silencing and development, and is homologous to the RNAi gene rde-1. These data indicate that at least two members of this family are required for complete and effective RNAi in C. elegans (Tijsterman, 2002).

The ppw-1 gene product is homologous to RDE-1. Because both proteins are required to trigger efficient RNAi in the germline upon environmentally administered dsRNA, it is suggested that both proteins play nonredundant roles in the RNAi reaction and perhaps stabilize the RNAi intermediates, i.e., siRNAs, at different steps in the reaction. A putative role for RDE-1 in stabilizing such intermediates comes from the observation that pos-1 siRNA species are not detected in rde-1 animals that were fed pos-1 dsRNA. These animals are, however, capable of siRNA production, because cell-free extracts prepared from rde-1 animals are capable of dicing dsRNA into siRNAs, and siRNA can be detected shortly (5 hr) after injection of radio-labeled dsRNA molecules into the gonad of rde-1 animals. This indicates that the Dicer reaction, by which dsRNA is diced into siRNAs, is not compromised by the absence of functional RDE-1, and that the absence of observable steady-state levels of these siRNAs is thus more likely a stability issue (Tijsterman, 2002).

Whether the Hawaiian strain, as a result of the defect in PPW-1, is more susceptible to invasive elements that threaten the genomic content, like DNA transposons or dsRNA viruses (at present, no viroid species are known to infect the nematode), is difficult to address, but it seems unlikely. (1) It is found that DNA transposition is still repressed in the germline of the Hawaiian strain, and (2) the genome of the Hawaiian strain is not loaded with more remnants of retroviruses, compared to the Bristol N2 strain. (3) Without apparent selection pressure in its natural habitat, multiple basepair changes accumulated in PPW-1 in the Hawaiian isolate without apparent loss of growth or fecundity (Tijsterman, 2002).

In summary, PPW-1, a member of the PIWI/RDE-1/Argonaute protein family, is involved in RNAi in the C. elegans germline, most noticeably when the trigger molecules, dsRNA, are environmentally provided, and one C. elegans isolate, which was obtained from Hawaiian soil, lacks this factor. The fact that loss of rde-1 as well as loss of ppw-1 results in an RNAi-defective phenotype (of which the defect in ppw-1 seems tissue specific) indicates that at least two members of this important protein family are required for complete and effective RNAi in C. elegans (Tijsterman, 2002).

Epigenetic regulation by diverse classes of small RNAs is mediated by the highly conserved Argonaute/Piwi family of proteins. Although Argonautes are broadly expressed, the Piwi subfamily primarily functions in the germ line. Piwi proteins are associated with germline-specific ribonucleoprotein (RNP) granules in Drosophila, zebrafish, and mouse. Depending on the species and on the specific family member, Piwis play important roles in spermatogenesis and/or in maintaining germ cell and stem cell totipotency. Piwis bind to a newly discovered class of small RNAs, called piRNAs. C. elegans contains a large set of Argonaute/Piwi-related proteins, including two closely related to piwi called prg-1 and prg-2. The function of prg-1 and prg-2 and whether piRNAs exist in C. elegans is unknown. This study demonstrates that the Piwi-like protein PRG-1 is localized to P granules in germ cells entering spermatogenesis and is required for successful spermatogenesis. Loss of prg-1 causes a marked reduction in expression of a subset of mRNAs expressed during spermatogenesis, and prg-1 mutant sperm exhibit extensive defects in activation and fertilization. Moreover, prg-1 activity is required for the presence of the small RNAs called 21U-RNAs. These data suggest that PRG-1 promotes expression, processing, or stability of 21U-RNAs, which, in turn or in concert with PRG-1, promote proper expression of spermatogenesis transcripts (Wang, 2008).

Piwi in planarians

Two genes have been identified, smedwi-1 and smedwi-2, expressed in the dividing adult stem cells (neoblasts) of the planarian Schmidtea mediterranea. Both genes encode proteins that belong to the Argonaute/PIWI protein family and that share highest homology with those proteins defined by Drosophila PIWI. RNA interference (RNAi) of smedwi-2 blocks regeneration, even though neoblasts are present, irradiation-sensitive, and capable of proliferating in response to wounding; smedwi-2(RNAi) neoblast progeny migrate to sites of cell turnover but, unlike normal cells, fail at replacing aged tissue. It is suggested that SMEDWI-2 functions within dividing neoblasts to support the generation of cells that promote regeneration and homeostasis. PIWI proteins may be universal regulators of the production of stem cell progeny competent for performing differentiated functions (Reddien, 2006).

Planarian regeneration, based upon totipotent stem cells, the neoblasts, provides a unique opportunity to study in vivo the molecular program that defines a stem cell. DjPiwi-1, a planarian homologue of Drosophila Piwi, is preferentially accumulated in small cells distributed along the midline of the dorsal parenchyma. DjPiwi-1 transcripts were not detectable after X-ray irradiation by whole mount in situ hybridization. Real time RTPCR analysis confirmed the significant reduction of DjPiwi-1 expression after X-ray treatment. However, the presence of residual DjPiwi-1 transcription suggests that, although the majority of DjPiwi-1-positive cells can be neoblasts, this gene is also expressed in differentiating/differentiated cells. During regeneration, DjPiwi-1-positive cells reorganize along the midline of the stump and no accumulation of hybridization signal was observed either in the blastema area or in the parenchymal region beneath the blastema. DjPiwi-1-positive cells, as well as the DjMCM2-expressing neoblasts located along the midline and those spread all over the parenchyma, showed a lower tolerance to X-ray with respect to the DjMCM2-expressing neoblasts distributed along the lateral lines of the parenchyma. Taken together, these findings suggest the presence of different neoblast subpopulations in planarians (Rossi, 2006).

Roles of Piwi proteins in transcriptional regulation mediated by HP1s in cultured silkworm cells

Piwi proteins are part of a superfamily of Argonaute proteins, which are one of the core components of the RNA silencing pathway in many eukaryotes. Piwi proteins are thought to repress the transposon expression both transcriptionally and post-transcriptionally. Drosophila melanogaster Piwi was recently reported to associate with chromatin and to interact directly with the Heterochromatin Protein 1 (HP1a). This study shows that silkworm Piwi proteins interact with HP1s in the nucleus. The silkworm, Bombyx mori, has two Piwi proteins, Ago3 and Siwi, and two typical HP1 proteins, HP1a and HP1b. HP1a was found to play an important role in the interaction between Ago3/Siwi and HP1b in the ovary-derived BmN4 cell line. This study also found that Ago3/Siwi regulates the transcription in an HP1-dependent manner. These results suggest that silkworm Piwi proteins function as a chromatin regulator in collaboration with HP1a and HP1b (Tatsuke, 2014).

Vertebrate Piwi homologs

Genes belonging to the piwi family are required for stem cell self-renewal in diverse organisms. Mouse homologs of piwi were cloned by RT-PCR using degenerative primers. The deduced amino acid sequences of mouse homologs MIWI and MILI show that each contains a well-conserved C-terminal PIWI domain and that each shares significant homology with PIWI and their human counterparts HIWI. Both miwi and mili are found in germ cells of adult testis by in situ hybridization, suggesting that these genes may function in spermatogenesis. Furthermore, mili was expressed in primordial germ cells (PGCs) of developing mouse embryos and may therefore play a role during germ cell formation. MIWI may be involved in RNA processing or translational regulation, since MIWI was found to possess RNA binding activity. The data suggest that miwi and mili regulate spermatogenesis and primordial germ cell production (Kuramochi-Miyagawa, 2001).

Subcellular localization analysis of Piwi shows that it is a nucleoplasmic protein. This implies two things: Piwi may be involved in post-transcriptional mRNA processing in the nucleus or, alternatively, Piwi may be involved in nuclear functions indirectly related to gene expression. The former possibility may be more likely, considering the functions of other members of the piwi/zwille family. Rabbit eukaryotic initiation factor 2C (eIF2C) is one of the family members. The protein fraction that includes eIFC2 stabilizes the ternary complex of Met-tRNA, GTP and eIF2 in the presence of mRNA. Recently, another new member, rde-1, was reported and is involved in RNA-mediated interference (RNAi). Still another family member, sting/aubergine of Drosophila is postulated to interfere with the 3' UTR of Oskar (Kuramochi-Miyagawa, 2001 and references therein).

Piwi is evidently localized in nucleus, but MILI and MIWI proteins are localized in cytoplasm. These data suggest that the proteins are involved in post-transcriptional regulation of mRNA in cytoplasm. Although circumstantial, these data raise the possibility that some of the family members may be involved in RNA post-transcriptional processing or translational regulation (Kuramochi-Miyagawa, 2001).

Based on the notion that MIWI or MILI may be involved in RNA processing, the RNA binding activity of these proteins was analyzed. Full length MIWI protein avidly binds to riboguanine polymers under high stringency and a fragment spanning amino acids between 541 and 678 is responsible for the binding. Cerutti (2000) advocates that Piwi family proteins commonly possess a Piwi domain that is a 300-amino-acid domain containing an ~40 amino-acid Piwi box in the middle of the domain. The N-terminus of the Piwi domain of MIWI is indispensable for its RNA binding activity. The region of C-terminus of Piwi domain is reported to be essential for Piwi function and its function might be the stabilization of the protein. Taken together, overall structure of Piwi domain would be necessary for the full function of MIWI. RNA binding activity of MILI could not be detected. Function may be different between MIWI and MILI, like their different expression patterns. However, the possibility of the RNA binding activity of MILI cannot be excluded. The sequence specificity of bound RNA and the function of MIWI in RNA processing and translational regulation remain open questions (Kuramochi-Miyagawa, 2001).

The piwi family genes, which are defined by conserved PAZ and Piwi domains, play important roles in stem cell self-renewal, RNA silencing, and translational regulation in various organisms. To reveal the function of the mammalian homolog of piwi, mice with targeted mutations in the Mili gene, which is one of three mouse homologs of piwi, were produced and analyzed. Spermatogenesis in the MILI-null mice is blocked completely at the early prophase of the first meiosis, from the zygotene to early pachytene, and the mice are sterile. However, primordial germ cell development and female germ cell production are not disturbed. Furthermore, MILI binds to MVH, an essential factor during the early spermatocyte stage. The similarities in the phenotypes of the MILI- and MVH-deficient mice and in the physical binding properties of MILI and MVH indicate a functional association of these proteins in post-transcriptional regulation. These data indicate that MILI is essential for the differentiation of spermatocytes (Kuramochi-Miyagawa, 2004).

The functions of the C. elegans homologs of piwi, prg-1 and prg-2, have been studied using RNA interference (RNAi); these genes are important for the mitotic ability of the germline nuclei and are essential for germline proliferation and maintenance. In this regard, the prgs are not only structural, but also functional homologs of piwi. It is intriguing to investigate whether the mammalian MILI and MIWI are functional homologs of Drosophila Piwi. A couple of lines of evidence suggest that the two mammalian structural homologs of Piwi, i.e., MILI and MIWI, inherit only a subset of piwi functions. One line of evidence comes from the analysis of gene targeting. In addition to its crucial roles in germline stem cells, Piwi has less-well characterized roles in early oogenesis and spermatogenesis, possibly including germline cyst mitosis, meiosis and egg chamber polarity. The expression of MIWI and MILI is restricted to germ lineages, and the gene-targeted animals show defective spermatogenesis. However, no defects of Mili^-/- Miwi^-/- mice are observed at the stage of testicular stem cells. Thus, Mili and Miwi may only represent a subset of piwi functions (Kuramochi-Miyagawa, 2004).

The other line of evidence is subcellular localization of MIWI and MILI. The Piwi protein can be localized either to the nucleoplasm in germline stem cells or in the cytoplasm co-localized with polar granules such as Vasa in early embryos. MILI and MIWI are found in the cytoplasm associated with MVH. This again reflects only a subset of the Piwi function. This function is also similar to Aubergine, another Drosophila Piwi family protein, that is recruited to the posterior pole in a vasa-dependent manner as a polar granule component. Interestingly, Aubergine remains exclusively in the cytoplasm even after pole cell formation. In addition, the levels of homology between MILI or MIWI and Aubergine (31.0% and 36.6%, respectively) are similar to those seen with Piwi (32.7% and 37.1%, respectively). Taking these data into consideration, it is conceivable that MILI and MIWI might be functionally more similar to Aubergine. Meanwhile, genome analysis has revealed the third mouse homolog of piwi and aubergine (Accession Number AY135692). An unanswered question remains: whether the third member will represent other functions of piwi and aubergine? (Kuramochi-Miyagawa, 2004).

The piwi family genes, defined by conserved PAZ and Piwi domains of unknown function, have been implicated in RNAi and related phenomena, such as post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) in several organisms. AGO1 (Argonaute) and QDE-2 are required for PTGS in Arabidopsis and Neurospora, respectively, and RDE-1 is required for RNAi in C. elegans. In Drosophila, mutations in piwi and aubergine block RNAi activation during egg maturation and perturb translational control during oogenesis. Aubergine has the ability to effect the silencing of Stellate, which is a tandemly repetitive gene. Furthermore, in Drosophila, AGO1 and AGO2 are involved in RNAi, and piwi is required for PTGS and for TGS, which is induced by multiple copies of Alcohol dehydrogenase. MILI and MIWI may be involved in similar silencing mechanisms required for spermatogenesis. The gene expression profiles between the control and the Mili^-/- testes are being compared. This could give some clues about the function on gene silencing (Kuramochi-Miyagawa, 2004).

Piwi proteins specify an animal-specific subclass of the Argonaute family that, in vertebrates, is specifically expressed in germ cells. Zebrafish Piwi (Ziwi) is expressed in both the male and the female gonad and is a component of a germline-specifying structure called nuage. Loss of Ziwi function results in a progressive loss of germ cells due to apoptosis during larval development. In animals that have reduced Ziwi function, germ cells are maintained but display abnormal levels of apoptosis in adults. In mammals, Piwi proteins associate with approximately 29-nucleotide-long, testis-specific RNA molecules called piRNAs. This study shows that zebrafish piRNAs are present in both ovary and testis. Many of these are derived from transposons, implicating a role for piRNAs in the silencing of repetitive elements in vertebrates. Furthermore, this study shows that piRNAs are Dicer independent and that their 3' end likely carries a 2'O-Methyl modification (Houwing, 2007).

Piwi proteins are essential for germline development, stem cell self-renewal, epigenetic regulation, and transposon silencing. They bind to a complex class of small noncoding RNAs called Piwi-interacting RNAs (piRNAs). Mammalian Piwi proteins such as Mili are localized in the cytoplasm of spermatogenic cells, where they are associated with a germline-specific organelle called the nuage or its derivative, the chromatoid body, as well as with polysomes. To investigate the molecular mechanisms mediated by Mili, Mili-interacting proteins were sought. This study reports that Mili specifically interacts with Tudor domain-containing protein 1 (Tdrd1), a germline protein that contains multiple domains. This RNA-independent interaction is mediated through the N-terminal domain of Mili and the N-terminal region of Tdrd1 containing the myeloid Nervy DEAF-1 (MYND) domain and the first two Tudor domains. In addition, Mili positively regulates the expression of the Tdrd1 mRNA. Furthermore, Mili and Tdrd1 mutants share similar spermatogenic defects. However, Tdrd1, unlike Mili, is not required for piRNA biogenesis. These results suggest that Mili interacts with Tdrd1 in the nuage and chromatoid body. This interaction does not contribute to piRNA biogenesis but represents a regulatory mechanism that is critical for spermatogenesis (Wang, 2009).

Other Piwi family proteins

Double-stranded (ds) RNA can induce sequence-specific inhibition of gene function in several organisms. However, both the mechanism and the physiological role of the interference process remain mysterious. In order to study the interference process, C. elegans mutants resistant to dsRNA-mediated interference (RNAi) have been selected. Two loci, rde-1 and rde-4, are defined by mutants strongly resistant to RNAi but with no obvious defects in growth or development. rde-1 is a member of the piwi/sting/argonaute/zwille/eIF2C gene family conserved from plants to vertebrates. Interestingly, several, but not all, RNAi-deficient strains exhibit mobilization of the endogenous transposons. Implications for the mechanism of RNAi and the possibility that one natural function of RNAi is transposon silencing are discussed (Tabara, 1999).

RNA interference (RNAi) is a cellular defense mechanism that uses double-stranded RNA (dsRNA) as a sequence-specific trigger to guide the degradation of homologous single-stranded RNAs. RNAi is a multistep process involving several proteins and at least one type of RNA intermediate, a population of small 21-25 nt RNAs (called siRNAs) that are initially derived from cleavage of the dsRNA trigger. Genetic screens in Caenorhabditis elegans have identified numerous mutations that cause partial or complete loss of RNAi. In this work, cleavage of injected dsRNA to produce the initial siRNA population is analyzed in animals mutant for rde-1 and rde-4, two genes that are essential for RNAi but that are not required for the organism's viability or fertility. These results suggest distinct roles for RDE-1 and RDE-4 in the interference process. Although null mutants lacking rde-1 show no phenotypic response to dsRNA, the amount of siRNAs generated from an injected dsRNA trigger was comparable to that of wild-type. By contrast, mutations in rde-4 substantially reduce the population of siRNAs derived from an injected dsRNA trigger. Injection of chemically synthesized 24- or 25-nt siRNAs can circumvent RNAi resistance in rde-4 mutants, whereas no bypass was observed in rde-1 mutants. These results support a model in which RDE-4 is involved before or during production of siRNAs, whereas RDE-1 acts after the siRNAs have been formed (Parrish, 2001).

Although translational regulation of maternal mRNA is important for proper development of the Drosophila embryo, few genes involved in this process have been identified. In this report, the role of aubergine in oskar translation is described. aubergine has been implicated in dorsoventral patterning, since eggs from aubergine mutant mothers are ventralized and seldom fertilized. Two new alleles of aubergine have been isolated in a novel genetic screen; it has been shown that aubergine is also required for posterior body patterning, since the small fraction of eggs from aubergine minus mothers that are fertilized develop into embryos that lack abdominal segmentation. Although aubergine mutations do not appear to affect the stability of either Oskar mRNA or protein, the level of Oskar protein is significantly reduced in aubergine mutants. Thus, aubergine is required to enhance Oskar translation. While aubergine-dependence is conferred upon Oskar mRNA by sequences in the Oskar 3' UTR, aubergine may influence Oskar translation through an interaction with sequences upstream of the Oskar 3' UTR (Wilson, 1996).

In Drosophila oocytes, activation of Oskar translation from a transcript localized to the posterior pole is an essential step in the organization of the pole plasm, specialized cytoplasm that contains germline and abdominal body patterning determinants. Oskar is a component of polar granules, large particles associated with the pole plasm and the germline precursor pole cells of the embryo. aubergine mutants fail to translate Oskar mRNA efficiently and are thus defective in posterior body patterning and pole cell formation. Aubergine protein is related to eukaryotic translation initiation factor 2C. Aubergine is recruited to the posterior pole in a vasa-dependent manner and is itself a polar granule component. Consistent with its presence in these structures, Aubergine is required for pole cell formation, independent of its initial role in Oskar translation. Unlike two other known polar granule components, Vasa and Oskar, Aubergine remains cytoplasmic after pole cell formation, suggesting that the roles of these proteins diverge during embryogenesis (Harris, 2001).

The sting mutation, caused by a P element inserted into polytene region 32D, was isolated by a screen for male sterile insertions in Drosophila melanogaster. This sterility is correlated with the presence of crystals in spermatocytes and spermatids that are structurally indistinguishable from those produced in males carrying a deficiency of the Y-linked crystal (cry) locus. In addition, their morphology is needle-like in Stellate (Ste) plus flies and star-shaped in Ste mutant flies, once again as observed in cry minus males. The sti mutation leads to meiotic drive of the sex chromosomes, and the strength of the phenomenon is correlated with the copy number of the repetitive Ste locus. The same correlation is also true for the penetrance of the male sterile mutation. A presumptive sti null allele results in male sterility and lethal maternal effect. The gene was cloned and shown to code for a putative protein that is 866 amino acids long. A C-terminal domain of 82 amino acids is identified that is well conserved in proteins from different organisms. The gene is expressed only in the germline of both sexes. The interaction of sting with the Ste locus can also be demonstrated at the molecular level. While an unprocessed 8-kb Ste primary transcript is expressed in wild-type males, in X/Y homozygous sti males, as in X/Y cry minus males, a 0.7-kb mRNA is produced (Schmidt, 1999).

Rabbit eIF2C (94kDa) has been shown to play important roles in the eukaryotic peptide chain initiation process. In this study, the primary structure of rabbit eIF2C is determined by cDNA cloning. A 3599-bp composite sequence, which contains a single open reading frame, translates into an 813-deduced amino acid sequence. Northern blot analysis of rabbit liver ploy(A)+ RNA yielded a single message species at approximately 4.6kb. Western blot analysis of rabbit reticulocyte lysate using polyclonal antibody against the 94kDa eIF2C detected a higher-molecular-weight polypeptide (140kDa). No 94kDa polypeptide was detected. Sequence analysis reveals that rabbit eIF2C has strong homology with a hypothetical protein in Caenorhabditis elegans (Zou, 1998).

A murine piwi gene (miwi) is essential for spermatogenesis. miwi encodes a cytoplasmic protein specifically expressed in spermatocytes and spermatids. miwi null mice display spermatogenic arrest at the beginning of the round spermatid stage, resembling the phenotype of CREM, a master regulator of spermiogenesis. Furthermore, mRNAs of ACT (activator of CREM in testis) and CREM target genes are downregulated in miwi null testes. Whereas MIWI and CREM do not regulate each other's expression, MIWI complexes with mRNAs of ACT and CREM target genes. Hence, MIWI may control spermiogenesis by regulating the stability of these mRNAs (Deng, 2002).

The miwi ORF predicts that the MIWI protein contains 862 amino acid residues, with a relative molecular mass (Mr) of 98,600 and an isoelectric point of 9.46. Except for a 100-200-amino acid stretch at the N terminus, MIWI shares significant homology over its entire length with other PIWI family proteins, such as MILI from mice, HIWI and HILI (41% identity) from humans, SEAWI from sea urchin (48% identity), PIWI (37% identity) and AUB (38% identity) from Drosophila, PRG-1 and PRG-2 from C. elegans (34% and 33% identity, respectively), and PAP from Paramecium (28% identity). Interestingly, most of these proteins are involved in the development of the germline or its equivalent. In addition, miwi shares significant homology to genes involved in RNA interference (rde-1 in C. elegans and qde-2 in Neurospora), meristem cell division (Zwille and Agonaute in Arabidopsis), and translational initiation (eIF2C1 and eIF2C2 in humans and GERp95 in rabbits). The homology is particularly high in the C-terminal PIWI domain and the 110-amino acid PAZ domain in the middle of this class of proteins, suggesting the potential importance of these regions for MIWI function (Deng, 2002).

How is MIWI involved in regulating spermiogenesis? Among the PIWI family proteins, AUB has been implicated in translational regulation in Drosophila. RDE-1 in C. elegans, QDE-2 in Neurospora, and eIF2C in mammalian systems are also involved in RNA-related processes. All PIWI family proteins share a highly conserved PIWI domain that is enriched with highly basic and highly charged residues. This region in MIWI can bind to ribohomopolymers in vitro and is essential for MIWI function. MIWI is found in ribonuclear protein complexes with mRNAs of ACT and CREM target genes. Thus, MIWI is likely an RNA binding protein that regulates its target RNAs by directly binding to them. The drastic reduction of ACE, RT7, TP1, MTEST82, and ACT mRNAs in the miwi null mutant further suggest that MIWI complexes with these mRNAs to ensure their stability. The stabilization of the ACT mRNA may be of particular importance, since ACT in turn activates the transcription of the postmeiotic genes. Interestingly, miwi null does not affect the expression of CREM proteins, suggesting that MIWI does not regulate postmeiotic genes by regulating CREM expression. The regulatory pathway proposed in this paper is likely to play a central role in spermiogenesis (Deng, 2002).

Developmentally regulated piRNA clusters implicate MILI in transposon control

Nearly half of the mammalian genome is composed of repeated sequences. In Drosophila, Piwi proteins exert control over transposons. However, mammalian Piwi proteins, MIWI and MILI, partner with Piwi-interacting RNAs (piRNAs) that are depleted of repeat sequences, which raises questions about a role for mammalian Piwi's in transposon control. A search for murine small RNAs that might program Piwi proteins for transposon suppression revealed developmentally regulated piRNA loci, some of which resemble transposon master control loci of Drosophila. Evidence of an adaptive amplification loop is found in which MILI catalyzes the formation of piRNA 5' ends. Mili mutants derepress LINE-1 (L1) and intracisternal A particle and lose DNA methylation of L1 elements, demonstrating an evolutionarily conserved role for PIWI proteins in transposon suppression (Aravin, 2007a).

Proteomic analysis of murine Piwi proteins reveals role for arginine methylation in specifying interaction with Tudor family members

In germ cells, Piwi proteins interact with a specific class of small noncoding RNAs, piwi-interacting RNAs (piRNAs). Together, these form a pathway that represses transposable elements, thus safeguarding germ cell genomes. Basic models describe the overall operation of piRNA pathways. However, the protein compositions of Piwi complexes, the critical protein-protein interactions that drive small RNA production and target recognition, and the precise molecular consequences of conserved localization to germline structures, call nuage, remains poorly understood. The three murine Piwi family proteins, MILI, MIWI, and MIWI2, were purified from mouse germ cells, and their interacting protein partners were characterized. Piwi proteins were found in complex with PRMT5/WDR77, an enzyme that dimethylates arginine residues. By immunoprecipitation with specific antibodies and by mass spectrometry, it was found that Piwi proteins are arginine methylated at conserved positions in their N termini. These modifications are essential to direct complex formation with specific members of the Tudor protein family. Recognition of methylarginine marks by Tudor proteins can drive the localization of Piwi proteins to cytoplasmic foci in an artificial setting, supporting a role for this interaction in Piwi localization to nuage, a characteristic that correlates with proper operation of the piRNA pathway and transposon silencing in multiple organisms (Vagin, 2009).

Comprehensive proteomic analysis of complexes containing mouse Piwi proteins, MIWI, MILI, and MIWI2, has revealed a new motif in mammalian transposon control formed by discrete axes of interaction between Piwi and Tudor domain-containing proteins. Previous studies have implicated Tudor domain proteins in RISC- or small RNA-related pathways. For example, a Tudor domain-containing nuclease Tudor-SN was identified as a component of Ago-RISC in Drosophila cells, with functional relationships to siRNA-mediated silencing in C. elegans. Spindle-E, the Drosophila ortholog of TDRD9, interacts with Piwi family proteins and plays critical roles in the piRNA pathway in germ cells. Other research has observed interactions between TDRD1 and MILI and TDRD6 and MIWI, though the determinants nor functional consequences of complex formation were not investigated. Copurification of a Tudor protein and Aubergine has also been reported in Drosophila (Thomson, 2008). This study reports pervasive interactions between specific Tudor domain proteins and Piwi family-binding partners in mice. These biochemical interactions are specific and are reflected at the level of developmental coexpression and patterns of colocalization to distinct nuage structures. Since patterns of specific interaction can be reproduced in ectopic settings in which no piRNA pathway operates and in which no nuage structures form, the specificity of interaction observed in germ cells are not simply a consequence of propinquity but instead must reflect intrinsic differences in affinity among different Tudor-Piwi pairs (Vagin, 2009).

Tudor proteins recognize symmetrically dimethylated arginine and lysine residues. Such modifications mediate interaction between SMN and Sm proteins, p53 and 53BP1, and between JMJD2A, 53BP1, and histone H3. This proteomic analyses indicated that all three mammalian Piwi family members complex with the PRMT5/WDR77 methylosome. During the preparation of this manuscript, arginine methylation of Aub and Ago3 by Capsuleen was also noted in flies (Kirino, 2009). Piwi family proteins, but not members of the Ago clade, contained conserved runs of RG/RA in their N termini that resemble known sites of recognition by the methylosome complex. Based on a series of analyses, it was possible to demonstrate the presence of both arginine monomethylation and symmetric dimethylation in MIWI and MILI. Detailed mapping of methylation sites indicated modification of conserved N-terminal residues. Through the use of both relatively general methylation inhibitors and through directed mutagenesis, it was possible to demonstrate that formation of specific Tudor domain-Piwi family interactions depended wholly on both methylation and the presence of specific target arginine residues. Thus, the evidence supports the notion that Tudor domain proteins read out a methylarginine code (perhaps similar to the histone code) that is written on Piwi family members by PRMT5 and perhaps also other methyltransferases (Vagin, 2009).

The functional relevance of this code is supported by genetic experiments in which disruption of the piRNA pathway impacts Tudor localization and reciprocally loss of Tudor proteins impact both the localization and function of Piwi proteins. Loss of TDRD1 has strong impacts on both the piRNA pathway and the localization of its components in gonocytes. Moreover, TDRD1 can bring MILI to discrete cytoplasmic foci in somatic cell lines. In contrast, loss of TDRD6 had minimal impacts on small RNA populations in meiotic germ cells, though this mutation has been reported to cause loss of MIWI from the chromatoid body. Reciprocally, loss of MIWI failed to impact localization of multiple Tudor proteins in the chromatoid body. The genetic interdependencies (or lack thereof) that were observed might reflect partial redundancy of Piwi proteins and the fact that Piwis are probably not the only binding partners of these Tudor family members in germ cells. In this regard, it was recently demonstrated that TDRD6 interacts with MVH (Vasileva, 2009), likely reflecting methylation of conserved arginine residues in the MVH N terminus. Indeed, MVH localizes in nuage granules in prospermatogonia as well as in the chromatoid body in round spermatids and is required for TDRD1, TDRD6, and TDRD7 localization (Vagin, 2009).

Inquiries into the functions of Tudor domain proteins such as SMN suggest that these can act as chaperones for the assembly of macromolecular proteins complexes. The large number of Tudor domain proteins involved, the complexity of their interaction patterns, and the interrelationships between the function and localization of Tudor and Piwi family members suggests a general role for arginine methylation and Tudor proteins in maintaining the integrity of small RNA pathways critical to germ cell preservation. Even ectopic expression of a single Tudor domain could disrupt the localization of chromatoid body components in round spermatids, suggesting that Tudor proteins could act as more general determinants of assembly of germline nuage. In some cases, Tudor-proteins appear critical for the localization of methylated compartment components to nuage and there appear to be reciprocal requirements for methylated proteins to bring Tudors to their native cellular locales. An example in Drosophila is Vasa, which acts upstream of Tudor in the pathway that assembles the pole plasm. The results suggest that MILI is important for localization of TDRD9 to nuage and, conversely, that TDRD1 is critical for localization of MIWI2 to these same structures (Vagin, 2009).

An emerging theme that is highlighted by the present work is that germ cells depend critically on compartmentalization of small RNA pathways. While these studies reflect this in the construction of the piRNA pathway, the observation of Argonaute-containing granules in other systems: for example, P-granules in C. elegans, and the recently demonstrated roles of small RNAs as mediators of epigenetic inheritance in Drosophila underscore the need for a tightly orchestrated and highly regulated pathway of RNP granule assembly. These data point to important roles for arginine methylation and Tudor family proteins in these processes (Vagin, 2009).

Heterochromatin-associated proteins HP1a and Piwi collaborate to maintain the association of achiasmate homologs in Drosophila oocytes

Accurate segregation of homologous chromosomes during meiosis depends on their ability to remain physically connected throughout prophase I. For homologs that achieve a crossover, sister chromatid cohesion distal to the chiasma keeps them attached until anaphase I. However, in Drosophila melanogaster wild-type oocytes, the 4th chromosomes never recombine and X chromosomes fail to cross over in 6-10% of oocytes. Proper segregation of these achiasmate homologs relies on their pericentric heterochromatin-mediated association, but the mechanism(s) underlying this attachment remains poorly understood. Using an inducible RNAi strategy combined with FISH to monitor centromere proximal association of the achiasmate FM7a/X homolog pair, this study analyzed whether specific heterochromatin-associated proteins are required for the association and proper segregation of achiasmate homologs in Drosophila oocytes. Upon knocking down HP1a, H3K9 methytransferases or the HP1a binding partner Piwi during mid-prophase, significant disruption of pericentric heterochromatin-mediated association of FM7a/X homologs was observed. Furthermore, for both HP1a and Piwi knockdown oocytes, transgenic co-expression of the corresponding wild-type protein is able to rescue RNAi-induced defects. Piwi is stably bound to numerous sites along the meiotic chromosomes, including centromere proximal regions. In addition, reduction of HP1a or Piwi during meiotic prophase induces a significant increase in FM7a/X segregation errors. The study presents a speculative model outlining how HP1a and Piwi could collaborate to keep achiasmate chromosomes associated in a homology dependent manner (Giauque, 2016)

Piwi is required during Drosophila embryogenesis to license dual-strand piRNA clusters for transposon repression in adult ovaries

Most piRNAs in the Drosophila female germline are transcribed from heterochromatic regions called dual-strand piRNA clusters. Histone 3 lysine 9 trimethylation (H3K9me3) is required for licensing piRNA production by these clusters. However, it is unclear when and how they acquire this permissive heterochromatic state. This study shows that transient Piwi depletion in Drosophila embryos results in H3K9me3 decrease at piRNA clusters in ovaries. This is accompanied by impaired biogenesis of ovarian piRNAs, accumulation of transposable element transcripts, and female sterility. Conversely, Piwi depletion at later developmental stages does not disturb piRNA cluster licensing. These results indicate that the identity of piRNA clusters is epigenetically acquired in a Piwi-dependent manner during embryonic development, which is reminiscent of the widespread genome reprogramming occurring during early mammalian zygotic development (Akkouche, 2017).

REFERENCES

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Akkouche, A., Mugat, B., Barckmann, B., Varela-Chavez, C., Li, B., Raffel, R., Pelisson, A. and Chambeyron, S. (2017). Piwi is required during Drosophila embryogenesis to license dual-strand piRNA clusters for transposon repression in adult ovaries. Mol Cell 66(3): 411-419. PubMed ID: 28457744

Aravin, A. A., Naumova, N. M., Tulin, A. A., Rozovsky, Y. M. and Gvozdev V. A. (2001). Double-stranded RNA-mediated silencing of genomic tandem repeats and transposable elements in Drosophila melanogaster germline. Curr. Biol. 11: 1017-1027. 11470406

Aravin, A. A., Sachidanandam, R., Girard, A., Fejes-Toth, K. and Hannon, G. J. (2007a). Developmentally regulated piRNA clusters implicate MILI in transposon control. Science 316(5825): 744-7. Medline abstract: 17446352

Aravin, A. A., Hannon, G. J. and Brennecke, J. (2007b). The Piwi-piRNA pathway provides an adaptive defense in the transposon arms race. Science 318: 761-764. Medline abstract: 17975059

Atikukke, G., Albosta, P., Zhang, H. and Finley, R. L. (2014). A role for Drosophila Cyclin J in oogenesis revealed by genetic interactions with the piRNA pathway. Mech Dev 133: 64-76. PubMed ID: 24946235

Batki, J., Schnabl, J., Wang, J., Handler, D., Andreev, V. I., Stieger, C. E., Novatchkova, M., Lampersberger, L., Kauneckaite, K., Xie, W., Mechtler, K., Patel, D. J. and Brennecke, J. (2019). The nascent RNA binding complex SFiNX licenses piRNA-guided heterochromatin formation. Nat Struct Mol Biol 26(8): 720-731. PubMed ID: 31384064

Bohmert, K., et al. (1998). AGO1 defines a novel locus of Arabidopsis controlling leaf development. EMBO J. 17: 170-180. PubMed Citation: 9427751

Brennecke. J., Aravin, A. A., Stark, A., Dus, M., Kellis, M., Sachidanandam, R. and Hannon, G. J. (2007). Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell 128(6): 1089-103. Medline abstract: 17346786

Brower-Toland, B., et al. (2007). Drosophila PIWI associates with chromatin and interacts directly with HP1a. Genes Dev. 21(18): 2300-11. PubMed citation

Carmell, M. A., et al. (2002). The Argonaute family: tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis. Genes Dev. 16: 2733-2742. 12414724

Cerutti, L., Mian, N., Bateman, A., 2000. Domains in gene silencing and cell differentiation proteins: the novel PAZ domain and redefinition of the Piwi domain. Trends Biochem. Sci. 25: 481-482. 11050429

Chambeyron, S., Popkova, A., Payen-Groschene, G., Brun, C., Laouini, D., Pelisson, A. and Bucheton, A. (2008). piRNA-mediated nuclear accumulation of retrotransposon transcripts in the Drosophila female germline. Proc. Natl. Acad. Sci. 105: 14964-14969. PubMed Citation: 18809914

Chen, D. and McKearin, D. (2005). Gene circuitry controlling a stem cell niche. Curr Biol. 15(2): 179-84. 15668176

Clark, J. P., Rahman, R., Yang, N., Yang, L. H. and Lau, N. C. (2017). Drosophila PAF1 modulates PIWI/piRNA silencing capacity. Curr Biol 27(17): 2718-2726.e2714. PubMed ID: 28844648

Cox, D. N., et al. (1998). A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal. Genes Dev.12(23): 3715-3727. PubMed Citation: 9851978

Cox, D. N., Chao, A. and Lin, H. (2000). piwi encodes a nucleoplasmic factor whose activity modulates the number and division rate of germline stem cells. Development 127: 503-514. 10631171

Deng, W. and Lin, H. (2002). miwi, a murine homolog of piwi, encodes a cytoplasmic protein essential for spermatogenesis. Dev. Cell 2: 819-830. 12062093

Eastwood, E. L., Jara, K. A., Bornelov, S., Munafo, M., Frantzis, V., Kneuss, E., Barbar, E. J., Czech, B. and Hannon, G. J. (2021). Dimerisation of the PICTS complex via LC8/Cut-up drives co-transcriptional transposon silencing in Drosophila. Elife 10. PubMed ID: 33538693

Fabry, M. H., Ciabrelli, F., Munafo, M., Eastwood, E. L., Kneuss, E., Falciatori, I., Falconio, F. A., Hannon, G. J. and Czech, B. (2019). piRNA-guided co-transcriptional silencing coopts nuclear export factors. Elife 8. PubMed ID: 31219034

Fagard, M., Boutet, S., Morel, J. B., Bellini, C. and Vaucheret, H. (2000) AGO1, QDE-2, and RDE-1 are related proteins required for posttranscriptional gene silencing in plants, quelling in fungi and RNA interference in animals. Proc. Natl. Acad. Sci. 97: 11650-11654. 11016954

Fagegaltier, D., Falciatori, I., Czech, B., Castel, S., Perrimon, N., Simcox, A. and Hannon, G. J. (2016). Oncogenic transformation of Drosophila somatic cells induces a functional piRNA pathway. Genes Dev 30: 1623-1635. PubMed ID: 27474441

Fire, A., et al. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806-811. PubMed Citation: 9486653

Giauque, C.C. and Bickel, S.E. (2016). Heterochromatin-associated proteins HP1a and Piwi collaborate to maintain the association of achiasmate homologs in Drosophila oocytes. Genetics [Epub ahead of print]. PubMed ID: 26984058

Grewal, S. I. and Rice, J. C. (2004). Regulation of heterochromatin by histone methylation and small RNAs, Curr. Opin. Cell Biol. 16: 230-238. 15145346

Grimaldi, M. R., Cozzolino, L., Malva, C., Graziani, F. and Gigliotti, S. (2007). nup154 genetically interacts with cup and plays a cell-type-specific function during Drosophila melanogaster egg-chamber development. Genetics 175: 1751-1759. PubMed ID: 17277377

Grimaud, C., Bantignies, F., Pal-Bhadra, M., Ghana, P., Bhadra, U. and Cavalli, G. (2006). RNAi components are required for nuclear clustering of Polycomb group response elements. Cell 124(5): 957-71. 16530043

Grimson, A., et al. (2008). Early origins and evolution of microRNAs and Piwi-interacting RNAs in animals. Nature 455(7217): 1193-7. PubMed Citation: 18830242

Grishok, A., et al. (2001). Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106: 23-34. 11461699

Gu, T. and Elgin, S. C. (2013). Maternal depletion of Piwi, a component of the RNAi system, impacts heterochromatin formation in Drosophila. PLoS Genet 9: e1003780. PubMed ID: 24068954

Gunawardane, L. S., Saito, K., Nishida, K. M., Miyoshi, K., Kawamura, Y., Nagami, T., Siomi, H. and Siomi, M. C. (2007). A slicer-mediated mechanism for repeat-associated siRNA 5' end formation in Drosophila. Science. 315(5818): 1587-90. Medline abstract: 17322028

Haase, A. D., et al. (2010). Probing the initiation and effector phases of the somatic piRNA pathway in Drosophila. Genes Dev. 24(22): 2499-504. PubMed Citation: 20966049

Hamada-Kawaguchi, N., Nore, B. F., Kuwada, Y., Smith, C. I. and Yamamoto, D. (2014). Btk29A promotes Wnt4 signaling in the niche to terminate germ cell proliferation in Drosophila. Science 343: 294-297. PubMed ID: 24436419

Handler, D., Olivieri, D., Novatchkova, M., Gruber, F. S., Meixner, K., Mechtler, K., Stark, A., Sachidanandam, R. and Brennecke, J. (2011). A systematic analysis of Drosophila TUDOR domain-containing proteins identifies Vreteno and the Tdrd12 family as essential primary piRNA pathway factors. EMBO J 30: 3977-3993. PubMed ID: 21863019

Harris, A. N. and Macdonald, P. M. (2001). Aubergine encodes a Drosophila polar granule component required for pole cell formation and related to eIF2C. Development 128(14): 2823-32. 11526087

Hayashi, R., Handler, D., Ish-Horowicz, D. and Brennecke, J. (2014). The exon junction complex is required for definition and excision of neighboring introns in Drosophila. Genes Dev 28(16):1772-85. PubMed ID: 25081352

Houwing, S., et al. (2007). A role for Piwi and piRNAs in germ cell maintenance and transposon silencing in Zebrafish. Cell 129(1): 69-82. PubMed citation: 17418787

Huang, X. A., Yin, H., Sweeney, S., Raha, D., Snyder, M. and Lin, H. (2013). A major epigenetic programming mechanism guided by piRNAs. Dev Cell 24(5): 502-516. PubMed ID: 23434410

Iki, T., Yoshikawa, M., Nishikiori, M., Jaudal, M. C., Matsumoto-Yokoyama, E., Mitsuhara, I., Meshi, T. and Ishikawa, M. (2010). In vitro assembly of plant RNA-induced silencing complexes facilitated by molecular chaperone HSP90. Mol Cell 39: 282-291. PubMed ID: 20605502

Ilyin, A. A., Ryazansky, S. S., Doronin, S. A., Olenkina, O. M., Mikhaleva, E. A., Yakushev, E. Y., Abramov, Y. A., Belyakin, S. N., Ivankin, A. V., Pindyurin, A. V., Gvozdev, V. A., Klenov, M. S. and Shevelyov, Y. Y. (2017). Piwi interacts with chromatin at nuclear pores and promiscuously binds nuclear transcripts in Drosophila ovarian somatic cells. Nucleic Acids Res [Epub ahead of print]. PubMed ID: 28472469

Iwasaki, S., Kobayashi, M., Yoda, M., Sakaguchi, Y., Katsuma, S., Suzuki, T. and Tomari, Y. (2010). Hsc70/Hsp90 chaperone machinery mediates ATP-dependent RISC loading of small RNA duplexes. Mol Cell 39: 292-299. PubMed ID: 20605501

Iwasaki, Y. W., Sriswasdi, S., Kinugasa, Y., Adachi, J., Horikoshi, Y., Shibuya, A., Iwasaki, W., Tashiro, S., Tomonaga, T. and Siomi, H. (2021). Piwi-piRNA complexes induce stepwise changes in nuclear architecture at target loci. EMBO J: e108345. PubMed ID: 34337769

Janic, A., Mendizabal, L., Llamazares, S., Rossell, D. and Gonzalez, C. (2010). Ectopic expression of germline genes drives malignant brain tumor growth in Drosophila. Science 330: 1824-1827. Pubmed: 21205669

Jia, S., Noma, K. and Grewal, S. I. (2004). RNAi-independent heterochromatin nucleation by the stress-activated ATF/CREB family proteins. Science 304: 1971-1976. 15218150

Jones, B. C., Wood, J. G., Chang, C., Tam, A. D., Franklin, M. J., Siegel, E. R. and Helfand, S. L. (2016). A somatic piRNA pathway in the Drosophila fat body ensures metabolic homeostasis and normal lifespan. Nat Commun 7: 13856. PubMed ID: 28000665

Josse, T., et al. (2007). Telomeric trans-silencing: an epigenetic repression combining RNA silencing and heterochromatin formation. PLoS Genet. 3(9): 1633-43. 17941712

Karam, J.A., Parikh, R.Y., Nayak, D., Rosenkranz, D. and Gangaraju, V.K. (2017). Co-chaperone Hsp70/Hsp90 organizing protein (Hop) is required for transposon silencing and piRNA biogenesis. J Biol Chem [Epub ahead of print]. PubMed ID: 28193840

King. F. J., et al. (2001). Yb modulates the divisions of both germline and somatic stem cells through piwi- and hh-mediated mechanisms in the Drosophila ovary. Molec. Cell 7: 497-508. PubMed Citation: 11463375

Kirino, Y., et al. (2009). Arginine methylation of Piwi proteins catalysed by dPRMT5 is required for Ago3 and Aub stability. Nat. Cell Biol. 11: 652-658. PubMed Citation: 19377467

Klein, J. D., Qu, C., Yang, X., Fan, Y., Tang, C. and Peng, J. C. (2016). c-Fos repression by Piwi regulates Drosophila ovarian germline formation and tissue morphogenesis. PLoS Genet 12: e1006281. PubMed ID: 27622269

Ku, H. Y., Gangaraju, V. K., Qi, H., Liu, N. and Lin, H. (2016). Tudor-SN interacts with Piwi antagonistically in regulating spermatogenesis but synergistically in silencing transposons in Drosophila. PLoS Genet 12: e1005813. PubMed ID: 26808625

Kuramochi-Miyagawa, S., et al. (2001). Two mouse piwi-related genes: miwi and mili. Mech. Dev. 108: 121-133. 11578866

Kuramochi-Miyagawa, S., et al. (2004). Mili, a mammalian member of piwi family gene, is essential for spermatogenesis. Development 131: 839-849. 14736746

Li, C., et al. (2009). Collapse of germline piRNAs in the absence of Argonaute3 reveals somatic piRNAs in flies. Cell 137: 509-521. PubMed Citation: 19395009

Lim, R. S., Anand, A., Nishimiya-Fujisawa, C., Kobayashi, S. and Kai, T. (2014). Analysis of Hydra PIWI proteins and piRNAs uncover early evolutionary origins of the piRNA pathway. Dev Biol 386: 237-251. PubMed ID: 24355748

Lin, H. and Spradling, A. C. (1997). A novel group of pumilio mutationsaffects the asymmetric division of germline stem cells in the Drosophila ovary. Development 124: 2463-2476. PubMed Citation: 9199372

Lin, H., Chen, M., Kundaje, A., Valouev, A., Yin, H., Liu, N., Neuenkirchen, N., Zhong, M. and Snyder, M. (2015). Reassessment of Piwi binding to the genome and Piwi impact on RNA Polymerase II distribution. Dev Cell 32: 772-774. PubMed ID: 25805139

Liu, L., Qi, H., Wang, J. and Lin, H. (2011). PAPI, a novel TUDOR-domain protein, complexes with AGO3, ME31B and TRAL in the nuage to silence transposition. Development 138: 1863-1873. PubMed ID: 21447556

Lynn, K., et al. (1999). The PINHEAD/ZWILLE gene acts pleiotropically in Arabidopsis development and has overlapping functions with the ARGONAUTE1 gene. Development 126(3): 469-81. 9876176

Malone, C. D., et al. (2009). Specialized piRNA pathways act in germline and somatic tissues of the Drosophila ovary. Cell 137: 522-535. PubMed Citation: 19395010

Malone, C. D., Mestdagh, C., Akhtar, J., Kreim, N., Deinhard, P., Sachidanandam, R., Treisman, J. and Roignant, J. Y. (2014). The exon junction complex controls transposable element activity by ensuring faithful splicing of the piwi transcript. Genes Dev 28: 1786-1799. PubMed ID: 25104425

Mani, S. R., Megosh, H. and Lin, H. (2013), PIWI proteins are essential for early Drosophila embryogenesis. Dev Biol. 385(2): 340-9. PubMed ID: 24184635

Marinov, G. K., Wang, J., Handler, D., Wold, B. J., Weng, Z., Hannon, G. J., Aravin, A. A., Zamore, P. D., Brennecke, J. and Toth, K. F. (2015). Pitfalls of mapping high-throughput sequencing data to repetitive sequences: Piwi's genomic targets still not identified. Dev Cell 32(6): 765-771. PubMed ID: 25805138

Megosh, H. B., Cox, D. N., Campbell, C. and Lin, H. (2006). The role of PIWI and the miRNA machinery in Drosophila germline determination. Curr. Biol. 16(19): 1884-94. Medline abstract: 16949822

Minakhina, S., Changela, N. and Steward, R. (2014). Zfrp8/PDCD2 is required in ovarian stem cells and interacts with the piRNA pathway machinery. Development 141: 259-268. PubMed ID: 24381196

Miyoshi, T., Takeuchi, A., Siomi, H. and Siomi, M. C. (2010). A direct role for Hsp90 in pre-RISC formation in Drosophila. Nat Struct Mol Biol 17: 1024-1026. PubMed ID: 20639883

Mochizuki, K., et al. (2002). Analysis of a piwi-related gene implicates small RNAs in genome rearrangement in Tetrahymena. Cell 110: 689-699. 12297043

Murano, K., Iwasaki, Y. W., Ishizu, H., Mashiko, A., Shibuya, A., Kondo, S., Adachi, S., Suzuki, S., Saito, K., Natsume, T., Siomi, M. C. and Siomi, H. (2019). Nuclear RNA export factor variant initiates piRNA-guided co-transcriptional silencing. EMBO J: 38(17):e10287 PubMed ID: 31368590

Moussian, B., et al. (1998). Role of the ZWILLE gene in the regulation of central shoot meristem cell fate during Arabidopsis embryogenesis. EMBO J. 17: 1799-1809. PubMed Citation: 9501101

Murano, K., Iwasaki, Y. W., Ishizu, H., Mashiko, A., Shibuya, A., Kondo, S., Adachi, S., Suzuki, S., Saito, K., Natsume, T., Siomi, M. C. and Siomi, H. (2019). Nuclear RNA export factor variant initiates piRNA-guided co-transcriptional silencing. EMBO J: e102870. PubMed ID: 31368590

Noma, K., et al. (2004). RITS acts in cis to promote RNA interference-mediated transcriptional and post-transcriptional silencing, Nat. Genet. 36: 1174-1180. 15475954

Olivieri, D., Senti, K. A., Subramanian, S., Sachidanandam, R. and Brennecke, J. (2012). The cochaperone shutdown defines a group of biogenesis factors essential for all piRNA populations in Drosophila. Mol Cell 47: 954-969. PubMed ID: 22902557

Pal-Bhadra, M., Bhadra, U. and Birchler, J. A. (1997). Cosuppression in Drosophila: gene silencing of Alcohol dehydrogenase by white-Adh transgenes is Polycomb dependent, Cell 90: 479-490. 9267028

Pal-Bhadra, M., Bhadra, U. and Birchler, J. A. (1999). Cosuppression of nonhomologous transgenes in Drosophila involves mutually related endogenous sequences, Cell 99: 35-46. 10520992

Pal-Bhadra, M., Bhadra, U. and Birchler, J. A. (2002). RNAi related mechanisms affect both transcriptional and posttranscriptional transgene silencing in Drosophila. Molec. Cell 9: 315-327. 11864605

Pal-Bhadra, M., et al. (2004). Heterochromatic silencing and HP1 localization in Drosophila are dependent on the RNAi machinery, Science 303: 669-672. 14752161

Parikh, R. Y., Lin, H. and Gangaraju, V. K. (2018). A critical role for nucleoporin 358 (Nup358) in transposon silencing and piRNA biogenesis in Drosophila. J Biol Chem. Pubmed ID: 29735528

Parrish, S. and Fire, A. (2001). Distinct roles for RDE-1 and RDE-4 during RNA interference in Caenorhabditis elegans. RNA 7(10): 1397-402. 11680844

Parker, J. S., Roe, S. M., and Barford, D. (2004). Crystal structure of a PIWI protein suggests mechanisms for siRNA recognition and slicer activity. EMBO J. 23(24): 4727-37. 15565169

Reddien, P. W., et al. (2006). SMEDWI-2 is a PIWI-like protein that regulates planarian stem cells. Science 310(5752): 1327-30. 16311336

Reiss, D., Josse, T., Anxolabehere, D. and Ronsseray, S. (2004). Aubergine mutations in Drosophila melanogaster impair P cytotype determination by telomeric P elements inserted in heterochromatin. Mol. Genet. Genomics 272: 336-343. PubMed citation: 15372228

Peng, J.C., Valouev, A., Liu, N. and Lin, H. (2016). Piwi maintains germline stem cells and oogenesis in Drosophila through negative regulation of Polycomb group proteins. Nat Genet 48(3):283-91. PubMed ID: 26780607

Roche, S. E. and Rio, D. C. (1998). Trans-silencing by P elements inserted in subtelomeric heterochromatin involves the Drosophila Polycomb group gene, Enhancer of zeste. Genetics 149: 1839-1855. PubMed citation: 9691041

Ronsseray, S., Lehmann, M., Nouaud, D. and Anxolabehere, D. (1996). The regulatory properties of autonomous subtelomeric P elements are sensitive to a Suppressor of variegation in Drosophila melanogaster. Genetics 143: 1663-1674. PubMed citation: 8844154

Ronsseray, S., Boivin, A. and Anxolabehere, D. (2001). P-Element repression in Drosophila melanogaster by variegating clusters of P-lacZ-white transgenes. Genetics 159: 1631-1642. PubMed citation: 11779802

Rossi, L., et al. (2006). DjPiwi-1, a member of the PAZ-Piwi gene family, defines a subpopulation of planarian stem cells. Dev. Genes Evol. 216(6): 335-46. 16532341

Saito, K., et al. (2006). Specific association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome. Genes Dev. 20: 2214-2222. Medline abstract: 16882972

Saito, K., Sakaguchi, Y., Suzuki, T., Suzuki, T., Siomi, H. and Siomi, M. C. (2007). Pimet, the Drosophila homolog of HEN1, mediates 2'-O-methylation of Piwi- interacting RNAs at their 3' ends. Genes Dev. 21(13): 1603-8. Medline abstract: 17606638

Saito, K., et al. (2009). A regulatory circuit for piwi by the large Maf gene traffic jam in Drosophila. Nature 461(7268): 1296-9. PubMed Citation: 19812547

Saito, K., et al. (2010). Roles for the Yb body components Armitage and Yb in primary piRNA biogenesis in Drosophila. Genes Dev. 24(22): 2493-8. PubMed Citation: 20966047

Sarot, E., et al. (2004). Evidence for a piwi-dependent RNA silencing of the gypsy endogenous retrovirus by the Drosophila melanogaster flamenco gene. Genetics 166: 1313-1321. 15082550

Schmidt, A., et al. (1999). Genetic and molecular characterization of sting, a gene involved in crystal formation and meiotic drive in the male germ line of Drosophila melanogaster. Genetics 151(2): 749-60. 9927466

Schnabl, J., Wang, J., Hohmann, U., Gehre, M., Batki, J., Andreev, V. I., Purkhauser, K., Fasching, N., Duchek, P., Novatchkova, M., Mechtler, K., Plaschka, C., Patel, D. J. and Brennecke, J. (2021). Molecular principles of Piwi-mediated cotranscriptional silencing through the dimeric SFiNX complex. Genes Dev 35(5-6): 392-409. PubMed ID: 33574069

Senti, K. A., Jurczak, D., Sachidanandam, R. and Brennecke, J. (2015). piRNA-guided slicing of transposon transcripts enforces their transcriptional silencing via specifying the nuclear piRNA repertoire. Genes Dev 29: 1747-1762. PubMed ID: 26302790

Sienski, G., Batki, J., Senti, K. A., Donertas, D., Tirian, L., Meixner, K. and Brennecke, J. (2015). Silencio/CG9754 connects the Piwi-piRNA complex to the cellular heterochromatin machinery. Genes Dev 29: 2258-2271. PubMed ID: 26494711

Siomi, M. C., Mannen, T. and Siomi H. (2010). How does the royal family of Tudor rule the PIWI-interacting RNA pathway? Genes Dev. 24: 636-646. PubMed Citation: 20360382

Smulders-Srinivasan, T. K. and Lin, H. (2003). Screens for piwi suppressors in Drosophila identify dosage-dependent regulators of germline stem cell division. Genetics 165: 1971-1991. 14704180

Szakmary, A., Cox, D. N., Wang, Z. and Lin, H. (2005). Regulatory relationship among piwi, pumilio, and bag-of-marbles in Drosophila germline stem cell self-renewal and differentiation. Curr. Biol. 15(2): 171-8. 15668175

Tabara, H., et al. (1999). The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99(2): 123-32. 10535731

Tatsuke, T., Zhu, L., Li, Z., Mitsunobu, H., Yoshimura, K., Mon, H., Lee, J. M. and Kusakabe, T. (2014). Roles of Piwi proteins in transcriptional regulation mediated by HP1s in cultured silkworm cells. PLoS One 9: e92313. PubMed ID: 24637637

Thomson, T., Liu, N., Arkov, A., Lehmann, R. and Lasko, P. (2008). Isolation of new polar granule components in Drosophila reveals P body and ER associated proteins. Mech. Dev. 125(9-10): 865-73. PubMed Citation:

Tijsterman, M., et al. (2002). PPW-1, a PAZ/PIWI Protein required for efficient germline RNAi, is defective in a natural isolate of C. elegans Curr. Biol. 12: 1535-1540. 12225671

Vagin, V.V., Sigova, A., Li, C., Seitz, H., Gvozdev, V. and Zamore, P. D. (2006). A distinct small RNA pathway silences selfish genetic elements in the germline. Science 313(5785): 320-4. 16809489

Vagin, V. V., et al. (2009). Proteomic analysis of murine Piwi proteins reveals role for arginine methylation in specifying interaction with Tudor family members. Genes Dev. 23(15): 1749-62. PubMed Citation: 19584108

Vasileva, A., et al. (2009). TDR: d6 is required for spermiogenesis, chromatoid body architecture, and regulation of miRNA expression. Curr. Biol. 19: 630-639. PubMed Citation: 19345099

Verdel, A. et al. (2004). RNAi-mediated targeting of heterochromatin by the RITS complex, Science 303: 672-676. 14704433

Wang, G. and Reinke, V. (2008). A C. elegans Piwi, PRG-1, regulates 21U-RNAs during spermatogenesis. Curr. Biol. 18(12): 861-7. PubMed Citation: 18501605

Wang, H., Ma, Z., Niu, K., Xiao, Y., Wu, X., Pan, C., Zhao, Y., Wang, K., Zhang, Y. and Liu, N. (2015). Antagonistic roles between Nibbler and Hen1 modulate piRNA 3' ends in Drosophila. Development 143(3):530-9. PubMed ID: 26718004

Wang, J., Saxe, J. P., Tanaka, T., Chuma, S. and Lin, H. (2009). Mili interacts with tudor domain-containing protein 1 in regulating spermatogenesis. Curr. Biol. 19(8): 640-4. PubMed Citation: 19345100

Wilson, J. E., Connell, J. E. and Macdonald, P. M. (1996). aubergine enhances oskar translation in the Drosophila ovary. Development 122(5): 1631-9. 8625849

Yashiro, R., Murota, Y., Nishida, K. M., Yamashiro, H., Fujii, K., Ogai, A., Yamanaka, S., Negishi, L., Siomi, H. and Siomi, M. C. (2018). Piwi nuclear localization and its regulatory mechanism in Drosophila ovarian somatic cells. Cell Rep 23(12): 3647-3657. PubMed ID: 29925005

Yu, Y., Gu, J., Jin, Y., Luo, Y., Preall, J. B., Ma, J., Czech, B. and Hannon, G. J. (2015). Panoramix enforces piRNA-dependent cotranscriptional silencing. Science 350: 339-342. PubMed ID: 26472911

Yu, Y., Gu, J., Jin, Y., Luo, Y., Preall, J. B., Ma, J., Czech, B. and Hannon, G. J. (2015). Panoramix enforces piRNA-dependent cotranscriptional silencing. Science 350: 339-342. PubMed ID: 26472911

Zamparini, A. L., Davis, M. Y., Malone, C. D., Vieira, E., Zavadil, J., Sachidanandam, R., Hannon, G. J. and Lehmann, R. (2011). Vreteno, a gonad-specific protein, is essential for germline development and primary piRNA biogenesis in Drosophila. Development 138: 4039-4050. PubMed ID: 21831924

Zhang, Y Liu, W., Li, R., Gu, J., Wu, P., Peng, C., Ma, J., Wu, L., Yu, Y. and Huang, Y. (2018). Structural insights into the sequence-specific recognition of Piwi by Drosophila Papi. Proc Natl Acad Sci U S A. PubMed ID: 29531043

Zou, C., et al. (1998). Molecular cloning and characterization of a rabbit eIF2C protein. Gene 211(2): 187-94. 9602122

date revised: 21 June 2024

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