InteractiveFly: GeneBrief

Argonaute 2: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

In mammalian cells, both microRNAs (miRNAs) and small interfering RNAs (siRNAs) are thought to be loaded into the same RNA-induced silencing complex (RISC), where they guide mRNA degradation or translation silencing, depending on the complementarity of the target. In Drosophila, Argonaute2 (AGO2) was identified as part of the RISC complex. AGO2 is an essential component for siRNA-directed RNA interference (RNAi) response and is required for the unwinding of siRNA duplex and in consequence, assembly of siRNA into RISC in Drosophila embryos. However, Drosophila embryos lacking AGO2, that are siRNA-directed RNAi-defective, are still capable of miRNA-directed target RNA cleavage. In contrast, Argonaute1 (AGO1), another Argonaute protein in fly, which is dispensable for siRNA-directed target RNA cleavage, is required for mature miRNA production that impacts on miRNA-directed RNA cleavage. The association of AGO1 with Dicer-1 and pre-miRNA also suggests that AGO1 is involved in miRNA biogenesis. These findings show that distinct Argonaute proteins act at different steps of the small RNA silencing mechanism and suggest that there are inherent differences between siRNA-initiated RISCs and miRNA-initiated RISCs in Drosophila (Okamura, 2004).

The Drosophila RISC activity has been purified to homogeneity from Drosophila Schnieder 2 cell extracts. Argonaute 2 is the sole protein component present in the purified, functional RISC. By using a bioinformatics method that combines sequence-profile analysis with predicted protein secondary structure, homology was found between the PIWI domain of Ago-2; endonuclease V and potential active-site amino acid residues within the PIWI domain of Ago-2 were identified (Rand, 2004).

Double-stranded (ds) RNA induces the sequence-specific posttranscriptional gene silencing of cognate genes in numerous organisms. The multidomain ribonuclease III enzyme Dicer excises long dsRNA into duplexes of 21-23 nucleotides (nt), termed short interfering RNAs (siRNAs), which direct the cleavage of complementary mRNA targets, a process known as RNA interference (RNAi). Prior to target mRNA recognition, an siRNA duplex goes through an ATP-dependent unwinding process and one strand over the other is often preferentially loaded onto the RNA-induced silencing complex (RISC), the multiple-turnover enzyme complex that mediates endonucleolytic cleavage in the RNAi pathway. The RISC is guided to cleave target mRNAs sharing perfect complementarity across the center of the complementary siRNA strand in the absence of high-energy cofactors. siRNAs are not the only products of Dicer. Natural dsRNA-encoding genes, named microRNA (miRNA) genes, encode RNA products of ~70 nt that are predicted to form imperfect hairpin structures and are processed by Dicer to mature 21-23-nt miRNAs. Only one Dicer enzyme is found in C. elegans and humans, therefore indicating that the same Dicer is required for both RNAi and for the processing of miRNA precursors in these organisms. The expression of miRNAs is often developmentally regulated, suggesting an important role for miRNAs in the regulation of endogenous gene expression. Target mRNAs containing sequences imperfectly complementary to the miRNA can be subject to translational repression without altering mRNA stability (Okamura, 2004 and references therein).

Recent findings point to a tight connection between miRNA and RNAi molecular machineries. Both miRNAs and siRNAs have been shown to be capable of target mRNA degradation or translation silencing in mammalian cells and plants. These findings imply that, regardless of the maturation process, once the small RNA is loaded, the RISC uses it to degrade or inhibit translation depending on the degree of complementarity between the small RNA and its mRNA target (Okamura, 2004 and references therein).

In C. elegans, genetic analyses suggest that RDE-1, a member of the Argonaute family of proteins, is required for the initiation of RNAi with injected dsRNAs, whereas Alg-1 and Alg-2, other Argonaute family members, are required for the accumulation of stable mature miRNAs in C. elegans, but not for RNAi driven by dsRNA. These results suggest that distinct members of the Argonaute family of proteins in C. elegans may provide specificity to their respective pathways, RNAi and translation inhibition. However, the underlying molecular mechanisms are not well understood (Okamura, 2004).

In Drosophila, it has been shown that Argonaute2 (AGO2) protein, a member of the Argonaute family of proteins, is essential for RNAi driven by exogenously introduced dsRNA and is the first protein component to be identified as part of the RISC complex in cultured Drosophila S2 cells (Hammond, 2001). Although AGO2 is known to associate with several proteins such as the Drosophila homolog (dFMR1) of the human fragile X mental retardation protein and Vasa intronic gene (Vig) (Caudy; 2002; Ishizuka; 2002), the precise role that AGO2 plays in RNAi is not well understood. In this paper, AGO2 deletion mutant flies have been produced, and it has been found that embryos lacking AGO2 are siRNA-directed RNAi defective but are still capable of miRNA-directed target RNA cleavage. AGO2 mutant embryos are impaired in the assembly of siRNA into RISC. In contrast, Argonaute1 (AGO1), another Argonaute protein in fly, is dispensable for siRNA-directed RNA cleavage but is necessary for the accumulation of stable mature miRNAs, and thus impacts on miRNA-directed target RNA cleavage. These findings suggest that distinct Argonaute proteins act at different steps of the small RNA silencing mechanism, and provide specificity to their respective pathways in Drosophila (Okamura, 2004).

In mammals, there appear to be no real distinctions between the siRNA and miRNA pathways downstream of Dicer. In contrast, genetic studies in C. elegans have shown that distinct Argonaute homologs appear to be dedicated to distinct RNAi and translation repression pathways. Using target RNA cleavage assays, this study shows that siRNA-directed RNA cleavage depends on AGO2 but does not require AGO1, whereas miRNA-directed RNA cleavage depends on AGO1 but does not need AGO2 in Drosophila embryos. These results suggest that maturation and the function of siRNAs and miRNAs have differential requirements for Argonaute proteins in Drosophila. These findings also suggest that Argonaute proteins regulate entry points of small RNAs to RISC but may not act as determinants for target RNA cleavage or translation repression. AGO2 is an essential component of RNAi and part of the RISC complex (Hammond, 2001). The particular function of AGO2 lies downstream of siRNA duplex production in the RNAi pathway. It has been shown that duplex siRNAs are incorporated in RISC precursors and ATPdependent unwinding of siRNAs converts RISC precursors into active RISC that then degrades the specific target mRNAs. Thus, AGO2 is thought to function at some or all of these steps in RNAi. Both the unwinding of siRNA duplex and the assembly of siRNA into functional RISC are impaired in AGO2 mutant embryos. Therefore, these results indicate that AGO2 functions at a step(s) in RISC assembly after binding of the siRNA duplex to RISC precursors. Recently, Liu (2003) demonstrated that Dicer-2 not only associates with siRNA production, but also, together with R2D2, facilitates siRNA loading onto RISC in Drosophila. These data suggest that AGO2, together with Dicer-2 and R2D2, assembles siRNA into functional RISC in Drosophila embryos (Okamura, 2004).

AGO2⁴¹⁴ flies are developmentally normal, which provides circumstantial evidence that AGO2 is not important for development and by inference, not essential for utilization and function of miRNA that often regulates the developmental pathways. In contrast, AGO1 is required for the stable production of mature miRNAs; this might explain the fact that AGO1 is essential for normal development, particularly in the nervous system. The physical association of AGO1 with Dicer-1 and premiRNA suggests that AGO1 is involved in miRNA biogenesis. In fact, recent genetic studies of Dicers in Drosophila have shown that mutations in Dicer-1 block processing of miRNA precursors (Lee, 2004). Together, these results suggest that in Drosophila, distinct pathways exist for siRNA and miRNA production and their concomitant assembly into RISC complexes. Do AGO1 and AGO2 functional specificities reflect physically distinct miRNA-associated RISC and siRNA-associated RISC, or differential processing and/or loading of small RNAs into generic RISC? Although there is no definitive evidence that there is a generic RISC for both small RNAs, recent studies have shown that the RISC containing active miRNAs and the RISC involved in siRNAdirected RNAi are very similar, if not identical, since endogenous miRNAs can cleave mRNAs with perfect complementarity, and exogenously introduced siRNAs can translationally repress mRNAs bearing imperfectly complementary binding sites. The fbindings show that the miRNA-associated RISC that cleaves RNA does not need AGO2, whereas siRNA-associated RISC does. This argues that there are inherent differences between siRNA-initiated RISC and miRNA-initiated RISC in Drosophila. Physically, they might be the same generic complex (for instance, both containing AGO2) but one does not need AGO2 activity. It is also possible that they might be physically distinct with respect to Argonaute proteins (Okamura, 2004).

AGO1 is not present in AGO2-associated complexes and can be biochemically separated from siRNA-loaded RISCs (Caudy, 2002). Therefore, AGO1 and AGO2, conceivably, are not incorporated into the same RISC. However, miRNAs and siRNAs are found, to some extent, in both AGO1 and AGO2 complexes, suggesting that association of Argonaute proteins with small RNAs is preferential rather than absolutely specific. Alternatively, a fraction of small RNAs might be exchangeable between the two complexes, probably during recycling of their silencing function. A recent study has shown that miRNA-associated RISCs and siRNA-associated RISC are different with respect to Dicers in Drosophila (Lee 2004). The current results clearly suggest that in conjunction with Dicers, Argonaute proteins regulate the formation of siRNA-associated RISC or miRNA-associated RISC, since their particular functions lie downstream of Dicers in a step(s) in the assembly of small RNAs into RISC (Okamura, 2004).

RNAi-independent role for Argonaute2 in CTCF/CP190 chromatin insulator function

A major role of the RNAi pathway in Schizosaccharomyces pombe is to nucleate heterochromatin, but it remains unclear whether this mechanism is conserved. To address this question in Drosophila, genome-wide localization of Argonaute2 (AGO2) by chromatin immunoprecipitation (ChIP)-seq was performed in two different embryonic cell lines; AGO2 was found to localize to euchromatin but not heterochromatin. This localization pattern is further supported by immunofluorescence staining of polytene chromosomes and cell lines, and these studies also indicate that a substantial fraction of AGO2 resides in the nucleus. Intriguingly, AGO2 colocalizes extensively with CTCF/CP190 chromatin insulators but not with genomic regions corresponding to endogenous siRNA production. Moreover, AGO2, but not its catalytic activity or Dicer-2, is required for CTCF/CP190-dependent Fab-8 insulator function. AGO2 interacts physically with CTCF and CP190, and depletion of either CTCF or CP190 results in genome-wide loss of AGO2 chromatin association. Finally, mutation of CTCF, CP190, or AGO2 leads to reduction of chromosomal looping interactions, thereby altering gene expression. It is proposed that RNAi-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome (Moshkovich, 2011).

This study provides the first evidence for an Argonaute protein functioning directly on euchromatin to effect changes in gene expression. The genome-wide binding profile of AGO2 displays striking overlap with insulator proteins. Genetic analysis revealed that AGO2, independent of its catalytic activity, promotes Fab-8 insulator activity. Like known insulator proteins, AGO2 also associates with promoters and can oppose PcG function. Genome-wide AGO2 recruitment to chromatin is dependent on CTCF and CP190 binding and may be partially achieved via looping interactions among cis-regulatory regions and promoters. It is proposed that AGO2 may act to facilitate or stabilize looping that is needed to partition the genome into independent transcriptional domains (Moshkovich, 2011).

These results suggest that the main function of AGO2 on chromatin resides in euchromatin and not in heterochromatin. Immunofluorescence localization of AGO2 on polytene chromosomes and cell lines indicates exclusion from heterochromatic and HP1-enriched regions. Furthermore, the majority of chromatin-associated AGO2 resides in nonrepetitive euchromatic but not repeat-rich regions, as determined by genome-wide ChIP-seq. It is suggested that the role of AGO2 in RNAi-dependent silencing of TEs occurs primarily at the post-transcriptional level and that AGO2 harbors a second RNAi-independent activity to promote chromatin insulator function (Moshkovich, 2011).

Several observations suggest that AGO2 chromatin association is mainly, if not exclusively, independent of the RNAi pathway. First, AGO2 chromatin association does not correspond to regions of the genome that produce high levels of endo-siRNAs, which are dependent on Dcr-2 and AGO2. Second, AGO2, but not Dcr-2, is required for Fab-8 insulator function. Finally, a catalytically inactive AGO2 protein, which is defective for RNAi, retains the ability to associate with chromatin and is functional with respect to both TrxG function and Fab-8 insulator activity (Moshkovich, 2011).

An intriguing question raised by these findings is whether or not the functions of AGO2 in RNAi and chromatin insulator activity are completely distinct. CP190 mutants were found to remain competent for silencing, suggesting that AGO2 chromatin association is not required for RNAi. Nevertheless, it remains possible that chromatin-associated AGO2 is loaded with siRNA. Future work will address how AGO2 subcellular localization and seemingly disparate functions in RNAi and chromatin insulator activities are regulated (Moshkovich, 2011).

A unique positive role for AGO2 but not other RNA silencing factors was identified in Fab-8 insulator function. Importantly, a catalytically inactive mutant form of AGO2 expressed at wild-type levels retains insulator activity, further suggesting that the RNAi pathway is dispensable for Fab-8 insulator function. A significant fraction of AGO2 resides in the nucleus, and physical interaction is observed between AGO2 and CP190. This interaction is insensitive to RNaseA, suggesting that RNA does not mediate the interaction between AGO2 and CP190. It remains possible that AGO2 can interact with siRNA or other RNA while associated with the insulator complex, although there is no evidence to support this hypothesis (Moshkovich, 2011).

This study shows that chromosomal looping in the Abd-B locus is dependent on CTCF, CP190, and AGO2. Confirming and extending previous studies, it was found that the Abd-B RB promoter interacts frequently with Fab-7, Fab-8, and the iab-8 enhancer and, moreover, that the Fab-8 region also contacts Fab-7 as well as multiple Abd-B promoters. Currently, the significance of insulator protein promoter association is unclear, but insulators may be thus situated to control looping interactions between promoters and cis-regulatory elements. Depletion of CP190 or CTCF reduces these high-frequency looping interactions, and loss of this specialized chromatin configuration could result in disassociation of AGO2. Given this possibility, AGO2 may act to detect the insulator-dependent conformation of this locus (Moshkovich, 2011).

AGO2 is recruited to chromatin insulator sites as well as noninsulator sites in a CTCF/CP190-dependent manner. It is speculated that AGO2 chromatin association with insulator sites could result from physical interactions with CP190 complexes, while AGO2 recruitment to other sites may be achieved at least in part by chromatin looping mediated by CP190 and CTCF. In fact, it was recently shown that PcG proteins can be transferred from a PRE to a promoter as a result of intervening insulator-insulator interactions. Once recruited to chromatin, AGO2 could perform a primarily structural function to promote or stabilize the frequency of CTCF/CP190-dependent looping interactions (Moshkovich, 2011).

AGO2 appears to promote Fab-8 insulator activity independently of an effect on gypsy insulator body localization. Previous work showed that both the gypsy class and CTCF/CP190 insulators colocalize to insulator bodies, suggesting that these subnuclear structures may be important for both gypsy and Fab-8 activities. However, since Fab-8 activity is not affected by RNA silencing components that disrupt gypsy insulator body localization, this subnuclear structure appears to be dispensable for Fab-8 function. Recent work indicates that the BX-C harbors multiple redundant cis-regulatory elements that can maintain looping interactions of this locus, suggesting that the configuration of the BX-C may not require a nuclear scaffold such as the gypsy insulator body (Moshkovich, 2011).

AGO2 mutations suppress the Polycomb phenotype, indicating that AGO2 behaves similarly to trxG genes and opposes PcG function. A previous study proposed that RNA silencing factors promote long-range PRE-dependent chromosomal pairing as well as PcG body formation but did not examine AGO2. This study found that the AGO2^51B-null mutation has no effect on Fab-X PRE pairing-dependent silencing on sd as assayed in that study, and genetic results suggest that AGO2 is unlikely to promote PRE-dependent interactions or PcG body formation, which are both positively correlated with PcG function. Interestingly, it has recently been shown in the case of AGO2-associated Fab-7 and Mcp boundary elements that long-range interactions are dependent on insulator sequences and not PREs. Future studies will elucidate the complex interplay between PcG and insulator organization as well as the role of AGO2 in the regulation of these structures (Moshkovich, 2011).

It remains to be seen whether Drosophila AGO2 euchromatin association and function may be conserved in other organisms. In Caenorhabditis elegans, the nuclear NRDE RNAi pathway can block transcriptional elongation of Pol II on a target transcript when treated with exogenous complementary dsRNA. Interestingly, this negative transcriptional effect is contemporaneous with an increase in H3K9me3. Whether the Argonaute protein NRDE-3/WAGO-12, which lacks Slicer activity, associates with euchromatin to effect this repression is not yet known. Furthermore, the C. elegans Argonaute Csr-1, loaded with 22G endo-siRNAs antisense to mRNAs of holocentric chromosomes, may serve as chromosomal attachment points to promote efficient chromosome segregation. Recently, it has been shown that Schizosaccharomyces pombe Ago1 participates in surveillance mechanisms to prevent readthrough transcription of mRNA. However, the majority of Ago1 associates with heterochromatic regions, and it is not clear thus far whether Ago1 directly associates with euchromatin or acts post-transcriptionally. An emerging theme from studies of RNAi in various model systems is that genome integrity and control of gene expression may be achieved by multiple yet overlapping mechanisms (Moshkovich, 2011).

Two new and distinct roles for Drosophila Argonaute-2 in the nucleus: alternative pre-mRNA splicing and transcriptional repression

Transcription and pre-mRNA alternative splicing are highly regulated processes that play major roles in modulating eukaryotic gene expression. It is increasingly apparent that other pathways of RNA metabolism, including small RNA biogenesis, can regulate these processes. However, a direct link between alternative pre-mRNA splicing and small RNA pathways has remained elusive. This study shows that the small RNA pathway protein Argonaute-2 (Ago-2) regulates alternative pre-mRNA splicing patterns of specific transcripts in the Drosophila nucleus using genome-wide methods in conjunction with RNAi in cell culture and Ago-2 deletion or catalytic site mutations in Drosophila adults. Moreover, it was shown that nuclear Argonaute-2 binds to specific chromatin sites near gene promoters and negatively regulates the transcription of the Ago-2-associated target genes. These transcriptional target genes are also bound by Polycomb group (PcG) transcriptional repressor proteins and change during development, implying that Ago-2 may regulate Drosophila development. Importantly, both of these activities were independent of the catalytic activity of Ago-2, suggesting new roles for Ago-2 in the nucleus. Finally, the nuclear RNA-binding profile of Ago-2 was determined, It was found bound to several splicing target transcripts, and a G-rich RNA-binding site for Ago-2 was identified that was enriched in these transcripts. These results suggest two new nuclear roles for Ago-2: one in pre-mRNA splicing and one in transcriptional repression (Taliaferro, 2013).

Although mammalian argonaute proteins are primarily known for their cytoplasmic functions, it has become clear that they can spend at least part of their time in the nucleus and that once in the nucleus, they can perform important functions relevant to chromatin formation and transcriptional silencing. This study has now expanded that nuclear repertoire to Drosophila and shown that it includes direct transcriptional repression and regulation of alternative pre-mRNA splicing (Taliaferro, 2013).

Previous reports have linked Ago-2 to higher-order chromatin structures, insulator function through interaction with CP190 and CTCF, and transcriptional silencing of heat shock loci through small RNAs in Drosophila (Cernilogar, 2011; Moshkovich, 2011). Indeed, the studies on the effects of Ago-2 on the heat shock loci used Ago-2-associated small RNAs and suggest a role for Ago-2 in RNA polymerase II pausing or elongation. However, the transcriptional repression found in this study is independent of the catalytic activity of Ago-2 but is related to its chromatin binding. It is unlikely that Ago-2 is directly contacting the DNA to mediate this repression, since Ago-2 has no known DNA-binding domains. Formaldehyde is an efficient protein-protein cross-linker, and perhaps interaction with other proteins is required to mediate association with DNA. Although the enrichment observed in the ChIP-seq reads was modest, the binding sites are quite consistent with those previously reported (Moshkovich, 2011) and can be readily connected to repression of mRNA steady-state levels in Ago-2-null mutants but not in Ago-2 catalytic mutants (Taliaferro, 2013).

If Ago-2 is not directly binding DNA, it is likely colocalizing with other chromatin-associated DNA-binding factors, such as the PcG proteins, as was observed. In fact, previous reports have shown that members of the RNAi machinery are required for proper PcG function in Drosophila cells (Grimaud, 2006). The fact that the overwhelming majority of direct transcriptional targets of Ago-2 were repressive events seems to fit nicely with the PcG complex being a repressive chromatin complex. In fact, 80% of these direct transcriptional targets are bound by at least one Polycomb protein in addition to Ago-2. This repression may also be developmentally regulated, since the locations of Ago-2 chromatin binding change slightly between embryonically derived S2 cells and larvae (Taliaferro, 2013).

On average, the level of transcriptional depression that was observed upon loss of Ago-2 was approximately twofold. This level of depression is lower than has been observed upon loss of PcG proteins in other circumstances. However, in other cases, loss of particular PcG complex members has resulted in a similar, modest derepression of PcG target genes (Taliaferro, 2013).

Importantly, the genes that show Ago-2 chromatin association events do not correlate with the target transcripts whose pre-mRNA splicing is affected upon RNAi knockdown of Ago-2. Therefore, the splicing effects observed are likely unrelated to the Ago-2 association with chromatin in mammals, where the formation of heterochromatin mediated by siRNAs is thought to slow RNA polymerase II and affect alternate splicing events (Allo, 2009; Ameyar-Zazoua, 2012). Moreover, in mammals, the effects of Ago-2 also require Dicer, which is not the case in Drosophila, since Dicer-1 and Dicer-2 RNAi knockdown shows no effects on pre-mRNA splicing. Instead, the data point to a more direct role of Ago-2 in the splicing changes that were observed and that are strongly supported by the direct RNA binding by Ago-2 from CLIP data, suggesting that the observed changes in pre-mRNA are due to Ago-2 binding of pre-mRNA and not chromatin, since no Ago-2 ChIP-seq peaks are found on the pre-mRNA splicing target genes. Furthermore, it is unlikely that splicing changes that occur upon Ago-2 knockdown are happening indirectly through a change in expression of another splicing factor, as neither the array experiments nor mRNA-seq experiments showed significant changes in mRNA transcripts encoding splicing factors, although it should be pointed out that mRNA levels, not protein levels, were measured (Taliaferro, 2013).

Small RNAs affecting splicing patterns have been reported. In particular, snoRNAs have been implicated in changing alternative splicing outcomes. This study observed Ago-2 strongly binding to several snoRNAs in CLIP assays, indicating one potential mechanism for the action of Ago-2 on alternative splicing (Taliaferro, 2013).

Although no general enrichment for CLIP clusters was observed near splice sites regulated by Ago-2, it was not possible to definitively link the position of Ago-2 binding relative to a splice site to either positive or negative regulation of that splice site. The enrichment of the G-rich motif within CLIP clusters was relatively modest. It is speculated that this may be at least partially due to the detection of a mixture of Ago-2-binding events, many of which are driven by a vast array of different small RNAs (Taliaferro, 2013).

Ago-2 binding to long stretches of RNA across many gene transcripts was observed in CLIP-seq assays. This was qualitatively different from what has been reported for the binding patterns for other splicing factors (Huelga, 2012). Although bound genes were enriched for being more highly expressed, many highly expressed genes were not bound at all, and control CLIP experiments did not yield the same extended patterns of binding. These findings suggest that the observed extended binding of Ago-2 across many transcripts may be legitimate. Further experimental work will be needed to determine the significance of this observation as well as a unified mechanism for the action of Ago-2 on alternative splicing and its interactions with other splicing factors and spliceosome components (Taliaferro, 2013).

Argonaute2 and LaminB modulate gene expression by controlling chromatin topology

Drosophila Argonaute2 (AGO2) has been shown to regulate expression of certain loci in an RNA interference (RNAi)-independent manner, but its genome-wide function on chromatin remains unknown. This study identified the nuclear scaffolding protein LaminB as a novel interactor of AGO2. When either AGO2 or LaminB are depleted in Kc cells, similar transcription changes are observed genome-wide. In particular, changes in expression occur mainly in active or potentially active chromatin, both inside and outside LaminB-associated domains (LADs). Furthermore, this study identified a somatic target of AGO2 transcriptional repression, no hitter (nht), which is immersed in a LAD located within a repressive topologically-associated domain (TAD). Null mutation but not catalytic inactivation of AGO2 leads to ectopic expression of nht and downstream spermatogenesis genes. Depletion of either AGO2 or LaminB results in reduced looping interactions within the nht TAD as well as ectopic inter-TAD interactions, as detected by 4C-seq analysis. Overall, these findings reveal coordination of AGO2 and LaminB function to dictate genome architecture and thereby regulate gene expression (Nazer, 2018).

Argonaute proteins correspond to an evolutionarily conserved protein family engaged in gene silencing. The well-studied RNA interference (RNAi) pathway effector protein Argonaute2 (AGO2) interacts with microRNAs (miRNAs) or short interfering RNAs (siRNAs) to regulate post-transcriptional gene silencing in the cytoplasm. In addition, several reports have shown that AGO2 is not restricted to the cytoplasm and can also function in the nucleus. In Drosophila, ChIP-seq analysis of AGO2 revealed association with active promoters, enhancers, and chromatin insulator sites. At the Hox gene Abd-B, AGO2 interacts with insulator proteins and exerts a positive role in gene expression. Consistent with a role in transcription, AGO2 was found to interact with the RNA Polymerase II (Pol II) core complex as well as Negative elongation factor (NELF), a key factor involved in transcriptional pausing. Finally, transcriptional profiling of AGO2 null but not catalytic mutants suggested that AGO2 functions primarily in transcriptional repression. These results suggest that AGO2 may harbor both positive and negative roles in transcriptional regulation (Nazer, 2018).

AGO2 has also been shown to affect chromatin topology and has been implicated in chromosome pairing. At Abd-B, AGO2 is required for proper looping between the promoter and its enhancer, resulting in activation of transcription. Moreover, AGO2 promotes long-range insulator-dependent pairing interactions within the nucleus. In addition to the aforementioned transcriptional profiling, all these studies showed that the function of AGO2 in controlling chromatin topology and transcription occurs independently of the RNAi pathway (Nazer, 2018 and references therein).

Transcription is not only regulated through interactions between promoter and cis-regulatory factors, but also through chromatin accessibility and interactions with distant regulatory elements. High-throughput chromosome conformation capture techniques have revealed that chromatin contacts take place within functional and structural domains termed topologically-associated domains (TADs. These structures consist of highly self-interacting genomic regions within the domain, each separated by adjacent genomic regions called domain partition sites (DPSs). TADs can be sub-classified as active or inactive depending on their protein content and transcriptional activity. A subset of inactive TADs are associated with the nuclear lamina, and these regions are referred to as Lamin-associated domains (LADs). LADs are broad regions defined by their interaction with the LaminB protein (associated with the BLACK chromatin state, and on average, they are 90 kb in length and tend to be gene poor. Less transcriptionally active in nature, LADs are enriched in repressive chromatin marks, such as Polycomb Group (BLUE) and heterochromatin factors (GREEN). Nevertheless, some hallmarks of active chromatin (RED and YELLOW) are also present within LADs (Nazer, 2018).

This study has elucidated genome-wide mechanisms of AGO2 transcriptional control in concert with LaminB. LaminB and Poll II as AGO2-associated factors in nuclear extracts. Nascent euRNA-seq (neuRNA-seq) experiments showed that depletion of AGO2 or LaminB produce highly similar transcriptome profiles, with both factors affecting transcription of genes located in active chromatin both inside and outside LADs. First revealed by transcriptional profiling of AGO2 mutants, it was found that both AGO2 and LaminB prevent transcription of nht, a master regulator of the spermatogenesis gene program, in somatic cells. Finally, 4C-seq analysis showed that AGO2 and LaminB modulate the chromatin topology of the TAD/LAD in which nht is located, thus contributing to transcriptional silencing of this key developmental regulator (Nazer, 2018).

This study has employ proteomics, neuRNA-seq, ChIP-seq, and 4C-seq to elucidate a novel genome-wide relationship between Drosophila AGO2 and LaminB to function in genome organization and thereby affect gene regulation. By examining AGO2 null mutants, focus was placed on a single somatic target of AGO2 repression, nht, which is a key activator of spermatogenesis genes. The nht locus is immersed in a LAD located within a repressive TAD, which is flanked by AGO2 binding sites. Finally, it was found that depletion of either AGO2 or LaminB results in a significant decrease in frequency of interactions within the TAD as well as increases in inter-TAD interactions. It is concluded that AGO2 and LaminB can work in concert to regulate gene expression by orchestrating overall genome organization (Nazer, 2018).

The majority of genes for which transcription is altered in AGO2- or LaminB-knockdowns are normally actively transcribed, but specificity of this effect was observed. In particular, up-regulated genes are enriched in RED compared to YELLOW chromatin, while down-regulated genes show the opposite pattern. Although AGO2 is present in both types of active chromatin, one major difference between these two active chromatin states is the absence of histone H3K36me3, a hallmark of elongation, in RED chromatin. Therefore, depletion of AGO2 may preferentially relieve attenuation of RED versus YELLOW chromatin. Furthermore, YELLOW chromatin generally corresponds to constitutively expressed genes, whereas RED is characteristic of developmentally regulated genes. Surprisingly, it was found that a substantial number of co-up-regulated genes within RED chromatin were distributed equally between LADs and non-LADs. Perhaps these RED domains are located within a subclass of LADs that allow gene expression at a certain level, likely to more efficiently respond to stimulus or developmental signals. Further analysis would be required in order to establish sub-classification of LADs in Drosophila and analyze their potential function, as has been recently performed for the mouse genome. Overall, the presence of RED chromatin within a LAD could correspond to an additional layer of regulation of developmentally controlled gene expression (Nazer, 2018).

Another non-exclusive possibility is that topological constraints imposed by AGO2 differentially affect the two chromatin types. For example, restriction of tissue-specific enhancer-promoter interactions present in RED chromatin might be relaxed by changes in topology while housekeeping genes could become ectopically subject to repression by surrounding chromatin from which it is normally insulated. The results suggest that AGO2 can exert either positive or negative effects on transcription depending on the chromatin context (Nazer, 2018).

While transcription changes observed in AGO2 and LaminB knockdowns genome-wide are most enriched for actively transcribed regions, some normally inactive or potentially active regions including within LADs, are also up-regulated. One such gene that is subject to tight tissue-specific regulation and repressed by AGO2 and LaminB is nht, which normally activates spermatogenesis specifically in primary spermatocytes. Since mRNA-seq profiling of AGO2 mutants was performed on whole female larvae, detection of up-regulation of normally silent genes was favored. Previous work showed that certain spermatogenesis gene clusters are associated with the nuclear periphery in somatic cells and become de-repressed and repositioned toward the interior upon depletion of LaminB. However, no significant detachment of nht from the nuclear periphery was observed upon depletion of AGO2 or LaminB. These results are in agreement with recent work showing that LaminB is not required to a large extent for LAD structure in mES cells. In AGO2- or LaminB-depleted somatic cells, nht partially escapes from repression, although it is not fully activated likely due to its presence in a silent BLACK domain and the absence of other required factors. It is important to note that despite being able to detect higher levels of a variety of spermatogenesis transcripts in AGO2- and LaminB-depleted somatic cells, increased protein levels were not observed. Strict regulation of spermatogenesis gene expression also occurs on the post-transcriptional level and helps ensure no phenotypic consequences when transcription becomes dysregulated (Nazer, 2018).

AGO2 and LaminB control the overall chromatin topology of the LAD and TAD in which nht is located, and these changes could be a key driver of observed transcriptional effects in AGO2- and LaminB-depleted cells. In the case of Abd-B, which is located outside of a LAD, AGO2 functions in concert with the insulator proteins CTCF and CP190 to promote or stabilize looping between the Abd-B promoter and iab-8 enhancer region, thus stimulating transcription. However, AGO2 itself does not associate with the nht promoter, so it is unlikely that AGO2 directly prevents the nht promoter from looping to nearby enhancers. Rather, AGO2 localizes to borders of the LAD/TAD encompassing nht, suggesting that it may play a larger role in constraining topology of the entire region. In fact, 3C and 4C-seq analyses show that interactions between nht and other sites within the TAD decrease in interaction frequency in both AGO2- and LaminB-depleted cells. At the same time, increased interactions are observed between the nht promoter and sequences located in other chromatin domains beyond 1 Mb away. In this structural view, AGO2 appears to function in a manner hypothesized for insulator proteins at TAD borders; however, no single insulator protein flanks this LAD/TAD. Large alterations in topology in AGO2- and LaminB-depleted cells could explain the resultant transcriptional increase of nht by allowing interaction with inappropriate enhancers or otherwise creating a more permissive transcriptional environment. The results are in agreement with recent work performed in mouse showing that the absence of lamins decreases inter-TAD interactions within constitutive LADs and increases inter-TAD interactions between TADs inside LADs and TADs outside LADs. Importantly, this topology remodelling correlates with changes in gene expression, overall suggesting an evolutionary conserved role of lamins to regulate transcription by controlling chromatin topology (Nazer, 2018).

Modulation of Ago2 loading by Cyclophilin 40 endows a unique repertoire of functional miRNAs during sperm maturation in Drosophila

In gene silencing, Hsp90 chaperone machinery assists Argonaute (Ago) binding and unwinding of silencing small RNA (sRNA) duplexes. This enables the formation of effector RNA-induced silencing complex (RISC) that often displays cargo preferences. Hence, in Drosophila, microRNAs (miRNAs) and small-interfering RNAs (siRNAs) are differentially sorted into Ago1-RISC and Ago2-RISC, respectively. This study identified fly Cyclophilin 40 (Cyp40) as a testis-specialized Hsp90 co-chaperone essential for spermatogenesis and for modulating Ago2-RISC formation. Testis-distinctive Ago-sorting and strand-selection mechanisms accumulate a unique set of miRNAs on Ago2. Cyp40 interacts with duplex-incorporating Ago2 through Hsp90 in vitro and selectively promotes the build-up of Ago2-bound miRNAs, but not endogenous siRNAs, in vivo. Moreover, one of Cyp40-dependent Ago2-sorted miRNAs is required for late spermatogenesis, unraveling the physiological relevance of the unconventional yet conserved Drosophila miRNA-Ago2 sorting pathway. Collectively, these results identify RISC-regulatory roles for Hsp90 machinery and, more generally, highlight the tissue-specific adaptation of sRNA pathways through chaperone diversification (Iki, 2020).

Gene silencing mediated by non-coding small RNAs (sRNAs) underlies diverse biological processes, including development, homeostasis, immunity, and reproduction in eukaryotes. Lines of evidence indicate that the expression and steady-state accumulation of regulatory sRNAs themselves are controlled by multiple, intertwined mechanisms, the misregulation of which has been implicated in disease. However, understanding the regulation of sRNA expression in vivo is still incomplete (Iki, 2020).

The sRNAs cooperate with Argonaute (Ago) family proteins, forming the core effector, namely, RNA-induced silencing complex (RISC). Many sRNAs are generated through the processing of double-stranded RNAs (dsRNAs) by RNase III enzymes. Most microRNAs (miRNAs) are derived from imperfectly base-paired hairpin structures within non-coding transcripts or intronic fragments, whereas short interfering RNAs (siRNAs) originate from highly base-paired dsRNA of both endogenous and exogenous origins. Cleavages by RNase III excise ~20 to 24-nucleotide (nt) mi/siRNAs as duplex intermediates, which upon loading onto Ago proteins, become unwound during RISC formation. One strand is selectively stabilized as a 'guide strand' that recognizes the target RNA displaying sRNA-complementary sites, enabling gene silencing. The complementary so called 'passenger strand,' by contrast, is eliminated. Depending on various cell/tissue/organ-specific features, including but not restricted to sRNA alternative processing, 3'-end tailing, or even editing, a guide strand can become a passenger strand and vice versa. Hence, RISC formation represents one one of the key steps determining the steady-state accumulation of specific sRNA cohorts in vivo (Iki, 2020).

Previous studies in both animals and plants demonstrated the fundamental requirement of the Hsp70/90 chaperone machinery for RISC formation. One of the core factors, Hsp90, is of particular importance by retaining Ago in an 'apo' state, an open conformation competent for sRNA duplex incorporation. By an open-close cycle driven by ATP binding and its hydrolysis, Hsp90 chaperone homodimers transiently interact with selected client proteins as well as distinct co-chaperones at particular steps. Hsp90 then assists multiple reactions, including the binding of small ligands to client proteins. As one of Hsp90 co-chaperones, Cyclophilin 40 (CYP40)/SQUINT promotes miRNA activities in plants by facilitating the binding of duplexes (ligands) to Ago1 (client). In mammalian cells, other Hsp90 co-chaperones, namely, FK506-binding protein (Fkbp)4/Fkbp52 and the homolog Fkbp5/Fkbp51, interact with Ago2 to promote RISC formation. However, a broader range of ligand-client binding reactions can be controlled by these Hsp90 co-chaperones. Indeed, both Cyp40 and Fkbp52 were first isolated as cofactors of nuclear receptors (clients) binding to steroid hormones (ligands) in animals. These interactions were functionally characterized in various studies involving cultured mammalian cells, yeast systems, or purified proteins. Consistently, Fkbp52 knockout mice exhibit reproductive defects accompanied with attenuated steroid sensitivity. In contrast, Fkbp52 Fkbp51 double-knockout individuals die in the early embryonic stage, implying other important, yet uncharacterized, functions for both co-chaperones, possibly including a role in RISC formation. However, the biological significance of Fkbp52- and Fkbp51-regulated RISC formation has yet to be addressed in vivo, and likewise, Cyp40 knockout animals have been unavailable except in the case of a protist model. Consequently, physiological functions for animal Cyp40 remain largely elusive (Iki, 2020).

Drosophila melanogaster provides an excellent model to study the commonalities and specificities of the miRNA versus siRNA pathways. Among the five Ago protein members encoded in the fly genome, Ago1 and Ago2 incorporate RNase III Dicer 1 (Dcr1)-dependent miRNA and Dcr2-dependent siRNA duplexes. The other three Ago proteins are dedicated to the loading of Piwi-interacting RNA accumulating prominently in the gonads in a Dcr-independent manner. Both structural and nucleotide sequence features of mi/siRNA duplexes underpin their sorting into Ago1 or Ago2, as well as the subsequent guide-strand selection process. In this respect, fly Ago1 hosts a wide range of miRNAs and is essential for development from early embryogenesis. In contrast, fly Ago2 is primarily required for antiviral defense by interacting with virus-derived siRNAs. Independent of its defensive roles, Ago2 is also loaded with endogenous siRNA (endo-siRNA) in testes to support the male reproductive functions. Apart from viral and endo-siRNAs, a smaller and selective fraction of miRNAs is preferentially sorted into Ago2 rather than Ago1, although the biological roles of these rarer Ago2-bound miRNAs has remained largely unexplored (Iki, 2020).

Whether, in addition to their key role in activating the apo-Ago conformation, (co-)chaperones exhibit RISC regulatory activities influencing sRNA steady-state accumulation remains an open question, as does the potential tissue- or cell-type specificity of these putative regulatory functions. To explore these issues and, more generally, better understand the physiological roles of Hsp90 co-chaperones in animals, the loss-of-function alleles of cyp40, fkbp4, and fkbp5 orthologs were generated and characterized in D. melanogaster. Although the fkbp mutant died at the pupal stage, the cyp40 mutant was eclosed but the male exhibited infertility, a phenotype resembling that of the ago2 mutant. These detailed analyses of Ago-bound sRNAs in testes show that Cyp40 is selectively required for the accumulation of testis-unique and functional miRNAs sorted to Ago2. The significance of these results is discussed in a broader context of how regulation on specific sRNA repertoires might be enabled by tissue-specialized chaperone expression (Iki, 2020).

This study uncovered an essential function of Cyp40 as an Hsp90 co-chaperone during late spermatogenesis in Drosophila. The in vitro physical interaction analyses and in vivo Ago-bound sRNA profiling revealed a molecular activity of Cyp40 in regulating the steady-state accumulation of miRNAs uniquely forming RISC with Ago2 in testes. Although the Drosophila miRNA-sorting mechanism is now well established, the biological basis underlying the binding of a cohort of miRNAs to Ago2 had remained elusive. This study has uncovered one such mechanism and biological circumstance by showing how, under the control of germline Cyp40, Ago2 efficiently binds testis-unique miRNAs. Because the genetic ablation of one of these miRNAs suffice to noticeably impair proper spermatid differentiation, these findings also support the notion that the miRNA-Ago2 sorting pathway, which is conserved in Drosophila, is physiologically relevant (Iki, 2020).

Although miRNA hairpins are usually considered to spawn a single dominant guide strand, previous studies reported guide-strand alteration, or arm switching, in a tissue- and/or developmental-stage-specific manner. This study has identified several such miRNAs that are subjected not only to altered guide-strand selection but also to unconventional Ago sorting in a testis-unique manner. It was further demonstrated the functional relevance of these alternative events with the example of miR-316. What mechanisms might underlie the strand/Ago-switching phenomenon? The central unpaired nucleotides, terminal instabilities of sRNA duplexes, and 5' nucleotide identities largely govern, together, Ago sorting and strand selection in Drosophila. In both plants and metazoans, such duplex-intrinsic features are, however, modifiable through alternative precursor processing by RNase III enzymes. As a result, excised duplex isoforms acquire novel fates through RISC formation. In the case of miR-316, the majority of strands share an identical 5' end, reflecting a fixed, as opposed to flexible, processing mechanism. Thus, the switching strand-selection behavior of miR-316 is most likely attributable to other mechanisms. They could include RNA editing, as observed in mammals; although, testing this possibility in flies will require further work. Notably, loss of cyp40 function perturbed Ago2 binding to miR-316-3p, and yet, neither abrogated the 3'-over-5' strand preference nor affected Ago1 sorting. Hence, Cyp40 might in part influence the 5'/3'-strand use bias, but it is unlikely to regulate strand switching per se (Iki, 2020).

Besides these considerations of the strand selection of some miRNA species, Cyp40 had a profound, global effect on discriminating miRNAs from endo-siRNAs bound to Ago2. In flies, miRNA and siRNA duplexes are delivered to Ago2 by loading complexes constituted of Dcr-2 and co-partners R2D2 or Loqs-PD. By asymmetry sensing, the complexes preferentially bind near-perfectly base-paired siRNA duplexes to enable their efficient loading, whereas they simultaneously disfavor duplexes containing central instability, a feature of many miRNAs. The process is thought, among other possibilities, to avoid the saturation of Ago2 by miRNAs during virus infections, which would presumably compromise siRNA-mediated antiviral defenses. This circumstance added to the fact that many arthropod viruses encode silencing suppressor proteins probably explain why Ago2 and Dcr2, unlike Ago1 and Dcr1, are among the fastest evolving 3% of all Drosophila genes, owing to positive selection as a consequence of the never ending host-pathogen arms race. Nonetheless, these results are in line with the observation that the Drosophila argo2 gene has also evolved under the adaptive basis of testis specialization, most likely independently of antiviral roles. Indeed, as much as Ago2-bound hp-siRNAs have co-evolved with target genes and assist male fertility, the data suggest that miRNAs, such as miR-316, can also gain functional roles through unconventional Ago2 sorting in testes. However, as typical miRNAs, Cyp40-dependent duplexes displayed central bulges or mismatches, of which a likely consequence is their predicted low loading efficacy into Ago2. This might have constituted one of the driving forces for the deployment of a germline Cyp40, which, together with Hsp90, likely improves the affinity of Ago2 for the kinetically less favorable miRNA members (Iki, 2020).

Together with the TPR domain tethering Cyp40 to Hsp90 during RISC formation, the PPIase domain is required to form functional chaperone complexes in both flies and plants. The PPIase domain of human Cyp40/PPID maintains the activity for the cis-trans isomerization of peptidyl prolines. Further biochemical and structural analyses could help decipher how this, and perhaps other activities of the poorly characterized PPIase domain, enable fly and plant Cyp40 to regulate sRNA during RISC formation. In addition, Fkbp, among the Hsp90 co-chaperones regulating ligand-client binding, displays a similar domain architecture to Cyp40, but endows distinct physiological functions. The generated fkbp59-mutant-based study should help illuminate the molecular basis for such a difference (Iki, 2020).

Why is Cyp40 effective with Ago2 rather than Ago1? In testes, Ago2 appears to be relatively limited compared to Ago1, whereas its main endogenous cargoes, hairpin-siRNA duplexes, are actively produced. This likely creates a competition between miRNA and siRNA duplexes for a limited pool of Ago2. In the duplex-competing condition, Cyp40 activity could ensure Ago2 loading of suboptimal miRNA duplexes, which would otherwise be largely excluded from the effector phase of the testicular silencing pathway. However, Ago1 is inert for most endo-siRNAs and appears to be relatively abundant, compared to Ago2, in testes. This might allow Ago1 to naturally evade the testicular duplex-competing condition, explaining why Cyp40 interacts with Ago1 in vitro and yet is dispensable for steady-state accumulation of most Ago1-bound miRNAs in testes. Thus, the relative abundance of duplexes compared to that of the sRNA-free Ago proteins could be key for influencing the selectivity and effectiveness of Cyp40. The same rationale could also apply to other Hsp90 co-chaperones involved in RISC formation and sRNA regulation in other biological contexts and/or tissues (Iki, 2020).

The cyp40 mutant male showed a completely sterile phenotype, which appears to be exerted by the combined deficiencies of Ago2-sorted multiple miRNAs, including, but not restricted to, miR-316-3p. Similar to cyp40, argo2 mutant males are sterile in Drosophila simulans but are only semi-fertile in D. melanogaster, raising the question of how the loss of an Ago2 modulator, Cyp40, might cause a more severe phenotype than the loss of Ago2 itself. Perhaps the mis-accumulation of some sRNAs and the ensuring disparities among RISCs in the cyp40 mutant have deeper detrimental consequences than those caused by the mere depletion of the effector Ago2. For instance, it was shown that the reduction of selected miRNAs over-activates, in turn, other species, possibly engaging in a whole new boast of not primarily intended regulations. However, the possibility is not excluded that Cyp40 plays multiple roles in testes, in addition to the control of sRNA bound to Ago2. Also noteworthy as a caveat-inducing limitation of the approach taken in this study, Ago-bound sRNAs were isolated from whole testes, including somatic cells where cyp40 is dispensable. Refining this study using sRNA profiles from the purified germline cells, especially from the spermatocyte and spermatid stages, would likely confer more sensitivity to sRNA analyses and help more comprehensively decipher the sRNA-related molecular aberrations caused by loss of cyp40 function in the germline (Iki, 2020).

Ago2 protects Drosophila siRNAs and microRNAs from target-directed degradation, even in the absence of 2'-O-methylation

Target-directed microRNA (miRNA) degradation (TDMD), which is mediated by the protein ZSWIM8 (mammalian homolog of Dorado), plays a widespread role in shaping miRNA abundances across bilateria. Some endogenous small interfering RNAs (siRNAs) of Drosophila cells have target sites resembling those that trigger TDMD, raising the question as to whether they too might undergo such regulation by Dorado, the Drosophila ZSWIM8 homolog. This study finds that some of these siRNAs are indeed sensitive to Dora when loaded into Ago1, the Argonaute paralog that preferentially associates with miRNAs. Despite this sensitivity when loaded into Ago1, these siRNAs are not detectably regulated by target-directed degradation because most molecules are loaded into Ago2, the Argonaute paralog that preferentially associates with siRNAs, and with siRNAs and miRNAs loaded into Ago2 were found to be insensitive to Dora. One explanation for the protection of these small RNAs loaded into Ago2 is that these small RNAs are 2'-O-methylated at their 3' termini. However, 2'-O-methylation does not protect these RNAs from Dora-mediated target-directed degradation, which indicates that their protection is instead conferred by features of the Ago2 protein itself. Together, these observations clarify the requirements for regulation by target-directed degradation and expand understanding of the role of 2'-O-methylation in small-RNA biology (Kingston, 2021).

Analyses of siRNAs in both wild-type and dora Drosophila S2 cells demonstrated that this class of small RNAs undergoes little regulation by conventional target-directed degradation (TDD). This finding does not support the suggestion that siRNAs loaded into Ago1 instead of Ago2 might be 'purified,' or removed from the cell, by TDD-a model put forth to help explain the high steady-state enrichment of siRNAs within Ago2. Although TDD of some Ago1-loaded siRNAs is seen, most siRNAs loaded in Ago1 escape such regulation. Furthermore, even for the TDD-sensitive siRNAs, up-regulation upon loss of Dora was undetectable when examining total-sRNA samples, because, for each siRNA, the Ago1-loaded fraction was minimal when compared to the Ago2-loaded fraction, and thus any increase in the Ago1-loaded fraction negligibly affected total siRNA levels. Thus, the known TDD pathway, which requires Dora, does not appear to be a major driving force in shaping the siRNA content of Drosophila S2 cells (Kingston, 2021).

Although methylation of small RNAs is proposed to protect these RNAs from TDD, this study observed both that loss of methylation does not make Ago2-loaded species susceptible to the known TDD pathway and that gained methylation does not protect Ago1-loaded species from this pathway. These observations indicate that features of Ago proteins, rather than modifications of small RNAs, dictate the ability of Ago-RNA complexes to be regulated by TDD. This importance of the Ago protein concurs with the new model for TDD, in which Ago proteins must interact with and be ubiquitinated by the ZSWIM8/Dora for TDD to occur (Han. 2020; Shi; 2020). Whereas Ago1 can engage with Dora in a TDD-competent manner, low sequence similarity between Ago2 and Ago1 supports the idea that Ago2 might lack the features necessary for Dora recognition and polyubiquitination. With respect to the sites of polyubiquitination, studies of human AGO2 implicate K493 and at least one other lysine within a cluster of 17 surface lysines as required for maximal ZSWIM8-mediated regulation, of which K493 and 12 of the other candidates sites are conserved in Drosophila Ago1, whereas K493 and all but two of the other candidates sites are not conserved in Drosophila Ago2. By analogy, it is speculated that if piRNAs are also protected from TDD, then this protection would also be conferred by the inability of PIWI proteins to interact with and be ubiquitinated by the ZSWIM8/Dora (Kingston, 2021).

Across many species, 2'-O-methylation occurs on guide RNAs that have extensive pairing to their targets, such as plant miRNAs, but not on guide RNAs that lack extensive pairing to most of their targets, such as metazoan miRNAs. Loss of this methylation leads to increased tailing and trimming that, at least for plant miRNAs, Tetrahymena piRNAs, nematode 26G siRNAs, and some Drosophila siRNAs, is associated with small-RNA destabilization. The realization that the identity of the Ago protein rather than the methylation status of the small RNA dictates susceptibility to TDD reopens the mystery as to why the tendency to be methylated correlates with the degree of complementarity of typical sites for a given class of small RNA (Kingston, 2021).

As a new solution to this mystery, it is suggested that these classes of small regulatory RNAs with highly complementary sites reside in Ago/PIWI proteins that have intrinsically weaker interactions with the 3' termini of their guide RNAs. This weaker intrinsic binding to guide-RNA 3' termini is expected for these proteins because it would favor formation of extensive target pairing, as release of the 3' terminus appears to be required to accommodate pairing to the central region of the guide RNA. In contrast, stronger intrinsic binding to guide-RNA 3' termini is expected for proteins that associate with metazoan miRNAs, as release of the 3' terminus is not required to accommodate target recognition typical of these small RNAs, i.e., seed pairing or seed pairing plus conventional 3'-supplementary pairing. The weaker intrinsic binding proposed for Ago/PIWI proteins with guide RNAs that recognize highly complementarity sites would presumably leave the 3' termini of their guide RNAs constitutively vulnerable to tailing and trimming even when they are not paired to a target, thereby explaining the benefit of terminal 2'-O-methylation. This Hen1-mediated methylation, found in plants and animals, presumably emerged early in eukaryotic evolution and thus would have been available for incorporation into the nascent metazoan miRNA pathway. Indeed, some methylation has been reported on most Nematostella miRNAs. Perhaps, however, as exemplified by the bilaterian lineage, as the miRNA-associated Ago proteins adapted to recognize less extensively paired sites, they acquired greater affinity to their guide-RNA 3' termini, which reduced vulnerability to trimming and tailing thereby obviating a benefit for their methylation (Kingston, 2021).

Several observations support aspects of this model. First, mutations within human Ago2 that reduce binding to miRNA 3' termini promote tailing and trimming even in the absence of an extensively paired target, which confirms the assumption that weaker binding to small-RNA 3' termini imparts constitutive vulnerability to tailing and trimming. Second, the 3' termini of piRNAs and metazoan siRNAs are 2'-O-methylated after these guide RNAs are loaded into Ago/PIWI, implying that the methylation machinery has at least intermittent access to the guide-RNA 3' termini, as would be expected if these proteins have relatively weak binding to the 3' termini of their guide RNAs. Third, loss of Hen1 led to increased tailing and trimming of Ago2-associated siRNAs that were Dora-insensitive when associated with Ago1, supporting the conjecture that Ago2-associated RNAs are vulnerable to tailing and trimming even in the absence of highly complementary sites able to trigger TDD (Kingston, 2021).

The notion that small RNAs with 3' termini not stably protected within Ago are susceptible to increased tailing and trimming might also help explain the observation that methylated miR-7, when loaded in Ago1, undergoes increased trimming relative to unmethylated miR-7. Perhaps the terminal methyl group is not well-accommodated by Ago1, which is typically loaded with unmethylated miRNAs, leading to increased exposure of the 3' terminus of the methylated miR-7. Indeed, conformations of human AGO2 represented by the crystal structures would not accommodate a terminal methyl group, implying that terminal methyl modifications might similarly clash with the ground-state structure of Drosophila Ago1. Although methylation is thought to protect small RNAs from trimming in addition to tailing, the observation that trimming of methylated miR-7 is Nibbler sensitive suggests that, at least in the context of Drosophila Ago1, methylated species can still be trimmed (Kingston, 2021).

In summary, the observation that some classes of small regulatory RNAs are methylated and some are not can be at least partly explained without invoking a TDD phenomenon: Because piRNAs, siRNAs, and plant miRNAs must efficiently pair to targets throughout their length, their corresponding Ago/PIWI proteins might have reduced affinity to guide-RNA 3' termini, and this reduced affinity would render these guide RNAs more susceptible to tailing and trimming even when they are not paired to target-unless they are 2'-O-methylated (Kingston, 2021).

EVOLUTIONARY HOMOLOGS

For information of Argonaute 2 homologs see Argonaute 1 site.

REGULATION

Drosophila microRNAs are sorted into functionally distinct argonaute complexes after production by dicer-1

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide distinct classes of RNA-induced silencing complexes (RISCs) to repress mRNA expression in biological processes ranging from development to antiviral defense. In Drosophila, separate but conceptually similar endonucleolytic pathways produce siRNAs and miRNAs. Despite their distinct biogenesis, double-stranded miRNAs and siRNAs participate in a common sorting step that partitions them into Ago1- or Ago2-containing effector complexes. These distinct complexes silence their target RNAs by different mechanisms. miRNA-loaded Ago2-RISC mediates RNAi, but only Ago1 is able to repress an mRNA with central mismatches in its miRNA-binding sites. Conversely, Ago1 cannot mediate RNAi, because it is an inefficient nuclease whose catalytic rate is limited by the dissociation of its reaction products. Thus, the two members of the Drosophila Ago subclade of Argonaute proteins are functionally specialized, but specific small RNA classes are not restricted to associate with Ago1 or Ago2 (Forstemann, 2007).

Animal miRNAs are produced by the sequential action of two distinct RNase III endonucleases. Drosha converts primary miRNAs, most of which are full-length RNA polymerase II transcripts, into pre-miRNAs, 70 nt RNAs that fold into a stem-loop or hairpin structure. Dicer then excises the mature miRNA, bound to its miRNA* strand, from the pre-miRNA. In Drosophila, distinct Dicer enzymes produce siRNA and miRNA. Dicer-1 (Dcr-1) acts with a double-stranded RNA (dsRNA)-binding protein partner, Loquacious (Loqs), to convert pre-miRNA to a miRNA/miRNA* duplex, whereas Dicer-2 (Dcr-2) produces siRNA duplexes by cleaving long dsRNA. Dcr-2 also acts with its dsRNA-binding partner protein, R2D2, to load an siRNA duplex into Ago2, a function that is separable from its role in siRNA production (Forstemann, 2007).

Both siRNAs and miRNAs act as components of RNA-induced silencing complexes (RISCs); the core protein component of all RISCs is a member of the Argonaute family of small RNA-guided RNA-binding proteins. The Drosophila genome encodes five Argonaute proteins, which form two subclades. The Ago subclade comprises Ago1 and Ago2, which have been reported to bind miRNAs and siRNAs, respectively. Piwi, Aub, and Ago3 form the Piwi subclade of Argonaute proteins and bind repeat-associated siRNAs (rasiRNAs; also called piRNAs), which direct silencing of selfish genetic elements such as transposons (Forstemann, 2007).

In lysates from Drosophila embryos, in cultured Drosophila S2 cells, and in adult flies, miRNA can be loaded into both Ago1 and Ago2. The data suggest that sorting miRNAs into Ago1- and Ago2-RISC generates silencing complexes with distinct functional capacities: Ago1-RISC represses expression of targets with which its guide miRNA matches only partially, whereas Ago2 silences fully matched target RNAs. These differences result, in part, from the surprisingly different catalytic efficiencies of Ago1 and Ago2: only Ago2 catalyzes robust, multiple-turnover target cleavage (Forstemann, 2007).

In mammals, only Ago2 retains the ability to catalyze guide RNA-directed endonucleolytic cleavage of RNA; the three other mammalian Argonaute proteins, Ago1, Ago3, and Ago4, lack a functional active site that is presumed to have been present in the evolutionarily ancestral Argonaute protein. Why then has Drosophila Ago1 retained any endonuclease activity at all, if it is so inefficient at target cleavage that it cannot measurably contribute to small RNA-directed RNAi? One potential explanation is that the primary role of the Ago1 endonuclease activity is to facilitate loading of Ago1-RISC. That is, the predominant substrate for the Ago1 endonuclease is not target RNA but, rather, miRNA* strands and perhaps the occasional siRNA passenger strand. Because miRNA* strand cleavage would occur only in cis and only once per loaded Ago1-RISC, efficient, multiple-turnover cleavage of target RNA would not be required (Forstemann, 2007).

These data reveal an important biochemical difference between Ago2 and Ago1, but they do not explain the molecular basis for the inefficiency of Ago1-directed cleavage of target RNA. Two explanations can be envisioned for the more than 40-fold lower kcat of Ago1 compared to Ago2. First, the active site of Ago1 might be less well suited to catalyzing phosphodiester bond cleavage. Alternatively, Ago1 might be slow to assume a catalytically active conformation. In this second model, the rate of a conformational rearrangement would limit the speed of target RNA cleavage by Ago1 (Forstemann, 2007).

The genome of Drosophila contains no mRNA with complete complementarity to miR-277. Why then do flies load miR-277 into Ago2-RISC? Perhaps there are as yet unknown iral RNAs targeted by Ago2-loaded miR-277. Such an innate immune response function has previously been proposed for miRNAs in mammals. Regardless of the biological purpose for loading miR-277 into Ago2, miR-277 provides an important in vivo test of the controversial proposal that the production of small RNA duplexes by Dicer is uncoupled from the loading of Argonaute proteins. That Dcr-2 and R2D2 act in vivo to load Ago2 with miR-277, a miRNA produced by Dcr-1 and Loqs, confirms previous in vitro data suggesting that both ends of a small RNA duplex are available for examination by the Ago2 loading machinery. The results suggest that the miR-277/miR-277* duplex dissociates from Dcr-1 after the dicing of pre-miR-277 and is then bound by the Dcr-2/R2D2 heterodimer, which loads it into Ago2 (Forstemann, 2007).

It was reasoned that Ago1 loading is also uncoupled from dicing. In all animals, some miRNAs are found on the 5' and others on the 3' arm of their pre-miRNA stem loops. In contrast, the geometry of Dcr-1 with respect to the two arms of the pre-miRNA stem is essentially the same for all miRNAs: Dcr-1 always makes staggered cuts that separate the pre-miRNA loop from the miRNA/miRNA* duplex. If Dcr-1 were to load miRNAs directly into Ago1, without first releasing the miRNA/miRNA* duplex, it would be expected that all miRNAs would reside on the same arm of the pre-miRNA stem. The simplest explanation, and one most consistent with the partitioning of miR-277 into both Ago1- and Ago2-RISCs, is that miRNA/miRNA* duplexes are released from Dicer immediately after their production, then rebound by the Ago1- and Ago2-loading machineries. Such a model allows both the terminal thermodynamics of the miRNA/miRNA* duplex to determine the mature miRNA strand (rather than its position within the pre-miRNA) and the pattern of mismatches within the duplex to determine how the miRNA partitions between Ago1 and Ago2 (Forstemann, 2007).

In mammals, siRNAs produce off-target effects largely by acting like miRNAs. In flies, siRNAs loaded into Ago2 are believed to defend against viral infection. Virus-derived siRNAs might therefore trigger widespread, off-target silencing of host genes as flies mount an antiviral RNAi response. The partitioning of siRNAs into Ago2-RISC appears to circumvent this problem, because silencing by Drosophila Ago2 requires greater complementarity between the siRNA and its target than silencing by Ago1. It is tempting to speculate that a similar functional specialization among Argonaute proteins has gone undetected in mammals (Forstemann, 2007).

Sorting of Drosophila small silencing RNAs

In Drosophila, small interfering RNAs (siRNAs), which direct RNA interference through the Argonaute protein Ago2, are produced by a biogenesis pathway distinct from microRNAs (miRNAs), which regulate endogenous mRNA expression as guides for Ago1. siRNAs and miRNAs are sorted into Ago1 and Ago2 by pathways independent from the processes that produce these two classes of small RNAs. Such small-RNA sorting reflects the structure of the double-stranded assembly intermediates the miRNA/miRNA* and siRNA duplexes from which Argonaute proteins are loaded. The Dcr-2/R2D2 heterodimer acts as a gatekeeper for the assembly of Ago2 complexes, promoting the incorporation of siRNAs and disfavoring miRNAs as loading substrates for Drosophila Ago2. A separate mechanism acts in parallel to favor miRNA/miRNA* duplexes and exclude siRNAs from assembly into Ago1 complexes. Thus, in flies small-RNA duplexes are actively sorted into Argonaute-containing complexes according to their intrinsic structures (Tomari, 2007).

In Drosophila the structure of a small-RNA duplex determines its partitioning between Ago1- and Ago2-RISC. These data suggest a simple model for this partitioning, with a central unpaired region serving as both an antideterminant for the Ago2-loading pathway and a preferred binding substrate for the Ago1 pathway. Supporting this view, miRNAs that contain central mismatches, such as let-7 and bantam, assemble primarily into Ago1-RISC. miR-277, whose central region is base paired, partitions between Ago1 and Ago2 in vivo (Tomari, 2007).

A model for small silencing RNA sorting in Drosophila. Dcr-2/R2D2 bind well to highly paired small-RNA duplexes but poorly to duplexes bearing central mismatches; such duplexes are therefore disfavored for loading into Ago2. Ago1 favors small RNAs with central mismatches, but no Ago1-loading proteins have yet been identified. Ago1- and Ago2-loading compete each other, increasing the selectivity of small-RNA sorting. The partitioning of a small-RNA duplex between the Ago1 and Ago2 pathways reflects its structure. A typical miRNA/miRNA^* duplex, such as let-7 or bantam, loads mainly Ago1, whereas a standard siRNA duplex loads mostly Ago2. Some miRNA/miRNA^* duplexes containing extensively paired central regions, such as miR-277/miR-277^*, partition between Ago1 and Ago2. Sorting of small-RNA duplexes into Ago1 and Ago2 produces pre-RISC, in which the duplex is bound to the Argonaute protein. Subsequently, mature RISC, which contains only the siRNA guide or miRNA strand of the original duplex, is formed. The separation of the miRNA and miRNA^* or the siRNA guide and passenger strands also reflects the structure of the small-RNA duplex. For Ago1, it is hypothesized that mismatches between the miRNA and the miRNA^* or siRNA guide and passenger strands in the seed sequence are required for the efficient conversion of pre-RISC to mature RISC. For Ago2, such seed sequence mismatches are not needed because Ago2 can efficiently cleave the passenger or miRNA^* strand, liberating the guide or miRNA from the duplex (Tomari, 2007).

Both the Ago2- and Ago1-loading pathways are selective. For Ago2, the affinity of the Dcr-2/R2D2 heterodimer for a small-RNA duplex provides the primary source of small-RNA selectivity. In the absence of either the Ago2-loading machinery or Ago2 itself, Ago1 is nonetheless preferentially loaded with a miRNA/miRNA^* duplex; an siRNA duplex still loads poorly into Ago1. Thus, the Ago1-loading pathway is also inherently selective and not a default pathway that assembles small RNAs rejected by the Ago 2 pathway. It is not yet know if this selectivity is a direct property of Ago1, of an Ago1-loading machinery that remains to be identified, or both (Tomari, 2007).

Previous bioinformatic analyses noted that a central region of thermodynamic instability was a common feature of miRNA/miRNA^* duplexes. The current data ascribe a function in flies to this common miRNA/miRNA^* structural feature: directing the miRNA into Ago1 and away from Ago2. Mammalian miRNA/miRNA^* duplexes also typically contain a central unpaired region, but it is not yet known if they are preferentially loaded into one of the four mammalian Ago-subclade Argonaute proteins (Tomari, 2007).

What is the biological significance in flies of sorting miRNAs into Ago1 and siRNAs into Ago2? One idea is that Ago1 and Ago2 are functionally distinct, with only Ago2 silencing targets that possess extensive complementarity to the small-RNA guide and only Ago1 directing repression of targets that contain multiple but only partially complementary miRNA-binding sites. Sorting small RNAs between Ago1 and Ago2 may also prevent miRNAs from saturating the Ago2 machinery, which might compromise Ago2-mediated antiviral defense. Conversely, excluding from Ago1 siRNAs produced in response to viral infection may minimize competition between such antiviral siRNAs and endogenous miRNAs, protecting flies from misregulation of gene expression during a viral infection. Restricting a robust RNAi (i.e., target cleavage) response to siRNAs loaded into Ago2 may also minimize undesirable, miRNA-like regulation of cellular genes by virally derived siRNAs. Thus, small-RNA sorting ensures that miRNAs are largely restricted to Ago1, whose relaxed requirement for complementarity between a miRNA and a regulated mRNA target allows each miRNA to control many different mRNAs, and that siRNAs are restricted to Ago2, whose silencing activity requires more extensive complementarity between the target and the siRNA guide. Nonetheless, a final question remains unanswered: why do some iconoclastic miRNA/miRNA^* duplexes contain features that favor their loading into Ago2 (Tomari, 2007)?

The Ago2-RISC-assembly pathway; Hen1 modifies germline piRNAs and single-stranded siRNAs in RISC

Small silencing RNAs repress gene expression by a set of related mechanisms collectively called RNA-silencing pathways. In the RNA interference (RNAi) pathway, small interfering mRNA (siRNAs) defend cells from invasion by foreign nucleic acids, such as those produced by viruses. In contrast, microRNAs (miRNAs) sculpt endogenous mRNA expression. A third class of small RNAs, Piwi-interacting RNAs (piRNAs), defends the genome from transposons. This study reports that Drosophila piRNAs contain a 2'-O-methyl group on their 3' termini; this is a modification previously reported for plant miRNAs and siRNAs and mouse and rat piRNAs. Plant small-RNA methylation is catalyzed by the protein HEN1. Drosophila melanogaster Hen1 (DmHen1), the Drosophila homolog of HEN1, termed Pimet (piRNA methyltransferase) by Saito (2007) in a parallel study, methylates the termini of siRNAs and piRNAs. Without DmHen1, the length and abundance of piRNAs are decreased, and piRNA function is perturbed. Unlike plant HEN1, DmHen1 acts on single strands, not duplexes, explaining how it can use as substrates both siRNAs, which derive from double-stranded precursors, and piRNAs which do not. 2'-O-methylation of siRNAs may be the final step in assembly of the RNAi-enzyme complex, RISC, occurring after the Argonaute-bound siRNA duplex is converted to single-stranded RNA (Horwich, 2007; Saito, 2007).

In flies, both piRNAs (also known as repeat-associated siRNAs, rasiRNAs) and siRNAs, but not miRNAs, are modified at their 3' termini. The terminal nucleotide of Drosophila 0-2 hr embryo and mouse and bull testicular piRNAs was selectively labelled. The resulting ³²P-radiolabeled nucleoside 2' or 3'-monophosphates were resolved by 2D thin-layer chromatography (2D TLC) with a solvent system that can resolve nucleoside 2' monophosphates, nucleoside 3' monophosphates, and 2'-O-methyl nucleoside 3' monophosphates. Modified nucleoside monophosphates derived from the 3' termini of piRNAs were identified by comparison to modified and unmodified nucleoside 2' and 3' monophosphate standards. The terminal nucleotide of the piRNAs of all three animals comigrate with 2'-O-methyl nucleoside 3' monophosphate standards but not with any unmodified nucleoside monophosphate standard. Because mouse piRNAs were previously shown to contain 2'-O-methyl modified 3' termini by both mass spectrometry and a 2D TLC system, it is concluded that Drosophila and bull piRNAs also contain a 2'-O-methyl group at their 3' termini (Horwich, 2007).

In Arabidopsis, the RNA methyltransferase, HEN1, modifies the terminal 2' hydroxyl group of small silencing RNAs. In Drosophila, predicted gene CG12367, whose 1559 nucleotide mRNA encodes a 391 amino acid protein with a 220 amino acid evolutionarily conserved methyltransferase domain, most closely resembles Arabidopsis HEN1. For simplicity, this gene has been called Drosophila melanogaster (Dm) hen1. When homozygous, a piggyBac transposon insertion (PBac{WH}CG12367[f00810]) within the first intron of the fly hen1 gene reduces the accumulation of hen1 mRNA by 1000-fold in testes and by more than 40,000-fold in ovaries and can therefore be considered a null mutation, which is referred to as hen1^f00810 (Horwich, 2007).

The 3' termini of two types of highly abundant piRNAs were examined in the germline of flies heterozygous or homozygous for hen1^f00810. In testes, the Suppressor of Stellate [Su(Ste)] locus produces 24-27 nucleotide rasiRNAs, a subclass of piRNAs that directs silencing of the selfish genetic element Stellate. Su(Ste) rasiRNAs, like other Drosophila piRNAs, are modified at their 3' termini and therefore do not react with NaIO₄. In contrast, Su(Ste) rasiRNAs from hen1^f00810/hen1^f00810 mutant testes reacted with NaIO₄ and could therefore be β-eliminated to remove the last nucleotide of the RNA, thereby increasing their gel mobility and indicating that in the absence of DmHen1 protein, they are not modified. Similarly, rasiRNAs that guide silencing of roo, the most abundant retrotransposon in Drosophila melanogaster, were not modified in hen1^f00810 homozygous ovaries. The Su(Ste) and roo rasiRNAs were also shorter in the hen1^f00810 homozygotes. In contrast, the length and amount of miR-8, which is expressed in both the male and female germline, was unaltered in hen1^f00810 homozygotes. For both Su(Ste) and roo, rasiRNAs were on average shorter and less modified even in hen1^f00810 heterozygotes, compared to the wild-type, suggesting that the abundance of DmHen1 protein limits the stability or production of piRNAs in flies (Horwich, 2007).

Modification of the termini of Drosophila piRNAs plays a role in their function: mRNA expression from HeT-A, the element whose expression is most sensitive to mutations that disrupt piRNA-directed silencing in the female germline, quadrupled in hen1^f00810 heterozygotes and was increased by more than 11-fold in homozygotes, relative to wild-type tissue. It is concluded that Hen1 protein is required for piRNA-directed silencing in the Drosophila germline (Horwich, 2007).

To test whether DmHen1 is required for modification of the 3' termini of siRNAs, Hen1 was depleted by RNAi in cultured Drosophila S2 cells. The cells were transfected with long double-stranded RNA (dsRNA) targeting hen1 on day 1 and day 5, then cotransfected with both GFP dsRNA and hen1 dsRNA on day 8. Total RNA was harvested on day 9, probed for modification with NaIO₄/β-elimination, and analyzed by Northern hybridization with a 5' ³²P-radiolabeled DNA probe complementary to the most abundant GFP-derived siRNA. DsRNAs targeting two different regions of the fly hen1 mRNA both reduced the amount of GFP siRNA modified at its 3' terminus, whereas all the GFP siRNA remained modified when a control dsRNA was used (Horwich, 2007).

Surprisingly, RNAi-mediated depletion of Ago2, but not Ago1, prevented the GFP siRNA from being modified. This result suggests that Ago2, but not Ago1, plays a role in the modification of siRNAs by DmHen1. To test this idea, the modification status of the 3' terminus of miR-277, which partitions into both Ago1 and Ago2 complexes in vivo, was examined. Drosophila miRNAs associate predominantly or exclusively with Ago1 and have unmodified 3' termini. In contrast, approximately half the miR-277 in cultured S2 cells failed to react with NaIO₄, suggesting that approximately half of miR-277 is modified at its 3' terminus. The fraction of miR-277 that was modified was reduced when two different dsRNAs were used to deplete DmHen1 by RNAi. When the cells were treated with dsRNA targeting ago1, all detectable miR-277 was modified, whereas all miR-277 became unmodified when dsRNA targeting ago2 was used. In contrast, bantam, a miRNA that associates nearly exclusively with Ago1, was unmodified under all conditions (Horwich, 2007).

siRNA modification can be recapitulated in lysates of embryos, ovaries, or cultured S2 cells. Modification of siRNA in vitro was inhibited by S-adenosyl homocysteine, but not by S-adenosyl methionine, consistent with DmHen1 transferring a methyl group from S-adenosyl methionine to the terminal 2' hydroxyl group of the RNA, thereby generating S-adenosyl homocysteine as a product (Horwich, 2007).

Data from cultured S2 cells suggested that DmHen1 modifies that portion of miR-277 that enters the Ago2-RISC-assembly pathway, but not the population of miR-277 that assembles into Ago1-RISC. To further test the idea that small-RNA modification requires both Hen1 and the Ago2-RISC-assembly pathway, cytoplasmic lysates were prepared from dsRNA-treated cultured S2 cells. Lysate from control-treated cells modified the 3' terminus of a 5' ³²P-radiolabeled synthetic siRNA duplex but not lysate from hen1-depleted cells. The addition of either of two different preparations of purified, recombinant DmHen1, expressed in E. coli as a ~74 kDa glutathione S-transferase fusion protein (GST-DmHen1), restored the ability of the lysates to modify the siRNA, indicating that loss of DmHen1 caused the loss of siRNA modification. Moreover, lysates depleted for Ago2, but not Ago1, could not modify the ³²P-siRNA in vitro. These in vitro data, together with S2-cell experiments, suggest that modification of the 3' terminus of siRNAs and miRNAs is coupled to assembly into Ago2-RISC (Horwich, 2007).

Dcr-2 and R2D2 act to load double-stranded siRNAs into Ago2. Lysates were prepared from ovaries homozygous mutant for hen1, dcr-2, r2d2, and ago2 by using alleles that were unable to produce the corresponding protein. A 5' ³²P-radiolabeled siRNA duplex was incubated in each lysate to assemble RISC. At each time point, whether the siRNA was 3' terminally modified was determined by assessing its reactivity with NaIO₄. No modified siRNA accumulated when the duplex was incubated in hen1^f00810, dcr-2^L811fsX, r2d2¹, or ago2⁴¹⁴ mutant lysate. Adding 250 nM purified, recombinant GST-DmHen1 restored siRNA modification to the hen1^f00810 but not the ago2⁴¹⁴ lysate. It is concluded that the defect in ago2⁴¹⁴ reflects a requirement for Ago2 in small-RNA modification by DmHen1, rather than an indirect effect such as destabilization of DmHen1 in the absence of Ago2. GST-DmHen1 similarly rescued lysate from hen1(RNAi) but not ago2(RNAi)-treated S2 cells. Together, the results of experiments using cultured S2 cells -- a somatic-cell line -- and ovaries, which comprise mainly germline tissue, suggest that a functional Ago2-RISC-assembly pathway is required for siRNA modification in Drosophila (Horwich, 2007).

To test at which step in the Ago2-RISC-assembly pathway siRNAs become modified, it was determined whether siRNAs are 2'-O-methylated by DmHen1 as single strands or as duplexes. In vitro, assembly of siRNAs into Ago2-RISC follows an ordered pathway in which the siRNA duplex first binds the Dicer-2/R2D2 heterodimer to form the RISC-loading complex (RLC). The RLC determines which of the two siRNA strands will become the guide for Ago2 and which will be destroyed (the passenger strand). The siRNA is then loaded into Ago2 as a duplex. In this pre-RISC complex, the passenger strand occupies the same position as future target RNAs. Cleavage of the passenger strand by the Ago2 endonuclease domain converts pre-RISC to mature RISC. No single-stranded guide or passenger RNA is produced prior to this maturation step. Thus, all single-stranded siRNA produced in vitro or in vivo corresponds to mature RISC (Horwich, 2007).

Ago2-RISC was assembled in vitro by using an siRNA designed to load only one of its two strands into Ago2. Then the reaction was sampled over time, isolating the 5' ³²P-radiolabeled siRNA under conditions previously demonstrated to preserve its structure, and single- from double-stranded siRNA was separated by native gel electrophoresis. The RNAs were then isolated from the gel and tested for reactivity with NaIO₄ to determine the presence of modification at their 3' termini. At each time, total siRNA was analyzed in parallel. 3' terminal modification increased over the course of RISC assembly and, at all times, was restricted to single-stranded siRNA: Within the limits of detection, all double-stranded siRNA was unmodified, even after 3 hr. It is concluded that siRNA modification is coupled to RISC assembly and occurs only after the conversion of pre-RISC to mature RISC (Horwich, 2007).

Whereas Arabidopsis HEN1 contains an N-terminal double-stranded RNA-binding motif, DmHen1 does not. To test whether DmHen1 modifies double-stranded small RNAs, purified, recombinant GST-DmHen1 was incubated with either single-stranded or double-stranded siRNAs. Modification, evidenced by loss of reactivity with NaIO₄, was detected only for the single-stranded RNA, suggesting that DmHen1 modifies single-stranded substrates, but not siRNAs or blunt RNA duplexes. A preference for single-stranded RNA would explain how DmHen1 could act on both siRNAs, which are born double stranded, and piRNAs, which are not. It is noted that the purified, recombinant GST-DmHen1 protein was more than 50-fold less active on its own than when supplemented with ovary lysate from hen1^f00810 homozygous flies. It is speculated that the Ago2-RISC machinery is required for Hen1 function in flies, although the possibility cannot be excluded that the lysate contains a factor (e.g., a kinase) required for activating Hen1 (Horwich, 2007).

Modification of single-stranded siRNAs -- that is, those loaded in fully mature Ago2-RISC but not double-stranded siRNAs might allow cells to distinguish siRNAs loaded successfully into functional complexes from those that fail to assemble. For example, if a 3'-to-5' nuclease acts to degrade single-stranded siRNAs, 2'-O-methylation of single-stranded siRNAs in Ago2 RISC may protect them from destruction. Moreover, such a nuclease might trim the 3' end of piRNAs. 2'-O-methylation of the piRNA 3' terminus may occur only when the length of RNA extending beyond the Piwi-family protein is short enough to permit the simultaneous binding of the final ribose sugar to the active site of DmHen1 and the interaction of DmHen1 with the Piwi protein itself. Modification of the terminus of the trimmed piRNA would then block further 3'-to-5' trimming of the small RNA, generating its Piwi-, Aubergine-, or Ago3-specific length. The observation that piRNAs are shorter in hen1^f00810 mutants supports this model (Horwich, 2007).

It is noted that all 2'-O-methyl-modified small RNAs identified thus far are associated with RISC complexes that efficiently cleave their RNA targets, i.e., Ago1-associated plant miRNAs, animal piRNAs, and Ago2-associated siRNAs in flies, whereas Drosophila miRNAs are typically both unmodified and associated with Ago1 RISC, which does not catalyze mRNA target cleavage in vivo. It is speculated that DmHen1 is recruited to RISC complexes containing single-stranded small silencing RNAs according to the identity of their Argonaute protein. This model predicts that DmHen1 will bind only to complexes containing fly Ago2 or the three fly Piwi proteins, Piwi, Aubergine, and Ago3, but not Ago1. Clearly, future experiments will need to test this hypothesis (Horwich, 2007).

An endogenous small interfering RNA pathway in Drosophila

Drosophila endogenous small RNAs are categorized according to their mechanisms of biogenesis and the Argonaute protein to which they bind. MicroRNAs are a class of ubiquitously expressed RNAs of approximately 22 nucleotides in length, that arise from structured precursors through the action of Drosha-Pasha and Dicer-1-Loquacious complexes. These join Argonaute-1 to regulate gene expression. A second endogenous small RNA class, the Piwi-interacting RNAs, bind Piwi proteins and suppress transposons. Piwi-interacting RNAs are restricted to the gonad, and at least a subset of these arises by Piwi-catalysed cleavage of single-stranded RNAs. This study shows that Drosophila generates a third small RNA class, endogenous small interfering RNAs, in both gonadal and somatic tissues. Production of these RNAs requires Dicer-2, but a subset depends preferentially on Loquacious rather than the canonical Dicer-2 partner, R2D2. Endogenous small interfering RNAs arise both from convergent transcription units and from structured genomic loci in a tissue-specific fashion. They predominantly join Argonaute-2 and have the capacity, as a class, to target both protein-coding genes and mobile elements. These observations expand the repertoire of small RNAs in Drosophila, adding a class that blurs distinctions based on known biogenesis mechanisms and functional roles (Czech, 2008).

Drosophila expresses five Argonaute proteins, which segregate into two classes. The Piwi proteins (Piwi, Aubergine and AGO3) are expressed in gonadal tissues and act with Piwi-interacting RNAs (piRNAs) to suppress mobile genetic elements. The Argonaute class contains AGO1 and AGO2. AGO1 binds microRNAs (miRNAs) and regulates gene expression. The endogenous binding partners of AGO2 have remained enigmatic (Czech, 2008).

Transgenic flies were generated expressing epitope-tagged AGO2 under the control of its endogenous promoter. Tagged AGO2 localizes to the cytoplasm of germline and somatic cells of the ovary. Immunoprecipitated AGO2-associated RNAs differ in their mobility from those bound to AGO1. Deep sequencing of small RNAs from AGO1 and AGO2 complexes yielded 2,094,408 AGO1-associated RNAs and 916,834 AGO2-associated RNAs from Schneider (S2) cells, and 455,227 AGO2-associated RNAs from ovaries that matched perfectly to the Drosophila genome. Three libraries were sequenced derived from 18-29-nucleotide RNAs (936,833 sequences from wild-type ovaries, 1,042,617 sequences from Dicer-2 (Dcr-2) mutant ovaries, and 1,946,339 sequences from loquacious (loqs) mutant ovaries) and an 18-24-nucleotide library from wild-type testes (522,848 sequences). Finally, the analysis included 92,363 published sequences derived from 19-26-nucleotide RNAs from S2 cells (Czech, 2008).

Among the ~50% of AGO2-associated RNAs from S2 cells that did not match the genome, ~17% matched the flock house virus (FHV), a pathogenic RNA virus and reported target for RNAi in flies. These probably arose because of persistent infection of the S2 cultures (Czech, 2008).

After excluding presumed degradation products of abundant cellular RNAs, each of the total RNA libraries were divided into two categories: annotated miRNAs and the remainder. For the S2 cell library, the size distribution of these populations formed two peaks, with non-miRNAs lying at 21 nucleotides and miRNAs exhibiting a broader peak from 21 to 23 nucleotides. Libraries derived from AGO1 and AGO2 complexes almost precisely mirrored these two size classes. In the ovary library, this approach revealed three size classes. Whereas two reflected those seen in S2 cells, a third class comprised piRNAs. Again, RNA size profiles from AGO2 or Piwi family immunoprecipitates mirrored those within the total ovary library. These data demonstrate that AGO2 is complexed with a previously uncharacterized population of small RNAs (Czech, 2008).

Whereas known miRNAs comprised more than 97% of AGO1-associated RNAs in S2 cells, they made up only 8% or 20% of the AGO2-bound species in S2 cells or ovaries, respectively. The remaining small RNAs in AGO2 complexes formed a complex mixture of endogenous siRNAs (endo-siRNAs). Among these, transposons and satellite repeats contributed substantially to AGO2-associated small RNAs in S2 cells (27%) and ovaries (53%). The nature of the transposons giving rise to abundant siRNAs in ovaries and S2 cells differed substantially, probably reflecting differential expression of specific transposons in these tissues. Unlike piRNAs, neither somatic nor germline siRNAs exhibited a pronounced enrichment for sense or antisense species (Czech, 2008).

In accord with these findings, knockdown of AGO2 in S2 cells leads to increased expression of several mobile elements. In the germ line, the Piwi-piRNA system has been reported as the dominant transposon-silencing pathway. Nevertheless, several transposons, with a potential to be targeted by siRNAs, were substantially derepressed in AGO2 mutant or Dcr-2 mutant ovaries. Although comparisons of relative abundance were difficult, both piRNAs and siRNAs mapped to piRNA clusters, with the regions that generate uniquely mapping species generally overlapping. Thus, piRNA loci are a possible source for antisense RNAs matching transposons and might serve a dual function in small RNA generation. Considered together, these data suggest that endo-siRNAs repress the expression of mobile elements, in some tissues acting alongside piRNA pathways (Czech, 2008).

To probe the nature of the remaining endo-siRNAs, genomic sites, which give rise to multiple uniquely mapping RNAs that do not fall into heterochromatic regions, were computationally extracted. These generally segregated into two categories, termed structured loci or convergently transcribed loci (Czech, 2008).

Transcripts from structured loci can fold to form extensive double-stranded RNA directly. The two major loci, termed esi-1 and esi-2, gave rise to half of the 20 most abundant endo-siRNAs in ovaries and also generated siRNAs in embryos, larvae and adults. esi-1, annotated as CG18854, can produce an ~400-base pair (bp) dsRNA through interaction of its 5' and 3' untranslated regions. esi-2 overlaps with CG4068 and consists of 20 palindromic ~260-nucleotide repeats. All siRNAs derived from these two loci arise from one genomic strand. In some previously characterized instances (for example, Arabidopsis trans-acting-siRNAs) Dicer generates 'phased' siRNAs with 5' ends showing a 21-nucleotide periodicity. In all tissues examined, esi-1 and esi-2 produced phased siRNAs, consistent with a defined initiation site for Dicer processing. Phasing was not observed for viral or repeat-derived siRNAs. Finally, siRNAs from both loci also joined AGO1 in proportions greater than siRNAs produced from transposons and repeats, perhaps owing to the imperfect nature of the dsRNA that they produce (Czech, 2008).

AGO2 regulates gene expression by cleavage of complementary sites rather than by recognition of seed sites typical of AGO1-miRNA-mediated regulation. Possible targets of endo-siRNAs were sought by identifying transcripts with substantial complementarity. A highly abundant siRNA from esi-2 is highly complementary to the coding sequence of the DNA-damage-response gene mutagen-sensitive 308 (mus308). Using a modified rapid amplification of cDNA ends (RACE) protocol, mus308 fragments were detected with 5' ends corresponding precisely to predicted endo-siRNA cleavage sites. Moreover, AGO2 and Dcr-2 loss consistently increased mus308 expression in testis and to a lesser extent in ovaries, consistent with the relative abundance of esi-2 siRNAs in these tissues. Finally, a reporter gene containing two mus308 target sites was significantly derepressed in S2 cells on depletion of Dcr-2 or AGO2 but not of Dcr-1 or AGO1. Although extensive complementarity between other endo-siRNAs and messenger RNAs was rare, several esi-1-derived siRNAs complementary to CG8289 were found, suggesting a potential regulatory interaction in vivo (Czech, 2008).

A second group of siRNA-generating loci contained regions in which dsRNAs can arise from convergent transcription. If sorted for siRNA density, most of the top 50 ovarian and S2 cell siRNA loci lay in regions where annotated 3' UTRs or expressed-sequence-tags corresponding to convergently transcribed protein-coding genes overlap. Typically, siRNAs arise on both genomic strands but only from overlapping portions of convergent transcripts. Examining all 998 convergently transcribed gene pairs in the Drosophila genome with annotated overlapping transcripts, the peak abundance of ovarian siRNAs was found to be at the centre of the overlap, with sharp declines away from this region. In an alternative arrangement, Pgant35A produces sense and antisense siRNAs across its entire annotated transcript, consistent with expressed-sequence-tag support for antisense transcription traversing this locus (Czech, 2008).

Thus, a large number of Drosophila genes generate endogenous siRNAs, with most having perfect complementarity to the 3' UTRs of neighbouring genes. Relative levels of endo-siRNAs generated from each convergent transcription unit were low, and no or little change (up to a ~1.3-fold increase) was found in the expression of such genes in AGO2 mutant ovaries. Possibly, the level of small RNAs produced by this genomic arrangement is inconsequential, amounting to noise within silencing pathways. However, there are probably circumstances wherein regulation by such arrangements might substantially impact expression (Czech, 2008).

In S2 cells, two neighbouring loci encoded nearly 16% of AGO2-associated RNAs. These reside within a large intron of klarsicht and did not generate siRNAs in any other tissue. A similar locus, corresponding to CG14033, was found within an intron of thickveins and gave rise to testis-specific siRNAs. Although the function of both siRNA clusters is unclear, the thickveins cluster shares considerable complementarity to CG9203, and loss of AGO2 and Dcr-2 mildly increased CG9203 mRNA levels in testis but not in ovaries (Czech, 2008).

Dcr-2 has been implicated in the production of siRNAs from viral replication intermediates or exogenously introduced dsRNAs, whereas Dcr-1 has been linked to miRNA biogenesis. In agreement with these observations, all endo-siRNA classes were lost in Dcr-2 mutant ovaries. To obtain more insight into the genetic requirements for endo-siRNA biogenesis and stability, components of siRNA and miRNA pathways were depleted in S2 cells, and levels of abundant siRNAs derived from structured loci were analysed . Although depletion of Dcr-2 and AGO2 resulted in substantial reductions in siRNA levels, little or no changes were observed on Drosha, Pasha, Dcr-1 or AGO1 depletion. Unexpectedly, virtually no requirement was found for the Dcr-2 partner R2D2 but a strong requirement was found for the Dcr-1 partner Loquacious. Only one analysed siRNA exhibited partial dependence on R2D2, potentially correlating with the extensive dsRNA character of its precursor duplex. Artificial sensors for endo-siRNAs from esi-1 and esi-2 in S2 cells gave patterns of de-repression that matched analysis of endo-siRNA levels (Czech, 2008).

Analysis of the most abundant siRNA from esi-2 in flies mutant for Dcr-2, AGO2, r2d2 or loqs extended these findings from cell culture. To examine the unexpected requirement for loqs more broadly, small RNAs were sequenced from loqs-mutant ovaries and a near complete loss of endo-siRNAs from structured loci was observed. A much smaller impact of loqs was seen on endo-siRNAs derived from repeats and convergent transcription units. However, an involvement of Loqs and not R2D2 in the function of siRNAs derived from perfect dsRNA precursors was supported by analysing the impact of depleting siRNA/miRNA pathway components on the ability to suppress FHV replication in infected S2 cell cultures (Czech, 2008).

The results uncover an unanticipated role for Loqs in siRNA biogenesis and suggest that R2D2 has a lesser impact on at least two types of endogenous siRNAs. It is well established that Loqs partners with Dcr-1 for miRNA processing. To probe a molecular interaction with Dcr-2, Loqs binding partners were analyzed using quantitative proteomics. Dcr-1 and Dcr-2 were both abundant in Loqs immunoprecipitates from cultured cells and flies, supporting a physical interaction between Dcr-2 and Loqs (Czech, 2008).

Among animals, endo-siRNA pathways have so far been restricted to Caenorhabditis elegans. The current results extend the prevalence of such systems to Drosophila and parallel recent discoveries of an endo-siRNA pathway in mouse oocytes. These systems have many common features but also key differences. In both, siRNAs collaborate with piRNAs to repress transposons. Also, mouse and Drosophila both generate endo-siRNAs from structured loci. In mouse, dsRNAs can form by pairing of sense protein-coding transcripts with antisense transcripts from pseudogenes. Whether or not transcripts from unlinked sites lead to siRNA production in Drosophila is unclear. However, transposon sense transcripts may hybridize to antisense sequences transcribed from piRNA clusters to form endo-siRNA precursors. In flies, a much larger number of genic loci enter the pathway as compared to mice because convergent transcription of neighbouring genes frequently creates overlapping transcripts. Overall, annotation of the Drosophila genome indicates that a significant proportion is transcribed in both orientations, providing widespread potential for dsRNA formation. This property is shared by many other annotated genomes, raising the possibility that the RNAi pathway has broad impacts on gene regulation. Viewed in combination, these studies suggest an evolutionarily widespread adoption of dsRNAs as regulatory molecules, a property previously ascribed only to miRNAs (Czech, 2008).

Argonaute divides its RNA guide into domains with distinct functions and RNA-binding properties

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) guide Argonaute proteins to silence mRNA expression. Argonaute binding alters the properties of an RNA guide, creating functional domains. The domains established by Argonaute - the anchor, seed, central, 3' supplementary, and tail regions - have distinct biochemical properties that explain the differences between how animal miRNAs and siRNAs bind their targets. Extensive complementarity between an siRNA and its target slows the rate at which fly Argonaute2 (Ago2) binds to and dissociates from the target. Highlighting its role in antiviral defense, fly Ago2 dissociates so slowly from extensively complementary target RNAs that essentially every fully paired target is cleaved. Conversely, mouse AGO2, which mainly mediates miRNA-directed repression, dissociates rapidly and with similar rates for fully paired and seed-matched targets. These data narrow the range of biochemically reasonable models (see Model for RISC Function) for how Argonaute-bound siRNAs and miRNAs find, bind, and regulate their targets (Wee, 2012).

Argonaute divides a small RNA guide into anchor, seed, central, 3' supplementary, and tail functional domains. Nucleotides in the anchor (g1) and tail (g18-g21) facilitate Argonaute loading and help secure the siRNA or miRNA guide to Argonaute after the passenger or miRNA^∗ strand has been removed. But these terminal domains are unlikely to base pair with a target RNA, even when pairing is predicted by their sequences. In contrast, central base pairing (g9-g12) between the guide and target is required for efficient target cleavage. Mismatches in this central region prevent RISC from attaining a catalytically competent conformation. For fly Ago2-RISC, achieving this conformation takes more time than seed pairing alone. The data show that nearly every fly Ago2-RISC that reaches this conformation cleaves its RNA target rather than releasing it. For mouse AGO2-RISC, a slow catalytic rate often allows the target to escape before being sliced (Wee, 2012).

In contrast, most miRNA:Argonaute complexes rapidly bind to and dissociate from their RNA targets via their seed. Even when RISC binds a target through both its seed and 3' supplementary regions, it dissociates nearly as rapidly as for seed-only binding. Thus, the properties of RISC are essentially the same for both the typical seed-only and the less common seed plus 3' supplementary pairing targets. That the rates of association and dissociation are so similar for these two binding modes suggests that pairing between a target and the 3' supplementary region of a miRNA does not require winding the target RNA around the guide, side-stepping the topological problem that must be solved for siRNAs to direct RISC to cleave a target (Wee, 2012).

The finding that miRNAs use so little of their sequence to identify their regulatory targets surprised the biological community. The data show that miRNA-programmed RISC binds with a strength and binding site size similar to those of high affinity RNA-binding proteins. It is siRNA-programmed RISC whose behavior should surprise us: it binds highly complementary targets far less tightly than a comparable antisense RNA because Argonaute reduces the contribution of most of its nucleotides to target binding (Wee, 2012).

What do the physical properties of RISC teach about its cellular function? miRNAs and siRNAs are typically present in cells at dramatically different concentrations. For example, in flies in which the white gene is silenced by RNAi, the abundance of all antisense white siRNAs combined is less than that of any of 29 most abundant miRNAs. Previously, the ability of siRNAs to function at low abundance has been ascribed to the catalytic nature of RNAi. To achieve a concentration 10-fold greater than the K_D for siRNA-like binding (3.7 pM for fly Ago2-RISC) would require only ∼5 molecules of RISC in ovarian terminal filament cells (∼200 μm³ and ∼11 molecules in a cultured S2 cell (∼500 μm³). Thus, even for Argonaute proteins with no endonuclease activity, small numbers of molecules of RISC can repress highly complementary targets; endonuclease activity is only needed when a small amount of RISC must repress a larger amount of target. The combination of high affinity and catalytic turnover helps explain why the siRNA-directed RNAi pathway provides an effective defense against viral infection in plants and invertebrate animals (Wee, 2012).

Animal miRNAs nearly always repress their targets by binding rather than endonucleolytic cleavage. This explains why animal cells express miRNAs at such high levels. Recent data suggest that only the most abundant cellular miRNAs mediate target repression. The data provide a biochemical explanation for this observation (Wee, 2012).

Consider two abundant miRNAs in a cultured HeLa cell (∼5,000 μm³: miR-21 (4 nM; and the let-7 miRNA family, nine highly related miRNAs sharing a common seed sequence (∼3 nM). Both miRNAs are present at a concentration greater than the K_D measured for seed matched targets for fly (∼210 pM) or mouse (∼26 pM) Ago2-RISC. Assuming a mean target mRNA abundance of ten molecules per cell and 50 different mRNA targets per miRNA, miR-21 and let-7 each regulate ∼500 (170 pM) total target mRNA molecules per HeLa cell. Under these conditions, nearly every miR-21 or let-7 target mRNA (∼95%-99%) with an accessible seed match will be bound by the complementary miRNA-programmed RISC (Wee, 2012).

Target repression by miRNAs can be reduced by the presence of competitor RNAs containing miRNA binding sites that titrate miRNA-RISC away from the mRNAs it regulates. The fundamental properties of RISC make specific predictions about how the activity of specific miRNAs can be inhibited by the expression of these competitor transcripts. The effect of such competitor RNAs reflects the concentration of both the miRNA and miRNA-binding sites, as well as the affinity of miRNA-RISC for those sites. For abundant miRNAs such as miR-21 or the let-7 family, the expression of competitor RNAs containing miRNA binding sites -- even highly complementary binding sites -- will have little impact on the regulation of their target genes in flies or mammals. Doubling the expression of mRNAs repressed by miR-21, for example, would require ∼7.8 nM seed only competitor and ∼4.0 nM fully paired competitor for fly Ago2-RISC. For mouse AGO2-RISC, it would still require ∼7.7 nM seed only competitor and ∼7.2 nM of the fully paired competitor. Taken together, this translates to ∼22,400 copies of seed only competitor and ∼12,000-21,700 copies of fully paired competitor. If the competitor contained one miRNA-binding site, it would comprise 12%-50% of all the mRNA in the cell (Wee, 2012).

In contrast, doubling the expression of the mRNA targets for an intermediate (mir-93; ∼140 pM) or a low abundance miRNA (mir-24; 7.3 pM) would require just 600-800 additional seed-matching sites. For mir-93 whose abundance confers the ability to bind to ∼60% of all potential targets, the competitor must be as abundant as the sum of all the target mRNAs (∼500 copies). Low abundance miRNAs like mir-24 are unlikely to contribute much biologically meaningful regulation because they are present at a concentration less than their K_D for seed-matching targets in both flies and mammals: <4% of miR-24 targets are expected to be bound by the miRNA at any given time. Using the conservative assumption that every bound miRNA-RISC completely represses an mRNA target, miR-24 is predicted to reduce the expression of the average seed-matched target by <4% (Wee, 2012).

Thus, the proposal that 'competing endogenous RNAs' ('ceRNAs') sequester miRNAs, derepressing the authentic targets of that miRNA, applies only to a small subset of miRNAs whose cellular concentration and target abundance meet a narrow range of values. The miRNAs with the largest impact on gene expression -- the most abundant miRNAs -- are not predicted to be regulatable by endogenous, transcribed seed-matched competitor transcripts. Consistent with this view, viral and experimental inhibition of specific miRNA function by transcribed RNA requires the use of extensively complementary miRNA-binding sites that recruit a cellular pathway that actively degrades the targeted miRNA. Absent this target directed, catalytic destruction of miRNAs, RNAs of ordinary abundance are unlikely to compete with mRNAs for binding abundant, biologically functional miRNAs (Wee, 2012).

Hidden sequence specificity in loading of single-stranded RNAs onto Drosophila Argonautes

Argonaute proteins play important roles in gene regulation with small RNAs (sRNAs) serving as guides to targets. Argonautes are believed to bind sRNAs in a sequence non-specific manner. Argonautes selectively load endogenous single-stranded (ss) RNAs, suggesting that Argonaute loading may conform to sequence specificity. To identify sequences preferred for Argonaute loading, HIgh-throughput Sequencing mediated Specificity Analysis (HISSA) was developed. HISSA allows massively parallel analysis of RNA binding efficiency by using randomized oligos in in vitro binding assays and quantifying RNAs by deep-sequencing. Drosophila was chosed as a model system to take advantage of the presence of two biochemically distinct Argonautes, AGO1 and AGO2. The results revealed AGO2 loading to be strongly favored by G-rich sequences. In contrast, AGO1 showed an enrichment of the 'GAC' motif in loaded species. Reanalysis of published sRNA sequencing data from fly tissues detected enrichment of the GAC motif in ssRNA-derived small RNAs in the immunopurified AGO1-complex under certain conditions, suggesting that the sequence preference of AGO1-loading may influence the repertoire of AGO1-bound endogenous sRNAs. Finally, this study showed that human Ago2 also exhibited selectivity in loading ssRNAs in cell lysates. These findings may have implications for therapeutic ssRNA-mediated gene silencing (Goh, 2018).

Intertwined pathways for Argonaute-mediated microRNA biogenesis in Drosophila

Although Dicer is essential for general microRNA (miRNA) biogenesis, vertebrate mir-451 is Dicer independent. Instead, its short pre-miRNA hairpin is 'sliced' by Ago2, then 3'-resected into mature miRNAs. This study shows that Drosophila cells and animals generate functional small RNAs from mir-451-type precursors. However, their bulk maturation arrests as Ago-cleaved pre-miRNAs, which mostly associate with the RNAi effector AGO2. Routing of pre-mir-451 hairpins to the miRNA effector AGO1 was inhibited by Dicer-1 and its partner Loqs. Loss of these miRNA factors promoted association of pre-mir-451 with AGO1, which sliced them and permitted maturation into approximately 23-26 nt products. The difference was due to the 3' modification of single-stranded species in AGO2 by Hen1 methyltransferase, whose depletion permitted 3' trimming of Ago-cleaved pre-miRNAs in AGO2. Surprisingly, Nibbler, a 3'-5' exoribonuclease that trims 'long' mature miRNAs in AGO1, antagonized miR-451 processing. An in vitro reconstitution assay was used to identify a soluble, EDTA-sensitive activity that resects sliced pre-miRNAs in AGO1 complexes. Finally, deep sequencing was used to show that depletion of dicer-1 increases the diversity of small RNAs in AGO1, including some candidate mir-451-like loci. Altogether, this study has document unexpected aspects of miRNA biogenesis and Ago sorting, and provides insights into maturation of Argonaute-cleaved miRNA substrates (Yang, 2013).

Single-molecule analysis of the target cleavage reaction by the Drosophila RNAi enzyme complex

Small interfering RNAs (siRNAs) direct cleavage of complementary target RNAs via an RNA-induced silencing complex (RISC) that contains Argonaute2 protein at its core. However, what happens after target cleavage remains unclear. This study analyzed the cleavage reaction by Drosophila Argonaute2-RISC using single-molecule imaging and revealed a series of intermediate states in target recognition, cleavage, and product release. The data suggest that, after cleavage, RISC generally releases the 5' cleavage fragment from the guide 3' supplementary region first and then the 3' fragment from the seed region, highlighting the reinforcement of the seed pairing in RISC. However, this order can be reversed by extreme stabilization of the 3' supplementary region or mismatches in the seed region. Therefore, the release order of the two cleavage fragments is influenced by the stability in each region, in contrast to the unidirectional base pairing propagation from the seed to the 3' supplementary region upon target recognition (Yao, 2015).

Age-driven modulation of tRNA-derived fragments in Drosophila and their potential targets

Development of sequencing technologies and supporting computation enable discovery of small RNA molecules that previously escaped detection or were ignored due to low count numbers. This study describes Drosophila melanogaster tRFs, which appear to have a number of structural and functional features similar to those of miRNAs but are less abundant. As is the case with miRNAs, (1) tRFs seem to have distinct isoforms preferentially originating from 5' or 3' end of a precursor molecule (in this case, tRNA), (2) ends of tRFs appear to contain short "seed" sequences matching conserved regions across 12 Drosophila genomes, preferentially in 3' UTRs but also in introns and exons; (3) tRFs display specific isoform loading into Ago1 Ago2 and thus likely function in RISC complexes; (4) levels of loading in Ago1 and Ago2 differ considerably; and (5) both tRF expression and loading appear to be age-dependent, indicating potential regulatory changes from young to adult organisms. Drosophila tRF reads mapped to both nuclear and mitochondrial tRNA genes for all 20 amino acids, while previous studies have usually reported fragments from only a few tRNAs. These tRFs show a number of similarities with miRNAs, including seed sequences. Based on complementarity with conserved Drosophila regions such seed sequences and their possible targets were identified with matches in the 3'UTR regions. Strikingly, the potential target genes of the most abundant tRFs show significant Gene Ontology enrichment in development and neuronal function. The latter suggests that involvement of tRFs in the RNA interfering pathway may play a role in brain activity or brain changes with age (Karaiskos, 2016).

The cricket paralysis virus suppressor inhibits microRNA silencing mediated by the Drosophila Argonaute-2 protein

Small RNAs are potent regulators of gene expression. They also act in defense pathways against invading nucleic acids such as transposable elements or viruses. To counteract these defenses, viruses have evolved viral suppressors of RNA silencing (VSRs). Plant viruses encoded VSRs interfere with siRNAs or miRNAs by targeting common mediators of these two pathways. In contrast, VSRs identified in insect viruses to date only interfere with the siRNA pathway whose effector Argonaute protein is Argonaute-2 (Ago-2). Although a majority of Drosophila miRNAs exerts their silencing activity through their loading into the Argonaute-1 protein, recent studies highlighted that a fraction of miRNAs can be loaded into Ago-2, thus acting as siRNAs. In light of these recent findings, this study re-examined the role of insect VSRs on Ago-2-mediated miRNA silencing in Drosophila melanogaster. Using specific reporter systems in cultured Schneider-2 cells and transgenic flies, this study showed that the Cricket Paralysis virus VSR CrPV1-A but not the Flock House virus B2 VSR abolishes silencing by miRNAs loaded into the Ago-2 protein. Thus, the results provide the first evidence that insect VSR have the potential to directly interfere with the miRNA silencing pathway (Besnard-Guerin, 2015).

Protein Interactions

Argonaute2 is essential for RNAi driven by exogenously introduced dsRNA and is the first protein component to be identified as part of the RISC complex

Double-stranded RNA induces potent and specific gene silencing through a process referred to as RNA interference (RNAi) or posttranscriptional gene silencing (PTGS). RNAi is mediated by RNA-induced silencing complex (RISC), a sequence-specific, multicomponent nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to contain short RNAs (approximately 22 nucleotides) derived from the double-stranded RNA trigger, but the protein components of this activity are unknown. The RNAi effector nuclease has been purified from cultured Drosophila cells. The active fraction contains a ribonucleoprotein complex of approximately 500 kilodaltons. Protein microsequencing reveals that one constituent of this complex, Argonaute2, is a homolog of genes that are essential for gene silencing in Caenorhabditis elegans, Neurospora, and Arabidopsis. This observation begins the process of forging links between genetic analysis of RNAi from diverse organisms and the biochemical model of RNAi that is emerging from Drosophila in vitro systems (Hammond, 2001).

Posttranscriptional silencing phenomena have also been observed in plants (e.g., PTGS) and fungi (e.g., quelling): genetic studies indicate that these are likely to be mechanistically related to RNAi. Moreover, RNAi per se has been demonstrated in a variety of experimental systems, including insects, protozoans, and mammals. A synthesis of in vivo and in vitro experiments has led to a mechanistic model for RNAi/PTGS. Silencing is initiated by exposure of a cell to dsRNA. This 'trigger' may be introduced experimentally or may derive from endogenous sources such as viruses, transgenes, or cellular genes. Double-stranded RNAs are processed into discrete ~21- to 25-nucleotide (nt) RNA fragments known as siRNAs (small interfering RNAs). These small RNAs join a multicomponent nuclease complex, RISC, and guide that enzyme to its substrates through conventional base-pairing interactions. Recognition of mRNAs by RISC leads to their destruction (Hammond, 2001).

To date, mechanistic studies have approached RNAi/PTGS from two standpoints. Genetic studies have identified nearly a dozen genes that affect the dsRNA response. These include genes that encode putative nucleases (mut-7), helicases (qde-3, SDE3, mut-6), RNA-dependent RNA polymerases (e.g., ego-1, qde-1, SDE1/SGS2), and members of the Argonaute family (rde-1, qde-2, AGO1). Biochemical studies, carried out exclusively in extracts from Drosophila embryos and cultured cells, have identified enzymatic activities that are proposed to contribute to the interference process. However, links between biochemical and genetic studies of RNAi have yet to be made (Hammond, 2001 and references therein).

This study reports attempts to identify the proteins and RNAs that carry out RNAi in vitro as a step toward unifying biochemical and genetic data into a single mechanistic model. A ribonuclease III family enzyme, Dicer, is a candidate for processing long dsRNA silencing triggers into ~22-nt siRNAs. A requirement for Dicer in RNAi in vivo has been demonstrated in C. elegans. This study reports the biochemical purification of RISC, the effector nuclease of RNAi, and the identification of one subunit of this enzyme. This protein is a member of the Argonaute family, which has been linked to RNAi through genetic studies in several experimental systems (Hammond, 2001).

RNA interference can be provoked in cultured Drosophila S2 cells by transfection with dsRNA, or indeed by simply adding dsRNA to the culture media. Extracts from such cells contain a nuclease complex, RISC, that specifically degrades mRNAs that are homologous to the dsRNA trigger. The hypothesis that this nuclease constitutes the effector activity of RNAi is strengthened by the observation that RISC cofractionates with ~22-nt RNAs that are derived directly from the silencing trigger. Furthermore, this nuclease contains an essential nucleic acid subunit, which is presumably a siRNA (Hammond, 2001).

A biochemical fractionation protocol has been developed that permits the purification of RISC to near-homogeneity. RISC is bound to ribosomes in cell-free extracts; however, the biological relevance of this association remains to be established. Ribosomes can be concentrated from S2 lysates by high-speed centrifugation, and soluble RISC can be recovered from the ribosome pellet by extraction with high concentrations of salt (Hammond, 2001).

Size fractionation of soluble RISC yielded a single peak of sequence-specific nuclease activity. Thus, a single complex contains all the activities and information needed to identify and degrade cognate mRNAs. The large size of this complex (~500 kD) is consistent with its being composed of several subunits, which, according to previous studies, comprise both RNA and protein. A series of additional chromatographic steps were developed that yielded a fraction with a sequence-specific nuclease activity that was purified ~1:10,000 from the crude extract (Hammond, 2001).

Analysis of fractions from the hydroxyapatite column by SDS-polyacrylamide gel electrophoresis (PAGE) indicates that the complex has not been purified to complete homogeneity; however, several proteins clearly cofractionate with the active RISC fraction. Candidate proteins were excised from the gel and microsequenced using tandem mass spectroscopy. Two of four bands failed to produce protein sequence. However, numerous peptides were obtained from bands of ~87 and ~130 kD that matched a single Drosophila gene. Database and domain searches identified this peptide as a homolog of rde-1, a member of the Argonaute gene family, which is essential for RNAi in C. elegans. This gene has been named Argonaute2 (AGO2, Flybase annotation number CG7439) because of the prior assignment of Argonaute1 to another gene in the Drosophila genome. Although the Drosophila genome contains at least four Argonaute family members -- AGO1, AGO2, Piwi, and Sting, only AGO2 has been idenfied as a component of RISC in S2 cells. However, the possibility that other Drosophila Argonaute family members join the RISC complex in specific tissues or at specific times during development cannot be excluded (Hammond, 2001).

To verify the presence of AGO2 in RISC, AGO2-specific antibodies were generated. Western blotting of chromatography column fractions with affinity-purified anti-AGO2 shows precise cofractionation of a ~130-kD AGO2 protein and the active RISC fraction through each purification step. In addition, the association between AGO2 and other components of RISC, the siRNAs, was tested. A version of AGO2 was constructed that was tagged at its NH₂-terminus with both a T7 epitope and polyhistidine. This was expressed in cells in which RNAi had been induced against firefly luciferase. Tagged AGO2 protein cofractionates with endogenous AGO2, and with the active RISC fraction, in the 500-kD size range. RISC was affinity-purified from cell extracts on a polyhistidine-binding resin. Analysis of the imidazole elution profile from this column by Western blotting with a T7 antiserum and by Northern blotting with a luciferase probe indicates cofractionation of the tagged AGO2 and 22-nt siRNAs. Considered together, these data strongly support the hypothesis that AGO2 is a component of RISC (Hammond, 2001).

To test whether AGO2 is essential for RNAi in Drosophila S2 cells, RNA interference was used to suppress endogenous AGO2. Treatment of S2 cells with either of two different ~1000-nt dsRNAs homologous to AGO2 reduces the levels of this protein by a factor of >10. The ability of these cells to carry out RNAi was assessed by transfection with a mixture of firefly and Renilla luciferase expression plasmids (as an internal control) in combination with either a control dsRNA (green fluorescent protein, GFP) or a firefly luciferase dsRNA. Suppression of AGO2 expression correlates with a pronounced reduction in the ability of cells to silence an exogenous reporter by RNAi (Hammond, 2001).

The biochemical function of Argonaute family members is completely unknown. However, one domain of this protein, the PAZ domain, is shared with Dicer, which initiates RNAi by processing dsRNA silencing triggers into siRNAs. The possibility that Dicer and AGO2 might physically interact, perhaps through their shared PAZ domains, was therefore considered. Indeed, endogenous AGO2 can be coimmunoprecipitated with an epitope-tagged version of Dicer protein from transfected S2 cells. Dicer and RISC are biochemically separable, and none of the purified RISC fractions is able to process dsRNA into 22-nt fragments. One possibility is that Dicer is indeed a component of RISC but fails to process dsRNA when present in this complex. However, the current model is that the interaction between AGO2 and Dicer facilitates the incorporation of siRNAs into RISC complexes, which ultimately dissociate from Dicer and target cognate mRNAs for destruction (Hammond, 2001).

Previous genetic studies in three organisms have indicated that Argonaute family members are essential for RNAi/PTGS. The first link between Argonaute proteins and RNAi was shown by the isolation of C. elegans rde-1 in a screen for RNAi-deficient mutants. In Neurospora, another member of the Argonaute family, QDE-2, emerged from a selection of mutants that were defective in a transgene cosuppression phenomenon, termed 'quelling'. The founding member of this family (AGO1) was first identified in Arabidopsis in a screen for mutants with aberrant leaf morphology. Subsequently, ago1 was re-isolated in a screen for plants that were defective in transgene cosuppression (Hammond, 2001).

Argonaute proteins are typically members of multigene families. In Drosophila there are four annotated genes: Sting, Piwi, AGO1, and AGO2. Mutations in three family members (ago1, piwi, and sting) have previously been studied. Piwi is required for maintenance of cell proliferation in both the male and female germ line, and sting mutations produce spermatid defects and male sterility. Ago1 was identified in a screen for mutations in the wingless pathway, and null mutations in this gene cause defects in neurological development (Hammond, 2001 and references therein).

Thus, Argonaute family members have been linked both to gene silencing phenomena and to the control of development in diverse species. The critical question is whether these two roles of Argonaute proteins are mechanistically related. It is already clear that RNAi-related silencing pathways can control the activity of endogenous genetic elements (e.g., transposons). The possibility also exists that these same pathways may control the expression of endogenous protein-coding genes that regulate development. An answer to this question is likely to emerge both from further genetic studies of RNAi pathways and from a search for endogenous targets of RISC that may be identified via its internal RNA guides to substrate selection (Hammond, 2001).

Biochemical identification of Argonaute 2 as the sole protein required for RNA-induced silencing complex activity

RNA interference is carried out by the small double-stranded RNA-induced silencing complex (RISC). The RISC-bound small RNA guides the RISC complex to identify and cleave mRNAs with complementary sequences. The proteins that make up the RISC complex and cleave mRNA have not been unequivocally defined. This study reports biochemical purification of RISC activity to homogeneity from Drosophila Schnieder 2 cell extracts. Argonaute 2 (Ago-2) is the sole protein component present in the purified, functional RISC. By using a bioinformatics method that combines sequence-profile analysis with predicted protein secondary structure, homology was found between the PIWI domain of Ago-2 and endonuclease V and potential active-site amino acid residues within the PIWI domain of Ago-2 were identified (Rand, 2004).

RNA interference is the phenomenon in which long double-stranded (ds)RNA is able to silence cognate gene expression. First, the dsRNA is processed into small interfering RNA (siRNA) by a Dicer enzyme. In Drosophila cells, siRNA is retained by Dicer-2 and an associated protein, called R2D2. The Dicer-2-R2D2 complex is then able to facilitate the loading of siRNA onto a complex known as the RNA-induced silencing complex (RISC). The RISC-bound siRNA strand acts as a guide to identify mRNA targets with complementary sequence for nucleolytic cleavage. RISC activity is defined simply as siRNA-guided, site-specific cleavage of an mRNA target (Rand, 2004 and references therein).

The guide RNA in RISC is held by the PAZ domain of Argonaute 2. This protein was recognized when RNA interference-defective mutants were found in Caenorhabditis elegans. Ago-2 was also found to correlate with partially purified RISC activity from Drosophila and human cells. Furthermore, siRNAs were demonstrated to copurify with Ago-containing complexes. Finally, the PAZ domain of Ago-2 was determined to be an siRNA-binding domain by structural studies (Rand, 2004 and references therein).

Several characteristic features of RISC nucleolytic activity have been determined. RISC cleaves the target mRNA strand between positions 10 and 11, counted from the 5' to 3' end of the guide RNA. These cleavage products have a 5' phosphate and a 3' hydroxyl group. Finally, RISC activity is magnesium-dependent. Although a long list of proteins have been proposed to be RISC components, none has been identified that accounts for the nuclease activity of RISC (Rand, 2004 and references therein).

A biochemical fractionation approach was used to purify the RISC nuclease activity to homogeneity from Drosophila Schnieder 2 (S2) cells. RISC activity was found to be generated by a single protein, Ago-2. A sequence information comparison tool was used to predict that the PIWI domain of Ago-2 is similar to the endonuclease V in sequence and secondary structure, suggesting that the PIWI domain provides the missing nuclease activity of RISC. An alignment of PIWI domain with several endonucleases was used to predict three residues, aspartate 965 (GADVT), glutamate 1016 (TLEHL), and aspartate 1037 (YRDGV), as being magnesium-coordinating residues at the catalytic center of Ago-2 nuclease. The corresponding two aspartate residues have also been recognized as part of the nuclease active site in mammalian and archaebacterium Pyrococcus furiosus Ago-2 by structural and mutagenesis studies (Rand, 2004).

To study RISC activity, let-7 siRNA was added to S-100 extracts prepared from Drosophila S2 cells and siRNA-dependent cleavage of a complementary mRNA was generated. The sensitivity of RISC activity to high salt (up to 1 M) was analyzed both before and after siRNA addition. High salt exposure after siRNA addition reversibly masked the ability of RISC to cleave target substrate. After the salt was removed by dialysis, full RISC activity was recovered. However, upon exposure of S2 cell extracts to high salt prior to siRNA loading, RISC activity was irreversibly lost; it could not be recovered even after the salt was completely dialyzed away. The difference in salt sensitivity suggested that the assembly of RISC (by addition of siRNA to naive extracts) involves a component (or components) with a molecular conformation that is irreversibly damaged upon salt addition, and it also suggested that the salt-labile, functioning conformation is upstream of the siRNA-loaded RISC. The salt sensitivity made it difficult to purify the components needed for de novo RISC assembly. However, resistance of preloaded RISC to high salt exposure makes it a better subject for protein-purification procedures. Therefore, a procedure was developed for the purification of preloaded RISC nuclease activity to homogeneity (Rand, 2004).

The purification procedure began with the addition of a 3'-biotinylated siRNA to 200 ml of S-100 extract (1 g of total protein) from S2 cells. After incubation at room temperature for 120 min to load the siRNA into RISC, the RISC activity was collected by centrifugation at 200,000 x g. The pelleted RISC activity was then solubilized by extraction with 400 mM potassium acetate. After addition of ammonium sulfate to 20% saturation, the 100,000 x g supernatant, containing RISC activity, was loaded onto a phenyl-Sepharose column and eluted with a decreasing ammonium sulfate/potassium acetate gradient over 10 column volumes. The RISC activity was eluted in a sharp peak around fractions 19-22. The active fractions were pooled, dialyzed to 200 mM KOAc, and loaded onto a Q-Sepharose column. The RISC activity that flowed through this column was subsequently loaded onto a heparin agarose column, and the RISC activity was eluted by a 15%-100% step of 1 M KOAc. The eluate was dialyzed buffer, and the RISC activity was pulled down by using streptavidin-conjugated magnetic beads by overnight incubation with rotation at 4°C in the presence of 1% Triton X-100. The RISC activity pulled down by the beads was then split. An aliquot of 10% of the beads was used for the RISC assay, and the remaining 90% was used for protein identification. Before assaying, the beads were coated with Denhardt's reagent to keep the mRNA substrate from sticking to the beads, a step critical for assaying RISC activity directly on beads. The RISC activity was enriched and pulled down by the beads when biotin-labeled, but not normal, siRNA was used. Because the RISC activity was resistant to high salt, another five washes of high salt were added. The final high-salt wash did not affect the RISC activity on the beads, and the target RNA was completely processed (Rand, 2004).

Protein purifications are normally finished by running an SDS gel electrophoresis to identify correlating bands. However, proteins with unusual sizes, extremely positive charges, low abundance, or those that are poor substrates for staining (for instance, with silver stain) may be lost during this step. All of these potential pitfalls were avoided by directly digesting all of the proteins on the beads with trypsin and subjecting all resulting peptides directly to MS. Interestingly, if the beads were not subjected to the final high-salt wash, many proteins (including previously identified RISC component Ago-2) were identified. Most of other proteins were ribosomal and other RNA-binding proteins. However, when the beads were subjected to high-salt wash before digestion with trypsin, all of the 33 peptides recovered were from Ago-2. Importantly, not a single peptide from any other protein was detected under this condition. Also, all of the nonspecific proteins that were pulled down by the bead when normal siRNA was used for RISC assembly were washed away by high salt. Therefore, it was conclude that one protein, Ago-2, composes the core RISC nuclease activity (Rand, 2004).

Previous attempts to purify RISC nuclease from human and Drosophila systems have identified Ago-2 as well as several other proteins, including dFXMR, VIG, and tudor-SN, all associated with RISC. However, the roles of these proteins in the fundamental activity of RISC (siRNA-guided, site-specific endonucleolytic cleavage) are still unclear. RISC activity has also been purified to near homogeneity from HeLa cell extract. A correlation between only two proteins, eIF2C2 and eIF2C1 (two Ago family members), and RISC activity was seen. This work is especially important because it rules out many of the previously identified RISC-associated proteins as being required for RISC activity, and it suggests that RISC activity might be provided by an Ago family protein alone. It was reasoned that purification of RISC activity to homogeneity judged by the most rigorous standard would provide answers to what the actual composition of the RISC complex was and this led to identification of the RISC nuclease. Surprisingly, Ago-2 is the only detected protein with the recovered peptides covering most of the protein. This result provides strong evidence that Ago-2 alone is sufficient for siRNA-loaded RISC activity (Rand, 2004).

This finding was surprising because there had been no previous indication that Ago-2 had any nuclease domain. After a careful bioinformatics analysis based on sequence and predicted secondary structure, it was realized that the PIWI domain of Ago-2 shows similarity to endonuclease V. This alignment predicts three active-site residues. Two recently published articles reported a nearly identical conclusion from two different experimental systems. In the first article, the structure for the Ago protein from archaebacterium P. furiosus was solved (Song, 2004). The structure revealed that the PIWI domain fold is similar to RNase H, with two conserved active-site residues that fit perfectly with the bioinformatics-based prediction of the active site of PIWI to endonuclease V. Although no structure is available for endonuclease V, sequence analysis strongly suggests that it should possess an RNase H-like fold. RNase H hydrolyzes RNA from an RNA-DNA hybrid ds molecule, whereas endonuclease V nicks DNA near chemically damaged sites on dsDNA substrates. Endonuclease V also has the ability to cleave undamaged single-stranded DNA. Both proteins are identical to the RISC endonuclease in terms of magnesium dependence and the molecular ends produced after cleavage (Rand, 2004).

In the second article, site-directed mutagenesis on two of the structurally predicted and alignment-predicted active-site residues in mammalian Ago-2 protein abolished RISC activity when the mutant proteins were expressed in the Ago-2 knockout mouse embryonic fibroblasts (Liu, 2004). The third Mg2+ coordination site has not yet been experimentally determined. When considering the biochemical purification and the published structure analysis of Ago-2 together, it is clear the siRNA-Ago-2 complex alone is sufficient for RISC activity (siRNA-guided, site-specific cleavage of mRNA targets), with the PIWI domain of Ago-2 functioning as the nuclease (Rand, 2004).

R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway

Although only one Dicer enzyme is found in C. elegans and humans, two (DCR-1 and DCR-2) have been identified in Drosophila. It remains unclear how siRNA is transferred from Dicer to RISC. This problem was studied by purifying the siRNA-generating activity from the cytoplasmic (S100) extract of S2 cells through a six-step chromatographic procedure. A single major peak of activity was observed at all steps and was followed throughout purification. Two proteins, ~190 kD and ~36 kD, showed perfect correlation with the enzymatic activity after the final gel filtration step. They were identified by mass spectrometry to be DCR-2 and a previously unknown protein (Flybase CG7138 or R2D2), respectively. R2D2 bears 20.9% identity and 33.4% similarity to the C. elegans RNAi protein RDE-4, which also contains tandem dsRNA-binding domains and interacts with Dicer (Liu, 2003).

DCR-2/R2D2 not only generates siRNA from dsRNA but also binds to nascent siRNA and facilitates its loading onto RISC. The latter activity is dependent on the dsRNA-binding domains of R2D2. To confirm that DCR-2/R2D2 facilitates siRNA loading onto RISC, the association between AGO2, an essential component of RISC, and a 3'-biotinylated siRNA was followed by precipitation using streptavidin beads. The biotinylated siRNA was as active as unmodified siRNA in inducing RISC activities in S100. However, streptavidin beads precipitated AGO2 protein only when biotinylated siRNA was used, suggesting that the siRNA RISC activity was specific. RISC assays were then performed with the use of biotinylated siRNA in 10% PEG supernatant alone or in combination with recombinant DCR-2, DCR-2/R2D2, and DCR-2/R2D2^M proteins. Consistently, more AGO2 proteins were detected in the biotinylated siRNA precipitates when wild-type DCR-2/R2D2 complex was used instead of DCR-2 alone or the mutant complex. Together, these results indicate that DCR-2/R2D2 not only generates siRNA from dsRNA but also binds to nascent siRNA and facilitates its loading onto RISC. The latter activity is dependent on the dsRNA-binding domains of R2D2 (Liu, 2003).

A protein sensor for siRNA asymmetry

How does the RISC-loading complex (RLC), with the Dcr-2/R2D2 heterodimer positioned asymmetrically on the siRNA, progress to the RISC? Argonaute 2 is a ~130-kD protein that is a core component of the RISC and is required for siRNA unwinding. An ~130-kD protein was crosslinked to siRNA when the guide strand contained 5-iodouracil at p20. The ~130-kD protein was photocrosslinked only to the guide strand of the siRNA, which suggests that this protein is a component of the RISC. The ~130-kD protein was immunoprecipitated with antibodies to Ago2 but not to Ago1 and was not observed in embryos lacking both maternal and zygotic Ago2 (ago2⁴¹⁴). Thus, the ~130-kD protein is Ago2. When R2D2 and Ago2 were photocrosslinked to siRNAs that contain 5-iodouracil at p20 of the passenger or the guide strand, R2D2 is bound to the 3' end of the guide strand and Dcr-2 to the 3' end of the passenger strand at early times in the reaction. Later, binding of R2D2 and Dcr-2 decrease concurrently, accompanied by a corresponding increase in binding of Ago2 to the 3' end of the guide strand. In ago2⁴¹⁴ lysates, R2D2 binding to the 3' end of the guide strand and Dcr-2 binding to the 3' end of the passenger strand did not decrease with time; this finding suggests that binding of Ago2 facilitates the release of the heterodimer from siRNA (Tomari, 2004).

The siRNA bound by Ago2 is single-stranded, because Ago2, when photocrosslinked to siRNA, was captured by a tethered 2'-O-methyl oligonucleotide complementary to the siRNA guide strand. R2D2 is not captured by the 2'-O-methyl oligonucleotide, but is instead recovered in the supernatant, consistent with R2D2 binding of double-stranded siRNA (Tomari, 2004).

The data suggest a model for RISC assembly. Initially, R2D2 orients the Dcr-2/R2D2 heterodimer on the siRNA within the RLC. As siRNA unwinding proceeds, the heterodimer is exchanged for Ago2, the core component of the RISC. Indeed, single-stranded siRNA was not detected in the RLC assembled in ago2⁴¹⁴ lysate. It is hypothesized that unwinding occurs only when Ago2 is available, so that siRNA in the RLC is unwound only when the RISC can be assembled (Tomari, 2004).

Fmr1 is associated with Dicer, Argonaute2 and microRNAs (miRNAs) in vivo, suggesting that Fmr1 is part of the RNAi-related apparatus

In Drosophila, Fmr1 binds to and represses the translation of an mRNA encoding of the microtuble-associated protein Futsch. A Fmr1-associated complex has been isolated that includes two ribosomal proteins, L5 and L11, along with 5S RNA. The Fmr1 complex also contains Argonaute2 (AGO2) and a Drosophila homolog of p68 RNA helicase (Dmp68, otherwise known as Rm62). AGO2 is an essential component for the RNA-induced silencing complex (RISC), a sequence-specific nuclease complex that mediates RNA interference (RNAi) in Drosophila. Dmp68 is also required for efficient RNAi. Fmr1 is associated with Dicer, another essential component of the RNAi pathway, and microRNAs (miRNAs) in vivo, suggesting that Fmr1 is part of the RNAi-related apparatus. These findings suggest a model in which the RNAi and Frm1-mediated translational control pathways intersect in Drosophila. The findings also raise the possibility that defects in an RNAi-related machinery may cause human disease (Ishizuka, 2002).

The identification of AGO2 as a Fmr1-interacting protein is particularly striking. AGO2 is a member of the Argonaute gene family and is an essential component for the RNA-induced silencing complex (RISC), a sequence-specific nuclease complex that mediates RNAi in Drosophila. Therefore, the finding that Fmr1 forms a complex in vivo with AGO2 suggests that Fmr1 may function in RNAi. To test this, RNAi was used to suppress the endogenous proteins, much as had been done previously to establish a role for AGO2 in RNAi. Suppression of ribosomal proteins L5 and L11 with specific dsRNAs made S2 cells so sick that their roles in RNAi could not be assessed. However, treatment of S2 cells with dsRNAs homologous to AGO2, Fmr1, or Dmp68 markedly reduces the levels of these proteins. The ability of these cells to carry out RNAi was tested by transfection with enhanced green fluorescent protein (EGFP) expression plasmid in combination with an EGFP dsRNA. Suppression of AGO2 expression correlates with a pronounced reduction in the ability of cells to silence the reporter EGFP by RNAi. Interestingly, RNAi targeting Dmp68 results in inhibition of RNAi in S2 cells. These results suggest that the DEAD-box helicase Dmp68 not only interacts with Fmr1 but is also required for efficient RNAi in S2 cells. Dmp68 is a Drosophila ortholog of human p68, which has been demonstrated to unwind short but not long dsRNAs in an ATP-dependent manner. It is concluded that at least two of the Fmr1-interacting proteins, AGO2 and Dmp68, are required for RNAi in cultured Drosophila S2 cells. In contrast, depletion of Fmr1 did not appear to affect the EGFP silencing. Therefore, although Fmr1 interacts strongly with AGO2 and Dmp68 in vivo, Fmr1 does not appear to be essential for efficient RNAi (Ishizuka, 2002).

Recent work in numerous organisms has shown that RNAi shares features with a developmental gene regulatory mechanism that involves miRNAs. These small RNAs (siRNAs and miRNAs) are thought to be incorporated into silencing complexes that mediate mRNA destruction during RNAi and translational control during development, respectively. Therefore, it is suggested that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation. AGO2 and Dmp68 are essential for RNAi in Drosophila. However, Fmr1 appears to be a translation repressor. Because Fmr1 interacts with AGO2 and Dmp68 in vivo, it was of interest to examine whether Fmr1 is also present in an AGO2- and/or Dmp68-associated complex. To do this, TAP-tagged AGO2 (AGO2-TAP) or Dmp68 (Dmp68-TAP) were expressed in S2 cells. Cytoplasmic lysate of the cells expressing AGO2-TAP or Dmp68-TAP was prepared and subjected to TAP purifications. In reciprocal assays, endogenous Fmr1 and AGO2 were found to associate with each other. In addition, endogenous AGO2 was copurified with AGO2-TAP. Endogenous Fmr1 and AGO2 were also found to be present in the Dmp68-associated complex. Because AGO2 can be coimmunoprecipitated with Dicer, which initiates RNAi by processing dsRNA silencing triggers into siRNAs, and also processing miRNA precursors into mature miRNAs, the possibility was considered that Fmr1 might also interact physically with Dicer. Indeed, endogenous Dicer can be copurified not only with AGO2-TAP but also with Fmr1-TAP, and it was also shown that Fmr1 remains associated with AGO2 after RNAi induction. It is well established that siRNAs associate with AGO2 during RNAi in S2 cells. Therefore, these results indicate that Fmr1 may be a part of RISC. Finally, analogous to the human AGO2 ortholog (eIF2C2)-associated complex that contains a DEAD-box type RNA helicase and miRNAs, it was of interest to test whether miRNAs are also found in AGO2- and/or Fmr1-associated complexes. RNA molecules copurified with AGO2-TAP or Fmr1-TAP were recovered, dissolved on a 15% polyacrylamide denaturing gel, and subjected to Northern blot analysis. A known miRNA, miR-2b, in Drosophila S2 cells could be detected both in the AGO2- and Fmr1-associated complexes. Together, these data show that Fmr1 is present in a complex with components of RNAi and miRNAs in cultured Drosophila S2 cells (Ishizuka, 2002).

The interaction between Fmr1 and AGO2 remains constant before and after RNAi induction, suggesting that Fmr1 is part of RISC during RNAi. However, there is no evidence to support the notion that RISC formation is induced by treatment of S2 cells with dsRNA. As one of the functions of the RNAi apparatus is to silence transposons and repetitive sequences residing naturally in the Drosophila genome, these cells are therefore likely to be full of pre-formed RISC complexes, irrespective of dsRNA treatment. Therefore, it is possible that Fmr1 is part of the pre-formed RISC complexes and remains to be part of the active RISC after ATP-dependent siRNA unwinding (Ishizuka, 2002).

The involvement of another Fmr1-interacting protein, Dmp68, in RNAi further suggests the close association of Fmr1 with RNAi. The p68 RNA helicase was first identified by cross-reaction with a monoclonal antibody that was originally raised against SV40 large T antigen two decades ago. The helicase plays important roles in cell proliferation and organ maturation and belongs to a large family of highly evolutionarily conserved proteins, the so-called DEAD-box family of putative ATPases and helicases. Recent studies have revealed that several RNA helicases, including mut6, SDE3, mut14 , drh-1, and spindle-E are required for RNAi and related posttranscriptional gene silencing (PTGS) pathways. Dmp68 is similar to, but clearly not an ortholog of these proteins. Therefore, Dmp68 is a novel component of RNAi. Because ATP-dependent unwinding of the siRNA duplex remodels the RISC to generate an active RISC in the RNAi pathway, Dmp68 may mediate the unwinding process. It is also conceivable that Dmp68 may be involved in downstream events such as target RNA recognition, as an RNA chaperone or an RNPase (Ishizuka, 2002).

Induction and suppression of RNA silencing by animal virus infected Drosophila cells

RNA silencing is a sequence-specific RNA degradation mechanism that is operational in plants and animals. Flock house virus (FHV) is both an initiator and a target of RNA silencing in Drosophila host cells and FHV infection requires suppression of RNA silencing by an FHV-encoded protein, B2. These findings establish RNA silencing as an adaptive antiviral defense in animal cells. B2 also inhibits RNA silencing in transgenic plants, providing evidence for a conserved RNA silencing pathway in the plant and animal kingdoms (Li, 2002).

Focus was placed on the flock house virus (FHV) because its B2 gene shares key features, but not sequence similarity, with the plant cucumoviral 2b gene of cucumber mosaic virus (CMV), which encodes a known group of silencing suppressors. Both open reading frame (ORF) 2b and B2 overlap the carboxyl terminal region and occupy the +1 reading frame of the ORF encoding the viral RNA-dependent RNA polymerase and are translated in vivo by a subgenomic mRNA (Li, 2002).

The FHV B2 protein indeed exhibited a potent silencing-suppression activity in the Agrobacterium co-infiltration assay, established in transgenic plants that express green fluorescent protein (GFP). Transient B2 expression prevented RNA silencing of the GFP transgene, leading to a strong and prolonged green fluorescence examined under ultraviolet (UV) illumination, similar to suppression by the cucumoviral 2b proteins. In contrast, a broad red fluorescent zone surrounding the infiltrated patch becomes clearly visible 6 days after infiltration, when the co-infiltrated transgene directs translation of neither 2b nor B2 (Li, 2002).

RNA blot hybridizations confirmed that expression of either protein is associated with high accumulation levels of the GFP mRNA. In addition, the GFP-specific siRNAs, a hallmark of RNA silencing, remain at extremely low levels in the leaves where there is expression of either B2 or 2b. B2 is able to functionally substitute for 2b of cucumber mosaic virus in whole plant infections, as found previously for a CMV 2b homolog. B2 suppression of RNA silencing in plants explains why FHV is able to overcome the RNA silencing defense and establish systemic infections in transgenic plants that express a plant viral protein that facilitates virus cell-to-cell movement (Li, 2002).

The finding that an FHV-encoded protein suppresses RNA silencing in plants suggests a role for RNA silencing in FHV infections of animal hosts. FHV belongs to the Nodaviridae family, members of which naturally infect vertebrate and invertebrate hosts, and Drosophila cells support complete infection cycles of FHV. Infection of Drosophila S2 cells with FHV virions resulted in a rapid appearance of the FHV-specific siRNAs of both positive and negative polarities. Accumulation of the siRNAs trailed that of FHV genomic and subgenomic RNAs, which suggests that the decreased accumulation of FHV RNAs at later stages of FHV infection may be caused by an FHV-specific RNA silencing (Li, 2002).

To investigate this possibility, a full-length FHV RNA1 cDNA clone (pRNA1) was constructed, which, after transfection into S2 cells, directed RNA1 self-replication and transcription of RNA3, the subgenomic mRNA for B2. Depleting the mRNA for Argonaute2 (AGO2) by RNAi, an essential component of the RISC complex, led to a pronounced increase (two- to three-fold) in the accumulation of FHV RNAs 1 and 3, indicating that a functional RNA-silencing pathway naturally restricted FHV accumulation in the host cells. Furthermore, co-transfection of pRNA1 with a dsRNA targeting the 3'-terminal 500 nucleotides of FHV RNA1 completely prevented the accumulation of intact FHV RNA1 in S2 cells. These results collectively demonstrate that FHV is both an initiator and a target of RNA silencing in this animal host (Li, 2002).

Further studies showed that B2 was essential for FHV accumulation in Drosophila cells, which is in contrast to a previous study carried out in nonhost mammalian cells. A B2-knockout mutant of FHV RNA1, referred to as RNA1-Delta B2, which contains point mutations that convert the first and 58th codons of the B2 ORF into serine and stop codons, respectively, failed to accumulate to detectable levels after transfection into S2 cells. This defect was partially trans-complemented (up to 10% of the wild-type level) by co-transfection of a plasmid expressing either B2 or a His-tagged B2. Expression of the His-tagged B2 from the co-transfected plasmid was detected in S2 cells by Western blot analysis using an antibody recognizing the His tag. Reverse transcription-polymerase chain reaction and sequencing revealed that the introduced mutations were stably maintained in the progeny FHV RNAs extracted from infected cells, indicating that B2 was indeed expressed from the co-transfected plasmid rather than from a revertant RNA1 (Li, 2002).

Accumulation of RNA1-Delta B2 in S2 cells was efficiently rescued, up to 50% of the wild-type level, by co-transfection with the AGO2 dsRNAs, either singly or in combination. However, co-transfection with dsRNAs targeting mRNAs of the two Drosophila Dicer genes was not effective under the same conditions. This is possibly due to a more efficient mRNA depletion by RNAi for AGO2 than for Dicer, which is required to process the input dsRNA. Notably, the level of complementation by RNAi of AGO2 was higher than that achieved by the B2-expressing plasmid, although this level was still achieved less efficiently than B2 expression from wild-type RNA1. Therefore, in the absence of B2 expression, FHV RNAs 1 and 3 accumulate to substantial levels when the RISC complex is disrupted by AGO2 depletion. These data confirm the finding that B2 is not required for RNA1 self-replication and indicate that the essential function of B2 for FHV infection of the S2 host cells observed in this study is to suppress RNA silencing that targets FHV RNAs for degradation. Thus, the same protein blocks RNA silencing in both animals and plants, providing the first experimental evidence for a highly conserved RNA silencing pathway in different kingdoms (Li, 2002).

It is known that RNA silencing operates in animals, including mammals. This work demonstrates that infection of Drosophila cells with an RNA virus triggers strong virus RNA silencing and that the same virus is equipped with an effective silencing suppressor essential for infection. These data provide direct evidence that RNA silencing naturally acts as an adaptive antiviral defense in animal cells. The specificity mechanism of this adaptive defense is based on nucleic acid base pairing between siRNA and its target RNA and thus is distinct from cellular and humoral adaptive immunity based on peptide recognition. A prediction from this work is that heterologous sequences inserted into a replicating virus genome will lead to the production of a population of siRNAs capable of silencing other viral and cellular RNAs in trans that are homologous to the insert. Indeed, recent studies have shown that viral sequences inserted in alphavirus vectors give rise to virus resistance in mosquitoes, which is dependent on the inserted RNA sequence rather than on its protein product. It will be of interest to determine if RNA silencing also plays a role in observed protection against mammalian viruses, derived similarly from heterologous expression of RNA sequences from a replicating RNA virus vector (Li, 2002).

Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing

Homology-dependent RNA silencing occurs in many eukaryotic cells. Nodaviral infection triggers an RNA silencing-based antiviral response (RSAR) in Drosophila, which is capable of a rapid virus clearance in the absence of expression of a virus-encoded suppressor. Evidence is presented to show that the Drosophila RSAR is mediated by the RNA interference (RNAi) pathway; the viral suppressor of RSAR inhibits experimental RNAi initiated by exogenous double-stranded RNA and RSAR requires the RNAi machinery. RNAi also functions as a natural antiviral immunity in mosquito cells. Vaccinia virus and human influenza A, B, and C viruses each encode an essential protein that suppresses RSAR in Drosophila. The vaccinia and influenza viral suppressors, E3L and NS1, are distinct double-stranded RNA-binding proteins and essential for pathogenesis by inhibiting the mammalian IFN-regulated innate antiviral response. The double-stranded RNA-binding domain of NS1, implicated in innate immunity suppression, is both essential and sufficient for RSAR suppression. These findings provide evidence that mammalian virus proteins can inhibit RNA silencing, implicating this mechanism as a nucleic acid-based antiviral immunity in mammalian cells (Li, 2004).

The biochemical framework of the core RNA silencing pathway is based mostly on the experimental induction of RNA silencing by exogenous dsRNA, commonly referred to as RNAi. Thus, an investigation was carried out to see whether B2 of FHV, known to inhibit RNA silencing induced by viral RNA replication, also suppresses RNAi. To this end, Drosophila cells were transfected with a plasmid encoding either GFP or B2 fused with GFP (B2-GFP) under the control of an inducible promoter, and 1 day after induction of expression of GFP or B2-GFP, dsRNA targeting the GFP coding sequence was introduced. B2-GFP remained active in suppressing virus RNA silencing because cotransfection with either pB2-GFP or pB2 rescued the in vivo accumulation of the self-replicating FHV RNA1 mutant carrying an untranslatable B2, called FR1-DeltaB2. Suppression of AGO2 expression by the specific dsRNA also rescued the accumulation of FR1-DeltaB2 RNAs. In the GFP RNAi assay, targeting of the B2-GFP mRNA by RNAi occurred in cells that were also expressing B2-GFP. Northern blot hybridizations found that accumulation of GFP mRNA was significantly reduced 2 days after GFP dsRNA was introduced as compared with treatment with lacZ dsRNA, indicating an effective RNAi against GFP mRNA by this sequential transfection protocol. However, no obvious difference in the accumulation of B2-GFP mRNA was observed between cells treated with either GFP or lacZ dsRNA. Thus, the specific degradation of mRNA targeted by a homologous dsRNA did not occur in Drosophila cells expressing the B2 fusion protein, establishing FHV B2 as a viral suppressor of RNAi in an animal system (Li, 2004).

When GFP dsRNA was cotransfected with pB2-GFP, however, B2-GFP mRNA was effectively destroyed by RNAi. Thus, RNAi suppression requires B2 expression before dsRNA is introduced into cells to initiate RNAi, suggesting that B2 suppression may occur before the production or RISC loading of siRNAs. Collectively, these data show that RNA silencing triggered by either dsRNA or viral RNA replication not only is AGO2-dependent, but also is sensitive to B2 suppression, providing compelling evidence that the RNA silencing antiviral immunity detected in Drosophila cells is mediated by the RNAi pathway (Li, 2004).

Whether the RNA silencing antiviral response is elicited when Drosophila cells are challenged with another virus was investigated. NoV is the only member of the Alphanodavirus genus that can lethally infect both insects and mammals. However, the sequence identity between the encoded proteins of NoV and FHV is either low (44% for the viral RdRP) or minimal (<19% for B2). A full-length NoV RNA1 cDNA clone (pNR1) was constructed encoding a self-replicating RNA. After transfection into the Drosophila S2 cells and transcriptional induction of the viral cDNA, pNR1 directed RNA1 self-replication and transcription of RNA3, the subgenomic mRNA for B2. A point mutation, as in NR1-DeltaB1, that abolished translation of the overlapping B1 ORF from RNA3, corresponding to the C-terminal portion of the viral RdRP, had no effect on the accumulation of either RNA1 or RNA3. However, a B2-knockout mutant, referred to as NR1-DeltaB2, was hardly detectable in transfected Drosophila cells by Northern blot hybridizations. NR1-DeltaB2 contained point mutations that prevented B2 translation but did not change any amino acid in the -1 reading frame that codes for RdRP. The defect of NR1-DeltaB2 accumulation in WT Drosophila cells was complemented in trans by cotransfection of a plasmid expressing the NoV B2 protein (nB2), indicating that nB2 is required for NoV RNA accumulation (Li, 2004).

Several lines of evidence indicate that NoV RNAs were targeted for silencing by the AGO2-dependent RNAi pathway in Drosophila cells and that nB2 suppressed the RNAi antiviral response to ensure successful NoV RNA replication and transcription. (1) The FHV B2 protein (fB2), shown to suppress RNAi, rescued accumulation of NR1-DeltaB2 in Drosophila cells. (2) Depleting RISC by either dsRNA or siRNA targeting AGO2 efficiently rescued accumulation of NR1-DeltaB2. Such a rescue was not observed by cotransfection with an unrelated lacZ siRNA or dsRNA. These results indicate that rescue of NR1-DeltaB2 by cotransfection of dsRNA and siRNA targeting AGO2 is caused by a specific AGO2 depletion, rather than a nonspecific effect of dsRNA. Similar specific rescue of FR1-DeltaB2 in Drosophila cells by either the dsRNA or siRNA targeting AGO2, but not by the dsRNA and siRNA targeting lacZ, was also observed. In addition, nB2 suppressed RNA silencing targeted against both FR1-DeltaB2 in transfected Drosophila cells and a GFP transgene in transgenic plants (Li, 2004).

Remarkably, an effective suppression was found of RSAR targeting FR1-DeltaB2 in Drosophila by the tombusvirus 19-kDa protein (p19), but not after it was truncated . This finding demonstrates cross-kingdom suppression of RNA silencing in an animal system by a plant viral suppressor. Together with the observations that the B2 proteins encoded by two animal nodaviruses suppress RNA silencing in both Drosophila and tobacco plants, these results show that the RSAR mechanism is conserved between the plant and animal kingdoms (Li, 2004).

Because the genome of the mosquito A. gambiae, which transmits both malaria and viruses, encodes a functional RNAi pathway similar to Drosophila, whether RNAi also protects A. gambiae against NoV was investigated. Self-replication of NoV RNA1 and transcription of RNA3 were detected in cells after transfection with pONR1, which contained the NoV RNA1 cDNA under the control of the OplE2 promoter. pNR1 failed to initiate RNA replication in A. gambiae cells, possibly because of a lack of transcriptional induction. Neither RNA1 replication nor RNA3 transcription was detected in A. gambiae cells transfected with pONR1-DeltaB2, which encoded the B2-knockout mutant of NoV RNA1. However, the defect was rescued by cotransfection with either a plasmid expressing a B2 or a dsRNA corresponding to mRNA of the A. gambiae AGO2. The rescue of ONR1-DeltaB2 was also observed by cotransfection with an siRNA targeting AGO2, although with an efficiency ~50% lower than the long dsRNA. ONR1-DeltaB2 rescue by AGO2 depletion was specific, since cotransfection with dsRNA to neither lacZ nor AGO1 mRNA was effective. Thus, as found in Drosophila, a self-replicative NoV RNA also triggered the RNAi antiviral response in A. gambiae cells, which is both AGO2 dependent and sensitive to B2 suppression, establishing this immunity pathway in two different insect cell lines. This finding opens up the possibility of targeting this pathway to prevent transmission of mosquito-borne human viral diseases such as dengue (Li, 2004).

To facilitate screening for new animal RNAi suppressors, the coding sequence of GFP was fused in-frame with the start of the B2 ORF of FHV RNA1 to yield pFR1gfp. pFR1gfp was essentially a B2-knockout mutant carrying a visual marker because only the first 23 aa of the 106-aa residue fB2 were translated in the fusion protein. Indeed, GFP cells were not visible after pFR1gfp was transfected alone but became abundant when it was cotransfected with a B2-expressing plasmid. GFP expression was detected also when pFR1gfp was cotransfected with the AGO2 dsRNA. Replication and accumulation of FR1gfp in S2 cells cotransfected with either the AGO2 dsRNA or the fB2-expressing plasmid were confirmed by Northern blot detection of both positive- and negative-strand viral RNAs. Thus, FR1gfp was defective in suppressing RSAR in Drosophila cells as was FR1-DeltaB2. Importantly, B2 suppression of the Drosophila RNA silencing response targeting either FR1gfp or FR1-DeltaB2 did not require prior B2 expression, which was found to be necessary for RNA silencing induced by dsRNA. This finding is consistent with the natural situation in which the expression of B2 after viral RNA replication but early in infection is sufficient to allow productive FHV infection and establishes an easy cell-based assay for the identification of animal RNAi suppressors, simply by detection of GFP after cotransfection of pFR1gfp with a plasmid expressing a candidate protein (Li, 2004).

Molecular requirements for RNA-induced silencing complex assembly in the Drosophila RNA interference pathway

Complexes in the Drosophila RNA-induced silencing complex (RISC) assembly pathway can be resolved using native gel electrophoresis, revealing an initiator called R1, an intermediate called R2, and an effector called R3 (now referred to as holo-RISC). R1 forms when the Dicer-2/R2D2 heterodimer binds short interfering RNA (siRNA) duplexes. The heterodimer, alone, can initiate RISC assembly, indicating that other factors are dispensable for initiation. During assembly, R2 requires Argonaute2 to convert into holo-RISC. This requirement is reminiscent of the RISC-loading complex (RLC), which also requires Ago2 for assembly into RISC. R2 was compared to the RLC and the two complexes are similar in their sensitivities to ATP and to chemical modifications on siRNA duplexes, indicating that they are likely to be identical. The requirements for RISC formation were examined; the siRNA 5' termini are repeatedly monitored during RISC assembly, first by the Dcr-2/R2D2 heterodimer and again after R2 formation, before siRNA unwinding. The 2' position of the 5'-terminal nucleotide also affects RISC assembly because an siRNA strand bearing a 2'-deoxyribose at this position can inhibit the cognate strand from entering holo-RISC; in contrast, the 2'-deoxyribose-modified strand has enhanced activity in the RNA interference (RNAi) pathway (Pham, 2005).

To act as silencing triggers, double-stranded siRNAs must be channeled through an ordered RISC assembly pathway that results in the selection of one strand and the destruction of the other. The results indicate that siRNA ends are recognized at multiple steps in the pathway and that these recognition events determine whether the siRNAs will become incorporated into the RISC or blocked from further assembly. The first recognition event occurs at initiation, when Dcr-2 and R2D2 bind the siRNA to form the RDI complex. RDI formation requires 5'-phosphorylated siRNA; however, the siRNA need not be phosphorylated on both strands. siRNAs bearing a single 5'-phosphate group are ineffective at guiding mRNA cleavage. This is true for siRNAs phosphorylated only on the guiding strand as well as those phosphorylated only on the nonguiding strand. To explain these observations, a model has been involked in which R2D2 acts as a sensor for siRNA asymmetry. It has been suggest that a 5' phosphate on the thermodynamically disfavored 'passenger' strand is required for stable R2D2 binding, facilitating siRNA incorporation into the RLC and the RISC (Pham, 2005 and references therein).

When only one of two siRNA strands is phosphorylated, R2D2 and Dcr-2 can still avidly engage the siRNA, probably with R2D2 located at the phosphorylated end as predicted. Despite this, singly phosphorylated siRNA duplexes are still ineffective silencing triggers. The data indicate that a second 5'-recognition event occurs after R2D2 binding. RNA unwinding and activation can proceed, but only if the siRNA bears the second 5' phosphate at the end not occupied by R2D2. Since Ago2 is required both for siRNA unwinding and target mRNA cleavage, it may be the factor that recognizes the second 5' phosphate. An archaebacterial PIWI protein has been shown to have conserved residues that interact with the 5'-phosphate group of siRNA-like duplexes. Furthermore, when analogous residues were mutated in the human Ago2 PIWI domain, target mRNA cleavage is compromised. These results seem to implicate Ago2 in the second 5' recognition event. Alternatively, Ago2 may not engage the 5' phosphate until later in the assembly pathway. In this case, some other factor may interact with the second 5' phosphate upstream of Ago2, causing the RISC assembly defect that is observed with singly phosphorylated siRNA duplexes (Pham, 2005).

Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes

In the Drosophila and mammalian RNA interference pathways, siRNAs direct the protein Argonaute2 (Ago2) to cleave corresponding mRNA targets, silencing their expression. Ago2 is the catalytic component of the RNAi enzyme complex, RISC. For each siRNA duplex, only one strand, the guide, is assembled into the active RISC; the other strand, the passenger, is destroyed. An ATP-dependent helicase has been proposed first to separate the two siRNA strands, then the resulting single-stranded guide is thought to bind Ago2. This study shows that Ago2 instead directly receives the double-stranded siRNA from the RISC assembly machinery. Ago2 then cleaves the siRNA passenger strand, thereby liberating the single-stranded guide. For siRNAs, virtually all RISC is assembled through this cleavage-assisted mechanism. In contrast, passenger-strand cleavage is not important for the incorporation of miRNAs that derive from mismatched duplexes (Matranga, 2005).

The data suggest a new model for Ago2 RISC assembly in which siRNAs are initially loaded into Ago2 as duplexes, and then Ago2 cleaves the passenger strand of the siRNA, facilitating its displacement and leaving the siRNA guide strand bound stably to Ago2. In support of this cleavage-assisted assembly model, it was found that the passenger strand is cleaved during RISC assembly. Both the position of the cleavage site on the passenger strand and the stereospecificity of inhibition by phosphorothioates are diagnostic of siRNA-directed, Ago2-catalyzed cleavage. Both Ago2 and the RISC-loading complex (RLC) are required for this cleavage, and cleavage occurs before the passenger strand has dissociated from the guide strand -- before the formation of active RISC (Matranga, 2005).

A cleavage-assisted assembly mechanism is also consistent with the finding that early (5 min) in assembly, RISC contains considerable amounts of double-stranded siRNA. When the asymmetry rules for siRNA loading were first uncovered, they evoked the idea of a nonprocessive helicase that separated the two strands of the duplex, starting from the end that was less stably paired. However, subsequent studies showed that the R2D2/Dcr-2 heterodimer, the core of the RLC, binds asymmetrically to double-stranded siRNA, suggesting that a nonprocessive helicase does not sense siRNA thermodynamic asymmetry during RISC loading. Unlike Ago2 loaded via the RISC assembly pathway, affinity-purified or recombinant Ago2 can only be programmed with single-, not double-stranded siRNA, suggesting that the essential function of the RLC is to facilitate the directional loading of a double-stranded siRNA into Ago2. The idea that Ago2-mediated passenger-strand cleavage triggers siRNA strand dissociation also accounts for the previous observation that one passenger strand appears to be destroyed for every cycle of assembly of a guide strand into target-cleaving RISC. Passenger-strand cleavage would strongly reinforce siRNA functional asymmetry by coupling passenger-strand destruction to RISC assembly (Matranga, 2005).

Blocking Ago2-mediated passenger-strand cleavage by substituting a phosphorothioate for the scissile phosphate revealed a slower bypass mechanism that dissociates the two siRNA strands. Of course, phosphorothioate-substituted siRNAs do not occur in nature, but miRNA/miRNA* duplexes do often contain central mismatches predicted to block cleavage of the miRNA* strand, the analog of the siRNA passenger strand. Most, if not all, miRNAs are efficiently loaded into Ago2 in cultured human cells, and miR-127, miR-136, miR-196, miR-431, miR-433, and miR-434 are known to cleave their targets in vivo and are therefore presumed to function in an Ago2-containing RIS CMatranga, 2005).

It is envisioned that in the bypass pathway -- as in the cleavage-assisted pathway -- Argonaute proteins are loaded with double- rather than single-stranded siRNAs. Dissociation of the full-length passenger strand would then require breaking its interaction with the 'seed' region of the guide strand (nucleotides 2-7), a region proposed to mediate miRNA target pairing. For metazoan miRNAs, conserved Watson-Crick pairing to the seed is necessary and sufficient for accurate target prediction. Similarly, Watson-Crick pairing to the seed can be sufficient for miRNA-mediated repression, and pairing to this 5' region of the guide strand makes a far greater contribution to target binding affinity than does pairing to the 3' end. The tight binding to the 5' portion of the guide strand is what presumably prevents an appreciable amount of a standard siRNA duplex from being assembled into Ago2 through the bypass mechanism. In contrast, when presented to Ago2 as a miR-1/miR-1* duplex, miR-1 loads efficiently without need for passenger-strand cleavage, because a Watson-Crick pairing to the miRNA seed is disrupted. Indeed, far less disruption of seed pairing might be sufficient to enable the bypass mechanism to begin to play a substantial role, as hinted by results using the miR-1 'GU' siRNA, which has a single G:U wobble disrupting perfect Watson-Crick pairing to the seed. Passenger-strand cleavage appeared less important for this duplex than for the standard siRNA duplex, a result consistent with computational and experimental studies of miRNA target specificity imply that single G:U wobble pairs in the seed disproportionately perturb small RNA binding (Matranga, 2005).

In addition to loading miRNAs, the bypass mechanism might be used to load siRNAs into Argonaute proteins that have lost their catalytic amino acids, such as human Ago1, Ago3, and Ago4. Conversely, Arabidopsis thaliana Argonaute1, a miRNA-guided plant Argonaute protein with a functional endonuclease domain (Baumberger and Baulcombe, 2005 and Qi, 2005), might be loaded by a passenger-strand cleavage-assisted pathway. Although human Ago1 and Ago3 can bind standard siRNAs, endogenous Ago1 and Ago3 cannot support siRNA-directed RNAi in Ago2-knockout mouse embryonic fibroblasts. The function of Ago1, Ago3, and Ago4-containing RISC is not yet known. Perhaps the capacity for loading small RNAs via the cleavage-assisted pathway confers a degree of specificity to the function of different silencing complexes, with Ago2 able to use a broader range of small RNAs than those Argonaute proteins incapable of RNA cleavage (Matranga, 2005).

The mRNA-cleavage step of RNA interference is mediated by an endonuclease, Argonaute2 (Ago2), within the RNA-induced silencing complex (RISC). Ago2 uses one strand of the small interfering (si) RNA duplex as a guide to find messenger RNAs containing complementary sequences and cleaves the phosphodiester backbone at a specific site measured from the guide strand's 5' end. Both strands of siRNA get loaded onto Ago2 protein in Drosophila S2 cell extracts. The anti-guide strand behaves as a RISC substrate and is cleaved by Ago2. This cleavage event is important for the removal of the anti-guide strand from Ago2 protein and activation of RISC (Rand, 2005).

Slicer function of Drosophila Argonautes and its involvement in RISC formation

Argonaute proteins play important yet distinct roles in RNA silencing. Human Argonaute2 (hAgo2) was shown to be responsible for target RNA cleavage ('Slicer') activity in RNA interference (RNAi), whereas other Argonaute subfamily members do not exhibit the Slicer activity in humans. In Drosophila, AGO2 was shown to possess the Slicer activity. Here it is shown that AGO1, another member of the Drosophila Argonaute subfamily, immunopurified from Schneider2 (S2) cells associates with microRNA (miRNA) and cleaves target RNA completely complementary to the miRNA. Slicer activity is reconstituted with recombinant full-length AGO1. Thus, in Drosophila, unlike in humans, both AGO1 and AGO2 have Slicer functions. Further, reconstitution of Slicer activity with recombinant PIWI domains of AGO1 and AGO2 demonstrates that other regions in the Argonautes are not strictly necessary for small interfering RNA (siRNA)-binding and cleavage activities. It has been shown that in circumstances with AGO2-lacking, the siRNA duplex is not unwound and consequently an RNA-induced silencing complex (RISC) is not formed. Upon addition of an siRNA duplex in S2 lysate, the passenger strand is cleaved in an AGO2-dependent manner, and nuclease-resistant modification of the passenger strand impairs RISC formation. These findings give rise to a new model in which AGO2 is directly involved in RISC formation as 'Slicer' of the passenger strand of the siRNA duplex (Miyoshi, 2005).

Genome-wide analysis of mRNAs regulated by Drosha and Argonaute proteins in Drosophila melanogaster

RNA silencing pathways are conserved gene regulation mechanisms that elicit decay and/or translational repression of mRNAs complementary to short interfering RNAs and microRNAs (miRNAs). The fraction of the transcriptome regulated by these pathways is not known, but it is thought that each miRNA may have hundreds of targets. To identify transcripts regulated by silencing pathways at the genomic level, mRNA expression profiles were examined in Drosophila melanogaster cells depleted of four Argonaute paralogs (i.e., AGO1, AGO2, PIWI, or Aubergine) that play essential roles in RNA silencing. Cells depleted of the miRNA-processing enzyme Drosha were also examined. The results reveal that transcripts differentially expressed in Drosha-depleted cells have highly correlated expression in the AGO1 knockdown and are significantly enriched in predicted and validated miRNA targets. The levels of a subset of miRNA targets are also regulated by AGO2. Moreover, AGO1 and AGO2 silence the expression of a common set of mobile genetic elements. Together, these results indicate that the functional overlap between AGO1 and AGO2 in Drosophila is more important than previously thought (Rehwinkel, 2006).

Using microarray analysis of Drosophila cells depleted of Drosha and Argonaute proteins, this study shows that transcripts whose levels are likely to be directly regulated by silencing pathways (up-regulated transcripts) represent less than 20% of the Drosophila S2 cell transcriptome. Computational predictions of miRNA targets indicate that more than 30% of the transcriptome is targeted by miRNAs. There are several possible explanations for these seemingly contradictory observations. First, it was shown that not all authentic targets change levels in a detectable manner. This indicates that although microarrays are a valuable tool to identify miRNA targets, many targets may escape detection using this approach. Second, some miRNAs and targets are expressed in a tissue-specific manner, so it is likely that only a subset of miRNA/target pairs is expressed in S2 cells. Finally, current models of miRNA function suggest that miRNAs expressed in a given cell type target transcripts that are already expressed at low levels but avoid housekeeping genes or genes that are expressed in these cells at high levels. These targets may escape detection by microarray analysis. Nevertheless, among transcripts regulated by the Argonaute proteins several were found that are expressed at relatively high levels, suggesting that miRNAs not only silence the expression of undesirable, low-abundance transcripts but may also play a role in fine-tuning the expression of abundant mRNAs (Rehwinkel, 2006).

AGO1 and AGO2 are thought to have nonoverlapping functions in Drosophila. This study shows that these proteins regulate the expression levels of a common set of miRNA targets. The observation that Drosha also regulates these transcripts strongly supports the idea that regulation is mediated by miRNAs. In agreement with this, it was observed that AGO2 can associate with endogenous miRNAs, although less efficiently than does AGO1. In this way, AGO2 may also regulate the expression levels of a subset of miRNA targets. Nonetheless, when miRNA function were assayed by overexpressing miRNAs together with luciferase-based mRNA reporters, it was observed that miRNA-mediated translational repression requires AGO1 but not AGO2. It is therefore possible that in this assay the fraction of miRNAs incorporated into AGO2-containing RISC is too small to observe changes in the expression levels of the reporter. Dicer-1 is involved in miRNA biogenesis and is also required for the assembly of RISC complexes, so these observations suggest that Dicer-1 may load AGO2-containing RISCs with miRNAs, at least to some extent (Rehwinkel, 2006).

A partial functional overlap between AGO1 and AGO2 is also suggested by the observation that these proteins regulate the expression of a common set of transposable elements. It remains, however, to be established whether this regulation occurs via similar mechanisms and whether it happens at the transcriptional or posttranscriptional level (Rehwinkel, 2006).

Apart from the common regulated transcripts, transcripts regulated exclusively by AGO2 but not by Drosha or AGO1 have also been identified, suggesting that AGO2 may regulate the expression of these transcripts by an miRNA-independent mechanism that might involve endogenous siRNAs (Rehwinkel, 2006).

The levels of hid and reaper mRNAs (two experimentally validated miRNA targets increase in cells in which the miRNA pathway is impaired. Moreover, by analyzing changes in mRNA levels, additional miRNA targets have been identified and validated in Drosophila. The observation that miRNA targets change levels following inhibition of the miRNA pathway lends further support to the idea that miRNAs can reduce the levels of the targeted transcripts and not just the expression of the translated protein. Along these lines, it has recently been shown that miRNAs can trigger a strong reduction in target levels in C. elegans. Among the 136 core transcripts, 21% are between 1.5- and 2-fold up-regulated, 73% exhibited changes in the 2- to 5-fold range, and 6% were at least 5-fold up-regulated in AGO1-depleted cells. Thus, although changes in transcript levels can be used to validate miRNA targets, the effects can be modest and, as mentioned above, not all targets can be identified using this approach (Rehwinkel, 2006).

In human cells, the Argonaute proteins localize to P-bodies. These are specialized cytoplasmic foci in which the enzymes involved in mRNA degradation in the 5'-to-3' direction colocalize (e.g., the DCP1:DCP2 decapping complex and the 5'-to-3' exonuclease XRN1. In addition, mRNA decay intermediates, miRNA targets, and miRNAs have been observed in P-bodies, suggesting a functional link between P-bodies and RNA silencing pathways. Consistent with this, it has been shown that P-body components play a crucial role in silencing pathways. In particular, the RNA-binding protein GW182 (a P-body component in metazoa) and the DCP1:DCP2 decapping complex are required for miRNA-mediated gene silencing in Drosophila cells. Likewise, human GW182 plays a role in silencing mediated by miRNAs and siRNAs. Finally, the C. elegans protein AIN-1, which is related to GW182, is also required for regulation of a subset of miRNA targets. Together with the observation that miRNAs inhibit cap-dependent but not cap-independent translation initiation, these observations suggest a model in which miRNA targets are stored in P-bodies after translation inhibition, where they are maintained in a silenced state by associating with proteins that prevent translation or possibly by removal of the cap structure. Decapping or simply the storage of miRNA targets in P-bodies may make these mRNAs susceptible to degradation, providing a possible explanation for the reduction in mRNA levels. In agreement with this, depletion of a 5'-to-3' exonuclease in C. elegans partially restores the levels of miRNA targets (Rehwinkel, 2006).

Nevertheless, not all authentic miRNA targets change expression levels. Thus, it is possible that the extent of the degradation depends on the number of miRNA binding sites and/or the stability of the miRNA:mRNA duplexes. It is also possible that the rate of mRNA decay triggered by miRNAs for some targets does not exceed the rate of transcription and that thus the steady-state levels of these targets remain unchanged. It would therefore be of interest to determine whether miRNAs generally cause a reduction in the half-life of targeted transcripts (Rehwinkel, 2006).

Drosophila argonaute1 and argonaute2 employ distinct mechanisms for translational repression

microRNAs induce translational repression by binding to partially complementary sites on their target mRNAs. An in vitro system was established that recapitulates translational repression mediated by the two Drosophila Argonaute (Ago) subfamily proteins, Ago1 and Ago2. Ago1-RISC (RNA-induced silencing complex) was shown to represses translation primarily by ATP-dependent shortening of the poly(A) tail of its mRNA targets. Ago1-RISC can also secondarily block a step after cap recognition. In contrast, Ago2-RISC competitively blocks the interaction of eIF4E with eIF4G and inhibits the cap function. The finding that the two Ago proteins in flies regulate translation by different mechanisms may reconcile previous, contradictory explanations for how miRNAs repress protein synthesis (Iwasaki, 2009).

This study shows that in flies both Ago1 and Ago2 can induce translational repression. These findings contradict a previous proposal that only Ago1 can repress translation (Förstemann, 2007). This notion was based on the observation that in S2 cells a central-bulged reporter for endogenous miR-277, which partitions into both Ago1 and Ago2, was derepressed when Ago1, but not Ago2, was absent. However, in flies Ago1- and Ago2-RISC-loading pathways compete in vitro and in vivo; when one Ago is absent, another Ago can function more strongly with more small RNAs loaded. Because the translational repression by Ago1 is stronger, up to ∼8-fold repression for Ago1 and up to ∼2.5-fold repression for Ago2 in an in vitro system) and presumably more irreversible than that of Ago2, it is possible that, in the case of miR-277, the Ago1-mediated repression was enhanced to compensate for the loss of Ago2. In contrast, this study programmed Ago1-RISC or Ago2-RISC in an essentially exclusive manner, which allowed discrimination of their functions (Iwasaki, 2009).

Drosophila Ago1 shortens the poly(A) tail of the target mRNA, and miRNA-directed deadenylation has been reported in other organisms. Because miRNAs can shorten the poly(A) tail even when translation is blocked, deadenylation is not a consequence of translational repression, but a process independent of it. This study found that deadenylation by Ago1 requires ATP, even though ATP is dispensable for the cleavage activity of Ago1-RISC and for the association of Ago1 with GW182, Pop2/Caf1, and Ccr4. Deadenylation catalyzed by the Ccr4-Not complex is per se ATP independent. Thus, the ATP-dependent step after target recognition remains to be identified for Ago1-directed mRNA deadenylation. Such ATP-dependent deadenylation is reminiscent of regulation of the Drosophila mRNA nanos (Iwasaki, 2009).

Kinetic modeling analyses have shown that differences in the rate-limiting steps of translation can have large effects on the outcome of repression. The current data show that poly(A)⁻ mRNAs are less efficiently repressed by Ago1 compared with poly(A)⁺ mRNAs, agreeing with previous reports on miRNA-mediated translational repression in mammals. This can be simply explained by the fact that poly(A)⁺ mRNAs are better translated than poly(A)⁻ mRNAs. At the same time, this is also consistent with the idea that poly(A) and poly(A)-binding protein (PABP) influence the rate-limiting steps in translation, which can affect the ability of repressors to limit translation. Indeed, PABP is involved in multiple key steps of translation initiation, including cap recognition by eIF4E, 48S, and 80S formation and ribosome recycling. Some literature concludes that poly(A) tail is hardly required for miRNA-mediated repression, but the reason for the apparent discrepancy on the poly(A) dependence of repression is obscure. Perhaps repression at a step after cap recognition is significantly stronger and/or translation itself is less dependent on the poly(A) length under these assay protocols compared with other protocols. Such variations can be caused by a number of factors, including time scale of analysis and stoichiometry of RISC, target mRNA, and translational machinery (Iwasaki, 2009).

Most importantly, the current data show that, even when the same protocols and substrates were used (i.e., under conditions with identical rate-limiting steps in translation), there are striking differences between Ago1- and Ago2-mediated translational repression. (1) Repression by Ago1 is accompanied by deadenylation of the target mRNA, whereas repression by Ago2 has no such effect on the target mRNA. (2) Ago1 requires GW182 for translational repression and ATP-dependent deadenylation, whereas Ago2 represses translation independent of GW182. (3) Ago1-RISC blocks a step after cap recognition, whereas Ago2-RISC binds to eIF4E and specifically blocks eIF4E-eIF4G interaction. Of note is that the affinity of Ago2 to eIF4E is dramatically enhanced when Ago2-RISC binds to a target mRNA, which should, in theory, ensure that the Ago2-mediated repression is limited to translation of the cognate target. Because Ago2 exerts no influence on the target mRNA quantitatively and qualitatively, it is tempting to speculate that Ago2 provides rapid, short-lived repression that preserves the target mRNA, allowing its expression to be reactivated later, whereas repression mediated by Ago1 is more irreversible in cells where deadenylation triggers mRNA decay. Although it is not known whether mammalian Ago proteins similarly act by distinct mechanisms, these findings may reconcile previous, contradictory explanations for how miRNAs repress protein synthesis (Iwasaki, 2009).

Direct role for Hsp90 in pre-RISC formation in Drosophila

Heat-shock proteins (Hsps) are molecular chaperones that control protein folding and function. Argonaute 2 (Ago2), the effector in RNA interference (RNAi), is associated with Hsp90; however, its function in RNAi remains elusive. This study shows that Hsp90 is required for Ago2 to receive the small interfering RNA (siRNA) duplex from the RNA-induced silencing complex-loading complex in RNAi, suggesting a model where Hsp90 modifies Ago2 conformation to accommodate the siRNA duplex (Miyoshi, 2010).

In RNA interference (RNAi), small interfering RNA (siRNA) is associated with Argonaute 2 (Ago2) and guides the protein to its target mRNAs for silencing. In Drosophila melanogaster, siRNAs are processed from long double-stranded RNA precursors by Dicer2. Upon processing, siRNAs are still in a duplex form and are associated with Dicer2 and R2D2, which are major components of the RNA-induced silencing complex (RISC)-loading complex (RLC). siRNA duplexes are then transferred to Ago2 from the RLC to form the precursor of the RISC, or pre-RISC. It has been shown that pre-RISC formation occurs in an ATP-dependent manner. However, it remains unclear why ATP is needed for this process (Miyoshi, 2010).

In the pre-RISC, siRNA duplexes are 'unwound' by Ago2 endonuclease or Slicer activity; the passenger strand of the duplexes is cleaved by Slicer and displaced from Ago2. This step is known to occur in an ATP-independent manner. The guide strand of the duplex remains associated with Ago2. The resultant Ago2-siRNA complex is termed RISC and is now active and ready to bind and cleave target RNAs. In this way, genes targeted by the RNAi machinery are effectively silenced. Ago2 frequently co-purifies with heat-shock protein (Hsp) 90, a chaperone whose activity depends on ATP. Thus, Hsp90 may have a role in RNAi. However, the functional involvement of Hsp90 at the molecular level in RNAi remains unclear (Miyoshi, 2010).

To determine for which step Hsp90 is required in RNAi, the RNAi pathway was dissected into multiple steps and assays were performed for the individual steps in the presence or absence of geldanamycin, the specific Hsp90 inhibitor that mimics ATP binding with the protein. In siRNA processing assays, Flag-tagged Dicer2 isolated from S2 cells was incubated with ³²P-labeled dsRNA precursors with or without geldanamycin. The ability of Dicer2 to excise siRNA duplexes from the precursors was not affected by geldanamycin. Thus, Hsp90 is not necessary for siRNA excision from the precursor (Miyoshi, 2010).

Next, whether the siRNA-unwinding step in RNAi requires Hsp90 function was examined. siRNA duplexes, which were labeled with ³²P at the 5′ end of the guide siRNA within the duplexes, was incubated in S2 lysates with or without geldanamycin. In the presence of geldanamycin, siRNA duplexes remained as duplexes even after 1 h incubation. DMSO, an organic solvent used for dissolving geldanamycin in the lysates, did not affect the activity. Thus, Hsp90 is required for unwinding siRNA duplexes into single-stranded siRNAs (Miyoshi, 2010).

It was then speculated that the RISC would not be assembled in S2 lysates when Hsp90 was inhibited. To test this, RISC formation assays were performed. Whereas DMSO alone did not show any obvious effect, geldanamycin severely inhibited RISC formation. RLC formation was not affected by geldanamycin treatment. These results indicate that Hsp90 has an important role in the particular step(s) necessary for converting RLC into RISC. A recent study has shown that geldanamycin significantly reduces the levels of Argonaute proteins in mammalian cells and thus reduces the programming of the RISC. However, the stability of Ago2 was barely changed in S2 lysates even after geldanamycin was added. Thus, the reduced RISC-forming activity was likely not due to reduced Ago2 stability (Miyoshi, 2010).

siRNA duplexes remained as duplexes in the presence of geldanamycin, implying that geldanamycin inhibits Ago2 Slicer activity. To test this, target RNA cleavage assays were performed. After expressing Flag-tagged Ago2 or Flag-tagged enhanced green fluorescent protein (EGFP) in S2 cells, the cells were lysed and the RISC in the lysates was assembled by adding siRNA duplexes. Flag-Ago2 and Flag-EGFP were isolated from the lysates and target RNAs were added to the complexes in the presence or absence of geldanamycin. The Flag-tagged Ago2 complex cleaved the target RNA in both cases. Thus, Hsp90 was not required for Ago2 to cleave the target RNAs once a functional RISC was properly formed. An earlier report showed that Hsp90 inhibition impairs RNAi in Drosophila ovary extracts (Pare, 2009). However, in these experiments, siRNAs and geldanamycin were added simultaneously to the lysates to examine the inhibitory effect of the functional loss of Hsp90 (Miyoshi, 2010).

Conversion of the RLC into the pre-RISC requires siRNA duplexes to be transferred from the RLC to Ago2. At this step, Dicer2, the main component of the RLC, should interact with Ago2, although this interaction may be transient. Thus,whether inhibition of Hsp90 activity interferes with the association between Dicer2 and Ago2 was examined. Flag-tagged Ago2 interacted with Dicer2 similarly either with or without geldanamycin. This indicated that, at least in Drosophila cells Hsp90 has little or no effect on the Dicer2-Ago2 association. This is in contrast to mammalian cells, where Hsp90 activity was required for the Argonaute-Dicer interaction (Miyoshi, 2010).

Two models regarding Hsp90 function in RNAi can now be proposed: the first is that Hsp90 functions in displacing the siRNA duplex from the RLC, whereas the second is that Hsp90 is required for Ago2 to receive the siRNA duplex from the RLC, after the siRNA duplex has been properly displaced from the RLC. To examine which model is correct, siRNA-protein interaction experiments were performed using three siRNA duplexes, siRNA1, siRNA2 and siRNA3, each of which was composed of the same sequence but contained an iodine-uridine at different positions within the duplex. siRNA duplexes individually incubated in S2 lysates for different time periods and then the mixtures were exposed to UV light to cross-link the siRNA duplexes with the proteins physically associated with them. Previous studies have indicated that Dicer2 and R2D2, the second known component of the RLC, are mainly cross-linked at the 3' end of the guide and passenger strands of the duplex, respectively. Ago2 was shown to be cross-linked predominantly at the 5' end of the guide strand of the duplex. The association of R2D2 and Dicer2 with siRNA1 and siRNA2, respectively, was not significantly altered by Hsp90 inhibition. However, Hsp90 inhibition drastically affected siRNA duplex association with Ago2; in the presence of geldanamycin, Ago2 barely interacted with siRNA3. The inhibitory effect was observed when geldanamycin was added at a concentration of 10 μM or higher. Radicicol, another Hsp90 inhibitor, also interfered with siRNA duplex association with Ago2. A siRNA duplex mutant (siRNA3 mutant), in which the passenger strand was modified with 2'-O-methyl groups at the 9th and 10th nucleotides from the 5' end and thus interferes with Ago2 Slicer activity, behaved similarly to siRNA3. In addition, it was shown that Hsp90 was present in immunoprecipitated Ago2 complex. These results suggest that Hsp90 is required for Ago2 to receive siRNA duplexes from the RLC in the RNAi pathway. In siRNA-Ago2 binding assays, the guide siRNAs were barely associated with Ago2 when geldanamycin was added first to the lysates prior to the addition of the siRNA duplexes. These results further support the idea that Hsp90 is needed for Ago2 to bind the siRNA duplex. Structural analysis of the eubacterial Ago protein has revealed that the cavity in the Ago protein that accommodates the siRNA duplex is too small to be bound by an RNA molecule, and a conformational change in the Ago protein would be required for binding. In the current study, the results suggest that Hsp90 acts as the driving force for changing the conformation of Ago2, likely by hydolyzing ATP as an energy source, as geldanamycin is known to inhibit the ATPase activity of Hsp90 by occupying the N-terminal ATP binding pocket of the protein, enabling it to accommodate siRNA duplex from the RLC . This step is crucial in RNAi, and so, without Hsp90, the pre-RISC is not formed and RNAi is impaired (Miyoshi, 2010).

DEAD-box RNA helicase Belle/DDX3 and the RNA interference pathway promote mitotic chromosome segregation

During mitosis, faithful inheritance of genetic material is achieved by chromosome segregation, as mediated by the condensin I and II complexes. Failed chromosome segregation can result in neoplasm formation, infertility, and birth defects. Recently, the germ-line-specific DEAD-box RNA helicase Vasa was demonstrated to promote mitotic chromosome segregation in Drosophila by facilitating robust chromosomal localization of Barren (Barr), a condensin I component. This mitotic function of Vasa is mediated by Aubergine and Spindle-E, which are two germ-line components of the Piwi-interacting RNA pathway. Faithful segregation of chromosomes should be executed both in germ-line and somatic cells. However, whether a similar mechanism also functions in promoting chromosome segregation in somatic cells has not been elucidated. This study presents evidence that belle (vasa paralog) and the RNA interference pathway regulate chromosome segregation in Drosophila somatic cells. During mitosis, belle promotes robust Barr chromosomal localization and chromosome segregation. Belle's localization to condensing chromosomes depends on dicer-2 and argonaute2. Coimmunoprecipitation experiments indicated that Belle interacts with Barr and Argonaute2 and is enriched at endogenous siRNA (endo-siRNA)-generating loci. These results suggest that Belle functions in promoting chromosome segregation in Drosophila somatic cells via the endo-siRNA pathway. DDX3 (human homolog of belle) and DICER function in promoting chromosome segregation and hCAP-H (human homolog of Barr) localization in HeLa cells, indicating a conserved function for those proteins in human cells. These results suggest that the RNA helicase Belle/DDX3 and the RNA interference pathway perform a common role in regulating chromosome segregation in Drosophila and human somatic cells (Pek, 2011).

Although Vasa and Belle have been implicated in the piRNA and endo-siRNA pathways, respectively, it is not known whether DDX3 is also involved in the RNAi pathway. The fact that DDX3 is involved in viral RNA sensing offers the possibility that DDX3 may be a component of the RNAi pathway in humans. Furthermore, the DCR-dependent localization of DDX3, both during interphase and prophase, suggests that DDX3 may function downstream of DCR. Further investigation into the nature of the genomic loci and RNAi pathway components that associate with DDX3 and the nature of the noncoding RNAs involved in this process will provide greater insight into its molecular mechanism in human cells (Pek, 2011).

This study has indicated that the robust chromosomal localization of Barr/hCAP-H is regulated by the Vasa/Belle/DDX3 class of DEAD-box RNA helicases in both germ-line and somatic Drosophila cells and human somatic cells. This finding suggests the possibility of a common pathway that regulates chromosome segregation by the Vasa/Belle/DDX3 class of RNA helicases. Although chromosome segregation appears to be regulated by RNAi machinery, the necessary small RNA pathway components vary notably between the germ-line and somatic cells. The piRNA pathway components are required in the germ-line cells, whereas the endo-siRNA pathway components function as their somatic counterparts. This finding suggests that various cell types can use the existing small RNAs and RNAi factors to achieve a common goal of robust Barr/hCAP-H localization. This study also provides a framework for future studies investigating the molecular mechanism of the cooperation between the Vasa/Belle/DDX3 RNA helicases and the RNAi factors to ensure proper chromosome segregation (Pek, 2011).

The Bin3 RNA methyltransferase is required for repression of caudal translation in the Drosophila embryo

Bin3 was first identified as a Bicoid-interacting protein in a yeast two-hybrid screen. In human cells, a Bin3 ortholog (BCDIN3) methylates the 5' end of 7SK RNA, but its role in vivo is unknown. This study shows that in Drosophila, Bin3 is important for dorso-ventral patterning in oogenesis and for anterior-posterior pattern formation during embryogenesis. Embryos that lack Bin3 fail to repress the translation of caudal mRNA and exhibit head involution defects. bin3 mutants also show (1) a severe reduction in the level of 7SK RNA, (2) reduced binding of Bicoid to the caudal 3' UTR, and (3) genetic interactions with bicoid, and with genes encoding eIF4E, Larp1, polyA binding protein (PABP), and Ago2. 7SK RNA coimmunoprecipitated with Bin3 and is present in Bicoid complexes. These data suggest a model in which Bicoid recruits Bin3 to the caudal 3' UTR. Bin3's role is to bind and stabilize 7SK RNA, thereby promoting formation of a repressive RNA-protein complex that includes the RNA-binding proteins Larp1, PABP, and Ago2. This complex would prevent translation by blocking eIF4E interactions required for initiation. These results, together with prior network analysis in human cells, suggest that Bin3 interacts with multiple partner proteins, methylates small non-coding RNAs, and plays diverse roles in development (Singh, 2011).

The human homolog of Bin3, also called BCDIN3 or methylphosphate capping enzyme (MePCE), was shown to methylate the 5' γ-phosphate on 7SK RNA and to stabilize 7SK RNA in cell culture. This study found that Bin3 associates with and stabilizes 7SK RNA in ovaries and embryos. And, as in human cells, Bin3 activity was specific for 7SK RNA and did not affect U3 RNA or another RNA pol III product, U6 RNA, both of which are methylated by distinct mechanisms. It seem likely, therefore, that Drosophila Bin3 has a similar biochemical activity to its human counterpart despite differing in size and sequence outside the AdoMet binding domain and the highly conserved Bin3-homology domain. Prior attempts to demonstrate protein-arginine methyltransferase activity of Bin3 were negative, consistent with Bin3 methylating RNA rather than protein. In Drosophila, there are two other Bin3-like genes, CG11342 and CG1239, but each is more divergent from the human BCDIN3 within the conserved motif architecture (24% and 39% identity, respectively) than Bin3. It is possible that CG1239, which is expressed in early embryos, could have partially overlapping functions with Bin3 that might contribute to the incomplete penetrance of the bin3 mutations (Singh, 2011).

Putative Bin3 orthologs containing the two conserved motifs are found in at least 70 eukaryotic organisms ranging from the yeast, Schizosaccharomyces pombe to humans, and including Caenorhabditis elegans, Arabidopsis thaliana, Xenopus laevis, and Danio rerio. It is not known what any of these genes do, with the possible exception of the zebrafish bin3 gene which was shown by morpholino knockdown to be important for anterior development and to display RNA splicing defects. Similar defects were sought in splicing of bicoid, caudal, eIF4E, d4EHP, and a control gene, taf1, known to show alternative splicing. No splicing defects were found using a sensitive qRT-PCR approach. It is possible that the splicing defects in zebrafish result from aberrant 5' capping of non-coding RNAs important for splicing (Singh, 2011).

Mammalian 7SK RNA has been studied extensively, but Drosophila 7SK RNA has only been annotated, and prior to this study has not been characterized. This study shows that 7SK RNA is highly expressed in ovaries and embryos and is regulated by Bin3 in a manner similar to that in humans (by BCDIN3). 7SK RNA can be coimmunoprecipitated with Bin3 and Bicoid and may work as a scaffold in translation repression. This is the first indication that 7SK RNA has a function apart from its role in the regulation of the pTEFb transcription elongation factor. While this study focused on Bicoid-dependent regulation, it is likely that 7SK RNA also functions in transcription elongation in other stages of development. Indeed, it was found that Drosophila 7SK RNA mutants showed larval lethality at later stages of development (Singh, 2011).

Bin3 seems to play no role in Bicoid's gene activation function, but instead is crucial for Bicoid-dependent repression of caudal mRNA. Bin3 seems to stabilize Bicoid at the caudal BRE via a mechanism that involves 7SK RNA. As suggested by genetic interaction data, the Bicoid/Bin3/7SK RNA complex may include Larp1, PABP, and Ago2, and target the eIF4E initiation factor (Singh, 2011).

La-related proteins are not restricted to control of transcription elongation. In C. elegans, a Larp1 homolog was shown to be important for downregulation of translation of mRNAs in the Ras-MAPK pathway and to localize to P-bodies, known sites of mRNA degradation, while in mammalian cells, LARP4B plays a stimulatory role in translation initiation. In Drosophila, it has been shown that Larp1 associates directly with PABP independent of RNA and double mutants show enhanced lethality, suggesting that Larp1 facilitates mRNA translation. It is not surprising, therefore, that genetic interactions were observed between bin3 and larp1, as well as with pAbp in the context of caudal translation regulation. Note that it is though that PABP (and Larp1) plays a negative role in translation initiation, as does PABP in the repression of msl-2 mRNA by Sex-lethal (Singh, 2011).

In human cells, BCDIN3 and LARP7 interact cooperatively with 7SK RNA forming a stable core complex that associates transiently with HEXIMS, hnRNPs and the P-TEFb elongation complex (see Drosophila Hexim). An emerging theme is that 7SK RNA serves as a scaffold for stable association of protein partners. In fact, there is evidence that 5' γ-methylation of 7SK RNA by BCDIN3 may occur co-transcriptionally, but that the modified RNA remains associated with both BCDIN3 and LARP7, which bind 7SK RNA cooperatively. It is proposed, therefore, that Bin3 and Larp1 are associated with 7SK RNA at the caudal BRE, but that 5'-methylation does not necessarily occur there. Consistent with the idea of cooperative binding to 7SK RNA, it was found that larp1 mutation enhanced the bin3 mutant phenotype (Singh, 2011).

Some of the phenotypes observed for bin3 mutants were also observed in mutants of the microRNA miR-184, including oogenesis defects and a cellularization defect. This was the rationale behind including ago2 in the genetic analysis. However, no effect was found of bin3 mutation on levels of several miRNAs, including miR-184, it was surprising to observe a genetic enhancement (albeit mild) of the bin3 phenotype when combined with an ago2 mutation. Ago2 has been shown to bind eIF4E and interfere with mRNA circularization mediated by PABP. However, this occurs in the context of the miRNA/RISC complex, so whether and how Ago2 participates in Bicoid-Bin3 repression is not clear, but it could potentially involve the 7SK RNA component (Singh, 2011).

Finally, no interaction was detected between bin3 and D4EHP, which encodes a previously identified partner of Bicoid important for repressing caudal translation. D4EHP interacts with Bicoid and is thought to directly bind the m⁷G cap of caudal mRNA, thereby displacing eIF4E and blocking all subsequent steps of initiation. Perhaps the Bin3 mechanism works redundantly with the D4EHP mechanism or perhaps Bin3 helps recruit D4EHP, and so that mutation of bin3 would preclude binding of D4EHP to the initiation complex. Thus, bin3 mutation would be epistatic to the D4EHP^CP53 mutation. Further investigation will be needed to determine relationship between these two pathways (Singh, 2011).

Bin3 is unlikely to be a dedicated Bicoid interactor and probably has roles as an RNA methyltranferase in many distinct pathways throughout development. In adults, quantitative trait transcript analysis linked bin3 with sleep-wake cycling. While studying Bin3's role in embryonic patterning, strong oogenesis defects were observed, particularly in bin3 null mothers, although other allelic combinations also revealed similar defects, especially at 29°C. Specifically, bin3 loss-of-function mutants showed dorsalized egg shell phenotypes. Conversely, bin3 overepressing lines showed strong ventralized egg shell patterns that appear to result from a failure of the dorsal appendage primordium to resolve into two domains along dorsal midline. These defects are similar to those of early D-V patterning mutations in the grk pathway, and probably do not result from defects that occur in later during morphogenesis step (Singh, 2011).

bin3 loss-of-function mutants resembled mutations in capicua, squid, cup and fs(K10), among others, while bin3 overexpressing lines resembled grk and pAbp mutations. Interestingly, mechanisms for translation repression of unlocalized grk mRNA feature prominently in the D-V patterning pathway, with squid and cup playing a critical role in repression via interaction with eIF4E, and PABP55 being important for release of that repression. Staining of bin3 mutant ovaries revealed a delocalized signal for Gurken protein but not for grk mRNA. Given the role of Bin3 in translation regulation, and the egg shell phenotypes of bin3 mutations, it seems plausible that Bin3 plays a role in negative regulation of grk translation (Singh, 2011).

Results presented in this study show that Bin3 plays a critical role during both oogenesis and embryonic development. In embryos, Bin3 is required for Bicoid to establish the Caudal protein gradient. Bin3 binds 7SK RNA and likely works by methylating 7SK RNA and stabilizing a repressive complex that assembles on the Bicoid-response element in the 3' UTR of caudal mRNA. Bin3's role during oogenesis is less clear, but based on the observed eggshell phenotypes in bin3 mutants, and gurken expression, Bin3 could play a similar role to help ensure that grk mRNA is translated only in the anterior-dorsal region of the oocyte (Singh, 2011).

Drosophila Argonaute2 turnover is regulated by the ubiquitin proteasome pathway

Argonaute (AGO) proteins play a central role in the RNA interference (RNAi) pathway, which is a cytoplasmic mechanism important for post-transcriptional regulation of gene expression. In Drosophila, AGO2 also functions in the nucleus to regulate chromatin insulator activity and transcription. Although there are a number of studies focused on AGO2 function, the regulation of AGO2 turnover is not well understood. This study found that mutation of T1149 or R1158 in the conserved PIWI domain causes AGO2 protein instability, but only T1149 affects RNAi activity. Mass spec analysis shows that several proteasome components co-purify with both wildtype and mutant AGO2, and knockdown of two proteasome pathway components results in AGO2 protein accumulation. Finally, AGO2 protein levels increase after treatment with the proteasome inhibitor MG132. These results indicate that the ubiquitin-proteasome pathway is involved in AGO2 protein turnover (Chinen, 2017).

EFFECTS OF MUTATION

The Drosophila AGO2 protein is an essential factor for RNAi as a component of the RISC complex in cultured S2 cells (Hammond, 2001). However, the precise role that AGO2 plays in RNAi is not well understood. To gain an insight into the molecular functions of AGO2 in RNAi, fly strains that lack AGO2 were produced. To obtain fly strains bearing deletions in AGO2, a P{EP}element inserted in the first exon of the AGO2 gene on the EP(3)3417 chromosome was mobilized to produce several partial deletions of AGO2. One such mutation named AGO2⁴¹⁴ was selected for further characterization. Western blot analysis with anti-AGO2 antibody revealed that there is no AGO2 protein in homozygous AGO2⁴¹⁴ flies. The absence of AGO2 mRNA was also confirmed by Northern blot analysis in homozygous AGO2⁴¹⁴ flies. These results demonstrated that AGO2⁴¹⁴ are AGO2 null flies. Homozygous AGO2⁴¹⁴ flies proceed into adulthood, are fertile, and appear outwardly normal. Because it is not known whether AGO2 is required for RNAi in embryos, RNAi was tested in vivo by assaying the ability of long dsRNA corresponding to the fushi tarazu (ftz) gene to produce a ftz phenotype when injected into wild-type and AGO2 mutant embryos. AGO2 mutant adult females were used to make mutant eggs to remove the maternal contribution of AGO2 (simply called the 'AGO2 mutant embryo' or AGO2⁴¹⁴ embryos, hereafter). Wild-type embryos injected with ftz dsRNA exhibited segmentation defects in their cuticle. In contrast, AGO2 mutant embryos showed a complete absence of interference in response to ftz dsRNA, indicating that they were RNAi defective. This is consistent with previous findings made with cultured S2 cells (Hammond et al. 2001; Caudy, 2002; Ishizuka, 2002). To confirm that disruption of the AGO2 gene is directly responsible for the RNAi-defective phenotype, mutant flies were transformed with a P element containing wild-type AGO2 genomic sequences. The RNAi-defective phenotype was ameliorated by the introduction of this AGO2 minigene, demonstrating that the RNAi-defective phenotype is caused by the AGO2 mutation rather than a second site mutation elsewhere on the chromosome (Okamura, 2004).

To determine whether AGO2 is required for the formation of the siRNA duplex or for interference thereafter, synthetic ftz siRNA was injected into AGO2⁴¹⁴ embryos. siRNA produced a ftz phenotype in wild-type embryos. However, AGO2⁴¹⁴ embryos were not responsive to the ftz siRNA. Similarly, in vitro, AGO2⁴¹⁴ embryo lysate processed long ftz dsRNA into short ~21-nt fragments, similar to lysates from wild-type embryos. These findings suggest that AGO2 is necessary for RNAi after the formation of the siRNA duplex (Okamura, 2004).

Before target mRNA recognition, the siRNA duplex is unwound, and each RISC contains only one of the two strands of the siRNA duplex. Whether AGO2 activity is necessary for this unwinding process was tested. Unwound siRNA was detected in wild-type embryo lysate, whereas no such activity was detected in AGO2⁴¹⁴ embryo lysate, suggesting that AGO2 is required for the unwinding of the siRNA duplex. An alternative explanation of the results could be that the absence of AGO2 leaves the unwound single-stranded RNA exposed to nucleolytic degradation and therefore the single-stranded RNA could not be detected. However, this is unlikely because the amount of input siRNA duplex left in AGO2⁴¹⁴ embryo lysate was not significantly reduced even after 3 h incubation as opposed to that seen in wildtype embryo lysate, indicating that input siRNA duplex is indeed not unwound in AGO2 mutant embryo lysate. Using a recently developed RISC assembly assay, RISC formation was analyzed in AGO2 mutant embryo lysates. ds-siRNA was incubated with embryo lysates in a standard RNAi reaction, and then RISC complexes were resolved by electrophoresis through a native gel. Three major complexes (B, A, and RISC) were detected in wild-type embryo lysates. Complexes B and A are thought to be intermediate precursors to active RISC and contain ds-siRNA. Both complexes B and A were readily detected in AGO2 mutant embryo lysates. However, RISC formation was impaired in AGO2 mutant embryo lysates. Together, these results suggest that AGO2 is required in a step(s) in RISC assembly after binding of the siRNA duplex to RISC precursors (complexes B and A) (Okamura, 2004).

Both siRNAs and miRNAs have been shown to be associated with the same RISC and miRNAs can even direct cleavage of perfectly basepaired substrates in human cells. To see if this is also the case in Drosophila, whether the lack of AGO2 affects the miRNA-mediated cleavage of target RNAs was investigated. ³²P-radiolabeled target mRNA, containing a sequence fully complementary to an endogenous miRNA, miR-2b, as well as a sequence exactly complementary to one strand of a ftz siRNA duplex, was incubated with AGO2⁴¹⁴ embryo lysate in an in vitro RNAi reaction. Both siRNA-directed and miRNA-directed cleavage products are seen in the wild-type lysate. However, ftz siRNA did not direct cleavage in AGO2⁴¹⁴ embryo lysate; in contrast, miR-2b still directed cleavage of the target RNA in AGO2⁴¹⁴ embryo lysates. These results demonstrate that although AGO2 is an essential factor in siRNA-directed RNAi, AGO2 is dispensable for miRNA-directed RNA cleavage in RNAi (Okamura, 2004).

In this experiment, however, the timings of miRNA loading and siRNA loading into RISC are different. miR- 2b is already incorporated into the RISC by the embryo before the lysate is made, whereas siRNA is added to the reaction mixture. Therefore synthetic Drosophila melanogaster let-7 precursor RNA was added to an in vitro RNAi reaction mixture containing the let-7 complementary target RNA. It should be noted that the sequence of the synthetic let-7 precursor used in this study is slightly longer than that of naturally processed pre-let-7, which contains short 3' overhangs. However, the synthetic let-7 precursor has been previously shown to be processed and produce mature let-7 with precisely the same 5' end as authentic let-7 in the Drosophila embryo lysates. In this experiment, the synthetic let-7 precursor RNA was also converted to mature let-7 in vitro, not only in the wild-type embryo lysate, but also in AGO2⁴¹⁴ embryo lysate. In the in vitro RNAi assay, target RNA was cleaved within the let-7 complementary sequence in AGO2⁴¹⁴ embryo lysate as efficiently as in wild type. Thus, miRNA that is not preloaded by the embryo is still capable of entering into the RNAi pathway without AGO2 activity. These results demonstrate that AGO2 is not required for miRNA production or the loading of miRNAs into the RISC and suggest that AGO2 has a specific role for siRNA activity, likely as a specific unwinding and loading factor of siRNA into RISC, and possibly as a necessary component of an siRNA-initiated RISC (Okamura, 2004).

Whether siRNA- and miRNA-directed RNA cleavage pathways have differential requirements for AGO1 was tested. Mutations in AGO1 result in late embryonic/ early larval lethality with developmental defects (Kataoka, 2001). Although AGO1 has been shown necessary for efficient RNAi in embryos (Williams, 2002), AGO1 appears to have little or no effect on the efficiency of RNAi in cultured S2 cells (see also Caudy, 2002). AGO1^k08121 is a strong allele (Kataoka 2001). AGO1^k08121 was balanced over a CyO chromosome carrying a kruppel-Gal4 (Kr-Gal4) and a UAS-GFP transgene. Homozygous embryos were separated from heterozygous and CyO;Kr-Gal4;UAS-GFP homozygous embryos on the basis of GFP expression. Because there is strong reduction of AGO1 transcripts in AGO1^k08121 embryos (Kataoka, 2001) and AGO1 protein was not detected in lysate from 14- to 16-h staged AGO1^k08121 embryos, 14-16-h staged AGO1^k08121 embryo lysate was tested for its ability to direct cleavage of siRNA or miRNA complementary RNA targets. ftz siRNA directed its cleavage in AGO1^k08121 embryo lysate as effectively as in wild type. AGO1^k08121 embryo lysate also processed long ftz dsRNA into short ~21-nt fragments. Furthermore, RISC formation directed by siRNA duplex was not impaired in AGO1^k08121 embryo lysate as judged by the native gel shift assay. These findings suggest that AGO1 is not necessary for the production of the siRNA duplex, siRNA-initiated RISC formation, or siRNA-directed cleavage. However, cleavage of the target RNA directed by miR-2b was suppressed in AGO1^k08121 embryo lysate by a factor of fivefold compared with that in wild type, suggesting that AGO1 is necessary for efficient target RNA cleavage mediated by miRNAs. Residual miRNA activity seen in AGO1^k08121 could be due to some degree of functional complementation among Argonaute family proteins expressed in embryos (Williams, 2002). The in vitro experiments with AGO1^k08121 mutant embryo lysates argue against AGO1 being required for siRNA-mediated RNAi in embryos when injected with either long dsRNA or siRNA (Williams, 2002). Although the discrepancy is currently not understood, these findings are at least consistent with the findings (Caudy, 2002) using cultured S2 cells (Okamura, 2004).

It was hypothesized that AGO1 might be involved in the maturation and/or function of miRNAs. To directly determine the contribution of AGO1 and AGO2 to miRNA production in Drosophila cells, each was depleted from S2 cells by dsRNA soaking and Northern blot analyses were perfomed to monitor the abundance of bantam miRNA (miR-ban). A marked reduction of mature miR-ban was observed in AGO1-depleted S2 cells but not in AGO2-depleted S2 cells. It was also confirmed that the miRNA level was significantly reduced in the AGO1^k08121 embryos. The accumulation of pre-miR-ban was not observed in both cases. Similar results were obtained when the same RNA preparations were probed for the expression of another miRNA, miR-2b, which is also known to be expressed both in S2 cells and embryos. These results suggest that AGO1 is involved in the maturation and/or stability of miRNAs (Okamura, 2004).

Production of both siRNAs and miRNAs require Dicers, which interact with RISC components, suggesting that Dicer action is coupled to loading small RNAs onto the RISC. Two Dicers, Dicer-1 and Dicer-2, have been identified in Drosophila. siRNA production is associated with Dicer-2, but not Dicer-1. Dicer-2, together with the dsRNA-binding protein R2D2, facilitates siRNA loading onto RISC. It was found that the targeted destruction of only one of the two Dicers, Dicer-1, leads to an accumulation of the miR-ban precursor in S2 cells, consistent with the recent genetic studies showing that Dicer-1 is required for miRNA processing. The amount of pre-miR-ban was further increased after prolonged Dicer-1 knockdown for 8 d. In contrast, prolonged suppression of AGO1 expression did not result in the accumulation of pre-miR-ban. These findings suggest that mature miRNAs are processed from premiRNAs by Dicer-1 and are stabilized by AGO1. It was next asked whether AGO1 is physically associated with Dicer-1 by purifying AGO1-associated complexes from S2 cells using a TAP method. It was found that AGO1 was associated with Dicer-1. This interaction of AGO1 with Dicer-1 seems to be quite specific for AGO1 because Dicer-1 was not detected in AGO2- and in dFMR1-associated complexes under the conditions used for the purification. RNA preparations from AGO1-, AGO2-, and dFMR1-associated complexes were then probed for the presence of miR-ban. AGO2- and dFMR1-associated complexes contained the mature form of miR-ban. Only the AGO1-associated complex contained the pre-miR-ban and mature forms. The interaction of AGO1 with Dicer-1 and pre-miRNA further suggests that AGO1 is involved in miRNA biogenesis (Okamura, 2004).

Involvement of microRNA in AU-rich element-mediated mRNA instability: i>Ago1 and Ago2 are required for ARE-mediated RNA degradation

AU-rich elements (AREs) in the 3' untranslated region (UTR) of unstable mRNAs dictate their degradation. An RNAi-based screen performed in Drosophila S2 cells has revealed that Dicer1, Argonaute1 (Ago1) and Ago2, components involved in microRNA (miRNA) processing and function, are required for the rapid decay of mRNA containing AREs of tumor necrosis factor-alpha. The requirement for Dicer in the instability of ARE-containing mRNA (ARE-RNA) was confirmed in HeLa cells. miR16, a human miRNA containing an UAAAUAUU sequence that is complementary to the ARE sequence, is required for ARE-RNA turnover. The role of miR16 in ARE-RNA decay is sequence-specific and requires the ARE binding protein tristetraprolin (TTP). TTP does not directly bind to miR16 but interacts through association with Ago/eiF2C family members to complex with miR16 and assists in the targeting of ARE. miRNA targeting of ARE, therefore, appears to be an essential step in ARE-mediated mRNA degradation (Jing, 2005).

The ARE motif (AUUUA) is the most studied cis-acting element responsible for rapid turnover of unstable mRNAs in mammalian cells. In the quest for a genetic system that allows a comprehensive search for components involved in ARE-mediated decay of mRNA, Drosophila S2 cells were examined and it was found that the decay of ARE-containing RNA in S2 cells is regulated in a manner similar to that in mammalian cells. Inhibition of gene expression by RNAi is much easier and more cost effectively conducted in Drosophila S2 cells compared to mammalian cells: this allowed for an investigation of a large number of genes for their involvement in ARE-mediated RNA decay. Surprisingly, knockdown of Drosophila Dicer1 gene expression leads to stabilizing an ARE-RNA reporter. Further studies revealed that Drosophila Ago1 and Ago2 are required for ARE-mediated RNA degradation, suggesting involvement of the miRNA system. It was then confirmed that human Dicer is required in ARE-RNA degradation in HeLa cells, which implies that this underlying mechanism is conserved in the mammalian cells. Given the involvement of Dicer in HeLa cells, it was reasoned that miRNA(s) are involved in ARE-mediated RNA decay and a search was conducted for miRNAs that possess a complementary sequence to the canonical AUUUA sequence of ARE. miR16 is a potential candidate due to the presence of the sequence UAAAUAUU, and it was shown that downmodulation and overexpression of miR16 increases or decreases, respectively, the stability of a RNA reporter containing ARE of TNF or Cox2, but not uPAR. Furthermore, it was determined that the regulation of ARE^TNF-RNA decay by miR16 is sequence specific. Just as with Dicer, a function of Ago family members in ARE^TNF-RNA degradation is likely to be the processing of miR16. However, the interaction with the ARE binding protein TTP indicates that Ago/eiF2C family members also play a crucial role in the targeting of miR16 to ARE. These data demonstrate the involvement of miR16 in controlling ARE-RNA turnover and suggest that cooperation of miRNA and ARE binding proteins is essential in the recognition of ARE and in triggering mRNA degradation (Jing, 2005).

Studies have shown that the ability of miRNA to target mRNA is directed by the pairing of miRNA to mRNA. The ARE-complementary sequence in miR16 is indeed required for miR16 function in destabilizing ARE-RNA. However, pairing with no more than an eight-base ARE-sequence may not be sufficient for miR16 to target ARE-RNA. In addition, the pairing of miR16 to ARE is not in the 5′ region of miRNA, which is believed to be more critical in causing gene repression than the 3′ region. It is speculated, then, that TTP is a factor that assists miR16 targeting to ARE sequences due to its ability to interact with the ARE and RISC complex. This explains why miR16-mediated ARE-RNA instability requires TTP. In addition, the requirement of miR16 in TTP-mediated destabilization of ARE-RNA suggests that targeting of miR16 to ARE is a necessary step for RNA degradation (Jing, 2005).

ARE sequences from different mRNA can vary dramatically, with some containing multiple AU-rich elements that allow for simultaneous interaction with more than one miRNA. This could influence the ability of miRNA to promote RNA degradation because of the potential synergistic effect of miR16 to bind to multiple sites. This synergism has been demonstrated in a study that shows the addition of multiple binding sites of CXCR4 siRNA into 3′UTR of a reporter results in more translation inhibition than expected when summing up the individual effects of each binding site. The number of pairs that miR16 can form with different ARE sequences varies from five to eight, and the strength of interaction between miR16 and different AREs in a given mRNA may also vary. The number of miRNAs targeted to an mRNA and the strength of the interaction may both contribute to the quantitative control of mRNA turnover or translation. Perhaps since no more than six pairs can form between miR16 and ARE of uPAR and since uPAR has only one AUUUA motif in the 3′UTR, miR16 does not have a significant effect on the stability of mRNA containing uPAR 3′UTR (Jing, 2005).

miR16 is conserved in mammals. Although a homolog of miR16 has not been found in Drosophila, miR289 contains UAAAUAUUUA, and four other known Drosophila miRNAs contain a UAAAU sequence. Among them, at least miR277, miR289, and miR304 are expressed in S2 cells. 2′-O-methyl oligonucleotides were used to test for Drosophila miRNA that could be involved in ARE-RNA degradation in S2 cells. The anti-miR289 oligo significantly stabilizes mRNA containing TNF-α ARE, while the other four oligos have no or very modest effects on the stability of ARE^TNF-RNA. miR289 has a similar effect on the stability of ARE^IL-6-RNA and ARE^IL-8-RNA. Sequence comparisons showed that miR289 partially complements with ARE, but not the other regions of these 3′UTRs. Thus, miR289 is likely to be a miRNA that has a role in regulating ARE-RNA in S2 cells (Jing, 2005).

Though the association of miR16 with ARE-RNA in the presence of TTP and S-100 in vitro has been demonstrated, the exact mechanism of miRNA targeting of ARE and regulation of RNA degradation remains undetermined. Because of the similarity between siRNA and miRNA in regulating gene expression, miR16-mediated ARE-RNA degradation could be similar to siRNA-mediated mRNA decay. It is theoretically possible that the targeting of ARE with miRNA leads to mRNA cleavage at the targeting site since RISC has been shown to be an RNA endonuclease in vitro. However, translational suppression caused by miRNA or imperfect pairing of siRNA suggests that endonuclease activity is not always associated with RISC. Since ARE-RNA degradation is believed to be initiated by deadenylation and subsequent targeting by the exosome pathway, and since endocleaved ARE-RNA was not detected in the experimental system that was used, it is believed that the RISC involved in ARE-RNA decay is not associated with endonuclease activity. At the present, it is not clear if RISC can execute an exonuclease function, although an exonuclease, Tudor-SN, has been found in the RISC complex. TTP has been shown to bind to extended ARE sequences by virtue of its zinc finger and associates with components of exosomes; this study shows that TTP is also associated with eiF2C/Ago family members. A recent study reported that an exosome associated DexH box helicase facilitates ARE-RNA deadenylation and decay in mammalian cells. Interestingly, a C. elegens homolog of this DexH box protein has been shown to interact with a protein complex containing Dicer, RDE-1, and RDE-4. It appears that ARE binding proteins, miRNA, deadenylase, and exosomes cooperate with each other in regulating mRNA degradation. A model is favored in which TTP binds to an ARE and transiently interacts with the RISCs that scan mRNA. When a RISC containing miR16 encounters TTP, it stays with ARE and TTP due to base complementarity between miR16 and ARE. It is conceivable that RISC, in conjunction with TTP, serves to recruit proteins for deadenylation and/or exosomes for mRNA degradation (Jing, 2005).

Hundreds of miRNAs have been identified, but the targets of most miRNAs are unknown. Since perfectly or nearly perfectly paired sequences can only be found for a few miRNAs, computational as well as experimental approaches have been developed to identify potential miRNA targets that do not contain perfect complementary sequences. Although these approaches have been shown to be very useful, ARE was not identified as the target of miR16 through currently available computer programs. The current data suggest that additional factors, such as sequence-specific RNA binding proteins, needs to be considered in studying the function of miRNA. As in the case of miR16, many miRNAs may require specific proteins in binding to their mRNA targets. The role of many miRNAs may need to be studied, not only in the context of miRNA-mRNA interaction, but also the interaction of miRNA complexes with other proteins (Jing, 2005).

Drosophila argonaute-2 is required early in embryogenesis for the assembly of centric/centromeric heterochromatin, nuclear division, nuclear migration, and germ-cell formation

The RNA-induced silencing complex (RISC) or the RISC complex mediates RNAi and is comprised of proteins belonging to the dicer and Argonaute family proteins. Argonaute-2 is required for proper nuclear migration, pole cell formation, and cellularization during the early stages of embryonic development in Drosophila. These defects have been traced back to the nuclear division cycles. Unlike wild type, nuclear division is asynchronous in ago-2 embryos and there are defects in chromosome condensation, nuclear kinesis, and assembly of spindle apparatus. The aberrations in the nuclear division cycle are correlated with defects in the formation of centric/centromeric heterochromatin and point to a failure in the assembly of functional centromeres (Deshpande, 2005).

Since the RNAi machinery has been implicated in the formation of centric heterochromatin and mitosis in unicellular organisms, it was anticipated that a loss of ago-2 activity might influence the rapid nuclear divisions in early fly embryos. To investigate this possibility, wild-type and ago-2⁴¹⁴ mutant embryos stained with the DNA dye Hoechst were compared. Several abnormalities were evident in ago-2 embryos. The spacing between nuclei in wild type is relatively constant during the early cleavage cycles, and the nuclei are distributed uniformly through the central region of the embryo. In contrast, in ago-2⁴¹⁴ embryos, the distance from one nucleus to the next is quite variable, and nuclei are found clustered in some regions of the embryo while they are absent in other regions. When the nuclei migrate out to the cortex in wild type during cycles 8-9, they are spaced at regular intervals around the entire surface of the embryo. This is not the case in ago-2⁴¹⁴ embryos. Many ago-2⁴¹⁴ nuclei fail to migrate to the surface, while others appear to reach the surface but then fall back into the interior of the embryo. Consequently, the nuclear distribution at the surface is quite irregular. In some regions there is a high concentration of nuclei, often linked together in 'strings' of two, three, or four incompletely separated nuclei, while in other regions there are very few. There are also some very brightly stained nuclei that appear to have more than the normal complement of DNA, while other nuclei are smaller than normal (some are just small dots) and seem to be missing DNA. Defects in chromosome segregation are also evident prior to nuclear migration. Consistent with a requirement for ago-2 in the proper execution of the mitotic cycle, anomalous anaphase figures with incompletely condensed and irregularly positioned chromosomes are observed in the ago-2⁴¹⁴ mutant (Deshpande, 2005).

Similar defects in nuclear division, migration, and spacing were observed for a second ago-2^51b allele and for trans-combinations between the two ago-2 mutations. In all three cases, the frequency of embryos showing at least some defect in nuclear division and/or nuclear migration was between 50% and 60%. These abnormalities prompted a re-examination of the effects of ago-2 mutations on viability. 10%-15% of ago-2⁴¹⁴ embryos failed to hatch, while nearly 40% of ago-2^51B did not hatch. The viability of the trans-combination was intermediate between the homozygous mutants (Deshpande, 2005).

To further characterize the progression of the nuclear division cycles in ago-2, Histone H3 phosphorylation on Ser 10 was examined. In wild type, H3-Ser 10 phosphorylation can be detected nearly simultaneously in all nuclei when chromosome condensation commences at the onset of mitosis. High levels of H3 phospho-Ser 10 persist through mitosis and then disappear as the chromosomes start to decondense before the beginning of the next round of DNA synthesis. A variety of anomalies are evident in ago-2⁴¹⁴ embryos. In the early cleavage stage embryo, these include fragmented nuclei and nuclei that appear to have incompletely separated but are still entering mitosis. In the older embryo, the most striking defect is the asynchrony in the nuclear division cycles. Asynchrony can also sometimes be seen in incompletely separated nuclei (Deshpande, 2005).

The nuclear division defects suggested that ago-2 may be important for the assembly or functioning of the mitotic spindle apparatus. In the wild-type embryo, the chromosomes in each mitotic figure are aligned on the metaphase plate. Emanating from the chromosomes in opposite directions are microtubule arrays that converge at each centrosome. Nuclei that appear to be undergoing a normal mitosis are seen in ago-2⁴¹⁴ embryos. However, a variety of defects are also evident in more than half of the ago-2⁴¹⁴ embryos. Among the more striking are 'orphan' centrosome pairs. While the duplicated centrosomes occasionally remain in close proximity, most appear to migrate to 'opposite poles'. However, these orphan centrosome pairs are not associated with a mitotic spindle apparatus or with mitotic chromosomes, nor does there appear to be any nearby interphase nucleus. While other centrosome pairs appear to properly nucleate the mitotic spindle apparatus, the spindles are abnormally short and do not extend to the chromosomes or make connections with the centromeres. In these mitotic figures the chromosomes often appear to be displaced from their normal position in the center of the metaphase plate. In other cases there are DNA bridges that extend between two adjacent mitotic figures. There are also 'mitotic spindle' bridges that connect two different mitotic figures. Similar centrosomal and mitotic spindle defects are seen in ago-2^51b embryos (Deshpande, 2005).

The mitotic defects in S. pombe and T. brucei lacking Ago activity have been attributed to a disruption in centromere function because of a failure to properly assemble centromeric heterochromatin and the flanking centric heterochromatin. It therefore seemed possible that the abnormal nuclear division and the defects in the spindle apparatus evident in ago-2 embryos might also arise from a failure in the formation of heterochromatin across the centromeric repeats. Drosophila centromeric heterochromatin contains a centromere-specific histone H3 variant, called centromeric identifier (CID), that is critical for centromere function and assembly of the kinetochore. To examine centromeric heterochromatin, ago-2⁴¹⁴ embryos were probed with antibody against CID and Centrosomin. During anaphase the chromosomes on the metaphase plate begin moving toward the two centrosomes. At this point in the mitotic cycle, the centromeres are located in close proximity to the centrosomes, while the chromosomal arms extend back toward the metaphase plate. The centromeres can be visualized in wild-type chromosomes as several prominent dots of CID staining located near the base of the spindle apparatus in close proximity to each centrosome. While prominent CID dots can be seen on chromosomes throughout mitosis in wild-type embryos, this is not true in ago-2⁴¹⁴ embryos (Deshpande, 2005).

Flanking the centromere is the centric heterochromatin, which is marked by elevated levels of histone H3 methylated on Lys 9 (H3 meK9) and by the Swi6 homolog, Heterochromatin protein 1 (HP-1). Association of HP-1 with centric heterochromatin is abnormal in ago-2⁴¹⁴ precellular blastoderm embryos. There also appear to be defects in K9 methylation (Deshpande, 2005).

If ago-2 is required for the formation of centromeric/centric heterochromatin then ago-2 mutations should suppress silencing of mini-white transgenes inserted into pericentric heterochromatin. Males carrying mini-white transgenes inserted into pericentric regions of the second, third, or fourth chromosomes were mated to ago-2⁴¹⁴ females, and the eye color phenotype of ago-2⁴¹⁴/+ adults was examined. For inserts on all three chromosomes, it was found that mini-white expression is up-regulated in ~7% of the ago-2⁴¹⁴/+ flies. Significantly, the pattern of suppression points to a very early role for ago-2 in the establishment of silenced heterochromatin domains. Thus, while the eye color varies from one fly to the next, in the flies showing suppression, both eyes typically have very similar pigmentation. Moreover, instead of a highly variegated phenotype, the pigmentation is (with the exception of a few ommatidia) usually quite uniform across each eye. These results suggest that maternally derived ago-2 activity is required for establishing silenced heterochromatin domains in the paternal chromosomes at a point prior to nuclear migration (Deshpande, 2005).

The occurrence of 'orphaned' centrosomes prompted an examination of the cytoskeleton in ago-2 embryos with antibodies against Anillin, a component of the actin-myosin contractile ring, and Peanut, a septin. During interphase in wild-type Drosophila nuclear cycle 13-14 blastoderm embryos, cortical actin forms a cap on the apical surface of the embryo above the nucleus. Anillin is localized just underneath the actin cap in a ring that surrounds the nucleus, and in surface views it gives a quite regular mesh-like appearance. In ~50% of the ago-2⁴¹⁴ embryos this regular lattice is replaced by a broken network with irregularly shaped contractile rings that vary in thickness from one part of the ring to the next. Some of the anillin rings appear to contain multiple nuclei, while other rings have neither nuclei nor DNA. Similar irregularities are also observed in the Peanut lattice/network (Deshpande, 2005).

The anillin lattice is not present in wild-type embryos during earlier nuclear cycles, and instead there is a donut-like structure around each nucleus. While the anillin 'donut' is also seen around many nuclei in ago-2 embryos of a similar age, it is often absent altogether. In other cases, the DNA seems to surround the 'donut-hole', instead of being the 'donut-hole' as it is in wild type. Anillin can also be seen concentrated inside the nucleus. Interestingly, when defects are observed in the anillin donuts, they are typically associated with abnormalities in nuclear morphology or with fragmented or otherwise anomalous chromosomes. This finding would suggest that there is a link between the assembly and functioning of the mitotic apparatus and the proper organization of actin-myosin cytoskeleton. For example, the assembly of the anillin donuts/contractile rings may depend on the presence of a functional mitotic spindle and/or the reformation of the nuclear envelope at the end of mitosis. When the mitotic spindle is absent or disrupted, or if the nuclear envelope does not reform because of chromosome fragmentation, this could prevent the assembly of the regular anillin donuts/contractile rings (Deshpande, 2005).

Since proper nuclear migration is necessary for pole cell formation, the number of pole cells were counted at the syncytial blastoderm stage (nuclear cycles 12-13) in ago-2⁴¹⁴ embryos stained with Vasa antibody and counterstained with the DNA dye Hoechst. The total number of pole cells in ago-2⁴¹⁴ (eight per embryo; n = 20) is also much less than wild type (20 per embryo; n = 15). Many ago-2⁴¹⁴ embryos had only two or three pole cells, and the pole cells were often not positioned correctly at the very posterior or were separated from each other by somatic cells. There was also a great deal of variability in the number of pole cells in the ago-2 mutant that is not evident in wild type. While some ago-2⁴¹⁴ embryos had only two or three pole cells, there were a few that had near wild-type numbers. A similar, though not quite as dramatic reduction in pole cell number was observed for a second allele, ago-2^51B (12 per embryo). The reduction in the number of pole cells can be traced back to nuclear cycle 8-9 when migrating nuclei first enter the posterior pole plasm (Deshpande, 2005).

Two questions are posed by the findings. The first is the relationship between the phenotypes seen in early embryos and ago-2 function. Although the possibility that ago-2 participates in several distinct pathways, it is suspected that many of the phenotypes are attributable either directly or indirectly to a requirement for ago-2 in the proper execution of the nuclear division cycles. In principle, ago-2 could impact the division cycles by promoting the degradation and/or translational repression of mRNAs encoding factors that somehow impede the cleavage cycles. However, since the RNAi machinery has been implicated in the establishment of heterochromatin domains, an alternative possibility is that ago-2 is important for generating the appropriate chromatin organization of cleavage-stage chromosomes. In S. pombe, deletion of Ago-1 (the only Ago gene) disrupts the formation of transcriptionally silenced heterochromatin domains near the centromeres, and this impairs the assembly/functioning of the centromeres. Most of the abnormalities in the division cycles and mitotic apparatus evident in ago-2 embryos could also be explained by perturbations in the assembly/functioning of the centromeres. Probably the most compelling evidence in favor of this hypothesis is the defects in CID association with ago-2 chromosomes (Deshpande, 2005).

Dividing nuclei were observed in which a subset of chromosomes or in some cases all of the chromosomes have little or no detectable CID. Strikingly, in these anaphase figures the chromosomes that have little CID typically remain on the metaphase plate, while the chromosomes that have CID are seen to migrate toward the centrosomes. The effects of ago-2 are not limited to centromeric heterochromatin. It was found that the silencing of paternally inherited mini-white transgenes inserted into centric heterochromatin can be suppressed by reducing ago-2 activity in the mother. Significantly, the pattern of suppression -- a nearly uniform level of pigmentation that is the same in both eyes -- argues that ago-2 activity is required very early in development during the initial 'establishment' of silenced heterochromatin. Consistent with an early role in heterochromatin assembly, abnormalities were also found in HP1 localization. While these results do not provide insights into how Ago-2 might actually function in this process, the idea that the RNAi machinery plays some special role in the de novo assembly of centromeric/centric heterochromatin during the embryonic nuclear division cycles is supported by the finding that siRNAs are highly enriched in early embryos but not at later stages of development (Deshpande, 2005).

The second question is why do the ago-2 mutants show incomplete penetrance and expressivity. One reason may be the plasticity of early embryos that enables cells in regions of the embryo that are normal to compensate for cells in regions that have major defects. Consistent with this suggestion, mutations have been described in three genes, l(3)malignant brain tumor, shakleton, and out of sync, that have a range of nuclear division and migration phenotypes similar to those seen in the two ago-2 alleles. In spite of the defects observed in these mutants, most of the embryos hatch and go on to form fertile adults. Another reason is that the two mutants examined may not be complete nulls. Consistent with this possibility, a low level of Ago-2 protein was detected in mutant embryos. Finally, there could be other partially redundant mechanisms that ensure the formation and/or maintenance of centromeric/centric heterochromatin and functional centromeres. This idea is supported by the studies that found that like ago-2, mutations in two other ago-2-like proteins, piwi and aubergine, suppress heterochromatic silencing of mini-white transgenes and disrupt the localization of heterochromatin proteins HP1 and HP2. Thus, it is possible that either of these two genes or perhaps other components of the RNAi machinery are able to substitute for ago-2 and promote the establishment and/or maintenance of heterochromatin domains and functional centromeres in early embryos (Deshpande, 2005).

RNA interference directs innate immunity against viruses in adult Drosophila

Innate immunity against bacterial and fungal pathogens is mediated by Toll and immune deficiency (Imd) pathways, but little is known about the antiviral response in Drosophila. This study demonstrates that an RNA interference pathway protects adult flies from infection by two evolutionarily diverse viruses. The work also describes a molecular framework for the viral immunity, in which viral double-stranded RNA produced during infection acts as the pathogen trigger whereas Drosophila Dicer-2 and Argonaute-2 act as host sensor and effector, respectively. These findings establish a Drosophila model for studying the innate immunity against viruses in animals (Wang, 2006).

RNA interference (RNAi) silences gene expression through small interfering RNAs (siRNAs) and microRNAs (miRNAs). In Drosophila, Dicer-2 (Dcr-2) produces siRNAs, whereas Dicer-1 (Dcr-1) recognizes precursors of miRNAs. The small RNAs are assembled with an Argonaute (Ago) protein into related effector complexes, such as RNA-induced silencing complex (RISC), to guide specific RNA silencing (Wang, 2006).

RNA silencing provides an antiviral mechanism in plants and animals. Plant viruses have evolved diverse strategies for evading the RNA silencing immunity, and expression of viral suppressors of RNAi (VSRs) is essential for infection and virulence. However, it is unknown whether antiviral silencing in plants is controlled by a specific small RNA pathway targeted by plant VSRs. Bacterial and fungal infections of Drosophila induce Toll and immune deficiency (Imd) pathways, leading to transcriptional induction of antimicrobial peptide effectors via NF-KappaB)–like signaling processes. However, it has been unclear whether either pathway plays a role in Drosophila innate immunity against viruses. Previous work in cell culture has indicated that RNAi might mediate viral immunity in Drosophila. This study investigated whether RNAi indeed provides protection against virus infection in Drosophila embryos and adults (Wang, 2006).

Flock house virus (FHV) contains an RNA genome divided among two plus-strand molecules, RNAs 1 and 2. RNA2 (R2) encodes the single virion structural protein, whereas RNA1 (R1) encodes protein A, the viral RNA–dependent RNA polymerase (RdRP), and B2, a VSR expressed after RNA1 replication from its own mRNA, RNA3. In the absence of R2, R1 replicates autonomously, accumulates to high levels, and produces abundant RNA3 in wild-type (WT) Drosophila embryos 30 hours after injection with R1 transcripts synthesized in vitro. No FHV RNAs accumulate in WT embryos injected with R1fs transcripts that contain a frameshift mutation in the RdRP open reading frame (ORF). FHV RNAs are also not readily detected in WT embryos injected with a second mutant of R1, R1DeltaB2, which does not express the VSR. However, abundant accumulation of R1DeltaB2 but not FR1fs occurs in mutant Drosophila embryos that carry a homozygous null mutation in ago-2 (ago-2⁴¹⁴), which is essential for RNAi in Drosophila. These data indicated that viral RNA replication in Drosophila embryos triggers an RNAi-mediated virus clearance in an Ago-2–dependent manner and that effective RNAi suppression by B2 is necessary to achieve normal accumulation of FHV RNAs (Wang, 2006).

In Drosophila, Ago-2 acts downstream of Dicer-2 (Dcr-2) to recruit siRNAs, the products of Dcr-2 activity, into the siRNA-dependent RISC (siRISC). Thus, a genetic requirement for ago-2 in FHV RNA clearance implicates Dcr-2 in the RNAi antiviral effector mechanism. To test this hypothesis, R1, R1fs, and R1DeltaB2 transcripts were injected into embryos carrying a homozygous dcr-2 null mutation, dcr-2^L811fsX. Northern blot hybridizations showed that, although FHV RNAs remained undetectable in dcr-2^L811fsX embryos injected with R1fs, viral RNA accumulation is rescued in the dcr-2^L811fsX embryos injected with R1DeltaB2 transcripts. This result shows that Dcr-2 is required to initiate RNAi-mediated clearance of FHV RNAs in Drosophila embryos (Wang, 2006).

To investigate whether the RNAi pathway protects Drosophila from virus infection, adult flies of either WT or dcr-2^L811fsX genotype were injected with purified FHV virions. The FHV isolate was of low virulence in WT flies, because about 50% of infected flies survived 15 days postinoculation (dpi) despite a detectable virus load. Inoculation with the same dose of FHV resulted in 60% mortality by 6 dpi and 95% by 15 dpi in dcr-2^L811fsX flies. Mock inoculation with buffer had little effect on either WT or dcr-2^L811fsX flies for as long as observations were made. Both Northern and Western blot analyses revealed that the virus accumulated more rapidly and to much greater levels in dcr-2^L811fsX than WT flies. Thus, dcr-2 mutants exhibit enhanced disease susceptibility to FHV in comparison with WT flies, demonstrating that Dcr-2 is also required to mount an immune response that protects adult Drosophila against FHV infection (Wang, 2006).

R2D2 contains tandem double-stranded RNA (dsRNA)–binding domains and forms a heterodimer with Dcr-2 in vivo that is required for siRNA loading into RISC. Flies homozygous for a loss-of-function mutation in r2d2 exhibit a phenotype of enhanced disease susceptibility to FHV infection similar to that of dcr-2^L811fsX. Thus, R2D2 also participates in the innate immunity pathway that protects adult flies from FHV infection. Notably, although FHV accumulates to extremely high levels in both dcr-2 and r2d2 mutant flies, abundant viral siRNAs were detected only in r2d2 mutant flies, and viral siRNAs were below the level of detection in dcr-2^L811fsX flies. Thus, FHV infection is detected by Dcr-2, leading to production of FHV siRNAs. However, R2D2 is not required for the production but is essential for the function of viral siRNAs, which is consistent with the genetic requirements for processing the artificially introduced dsRNA (Wang, 2006).

To investigate whether the RNAi pathway in Drosophila is specific against nodaviruses and not other classes of RNA viruses, the susceptibility of WT, dcr-2^L811fsX, and r2d2 mutant flies to cricket paralysis virus (CrPV) was assessed. CrPV contains a nonsegmented plus-strand RNA genome but belongs to a group of picorna-like viruses. CrPV is substantially more virulent than FHV in Drosophila; injection of CrPV at much lower titers resulted in mortality of 70% of WT flies by 15 dpi. CrPV was also found to induce enhanced disease susceptibility in both dcr-2 and r2d2 mutant flies. About 60% of the infected mutant flies were dead by 6 dpi, and more than 95% were dead by 15 dpi. In addition, Northern blots indicated that the virus accumulated more rapidly and to greater levels in the mutant flies. Thus, both dcr-2 and r2d2 are required for protection of Drosophila against CrPV (Wang, 2006).

CrPV infection of cultured S2 cells induced antiviral silencing, illustrated by detection of CrPV-specific siRNAs. Antiviral silencing against FHV in S2 cells induced by FR1gfp is suppressed by CrPV superinfection, leading to derepression of green fluorescent protein (GFP). Two ORFs are encoded by the CrPV RNA genome. No suppression of antiviral silencing was observed in S2 cells cotransfected with a plasmid expressing either the entire downstream ORF of CrPV or the individual mature virion proteins processed from the polyprotein. In contrast, RNAi suppression was detected after cotransfection with a plasmid expressing either the entire upstream ORF of CrPV or its N-terminal 140 codons. However, the suppressor activity was not detected after a frameshift mutation was introduced into pA, thus identifying the N-terminal fragment of 140 amino acids of the CrPV nonstructural polyprotein as a VSR (Wang, 2006).

In Drosophila, Imd signaling is stimulated by Gram negative (Gram^–) bacterial infection, whereas Toll signaling is triggered by Gram positive (Gram⁺) bacterial infection. To determine whether loss of the RNAi pathway initiated by Dcr-2 has an impact on the Toll and Imd signaling processes, WT, dcr-2^L811fsX, and r2d2 mutant flies were subjected to immune challenge by inoculation with Escherichia coli (Gram^–) or Micrococcus luteus (Gram⁺). Northern blot hybridizations detected substantial transcriptional induction of the antimicrobial peptide gene Diptericin A 6 hours postimmune challenge (hpi) with either E. coli or M. luteus, which declined at 24 hpi as described. Similar induction patterns for Diptericin A were observed in dcr-2^L811fsX and r2d2 mutant flies inoculated with Gram⁺ and Gram^– bacteria. Furthermore, it was found that induction of either Attacin A or Drosomycin by Gram⁺ and Gram^– bacteria was also not altered in dcr-2^L811fsX and r2d2 mutant flies as compared to WT flies. These results indicate that induction of antimicrobial peptide genes via Toll and Imd signaling pathways is not compromised in dcr-2^L811fsX and r2d2 mutant flies (Wang, 2006).

Nodaviruses and the polio-like CrPV belong to two different superfamilies of animal RNA viruses. The same set of RNAi pathway genes is required for Drosophila defense against FHV and CrPV and both viruses encode a potent VSR. These results collectively show that RNAi pathway functions as a common viral immunity mechanism in Drosophila and that RNAi suppression represents a general counterdefensive strategy used by insect viruses. Furthermore, a genetic requirement for Dcr-2, R2D2, and Ago-2 in antiviral silencing establishes a molecular framework for the innate immunity against viruses in Drosophila. None of Dcr-2, R2D2, and Ago-2 plays a detectable role in either the production or function of miRNAs in Drosophila. Thus, this work identifies the dsRNA-siRNA pathway of RNAi as providing the innate immunity against virus infection in Drosophila and establishes that dsRNA produced during virus replication acts as the pathogen trigger whereas Dcr-2 and Ago-2 act as host sensor and effector of the immunity, respectively. These results support and extend the previous findings on antiviral silencing in C. elegans (Wang, 2006).

Although NF-KappaB-like signaling in the Toll and Imd pathways do not appear to play a role in the RNAi-directed viral immunity mechanism in Drosophila, the fly mutant defective in the Janus kinase (JAK) Hopscotch exhibit a modest increase in susceptibility to infection with Drosophila C virus, suggesting an antiviral role for JAK–signal transducer and activator of transcription (STAT) signaling. Nonetheless, it is believed that RNAi-based immunity is independent of JAK-STAT signaling, because virus infection is not known to induce the RNAi pathway in Drosophila and FHV induction of the JAK-STAT responsive gene vir-1 is unaltered in the dcr-2 and r2d2 mutants. Because the Toll and Imd pathways are highly conserved in vertebrates, the Drosophila model established for RNAi may also be useful for the analyses of the innate antiviral immunity in vertebrates (Wang, 2006).

Overlapping functions of Argonaute proteins in patterning and morphogenesis of Drosophila embryos

Argonaute proteins are essential components of the molecular machinery that drives RNA silencing. In Drosophila, different members of the Argonaute family of proteins have been assigned to distinct RNA silencing pathways. While Ago1 is required for microRNA function, Ago2 is a crucial component of the RNA-induced silencing complex in siRNA-triggered RNA interference. Drosophila Ago2 contains an unusual amino-terminus with two types of imperfect glutamine-rich repeats (GRRs) of unknown function. This study shows that the GRRs of Ago2 are essential for the normal function of the protein. Alleles with reduced numbers of GRRs cause specific disruptions in two morphogenetic processes associated with the midblastula transition: membrane growth and microtubule-based organelle transport. These defects do not appear to result from disruption of siRNA-dependent processes but rather suggest an interference of the mutant Ago2 proteins in an Ago1-dependent pathway. Using loss-of-function alleles, it is further demonstrated that Ago1 and Ago2 act in a partially redundant manner to control the expression of the segment-polarity gene wingless in the early embryo. These findings argue against a strict separation of Ago1 and Ago2 functions and suggest that these proteins act in concert to control key steps of the midblastula transition and of segmental patterning (Meyer, 2006).

This study characterizes the maternal-effect mutation drop out (dop), which causes specific developmental defects at the midblastula transition. The mutant embryos show a transient block in membrane growth and fail to undergo a developmental switch in the microtubule-based polarized transport of lipid droplets. Surprisingly, dop mutations represent special alleles of ago2. Two independently generated dop alleles reduce the copy number of the GRRs, providing the first evidence of a functional role of this domain. These mutations render Ago2 only partially defective in siRNA responses. However, these alleles interact genetically with Ago1, suggesting the possibility of crosstalk between Ago1- and Ago2-mediated pathways. This conclusion is further supported by double-mutant analysis using loss-of-function alleles of ago2 and ago1; it was demonstrated that the two gene products function redundantly in embryonic patterning. The results reveal novel functions of Argonaute proteins in early embryogenesis and suggest a regulatory role for the GRR domain of Ago2 (Meyer, 2006).

In Drosophila, two major molecular pathways of RNA silencing have been defined: miRNA-induced silencing and siRNA-induced RNAi. At the level of Argonaute family members, Ago1 has been implicated in miRNA function while Ago2 was shown to be essential for siRNA function. This analysis provides genetic and biochemical evidence that Ago1 and Ago2 have overlapping functions both in siRNA-triggered RNAi and during early embryogenesis (Meyer, 2006).

In addition to the PAZ and PIWI domains conserved in all family members, insect orthologs of Ago2 contain an amino-terminal GRR domain. The ago2^dop alleles allowed the function of this domain to be probed. Even the subtle alterations in these alleles have striking organismal phenotypes, but the absence of Ago2 (in the reported null alleles) does not. While the mutant Ago2 proteins still support siRNA function to some extent, they also interfere with Ago1-dependent processes (Meyer, 2006).

In other proteins, glutamine-rich domains have been implicated in protein aggregation, such as in certain neurodegenerative diseases that involve the formation of long-lived protein aggregates (e.g., the PolyQ domain of mutant Huntingtin). Extension of the glutamine-rich region promotes aggregation, and the length of the polyglutamine extension correlates with the severity of the disease. Glutamine-rich domains are also involved in the mechanism by which yeast prions switch between soluble and aggregated states. For the translation factor Sup35, e.g., increases in the copy number of GRRs in the prion domain favor the aggregated, inactive state; decreases in the copy number favor the soluble, active state. Genetic and molecular analyses of the ago2^dop alleles thus raise the tantalizing possibility that the GRRs regulate Ago2 by modulating its aggregation state. Unlike in the polyglutamine diseases, however, it is the reduction, rather than the expansion, of the GRR region that leads to an aberrant Ago2 protein. Drosophila Ago2 may therefore provide a unique inroad for dissecting the normal organismal function of glutamine-rich or PolyQ domains (Meyer, 2006).

Since Ago2 is an essential component of protein complexes, such as the RISC, control of its aggregation state is conceivably important for its function. Mammalian Argonaute proteins are localized to GW bodies, cytoplasmic compartments analogous to yeast P-bodies, which are centers of mRNA degradation. Central components of GW bodies, like GW182 and decapping enzymes DCP1:DCP2, have been shown to also be involved in miRNA-mediated gene silencing in Drosophila cultured cells. The presence of both Ago1 and Ago2 in GW bodies is consistent with the biochemical studies. An important next step for unraveling the molecular function of the Ago2 GRR domain will be to determine whether the ago2^dop alleles alter the recruitment of Ago2 to particular cytoplasmic mRNA degradation complexes. Such recruitment via glutamine-rich domains need not necessarily inactivate the protein: in the translation factor CPEB from Aplysia, a glutamine-rich prion-like amino-terminal domain promotes protein aggregation, and it is the aggregated form that has the greatest capacity to stimulate translation (Meyer, 2006).

Previous analyses have suggested a simple model of division of labor between Argonaute proteins in Drosophila, with Ago1 specific for miRNA-directed silencing and Ago2 involved in siRNA-triggered RNAi. However, the genetic data add to emerging evidence that these proteins play much broader roles. Ago2, for example, appears to have functions beyond siRNA-induced RNAi. It has been proposed that in larval neurons Ago2 is recruited via the dFMR1 protein to certain RNP complexes, including those containing the PPK1 mRNA. This recruitment is functionally important since in the ago2^51B allele PPK1 mRNA levels are not properly downregulated; thus, Ago2 may play a role in the turnover of specific transcripts (Meyer, 2006).

For Ago1, in contrast, it is well established that it has a function in miRNA-directed RNA silencing. But while in biochemical assays Ago1 is not essential for siRNA function, ago1 mutations impair the response of siRNA-triggered RNAi in vivo. The data provide further evidence for overlapping functions of Ago2 and Ago1 in siRNA-directed RNAi. It is possible that although Ago2 is in principle sufficient to promote siRNA-directed RNA decay, in vivo the two proteins act in concert to make this process more efficient (Meyer, 2006).

It is unlikely that the morphogenesis phenotypes of ago2^dop mutant embryos are simply caused by disturbing the function of Ago2 in RNAi. Unlike ago2^dop1 mutants, ago2 alleles that completely abolish experimental siRNA-induced responses do not cause these gross morphological defects and exhibit problems with nuclear migration only during syncytial stages; these phenotypes occur with a moderate penetrance such that animals homozygous for these alleles can be kept as a stock. Rather, genetic data suggest that ago2^dop mutations compromise the function of both Ago2 and Ago1 in controlling specific aspects of the MBT. A genome-wide analysis of mRNA targets regulated by Argonaute proteins has recently shown that Ago1 and Ago2 are required for the regulation of a common set of miRNA targets, despite the fact that only Ago1 is essential for miRNA function in vitro. In S2 cells, both Ago1 and Ago2 coprecipitate with specific miRNAs, suggesting that not only Ago1, but also Ago2, is able to bind miRNAs. Based on the results, it is conceivable that the interaction of miRNAs with Ago2 is indirect, namely that Ago2 coprecipitates those miRNAs that are bound to Ago1. While the exact mechanisms need to be resolved, the available data provide ample support for the conclusion that Ago1 and Ago2 act in a partially redundant fashion during early embryogenesis (Meyer, 2006).

It is conceivable that the ago2^dop mutations not only interfere with Ago1 and Ago2 function but might affect a common factor that is essential for both Ago1 and Ago2 or for Argonaute protein function in general. Preliminary observations suggest that mutations in other Argonaute family members, piwi or aubergine, might also interact genetically with ago2^dop alleles. The model is favored that disrupting both Ago1 and Ago2 function is sufficient to cause the observed defects at the MBT because ago2^dop1 mutants can be rescued by zygotic expression of either ago1 or ago2. A test of this notion will be to determine the phenotypic consequences for embryos when both the maternal and zygotic expression of ago1 and ago2 has been eliminated. In addition, the interactions of ago2^dop alleles with other components of RNA silencing pathways should be examined to further understand the genetic and molecular basis for the altered activity of Ago2^dop proteins during the MBT (Meyer, 2006).

Mutations in ago1 were originally discovered in a genetic screen for modifiers of the Wg pathway. Overexpression of ago1 rescues a defect in Wg signaling induced by depletion of cytoplasmic Arm in the wing imaginal disc. However, because embryos homozygous for a loss-of-function mutation in ago1 did not exhibit defects in segment polarity, the relevance of Ago1 for normal Wg signaling remained unclear. The data presented in this paper now provide an explanation for this result. By combining loss-of-function mutations in both ago1 and ago2, it is demonstrated that the two Argonaute genes have partially overlapping functions and together are required for establishing segment polarity (Meyer, 2006).

The requirement of Ago1 and Ago2 for the initial expression of Wg protein is striking. No other genes have been identified that are similarly essential for the general expression of Wg. Two possible explanations are proposed for this result. Ago1 and Ago2 might act to eliminate a general repressor of wg transcription or translation. In this case, it is conceivable that specific miRNAs exist that modulate wg expression by negatively regulating a repressive mechanism. Alternatively, Ago1 and Ago2 might be part of RNPs that contain wg mRNA, and the reduction in Argonaute function might interfere with the microtubule motor-driven localization of the transcripts. It is well established that compromising the apical localization of wg mRNA strongly affects the intracellular distribution and the signaling activity of the protein. A detailed analysis of the expression and the localization of wg transcripts will be required to discriminate between these possibilities (Meyer, 2006).

Although no direct evidence was found that any of the ago2 alleles interfere with miRNA function in vivo or in vitro, it is interesting to note that ago1;Dcr-1 double mutants exhibit the same segment polarity phenotypes as ago1, ago2 double mutants. This result further strengthens the notion that in the embryo Ago1 and Ago2 might both be important for miRNA function. An eye reporter assay was employed to test if ago2^dop alleles interfere with the function of the bantam miRNA, no interactions were detected. This result might be due to the observed redundancy of Ago2 with Ago1 function; such a redundancy was recently described for S2 cells. Future studies to identify the miRNAs involved and their targets might yield novel insight into the regulation of Wg expression (Meyer, 2006).

An alternative explanation is that this analysis has uncovered a novel function of Argonaute protein family members. Intriguingly, ectopically expressed Ago1 constructs can suppress Wg pathway defects even if they lack a functional PIWI domain. This result may suggest that Ago1 function in Wg signaling does not involve its PIWI domain, hinting at an uncharacterized biochemical property of Ago1. Although too little is known at this point to speculate what such a new function might entail, it is interesting to note that there are intriguing connections between microtubules and the RNA silencing machinery: Armitage, a putative helicase required to assemble Ago2-containing RISC, is associated with microtubules in developing oocytes; the dop alleles of Ago2 interfere with microtubule-based processes at the MBT; and it is conceivable that Ago1 and Ago2 control the microtubule-dependent localization of wg mRNA. Whether or not these phenomena are explained by a shared molecular mechanism remains to be established (Meyer, 2006).

In summary, the genetic interactions described in this paper are not easily reconciled with the model that different pathways in gene silencing are strictly separated. Rather, the data suggest that in the living organism these pathways, or at least crucial components of these pathways, might act in concert. Observation that ago1 and ago2 cooperate in Wg signaling provides a powerful new tool to resolve some of these issues since now the function of these Argonaute proteins can be assessed using a clearly defined phenotype of a well-characterized signaling pathway (Meyer, 2006).

Freshly laid Drosophila embryos contain large amounts of maternally supplied mRNAs that encode proteins essential for the earliest stages of embryogenesis. As development proceeds, these maternally supplied transcripts need to be replaced by transcripts synthesized by the zygote. This process is a hallmark of the MBT. Maternal transcripts are degraded via two pathways: a maternal pathway switched on at egg activation, and a zygotic pathway activated at the MBT. Genetic analysis has shown that although ago2^dop alleles represent maternal-effect mutations, they specifically perturb processes shortly after the onset of zygotic transcription at the MBT. It is therefore proposed that Ago1 and Ago2 are key mediators of the zygotic pathway of maternal transcript degradation. Precedence for such a scenario has recently been provided by the identification of the miR-430 miRNA family in zebrafish. miR-430 expression is strongly upregulated at the MBT and is required to specifically downregulate a set of maternal mRNAs. Conversely, embryos deficient for Dicer activity display defects shortly after the MBT. It remains to be determined whether miRNAs are also required for maternal transcript degradation in Drosophila (Meyer, 2006).

The known functions and structural features of Argonaute proteins suggest a model for the underlying molecular mechanisms. It is well established that Argonaute proteins can act as ribonucleases and provide slicer activity in RISC. During early development, Ago2 and Ago1 might act as ribonucleases that cleave maternal transcripts at the MBT. Abnormal persistence of maternal mRNAs could then interfere with the morphogenetic events usually triggered by zygotic transcription, such as membrane growth during cellularization and correct directionality of lipid-droplet transport. Alternatively, Argonaute proteins might regulate the translation of such maternal or zygotic transcripts. Since no significant changes in the expression pattern of known regulators of membrane growth and droplet transport (Halo, Slam, Klar) were detected, the relevant targets are likely novel components of these regulatory pathways. Identifying them should not only give insight into the regulation of these fundamental cell-biological processes but will also shed light on the mechanisms by which the Argonaute proteins Ago1 and Ago2 work together to control developmental events (Meyer, 2006).

RNA interference machinery influences the nuclear organization of a chromatin insulator

RNA interference (RNAi) is a conserved silencing mechanism that can act through alteration of chromatin structure. Chromatin insulators promote higher-order nuclear organization, thereby establishing DNA domains subject to distinct transcriptional controls. Evidence is presented for a functional relationship between RNAi and the gypsy insulator of Drosophila. Insulator activity is decreased when Argonaute genes required for RNAi are mutated, and insulator function is improved when the levels of the Rm62 helicase, involved in double-stranded RNA (dsRNA)-mediated silencing and heterochromatin formation, are reduced. Rm62 interacts physically with the DNA-binding insulator protein CP190 in an RNA-dependent manner. Finally, reduction of Rm62 levels results in marked nuclear reorganization of a compromised insulator. These results suggest that the RNAi machinery acts as a modulator of nuclear architecture capable of effecting global changes in gene expression (Lei, 2006).

These results suggest the existence of an RNA species required for the formation or integrity of insulator bodies, perhaps a product of processing by Argonautes and the other RNAi machinery. The putative RNA helicase Rm62 may be recruited to insulator complexes through physical interaction with CP190 and RNA. Although it is unknown at what mechanistic step Rm62 acts in RNAi, Rm62 may act downstream of Argonautes to unwind or remodel RNA-insulator protein complexes, thereby disrupting gypsy insulator activity and nuclear organization. Proper insulator body localization requires an intact nuclear matrix, and early observations identified RNA as an important component of this nuclear scaffold. Future studies should determine the identity of putative gypsy insulator associated RNAs. These results suggest a previously unknown function of the RNAi machinery in the control of nuclear architecture to effect changes in gene expression (Lei, 2006).

P-body formation is a consequence, not the cause, of RNA-mediated gene silencing; AGO2 is required for P-body integrity

P bodies are cytoplasmic domains that contain proteins involved in diverse posttranscriptional processes, such as mRNA degradation, nonsense-mediated mRNA decay (NMD), translational repression, and RNA-mediated gene silencing. The localization of these proteins and their targets in P bodies raises the question of whether their spatial concentration in discrete cytoplasmic domains is required for posttranscriptional gene regulation. This study shows that processes such as mRNA decay, NMD, and RNA-mediated gene silencing are functional in cells lacking detectable microscopic P bodies. Although P bodies are not required for silencing, blocking small interfering RNA or microRNA silencing pathways at any step prevents P-body formation, indicating that P bodies arise as a consequence of silencing. Consistently, releasing mRNAs from polysomes is insufficient to trigger P-body assembly: polysome-free mRNAs must enter silencing and/or decapping pathways to nucleate P bodies. Thus, even though P-body components play crucial roles in mRNA silencing and decay, aggregation into P bodies is not required for function but is instead a consequence of their activity (Eulalio, 2007).

The first proteins found in P bodies are those functioning in the degradation of bulk mRNA. In eukaryotes, this process is initiated by removal of the poly(A) tail by deadenylases. There are several deadenylase complexes in eukaryotes: the PARN2-PARN3 complex is thought to initiate deadenylation, which is then continued by the CAF1-CCR4-NOT complex. Following deadenylation, mRNAs are exonucleolytically digested from their 3' end by the exosome, a multimeric complex with 3'-to-5' exonuclease activity. Alternatively, the cap structure is removed by the decapping enzyme DCP2 after deadenylation, rendering the mRNA susceptible to 5'-to-3' degradation by the major cytoplasmic exonuclease XRN1 (Eulalio, 2007).

Decapping requires the activity of several proteins generically termed decapping coactivators, though they may stimulate decapping by different mechanisms. In the yeast Saccharomyces cerevisiae, these include DCP1, which forms a complex with DCP2 and is required for decapping in vivo, the enhancer of decapping-3 (EDC3 or LSm16), the heptameric LSm1-7 complex, the DExH/D-box RNA helicase 1 (Dhh1, also known as RCK/p54 in mammals), and Pat1, a protein of unknown function that interacts with the LSm1-7 complex, Dhh1, and XRN1. In human cells, DCP1 and DCP2 are part of a multimeric protein complex that includes RCK/p54, EDC3, and Ge-1 (also known as RCD-8 or Hedls), a protein that is absent in S. cerevisiae (Eulalio, 2007).

The decapping enzymes, decapping coactivators, and XRN1 colocalize in P bodies. Additional P-body components in multicellular organisms include the protein RAP55 (also known as LSm14; Drosophila homolog - Trailer hitch), which has a putative role in translation regulation, and GW182, which plays a role in the microRNA (miRNA) pathway (Eulalio, 2007).

The P-body marker GW182 localizes to cytoplasmic foci in Drosophila S2 cells together with the decapping enzyme DCP2 and the decapping coactivator DCP1, suggesting that these foci represent P bodies. To characterize D. melanogaster P bodies further, antibodies were raised to the Drosophla orthologs of two proteins found in human-cell P bodies. These correspond to Ge-1 and Tral (LSm15), which is closely related to human RAP55 (or LSm14) (see Tanaka, 2006). Both antibodies stained the cytoplasm diffusely and also stained discrete cytoplasmic foci with a diameter ranging from 100 nm to 300 nm. The antibody signals are specific, as they are lost in cells in which the cognate proteins were depleted. The foci are present in about 95% of the cell population and are readily detectable because the concentration of Tral or Ge-1 in these foci is significantly higher than that in the surrounding cytoplasm (Eulalio, 2007).

The distribution of green fluorescent protein (GFP)-tagged versions of proteins found in P bodies was examined in yeast and/or human cells. These include DCP1, DCP2, GW182, Me31B (the D. melanogaster ortholog of S. cerevisiae Dhh1 and vertebrate RCK/p54), CG5208 (the D. melanogaster homolog of S. cerevisiae Pat1, referred to as HPat hereafter), and EDC3 (also known as LSm16). All of these proteins formed cytoplasmic foci that costained with the anti-Tral or anti-Ge-1 antibodies. Importantly, the expression of the GFP-tagged proteins did not significantly alter the number and size of endogenous P bodies. Together, these results indicate that the localization of decapping enzymes and decapping coactivators into P bodies is evolutionarily conserved. The localization of GW182 in Drosophila P bodies is in agreement with the proposal that GW-bodies and P bodies overlap, as reported for mammalian cells (Eulalio, 2007).

The localization of proteins implicated in translational regulation was examined in Drosophila oocytes whose corresponding transcripts are detectable in S2 cells, in particular, Smaug and the dsRNA binding protein Staufen. Smaug is a translational repressor that also promotes deadenylation of bound mRNAs by recruiting the CAF1-CCR4-NOT1 complex (Zaessinger, 2006). Both proteins localized to P bodies with endogenous Tral. Strikingly, P bodies increased in size in cells expressing Staufen at high levels but not in cells overexpressing GFP fusions of Smaug, suggesting that Staufen promotes P-body formation. Drosophila Staufen, Tral, DCP1, DCP2, XRN1, and Me31B have also been detected in RNP granules in neuronal cells and/or in oocytes, indicating that P bodies and other RNP granules observed in neuronal cells or during development share common components (Eulalio, 2007).

P-body formation requires nontranslating mRNPs and/or mRNPs undergoing decapping. A conserved feature of P bodies in human and yeast cells is that their formation depends on RNA and is enhanced in cells in which the concentration of nontranslating mRNAs or of mRNAs undergoing decapping increases. These observations indicate that mRNAs must exit the translation cycle to localize to P bodies. In agreement with this, it was observed that Drosophila P bodies decline when cells are treated with RNase A or with cycloheximide (which inhibits translation elongation and stabilizes mRNAs into polysomes). In contrast, P-body sizes increase in cells treated with puromycin, which causes premature polypeptide chain termination and polysome disassembly. Both puromycin and cycloheximide inhibit protein synthesis in S2 cells, as judged by the reduction of F-Luc and R-Luc activities after the treatment of cells transiently expressing these proteins with these drugs (Eulalio, 2007).

The size of Drosophila P bodies also depends on the fraction of mRNAs undergoing decapping, in agreement with the results reported for yeast and human cells. Indeed, blocking mRNA decay at an early stage, for instance, by preventing deadenylation in cells in which NOT1 (a component of the CAF1-CCR4-NOT deadenylase complex) is depleted, leads to the dispersion of P bodies, whereas P bodies are on average more prominent in cells from which DCP2 or XRN1 is depleted (in which decapping and subsequent 5'-to-3' mRNA decay are inhibited) (Eulalio, 2007).

Several lines of evidence show that P bodies do not serve as storage sites for the effectors of posttranscriptional process but are sites where mRNA degradation and silencing can take place. For instance, P-body formation is RNA dependent, and decay intermediates, siRNAs, and miRNAs and their targets are detected in P bodies. Moreover, the size and number of P bodies depends on the fraction of mRNAs undergoing decapping. However, the question of whether mRNA decay and silencing require the environment of microscopic, wild-type P bodies to occur or whether these processes can also occur outside of P bodies in soluble protein complexes remains open. This study shows that formation of large P bodies visible in the light microscope as observed in wild-type cells is not required for several processes associated with P-body components, including NMD, mRNA decay, and RNA-mediated gene silencing (Eulalio, 2007).

The question addressed in this study was whether the environment of macroscopic P bodies is required for posttranscriptional regulation. P bodies are defined as the large cytoplasmic foci visible by light microscopy in wild-type cells. These foci are on average 100 to 300 nm in diameter and are readily detected as bright cytoplasmic dots because the concentration of proteins in these foci is significantly higher than in the surrounding cytoplasm. Nevertheless, most P-body components are also detected diffusely throughout the cytoplasm. For a limited number of examples that have been analyzed, it has been shown that P-body components are not confined to these structures but dynamically exchange with the cytoplasmic pool. Quantitative information regarding the fractionation of P-body components between P bodies and the cytoplasm is still lacking, but given the volume of P bodies relative to that of the cytoplasm, it is likely that the diffuse cytoplasmic fraction is significantly larger. This suggests that posttranscriptional processes are likely to occur and may even be initiated in the diffuse cytoplasm or in soluble protein complexes that aggregate to form P bodies. Whether these processes take place in submicroscopic aggregates or soluble protein complexes in the absence of detectable microscopic P bodies remains to be solved. However, it is considered that aggregates or large multiprotein assemblies that are not detectable by light microscopy cannot be defined as bodies (Eulalio, 2007).

Translation factors or ribosomes are generally not present in P bodies (with the exception of cap binding protein eIF4E), indicating that mRNAs leave the translation cycle prior to entering P bodies. Consistently, releasing mRNAs from polysomes leads to increases in P-body sizes and numbers, whereas the stabilization of mRNAs into polysomes disrupts P bodies. These observations suggest that a critical step in P-body formation is the release of mRNPs from a translationally active state associated with polysomes to a translationally inactive state. This paper has shown that releasing mRNAs from polysomes by puromycin treatment is not sufficient to elicit P-body formation and that functional silencing pathways or proteins generically termed decapping coactivators are required for P-body assembly. These proteins include Me31B (Dhh1 in yeast), HPat (Pat1 in yeast), Ge-1, and the LSm1-7 complex (Eulalio, 2007).

What could be the role of these proteins in P-body formation? Me31B is an RNA helicase which could facilitate rearrangements in mRNP composition upon release from polysomes. The role of HPat is unclear, but the yeast ortholog interacts with Dhh1, XRN1, and the heptameric LSm1-7 complex. Coimmunoprecipitation assays indicate that the interaction between Dhh1 and Pat1 orthologs (i.e., Me31B and HPat) is conserved in Drosophila. Finally, the LSm1-7 complex associates with deadenylated mRNAs and stimulates decapping. Clearly, many details regarding the precise molecular function of these proteins remain to be discovered, but their requirement for P-body assembly indicates that mRNAs that are not actively translated do not enter into P bodies by default: the activity of a defined set of proteins is required. Alternatively, nontranslating mRNAs may enter silencing pathways, and this would also lead to changes in mRNP composition due to the recruitment of Argonaute proteins and binding partners, which include P-body components such as GW182, decapping enzymes, and RCK/p54 (Eulalio, 2007).

Once P-body components are bound to an RNP, P-body formation may then be triggered by protein-protein interactions. Indeed, proteins required for P-body assembly are known to interact to form multimeric protein complexes. Consistently, in addition to the interactions mentioned above, DCP1, DCP2, Ge-1, RCK/p54, and EDC3 form a multimeric protein complex in human cells. The absolute requirement of RNA for P-body formation could be explained if affinities between these proteins increased upon RNA binding. Additionally, proteins like GW182 and Ge-1 are multidomain proteins that could bind more than one RNP simultaneously, bringing into close proximity several components and thus nucleating the formation of P bodies (Eulalio, 2007).

RNAs targeted by silencing pathways nucleate P bodies. In this study, it is shown that both the RNAi and miRNA pathways contribute to the generation of a pool of nontranslating mRNPs and/or of mRNPs committed to decay which are required for P-body formation. Nevertheless, silencing can occur in the absence of microscopic P bodies. The results provide support to previous models proposing that silencing is initiated in the cytoplasm and that the localization of the silencing machinery into P bodies is a consequence, rather than the cause, of silencing (Eulalio, 2007).

An unexpected observation from these studies is that AGO2 and Dicer-2, which function in siRNA-mediated gene silencing in Drosophila, are required for P-body integrity. The role of these proteins in P-body assembly is unlikely to be structural, because P bodies are restored upon puromycin treatment in cells from which AGO2 or Dicer-2 is depleted. The most likely explanation for the requirement of these proteins is, therefore, that silencing by siRNAs also generates RNPs that elicit P-body formation. The requirement for AGO2 could be at least partially explained by the observation that the expression levels of a small subset of endogenous miRNA targets are affected in AGO2-depleted cells, suggesting that some miRNAs may be loaded into AGO2-containing RNA-induced silencing complexes. Furthermore, the AGO1 and AGO2 genes interact, although it is unclear how this interaction affects the activities of these proteins (Eulalio, 2007).

The requirement for Dicer-2 in P-body assembly, however, suggests that endogenous siRNA targets also contribute to P-body formation. Because the levels of dsRNA synthesis from endogenous loci that could provide precursors for the production of endogenous siRNAs are currently unknown, the fraction and origin of transcripts regulated by endogenous siRNAs cannot be estimated. Nonetheless, a possible source of endogenous dsRNAs is the bidirectional transcription of pseudogenes and transposable elements, in agreement with the role of the RNAi pathway as a defense mechanism against RNA viruses and mobile genetic elements (Eulalio, 2007).

The essential role of silencing pathways in P-body formation in Drosophila, and presumably in human cells, raises the question of how P bodies are assembled in S. cerevisiae, which lacks silencing pathways. One possibility is that other posttranscriptional processes generate nontranslating mRNPs required to nucleate P bodies. For instance, the NMD pathway contributes to P-body assembly in yeast cells, because depletion of Upf2 or Upf3 leads to increases in P-body size and number in a Upf1-dependent manner, whereas similar experiments with Drosophila cells do not affect P bodies (Eulalio, 2007).

With the exception of the proteins involved in silencing, the composition of P bodies and the effects of drugs such as cycloheximide and puromycin on P-body size and number are strikingly similar in yeast, Drosophila, and human cells, raising the question of what the role of these structures accounting for their conservation in eukaryotic cells could be. The results show that the environment of microscopic P bodies is not essential for mRNA decay or silencing but do not exclude that the formation of P bodies confers a kinetic advantage. Moreover, the results do not rule out a role for large P bodies in sequestering a specific set of nontranslating mRNPs and reinforcing their repression by shielding them from the translation machinery (Eulalio, 2007).

Finally, the conservation of P bodies may reflect a role for these structures in other cellular processes that is not yet fully appreciated. A role in some steps of retroviral or retrotransposon life cycles is suggested by the localizations of the antiretroviral proteins APOBEC3G and APOBEC3F in human cell P bodies and of the protein and RNA components of the retrovirus-like element Ty3 in yeast P bodies. A link between P bodies and the regulation of retrotransposition would be consistent with the role of RNAi pathways in silencing the expression of transposable elements. Because all known essential P-body components play roles in decapping and/or silencing and proteins playing an exclusively structural role in P-body assembly have not yet been identified, it is currently not possible to evaluate the role of P bodies for cell, tissue, or organism survival (Eulalio, 2007).

Endogenous RNA interference provides a somatic defense against Drosophila transposons

Because of the mutagenic consequences of mobile genetic elements, elaborate defenses have evolved to restrict their activity. A major system that controls the activity of transposable elements (TEs) in flies and vertebrates is mediated by Piwi-interacting RNAs (piRNAs), which are ~24-30 nucleotide RNAs that are bound by Piwi-class effectors. The piRNA system is thought to provide primarily a germline defense against TE activity. This study describes a second system that represses Drosophila TEs by using endogenous small interfering RNAs (siRNAs), which are 21 nucleotide, 3'-end-modified RNAs that are dependent on Dicer-2 and Argonaute-2. In contrast to piRNAs, the TE-siRNA system is active in somatic tissues, and particularly so in various immortalized cell lines. Analysis of the patterns and properties of TE-derived small RNAs reveals further distinctions between TE regions and genomic loci that are converted into piRNAs and siRNAs, respectively. Finally, functional tests show that many transposon transcripts accumulate to higher levels in cells and animal tissues that are deficient for Dicer-2 or Argonaute-2. It is concluded that Drosophila utilizes two small-RNA systems to restrict transposon activity in the germline (mostly via piRNAs) and in the soma (mostly via siRNAs) (Chung, 2008).

Although the Drosophila RNAi pathway produces regulatory siRNAs in response to viral invasion, exogenous dsRNA, or IR transgenes, relatively little is historically known about the endogenous usage of Drosophila RNAi. This study describes a rich set of bona fide siRNAs that derive from transposable elements in Drosophila. These data add to a host of concurrent studies that recently elucidated multiple classes of siRNAs that derive from the host genome, not only from TEs, but also from 3' cis-natural antisense gene pairs, long IR transcripts, and two unique intronic and exonic clusters localized to the klarsicht and thickveins genes. Although these myriad siRNAs differ in origin, with some derived from bidirectional transcription and others from intramolecular dsRNA, they are united by their dependence on Dcr-2 and Ago2, their 3'-end modification, and, for at least some members of each class, an appreciable dependence on Loqs (Chung, 2008).

TE-siRNAs may be confidently distinguished from previously described TE-piRNAs on the basis of their characteristic sizes, genomic origins, tissue distribution, and origin from within a given TE. Both types of small-RNA pathways are demonstrably required to restrict TE transcript accumulation, and their separable roles correlate with their distinct tissue requirements. The germline is highly active in TE-piRNA production and uses piRNA components to restrict TE accumulation, whereas somatic tissues such as adult heads specifically produce TE-siRNAs and use RNAi components to restrict TE levels. Similar conclusions on TE-siRNA biogenesis and function have been reached in the concurrent studies of other groups. Curiously, whereas the mouse male germline depends strongly on piRNAs to restrict transposon activity, the mouse female germline appears to use both piRNAs and siRNAs to control TE activity. Therefore, there has been evolutionary flux in how these conserved small-RNA pathways are used to control TEs in animals (Chung, 2008).

Curiously, it was found that independently derived lines of cultured cells, namely S2 and Kc, exhibit pronounced siRNA responses to a subclass of LTR retrotransposons. This can be directly correlated with the fact of deregulation and genomic amplification of these particular TEs. It is possible that transposon deregulation was a direct consequence of the process of cell immortalization. However, one could speculate that their deregulation was a gradual consequence of divorcing these cells from piRNA control, which in the animal occurs mostly in the germline and is transmitted from generation to generation via maternal deposition of piRNA complexes into the embryo. In either case, the stronger TE-siRNA response in cultured cells may be viewed as an adaptive response to deregulated transposons, as proposed for the piRNA pathway (Chung, 2008).

The endogenous siRNA pathway is involved in heterochromatin formation in Drosophila

A new class of small RNAs (endo-siRNAs) produced from endogenous double-stranded RNA (dsRNA) precursors was recently shown to mediate transposable element (TE) silencing in the Drosophila soma. These endo-siRNAs might play a role in heterochromatin formation. This has been shown in S. pombe for siRNAs derived from repetitive sequences in chromosome pericentromeres. To address this possibility, the viral suppressors of RNA silencing B2 and P19 were used. These proteins normally counteract the RNAi host defense by blocking the biogenesis or activity of virus-derived siRNAs. It was hypothesized that both proteins would similarly block endo-siRNA processing or function, thereby revealing the contribution of endo-siRNA to heterochromatin formation. Accordingly, P19 as well as a nuclear form of P19 expressed in Drosophila somatic cells were found to sequester TE-derived siRNAs whereas B2 predominantly bound their longer precursors. Strikingly, B2 or the nuclear form of P19, but not P19, suppressed silencing of heterochromatin gene markers in adult flies, and altered histone H3-K9 methylation as well as chromosomal distribution of histone methyl transferase Su(var)3-9 and Heterochromatin Protein 1 in larvae. Similar effects were observed in dcr2, r2d2, and ago2 mutants. These findings provide evidence that a nuclear pool of TE-derived endo-siRNAs is involved in heterochromatin formation in somatic tissues in Drosophila (Fagegaltier, 2009).

This study implicates components of the RNAi pathway in heterochromatin silencing during late Drosophila development. The study also provides correlative evidence supporting a functional link between endo-siRNAs and the formation or maintenance of somatic heterochromatin in flies. The viral proteins NLS-P19 and B2 suppress the silencing of PEV markers and induce aberrant distribution of H3m2K9 and H3m3K9 heterochromatic marks as well as histone H3 methylase Su(var)3-9 in larval tissues. Dcr2 and Ago2 mutations have similar effects. In striking contrast, cytoplasmic P19 has no noticeable effect on chromatin. It is proposed that B2 inhibits Dcr2-mediated processing of double-stranded TE read-through transcripts in the cytoplasm; it is further proposed that NLS-P19 sequesters TE-derived siRNA duplexes. This model implies that part of the cytoplasmic pool of TE-derived endo-siRNA (which might be involved in PTGS events) is translocated back into the nucleus to exert chromatin-based functions. In C. elegans, silencing of nuclear-localized transcripts involves nuclear transport of siRNAs by an NRDE-3 Argonaute protein. A similar siRNA nuclear translocation system, possibly mediated by Ago2, may also exist in flies. Alternatively, an as yet unidentified siRNA duplex transporter may be involved. Deep sequencing analyses show that the fraction of siRNAs sequestered by NLS-P19 is smaller as compared with the one bound by P19 in the cytoplasm. Thus, the poor effects of P19 on nuclear gene silencing may be explained if the cytoplasmic pool of siRNA competes with the pool of siRNA to be translocated in the nucleus (Fagegaltier, 2009).

The Dcr-1 partner Loquacious (Loqs), but not the Dcr-2 partner R2D2, was unexpectedly found to be required for biogenesis of siRNA derived from fold-back genes that form dsRNA hairpins. By contrast, it is noteworthy that loqs mutations had little or no impact on the accumulation of siRNA derived from TE. The finding that r2d2 but not loqs mutation suppresses the silencing of PEV reporters and delocalizes H3m2K9 and H3m3K9 heterochromatic marks agrees with these results and further suggests that siRNA involved in heterochromatin formation and siRNA derived from endogenous hairpins arise from distinct r2d2- and loqs-dependent pathways, respectively. One possible mechanism by which TE- or repeat-derived endo-siRNAs could promote heterochromatin formation is by tethering complementary nascent TE transcripts and guiding Su(var)3-9 recruitment and H3K9 methylation. Identifying which enzymes tether siRNAs to chromatin in animals is a future challenge. In addition, some endo-siRNAs could also impact on heterochromatin formation by posttranscriptionaly regulating the expression of chromatin modifiers, such as Su(var)3-9. In any case, the current results demonstrate the value of viral silencing suppressor proteins in linking siRNAs to heterochromatin silencing in the fly soma, as established in S. pombe and higher plants. Because silencing suppressors are at the core of the viral counterdefensive arsenal against antiviral RNA silencing in fly, whether they also induce epigenetic changes in chromatin states during natural infections by viruses deserves further investigation (Fagegaltier, 2009).

The let-7-Imp axis regulates aging of the Drosophila testis stem-cell niche

Adult stem cells support tissue homeostasis and repair throughout the life of an individual. During ageing, numerous intrinsic and extrinsic changes occur that result in altered stem-cell behaviour and reduced tissue maintenance and regeneration. In the Drosophila testis, ageing results in a marked decrease in the self-renewal factor Unpaired (Upd), leading to a concomitant loss of germline stem cells. This study demonstrates that IGF-II messenger RNA binding protein (Imp) counteracts endogenous small interfering RNAs to stabilize upd (also known as os) RNA. However, similar to upd, Imp expression decreases in the hub cells of older males, which is due to the targeting of Imp by the heterochronic microRNA let-7. In the absence of Imp, upd mRNA therefore becomes unprotected and susceptible to degradation. Understanding the mechanistic basis for ageing-related changes in stem-cell behaviour will lead to the development of strategies to treat age-onset diseases and facilitate stem-cell-based therapies in older individuals (Toledano, 2012).

Many stem cells lose the capacity for self-renewal when removed from their local microenvironment (or niche), indicating that the niche has a major role in controlling stem-cell fate. Changes to the local and systemic environments occur with age that result in altered stem-cell behaviour and reduced tissue maintenance and regeneration. The stem-cell niche in the Drosophila testis is located at the apical tip, where both germline stem cells (GSCs) and somatic cyst stem cells are in direct contact with hub cells. Hub cells express the self-renewal factor Upd, which activates the JAK-STAT signalling pathway to regulate the behaviour of adjacent stem cells. Ageing results in a progressive and significant decrease in the levels of upd in hub cells. However, constitutive expression of upd in hub cells was sufficient to block the age-related loss of GSCs, suggesting that mechanisms might be in place to regulate upd and maintain an active stem-cell niche (Toledano, 2012).

To identify potential regulators of upd, a collection of transgenic flies carrying green fluorescent protein (GFP)-tagged proteins was screened for expression in hub cells. The Drosophila homologue of Imp protein is expressed throughout the testis tip in young flies (Fabrizio, 2008); however, antibody staining revealed a decrease (~50%) in Imp expression in the hub cells of aged males. Imp is a member of a conserved family of RNA-binding proteins that regulate RNA stability, translation and localization (Yisraeli, 2005). Given the similarity in the ageing-related decline in Imp protein and upd mRNA in hub cells, it is proposed that Imp could be a new regulator of upd (Toledano, 2012).

To address whether Imp acts in hub cells to regulate upd, the bipartite GAL4-UAS system was used in combination with RNA-mediated interference (RNAi) to reduce Imp expression exclusively in hub cells. Fluorescence in situ hybridization (FISH) to detect upd mRNA was used in combination with immunofluorescence microscopy to determine whether the loss of Imp expression affects upd levels. The loss of Imp specifically in hub cells resulted in reduced expression of upd, as well as a significant reduction in GSCs and hub cell), when compared with controls. Consistent with a reduction in JAK-STAT signalling, decreased accumulation of STAT was observed when Imp levels were reduced by RNAi in hub cells (Toledano, 2012).

RNA-binding proteins characteristically target several RNAs; therefore, it was of interest to determine whether upd is a physiologically relevant target of Imp. Expression of upd together with an Imp RNAi construct was sufficient to completely rescue the defects caused by reduced Imp expression in hub cells, suggesting that Upd acts downstream of Imp to maintain GSCs and niche integrity. Importantly, the constitutive expression of upd alone in hub cells did not lead to an increase in GSCs in testes from 1-day-old males. These data suggest that Imp acts in hub cells to promote niche integrity and GSC maintenance, at least in part, by positively regulating upd (Toledano, 2012).

If Imp acts in hub cells in adult testes to regulate upd mRNA, it is speculated that the loss of Imp function during development might lead to a decrease in upd and a subsequent reduction in GSCs. Null mutations in Imp result in lethality at the pharate adult stage; therefore, testes from third instar larvae (L3) carrying Imp null alleles, Imp⁷ and Imp⁸, were examined. Deletion of the Imp locus was verified by PCR of genomic DNA. Combined immunofluorescence and FISH showed that although Fas3⁺ hub cells were easily detected, the expression of upd was significantly reduced: 24% of Imp⁷ mutants and 15% of Imp⁸ mutants had no detectable upd at this stage. In addition, the average number of GSCs and hub cells in testes from Imp mutants was significantly reduced when compared with control L3 testes. Notably, the re-expression of Imp in somatic niche cells was sufficient to rescue upd expression in Imp mutants to comparable levels to controls, and the reduction in the average number of GSCs and hub cells in Imp mutants was also reversed (Toledano, 2012).

Imp family members contain conserved KH domains that mediate direct binding to RNA targets. To determine whether Imp could associate directly with upd mRNA in vivo, testes were dissected from young flies expressing GFP-tagged Imp. Immunoprecipitation of Imp with anti-GFP antibodies, followed by quantitative reverse transcriptase PCR (qRT-PCR) analysis, showed a significant enrichment (~208-fold) of associated upd mRNA relative to control antibodies. Minimal enrichment for the ubiquitously expressed RNAs rp49 (also known as RpL32; ~4-fold) and GapDH (also known as Gapdh1; ~8 fold) or for the negative control med23 (~4-fold), was observed after Imp immunoprecipitation, indicating that the interaction between Imp and upd mRNA in hub cells is specific. Consistent with these observations, Imp protein and upd RNA co-localized in hub cells within perinuclear foci, probably ribonucleoprotein particles (Toledano, 2012).

An in vitro protein-RNA binding assay showed that Imp associates with the upd 3' untranslated region (UTR), specifically the first 250 base pairs (region 1), as no substantial binding to other portions of the upd 3'UTR was detected. Moreover, Imp did not bind the 5' untranslated or coding regions of upd or to the med23 3'UTR. Notably, a putative consensus binding sequence CAUH (in which H denotes A, U or C) for the mammalian IMP homologues (IGF2BP1- 3) occurs 22 times within the upd 3'UTR, including a cluster of four tandem repeats within the first 35 nucleotides of region 1. To test whether this motif mediates binding between Imp and upd, the first 33 nucleotides were removed to generate a sequence excluding the CAUH repeats, which resulted in a reduction in binding, compare domain 1 with domain 2. Point mutations in the third nucleotide of each motif (U = G) did not abolish the binding; however, point mutations in the consensus motif of MRPL9 RNA, a target of mammalian IGF2BPs, also did not abolish binding, suggesting that secondary structures probably mediate the association between IGFBP family members and their target RNAs. Altogether, the data identify the first 33 base pairs of the upd 3'UTR as a putative target sequence for Imp, and support observations that Imp associates specifically with upd in vivo (Toledano, 2012).

To gain further insight into the mechanism by which Imp regulates upd, a GFP reporter was constructed that contained the 3'UTR from either upd or med23. Transcript levels for gfp were fivefold higher in Drosophila Schneider (S2) cells that co-expressed Imp with the gfp-upd-3'UTR reporter than in cells that co-expressed Imp with the gfp-med23-3'UTR reporter. The significant increase in reporter mRNA levels indicates that it is likely that Imp regulates upd mRNA stability (Toledano, 2012).

RNA-binding proteins, including mammalian IGF2BP1, have been shown to counter microRNA (miRNA)-mediated targeting of mRNAs. However, no consensus miRNA seeds were located within the first 34 base pairs of domain 1 of the upd 3'UTR. It is speculated that if Imp binding blocks small RNA-mediated degradation of upd, polyadenylated, cleaved upd degradation intermediates would be detected in the testes of older males, when Imp expression in hub cells is reduced. Using a modified rapid amplification of complementary DNA ends (RACE) technique, a specific cleavage product was identifed starting at nucleotide 33 of the upd 3'UTR in the testes of 30-day-old flies, but not in RNA extracts from the testes of 1-day-old males. Importantly, the same degradation product of upd was also detected in the testes of young flies when Imp was specifically depleted from hub cells using RNAi-mediated knockdown. As a positive control, the esi-2-mediated cleavage product of mus308 was detected in testes from both 1- and 30-day-old flies (Toledano, 2012).

To test whether small RNAs might mediate upd cleavage, small RNA libraries generated from the testes of 1- and 30-day-old flies were cloned and deep-sequenced. Although no small RNAs with exact pairing to the upd degradation product were identified, two short interfering RNAs (siRNAs; termed siRNA1 and siRNA2) with high sequence complementarity to the predicted target site in the upd 3'UTR were present in the testis library generated from 30-day-old males. Using qRT- PCR for mature small RNAs, it was found that the siRNA2 levels in the testes, relative to the levels of the control small RNAs bantam and mir-184, were similar in young and old males (deep sequencing analysis demonstrated that expression of these two control miRNAs did not change with age). The source of siRNA2 is the gypsy5 transposon, which is inserted at several loci throughout the fly genome and is conserved in numerous Drosophila species (Toledano, 2012).

To gain further insight into the mechanism by which Imp and siRNA2 regulate upd, the levels of the upd GFP reporter (gfp-upd-3'UTR) in the presence or absence of Imp and siRNA2 was investigated in S2 cells. To generate a reporter that should not be susceptible to siRNA-mediated degradation, the cleavage site in the upd 3'UTR that was identified by RACE (³²AUU = CGG; gfp-upd-3'UTR^mut) was mutated. Cells were transfected with either of the GFP reporter constructs, with or without haemagglutinin-tagged Imp (Imp- HA), and subsequently transfected with siRNA2; gfp expression was quantified by qRT- PCR (Toledano, 2012).

The co-expression of siRNA2 and the gfp-upd-3'UTR reporter resulted in a significant decrease in gfp transcript levels. Conversely, the co-expression of Imp blocked siRNA2-mediated reduction of gfp mRNA such that gfp levels were higher than in control cells. Furthermore, mutation of the putative cleavage site rendered the upd 3'UTR resistant to siRNA2-mediated degradation. These data, in combination with the in vitro binding data, suggest that Imp binds to and protects the upd 3'UTR from endogenous and exogenous siRNA2 in S2 cells. Thus, endo-siRNA2 is a bona fide candidate that could direct upd degradation when Imp is absent or its levels are reduced, although targeting by other small RNAs cannot be excluded (Toledano, 2012).

In Drosophila, Argonaute-1 (AGO1) is the principle acceptor of miRNAs and primarily regulates targets in a cleavage-independent mode, whereas AGO2 is preferentially loaded with siRNAs and typically regulates targets by mRNA cleavage. AGO2 expression was detected throughout the tip of the testis, as verified by immunostaining of testes from transgenic flies expressing 3×Flag-HA-tagged AGO2. To test whether AGO2 binds to upd mRNA in vivo, thereby potentially regulating upd levels directly, testes were dissected from aged (30-day-old) 3×Flag- HA- AGO2 males. Immunoprecipitation of AGO2, followed by qRT- PCR, showed significant enrichment (~102-fold) of upd mRNA bound to AGO2. Negligible binding of a negative control, rp49, to AGO2 was detected, suggesting specific association of AGO2 with upd mRNA in vivo and supporting a previous findings that upd is probably targeted by the siRNA pathway (Toledano, 2012).

To test whether Imp can impede the binding of AGO2 to the upd 3'UTR, S2 cells stably expressing Flag-tagged AGO2 were transfected with the gfp-upd-3'UTR reporter. Consistent with our previous observations, transcript levels of gfp-upd-3'UTR increased ~18-fold when Imp was co-expressed. Despite increases in the overall levels of gfp mRNA, the presence of Imp markedly reduced the association of AGO2 with the upd 3'UTR, indicating that Imp antagonizes the ability of AGO2 to bind the upd 3'UTR (Toledano, 2012).

Similar to the AGO family, Drosophila encodes two Dicer proteins that seem to have distinct roles in small RNA biogenesis. Dicer-1 (Dcr-1) is essential for the generation of miRNAs, and Dcr-2 is required for siRNA production from exogenous and endogenous sources. If siRNAs were involved in upd degradation in older males, it would be predicted that the loss of Dcr-2 would suppress the ageing-related decline in upd and GSCs. Consistent with a role for Dcr-2 in the generation of siRNAs, siRNA2 levels were significantly reduced in Dcr-2 homozygous mutants relative to heterozygous controls. Testes from 30- and 45-day-old Dcr-2 mutant flies showed increased levels of upd by qRT- PCR when compared with controls. Whereas a ~90% reduction of upd is observed in the testes from aged Dcr-2 heterozygous controls, only a ~45% reduction in upd was observed in testes from age-matched, Dcr-2 homozygous mutants, indicating that upd levels are higher when Dcr-2 function is compromised. Furthermore, the testes from aged Dcr-2 mutants contained more GSCs, on average, when compared with controls. Conversely, the forced expression of Dcr-2 in hub cells resulted in a reduction in the average number of GSCs and led to a significant reduction in upd levels, as detected using qRT- PCR and combined immunofluorescence and FISH, which seemed to be specific, as no significant change in Imp transcript levels was observed. Expression of Imp in combination with Dcr-2 resulted in a significant increase in upd levels. These observations indicate that Imp can counter the decrease in upd levels resulting from forced Dcr-2 expression, providing further evidence that Imp protects upd from targeted degradation by the siRNA pathway (Toledano, 2012).

The data suggest that Imp has a role in stabilizing upd in hub cells; therefore, the ageing-related decline in Imp would be a major contributing factor to the decrease in upd mRNA in the hub cells of aged males. To investigate the mechanism that leads to the decline in Imp expression with age, the Imp 3'UTR was examined for potential instability elements. Within the first 160 base pairs there is a canonical seed sequence for the heterochronic miRNA let-7. Expression of a reporter gene under the control of the let-7 promoter showed that let-7 expression increases in hub cells of ageing male, which was confirmed by let-7 FISH of testes from aged males. In addition, mature let-7 miRNA was enriched twofold in the testes from 30-day-old flies, relative to 1-day-old males. Therefore, an age-related increase in let-7 is one mechanism by which Imp expression could be regulated in an ageing-dependent manner in testes from older males (Toledano, 2012).

Consistent with these observations, the forced expression of let-7 specifically in hub cells led to a decrease in Imp. In addition, let-7 expression in S2 cells reduced the levels of a heterologous gfp-Imp-3'UTR^WT reporter. S2 cells were transfected with a let-7 mimic or with negative control miRNA, and gfp expression was quantified by qRT- PCR. There was a 70% reduction in gfp-Imp-3'UTR^WT expression in the presence of let-7, relative to control miRNA. A gfp-Imp-3'UTR^mut reporter with mutations in the canonical seed for let-7 (at nucleotide 137) was unaffected by let-7 expression, indicating that mutation of the let-7 seed rendered the RNA resistant to degradation. These data confirm that let-7 can destabilize Imp through sequences in the 3'UTR. However, further increasing the levels of let-7 resulted in a decrease in gfp expression from the mutated 3'UTR, indicating that other, putative let-7 seeds in the Imp 3'UTR can be targeted by let-7 (Toledano, 2012)

If the age-related decrease in Imp contributes to a decline in upd and subsequent loss of GSCs, it is proposed that re-expression of Imp in hub cells would rescue the ageing-related decrease in upd. Therefore, flies in which Imp was constitutively expressed in hub cells were aged, and upd levels were quantified by qRT- PCR. The expression of an Imp construct containing a truncated 3'UTR (Imp-KH- HA) lacking let-7 target sequences specifically in hub cells was sufficient to suppress the ageing-related decline in upd, with concomitant maintenance of GSCs, similar to what was observed by re-expressing upd in the hub cells of aged males. Maintenance of Imp-KH- HA expression in aged males was verified by staining with an anti-HA antibody. Conversely, the expression of an Imp construct that is susceptible to degradation by let-7 (Imp^T21) did not lead to an accumulation of Imp in the testes of 30- and 50-day-old flies, as levels were similar to the levels of endogenous Imp at later time points. Consequently, the expression of this construct was not sufficient to block the ageing-related decline in GSCs. These data indicate that let-7-mediated regulation of Imp contributes to the decline in Imp protein in older flies, and supports a model in which an ageing-related decline in Imp, mediated by let-7, exposes upd to degradation by siRNAs. Thus, both the miRNA and siRNA pathways act upstream to regulate the ageing of the testis stem-cell niche by generating let-7 and siRNA2, which target Imp and upd, respectively (Toledano, 2012).

Drosophila has proven to be a valuable model system for investigating ageing-related changes in stem-cell behaviour. Cell autonomous and extrinsic changes contribute to altered stem-cell activity; thus, determining the mechanisms underlying the ageing-related decline of self-renewal factors, such as the cytokine-like factor Upd, may provide insight into strategies to maintain optimal niche function (Toledano, 2012).

The data indicate that Imp can regulate gene expression by promoting the stability of selected RNA targets by countering inhibitory small RNAs. Therefore, rescue of the aged niche by Imp expression may be a consequence of effects on Imp targets, in addition to upd, in somatic niche cells. Furthermore, as Imp is expressed in germ cells, it could also act in an autonomous manner to regulate the maintenance of GSCs. The canonical let-7 seed in the Imp 3'UTR is conserved in closely related species, and reports have predicted that the let-7 family of miRNAs target mammalian Imp homologues (IGF2BP1- 3). Given the broad role of the let-7 family in ageing, stem cells, cancer and metabolism, the regulation of Imp by let-7 may be an important, conserved mechanism in numerous physiological processes (Toledano, 2012).

Non-coding RNAs can ensure biological robustness and provide a buffer against relatively small fluctuations in a system. However, after a considerable change, a molecular switch is flipped, which allows a biological event to proceed unimpeded. In the current model, Imp preserves niche function in young flies until a time at which miRNAs and siRNAs act together to trigger an 'ageing' switch that leads to a definitive decline in upd and, ultimately, in stem-cell maintenance. Therefore, targeting signalling pathways at several levels using RNA-based mechanisms will probably prove to be a prevalent theme to ensure robustness in complex biological systems (Toledano, 2012).

Repeated duplication of Argonaute2 is associated with strong selection and testis specialization in Drosophila

Argonaute2 (Ago2) is a rapidly evolving nuclease in the Drosophila melanogaster RNA interference (RNAi) pathway that targets viruses and transposable elements in somatic tissues. This study reconstruct the history of Ago2 duplications across the Drosophila obscura group, and patterns of gene expression were used to infer new functional specialization. Some duplications were shown to be old, shared by the entire species group, and losses may be common, including previously undetected losses in the lineage leading to D. pseudoobscura. While the original (syntenic) gene copy has generally retained the ancestral ubiquitous expression pattern, most of the novel Ago2 paralogues have independently specialized to testis-specific expression. Using population genetic analyses, it was shown that most testis-specific paralogues have significantly lower genetic diversity than the genome-wide average. This suggests recent positive selection in three different species, and model-based analyses provide strong evidence of recent hard selective sweeps in or near four of the six D. pseudoobscura Ago2 paralogues. It is speculated that the repeated evolution of testis-specificity in obscura group Ago2 genes, combined with their dynamic turnover and strong signatures of adaptive evolution, may be associated with highly derived roles in the suppression of transposable elements or meiotic drive. This study highlights the lability of RNAi pathways, even within well-studied groups such as Drosophila, and suggests that strong selection may act quickly after duplication in RNAi pathways, potentially giving rise to new and unknown RNAi functions in non-model species (Lewis, 2016).

Drosophila tsRNAs preferentially suppress general translation machinery via antisense pairing and participate in cellular starvation response

Transfer RNA-derived small RNAs (tsRNAs) are an emerging class of small RNAs, yet their regulatory roles have not been well understood. The molecular mechanisms and consequences of tsRNA-mediated regulation in Drosophila was studied. By analyzing 495 public small RNA libraries, it was demonstrated that most tsRNAs are conserved, prevalent and abundant in Drosophila. By carrying out mRNA sequencing and ribosome profiling of S2 cells transfected with single-stranded tsRNA mimics and mocks, this study showed that tsRNAs recognize target mRNAs through conserved complementary sequence matching and suppress target genes by translational inhibition. The target prediction suggests that tsRNAs preferentially suppress translation of the key components of the general translation machinery, which explains how tsRNAs inhibit the global mRNA translation. Serum starvation experiments confirm tsRNAs participate in cellular starvation responses by preferential targeting the ribosomal proteins and translational initiation or elongation factors. Knock-down of AGO2 in S2 cells under normal and starved conditions reveals a dependence of the tsRNA-mediated regulation on AGO2. The repressive effects of representative tsRNAs on cellular global translation and specific targets was evaluated with luciferase reporter assays. This study suggests the tsRNA-mediated regulation might be crucial for the energy homeostasis and the metabolic adaptation in the cellular systems (Luo, 2018).

REFERENCES

Search PubMed for articles about Drosophila Argonaute 2

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date revised: 20 October 2023

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