InteractiveFly: GeneBrief

spindle E/homeless : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

homeless, now termed spindle E, was initially detected in a P element insertion screen: a female sterile line was obtained in which the insertion mapped at 80A5-6. spindle E mutants contain mislocalized oocytes in a small percentage of their vitellogenic egg chambers. Ovaries dissected from mutants contain a range of late-stage phenotypes. A wild-type egg chamber at stage 14 of oogenesis possesses two dorsal eggshell respiratory appendages, just lateral to the dorsal midline. Ninety to ninety-five percent of the mutant egg chambers show aberrant appendage formation: the majority possess only one appendage or fused appendages emerging from one base on the dorsal midline. The dorsal appendage phenotype suggests that spn-E plays a role in dorsalization of the oocyte, while the mislocalization phenotypes suggest that spn-E is involved in an even earlier role in egg polarity (Gillespie, 1995).

spindle E encodes a DEAD box protein that is likely to function as an RNA helicase (an RNA unwinding function). The similarity of Spn-E to proteins that function through binding RNAs suggests a possible role for Spn-E in RNA processing, transport, or stabilization. The localization patterns of seven mRNAs known to be localized during oogenesis were examined. These transcripts fall into three classes: (1) those that fail to be transported or localized correctly in some fraction of spn-E egg chambers; (2) those that are localized correctly but are reduced in amount, and (3) those that remain unaffected in spn-E mutants. Gurken mRNA is localized appropriately in the majority of stage 9 and stage 10 spn-E mutant egg chambers. However, about 30% of hls mutant chambers reveal a defective pattern. Some show no GRK mRNA localization, some show an anterior ring, and some show a dorsal patch that is broader than normal. Although Oskar mRNA is transported to the oocyte normally in these early stages, later localization to the posterior pole is defective. In the majority of S10 egg chambers, OSK mRNA is diffuse throughout the oocyte. In spn-E mutant egg chambers clearly aberrant Bicoid mRNA distribution is observed in stage 8. In most egg chambers, some BCD mRNA is transported to the oocyte, but the majority remains in the nurse cells, concentrated at the apical cortex. In addition, much of the BCD message within the oocyte is not transported laterally toward the periphery but instead remains centrally located. The failure to transport BCD and OSK mRNAs is the earliest defect in RNA transport observed in spn-E mutants (Gillespie, 1995).

Examination of two other anteriorly localized transcripts, the K10 and ORB mRNAs, identifies a second class of messages. In spn-E mutants, K10 transcripts are transported to the oocyte and later localized to the anterior edge, but the level of transcript is greatly decreased relative to wild type, specifically in vitellogenic stages. In spn-E mutants ORB mRNA accumulates normally in oocytes, but the anterior localization, while present, is significantly weaker in stage 8 to stage 10 oocytes. Bic-D and HTS mRNAs are unaffected in spn-E mutants (Gillespie, 1995).

Because the Spn-E protein has homology to RNA-binding proteins of the DE-H family, it seems likely that Spn-E is acting directly to localize RNAs in oogenesis. It is possible, however, that mutations in the gene are affecting RNA localization by disrupting the microtubule architecture that is a common component of the RNA's localization mechanism. Hence, the localization of a Kinesin heavy chain:beta-Galactosidase fusion protein was examined in a spn-E mutant background to analyze one aspect of microtubule structure and function. In wild-type stage 8 and stage 9 egg chambers, microtubule organizing centers (MTOCs) are located at the anterior of the oocyte and direct the formation of a gradient of microtubules whose plus ends extend toward the posterior pole. The fusion protein is thus propelled by the plus-end-directed kinesin motor function and is present in a band at the posterior of the oocyte, as detected by beta-Gal activity on an X-Gal substrate. In spn-E mutants, the fusion protein is detected in the center of S8 oocytes and in central and lateral portions of S9 oocytes, suggesting that microtubule structure or function is disrupted. Microtubule organization was examined directly in spn-E mutant egg chambers by comparing anti-alpha-Tubulin immunofluorescence in wild-type and mutant ovaries. In a majority of stage 8 spn-E chambers, a dense network of microtubules is present throughout the oocyte in place of the normal anterior MTOC localization. In all cases, the oocyte nucleus is correctly positioned at the dorsal anterior corner of the oocyte. Despite the presence of an aberrant network in stage 8 and 9 egg chambers, a normal rearrangement was observed in stage 10b spn-E oocytes. Thus the inappropriate formation of an extensive microtubule meshwork is confined to stage 8 and stage 9 egg chambers (Gillespie, 1995).

Spn-E could be functioning in one of at least two ways: (1) Spn-E could act downstream of the signaling pathways that induce correct microtubule reorganization during stages 8 and 9, actively interacting in microtubule reorganization, or, (2) Spn-E may be required for efficient transcription, pre-mRNA processing, localization, or translational regulation of products that control the kinetics of microtubule assembly, or to direct the reorganization of microtubule structures. The similarity of Spn-E protein to members of the DE-H family makes this second hypothesis attractive (Gillespie, 1995).

Subsequent studies reveal that spn-E phenotypes resemble a family of Drosophila mutants, all involved in a number of polarity determining steps during oogenesis. During wild-type oogenesis, the two cells in each germline cyst appear to be equivalent: these are the progeny of the first division of the cystoblast, derived from asymmetric division of a germ-line stem cell. Both cells enter meiosis to become pro-oocytes in region 2a of the germarium. In region 2b, one of these two cells is selected to develop as the oocyte and remains in meiosis, while the other exits meiosis and reverts to the nurse cell pathway of development. The event gives rise to the first asymmetry in egg development, the selection of one of two cells to become the oocyte. Later in oogenesis, anterior-posterior polarity originates when the oocyte comes to lie posterior to the nurse cells and signals through the Gurken/Egfr pathway to induce the adjacent follicle cells to adopt a posterior fate. This directs the movement of the germinal vesicle and associated Gurken mRNA from the posterior to an anterior corner of the oocyte, where Gurken protein signals for a second time to induce the dorsal follicle cells, thereby polarizing the dorsal-ventral axis. A group of five genes, the spindle loci, is described which is required for each of these polarizing events. The five spindle genes were originally identified in a screen for maternal-effect mutants on the third chromosome because homozygous mutant females lay ventralized eggs.

Mutations in spn-E give rise to an oocyte displacement phenotype, but also affect the oocyte cytoskeleton and mRNA localization, even when the oocyte is at the posterior of the egg chamber. Double spindle mutants reveal a phenotype even earlier in oogenesis, one where both pro-oocytes develop as oocytes, by delaying the choice between these two cells. spindle mutants inhibit the induction of both the posterior and dorsal follicle cells by disrupting the localization and translation of Gurken mRNA. The transient mislocalization of Gurken mRNA to an anterior ring in spn mutant stage 9 egg chambers is very similar to the mislocalization of Gurken mRNA observed in fs(K10) mutants. However, K10 mutations produce a dorsalization of the egg chamber rather than a ventralisation, because the mislocalization of Gurken mRNA directs Gurken signaling to the follicle cells on all sides of the oocyte. In different spindle mutants, from 19% to 100% of egg chambers show a strong reduction or a complete absence of Gurken protein in the oocyte membrane. The oocyte often fails to reach the posterior of mutant egg chambers and it differentiates abnormally. This analysis of spindle phenotypes suggests that spindle genes are likely to be involved in the localization and/or translation of Gurken mRNA without having any discernible effect on the Gurken mRNA level, yet dramatically reduced amounts of Gurken protein are produced. K10 mutants cause a similar mislocalization of Gurken mRNA without significantly affecting protein expression. Thus, spindle mutants reveal a novel link between oocyte selection, oocyte positioning and axis formation in Drosophila, leading to a proposal that the spindle genes act in a process that is common to several of these events (Gonzalez-Reyes, 1997).

How the asymmetry between the two pro-oocytes arises is unknown, but it has been proposed that it could be generated during the first division of the cystoblast to give rise to a two-cell cyst. During this division, a vesicular structure called the spectrosome (see Drosophila Spectrin) associates with one pole of the mitotic spindle and is asymmetrically partitioned between the two daughter cells. Since each of these cells gives rise to one pro-oocyte and seven nurse cells, this asymmetry might determine which pro-oocyte is fated to become the oocyte. Whatever mechanism generates the initial asymmetry, it seems that the key step in the selection of the oocyte is the accumulation of Bicaudal-D and Egalitarian proteins in a single cell. Null mutants in either gene block the localization of the protein encoded by the other to the presumptive oocyte and prevent all other known steps in oocyte differentiation, such as the formation of an active MTOC in this single cell and the subsequent microtubule-dependent localization of oocyte-specific transcripts, such as Oskar. Although it is unclear at what point in the pathway of oocyte selection the spindle genes act, they must function upstream of the process that results in the localization of Bic-D (and presumably Egl) to a single cell. It is most likely that the spindle proteins are directly involved in this process: (1) the spn double mutant combinations delay but do not block the choice between the two pro-oocytes, suggesting that they do not remove the initial asymmetry, but slow down its expression. (2) A reduction in Bic-D or Egl activity later in oogenesis leads to the same ventralized phenotype that is produced by the single spn mutations. This raises the possibility that the spn gene products interact with Bic-D and Egl at two different stages of oogenesis, first to select the oocyte and then to regulate Gurken expression once the oocyte has formed (Gonzalez-Reyes, 1997).

Tejas functions as a core component in nuage assembly and precursor processing in Drosophila piRNA biogenesis

PIWI-interacting RNAs (piRNAs), which protect genome from the attack by transposons, are produced and amplified in membraneless granules called nuage. In Drosophila, PIWI family proteins, Tudor-domain-containing (Tdrd) proteins, and RNA helicases are assembled and form nuage to ensure piRNA production. However, the molecular functions of the Tdrd protein Tejas (Tej) in piRNA biogenesis remain unknown. This study conducted a detailed analysis of the subcellular localization of fluorescently tagged nuage proteins and behavior of piRNA precursors. The results demonstrate that Tej functions as a core component that recruits Vasa (Vas) and Spindle-E (Spn-E) into nuage granules through distinct motifs, thereby assembling nuage and engaging precursors for further processing. This study also reveals that the low-complexity region of Tej regulates the mobility of Vas. Based on these results, it is proposed that Tej plays a pivotal role in piRNA precursor processing by assembling Vas and Spn-E into nuage and modulating the mobility of nuage components (Lin, 2023).

Transposons (transposable elements, TEs) are mobile genetic elements that exist in the genomes of all eukaryotic organisms and they occupy a substantial portion of genomes. They directly impair genomes by causing double-strand breaks, promoting ectopic recombination, and abolishing gene expression. PIWI-interacting RNAs (piRNAs), a class of 23-29-nt gonad-specific small RNAs, protect genome integrity by mitigating any catastrophes in germline cells that will be transmitted to the next generations. piRNAs are quite conserved and widely found among animals, and the model animal system, Drosophila, has been used to investigate and dissect the molecular mechanisms of piRNAs (Lin, 2023).

Drosophila piRNAs are processed from long piRNA precursor transcripts derived from genomic loci called piRNA clusters, where inactive or fragmented transposons are deposited. Discrete piRNA clusters are active in gonads, where they produce dual-strand piRNA precursors in germline cells or unistrand piRNA precursors in somatic gonadal cells. In germline cells, nascent piRNA precursors are transported to a unique, germline-specific membraneless structure called nuage in the perinuclear region via the Nxf3-Nxt1 pathway. Nuage consists of precursors and transposon RNAs being processed, two PIWI family proteins-Aub and Ago3-and other relevant components, DEAD-box RNA helicase Vasa (Vas), DEAH box helicase RNA helicase Spindle-E (Spn-E), and a group of Tudor domain-containing proteins (Tdrds), Krimper (Krimp), Tejas (Tej), Tudor, Tapas (Tap), Qin/Kumo, and Vreteno. After loading long piRNA precursors and transposon RNAs onto Aub and Ago3, they are cleaved and sliced into mature piRNAs, leading to the formation of antisense and sense piRNAs with a 10-nt complementarity. These processed piRNAs are further amplified in nuage in a feed-forward amplification cycle called the ping-pong cycle. However, the molecular mechanisms of nuage assembly are still unclear (Lin, 2023).

Although Tdrds are multifunctional, their overall activities are not fully understood. They interact with symmetrically demethylated arginine (sDMA), which is usually present at the N-terminus of PIWI family proteins, through the Tudor domain, thereby promoting aggregate formation in mammalian cells. This behavior implies the importance of molecular associations of Tdrds for nuage formation. Membraneless organelles composed of RNA and proteins are responsible for diverse RNA processing, including P-body and Yb body in Drosophila, which modulate the molecular organization in a process called phase separation. Two Tdrds localized in Drosophila nuage-Tej and Tap-contain an extended Tudor domain (eTudor) and an additional Lotus domain that is conserved from bacteria to eukaryotes. The Lotus domain was previously reported to interact with Vas, which is required for the piRNA pathway (Lin, 2023).

Of these two proteins, Tej/Tdrd5 is one of the key factors in the piRNA pathway in both Drosophila and mice (Patil, 2014; Patil, 2010; Yabuta, 2011). piRNAs are massively reduced with the displacement of other components from nuage in the absence of Tej/Tdrd5; however, the molecular functions of Tej remain elusive. This study identified the domains of Tej that interact with Vas and Spn-E, which are required for proper nuage formation and piRNA precursor processing, in addition to the contribution of the intrinsically disordered region (IDR) to the dynamics of other nuage components. It is proposed that Tej plays a pivotal role in piRNA precursor processing by recruiting Vas and Spn-E for nuage and modulating their dynamics for nuage assembly (Lin, 2023).

The piRNAs in Drosophila germline cells are produced and amplified in the membraneless organelle, nuage, which is assembled by orderly recruitment of the corresponding components to ensure its proper function. Although its precise function has not been clarified, the findings of this study demonstrate that Tej plays a crucial role in recruiting RNA helicases Vas and Spn-E to nuage through distinct domains, namely, Lotus and SRS. The results provide new insights into the regulation of stepwise piRNA precursor processing by Tej, Spn-E, and Vas in the initial phase of piRNA biogenesis prior to the ping-pong amplification cycle. Tej recruits these helicases for the engagement of the precursors involved in further processing of nuage, thereby also controlling the dynamics of these nuage components (Lin, 2023).

The results confirmed that the Tej Lotus domain recruited Vas to nuage, which is consistent with the fact that it enables Vas to hydrolyze ATP for RNA release (Jeske, 2017). This study newly identified that the SRS motif in Tej is responsible for Spn-E recruitment to nuage. Full deletion or single amino acid substitution of SRS significantly disrupted Spn-E recruitment to Tej granules in S2 cells, whereas further deletions of eight amino acids other than SRS, eSRS, were critical for recruiting Spn-E to nuage in the ovaries. This result raises a possibility that Tej, as well as other factors, may assist the recruitment of Spn-E to nuage in the ovaries. Another protein known as Tap, which is a fly counterpart of TDRD7 and harbors Lotus and eTudor domains, has previously been reported to participate in the piRNA pathway and interact with Vas (Jeske, 2017; Patil, 2014). However, since Tap lacks the SRS found in Tej, it is unlikely to be involved in the recruitment of Spn-E. The mouse homolog of Spn-E (TDRD9) is localized in both nuage and the nucleus in prespermatogonia, and might perform different functions that remain elusive. This finding suggests a possibility that the intrinsically nuclear protein Spn-E was deliberately recruited to nuage via Tej to exert a unique function, such as piRNA precursor processing. In contrast, the eTudor domain mainly contributes to Tej aggregation, which is consistent with previous studies showing that the eTudor domain is engaged in granulation by binding to its ligand sDMA (Lin, 2023).

Despite the unusual nuage granules of Tej-ΔeTudor, it mildly suppressed transposon expression. Notably, Tej-ΔeTudor displays interaction with Vas and Spn-E, albeit to a lesser extent, especially with Spn-E. The CL-IP results also supported these interactions as reported in S2 cells (Patil, 2010). Alternatively, Tej-ΔeTudor possibly may facilitate the association of other components with nuage activity for piRNA processing. Unlike the mutation of precursor transporter, nxf3, and the ping-pong cycle assistant, krimp, tej, as well as spn-E and vas mutants, exhibited the accumulation of piRNA precursors in the perinuclear region and a collapse of the ping-pong amplification. These results suggest that they function upstream during ping-pong amplification. Stalling of piRNA precursors was also observed when the recruitment of Vas or Spn-E to nuage was abolished by the loss of the Lotus or eSRS domains, respectively. Precursor accumulation was concentrated in the malfunctioning nuage or perinuclear region, which would result in a failure in precursor processing and cause TE upregulation (Lin, 2023).

Genetic analysis of nuage organization revealed that Spn-E and Tej occupy a higher hierarchical position than Vas at an earlier stage, which is inconsistent with a previous observation (Patil, 2010), possibly due to the fluctuation of nuage assembly and/or structure at a later stage in the mutants. In contrast, Tej and Spn-E are mutually dependent for the proper assembly of nuage granules because Spn-E is required for the proper localization of Tej within nuage. Moreover, Tej may form a relatively stable scaffold with Spn-E for nuage assembly, while a mobile fraction of Tej may contain Vas. These results suggest that Tej may facilitate the compartmentalization of Vas and Spn-E, as shown in CL-IP experiments and also reported in Bombyx germ cells, while the possibility cannot be excluded of simultaneous binding among these proteins. Further results with S2 revealed that the weak hydrophobic interaction between the proteins may contribute to the formation and regulation of membraneless structures on nuage. DEAD-box RNA helicase family members, including Vas homolog, reportedly form non-membranous, phase-separated organelles in both prokaryotes and eukaryotes, and the large IDR at the N-terminal region facilitates their aggregation by LLPS. In addition, the loss of IDR in Tej significantly suppressed the mobility of Tej and Vas; nevertheless, the TE repression was only mildly attenuated. Thus, Tej-ΔIDR may remain colocalized with Vas and Spn-E, facilitating the processing of piRNAs. Alternatively, the reduction of Vas mobility by the loss of Tej IDR could be compensated by other components in nuage. Only the localization of Vas was remarkably changed upon 1,6-hexanediol (1,6-HD) treatment in S2 cells, further supporting the finding that weak hydrophobic interaction controlled the dynamics of Vas, although a possibility of the unexpected effects by the 1,6-HD treatment cannot be excluded. It also cannot be excluded that 1,6-HD treatment might have impaired kinase and/or phosphatase activity. Hence, localization might have been affected by the changes in their phosphorylation status. The behavior of these proteins is seemingly influenced by their respective binding modes and properties with Tej. The interaction of Vas with Tej is affected by 1,6-HD and IDR region of Tej through the hydrophobic association, whereas that of Spn-E with Tej is more rigid, possibly contributing to the formation of the scaffold of nuage. In conclusion, Tej utilizes the eTudor domain for granule formation, whereas the IDR of Tej appears to maintain the assemble of Tej granules, controlling the mobility of Vas in nuage (Lin, 2023).

Membraneless macromolecular nuage contains more than a dozen components, including Vas and Tej that harbor IDRs, which could contribute to the dynamics of nuage and impact the efficient production of piRNAs. Nuage also contains piRNA precursors and TE RNAs that are processed therein; their unique or specific propensities may affect nuage assembly and function. Further investigation of those proteins and RNA components will shed light on the regulatory mechanisms underlying the formation and dynamics of nuage to promote each sequential step of piRNA biogenesis (Lin, 2023).

Telomere elongation is under the control of the RNAi-based mechanism in the Drosophila germline; mutations in the spn-E and aub cause an increase in the frequency of telomeric element retrotransposition to a broken chromosome end

Telomeres in Drosophila are maintained by transposition of specialized telomeric retroelements HeT-A, TAHRE, and TART instead of the short DNA repeats generated by telomerase in other eukaryotes. This study implicates the RNA interference machinery in the control of Drosophila telomere length in ovaries. The abundance of telomeric retroelement transcripts is up-regulated owing to mutations in the spn-E and aub genes, encoding a putative RNA helicase and protein of the Argonaute family, respectively, which are related to the RNA interference (RNAi) machinery. These mutations cause an increase in the frequency of telomeric element retrotransposition to a broken chromosome end. spn-E mutations eliminate HeT-A and TART short RNAs in ovaries, suggesting an RNAi-based mechanism in the control of telomere maintenance in the Drosophila germline. Enhanced frequency of TART, but not HeT-A, attachments in individuals carrying one dose of mutant spn-E or aub alleles suggests that TART is a primary target of the RNAi machinery. At the same time, enhanced HeT-A attachments to broken chromosome ends were detected in oocytes from homozygous spn-E mutants. Double-stranded RNA (dsRNA)-mediated control of telomeric retroelement transposition may occur at premeiotic stages, resulting in the maintenance of appropriate telomere length in gamete precursors (Savitsky, 2006).

The problems of end-under-replication and stability of linear chromosomes are resolved by telomeres. The lengthening of terminal regions of linear eukaryotic chromosomes is often provided by RNA-templated addition of repeated DNA by reverse transcriptase enzyme, telomerase. In most eukaryotes, telomeric DNA is maintained by the action of telomerase, which is responsible for the synthesis of short 6-8-nucleotide (nt) arrays using an RNA component as a template. In contrast, telomeres of Drosophila are maintained as a result of retrotranspositions of specialized telomeric non-long-terminal repeat (LTR) HeT-A, TAHRE, and TART retrotranspositions (Biessmann, 1992b; Levis, 1993; for review, see Pardue, 2003; Abad, 2004b). Retrotransposons are also found in telomeric regions of such diverse organisms as Bombyx mori, Chlorella and Giardia lamblia. HeT-A, TAHRE, and TART are found at Drosophila telomeres in tandem arrays. HeT-A, the most abundant Drosophila telomeric element, contains a single ORF encoding a Gag-like RNA-binding protein, but lacks reverse transcriptase (RT). It is proposed that the RT necessary for its transposition might be provided in trans, perhaps by TART (Rashkova, 2002). TART ORF2 encodes a reverse transcriptase related to the catalytic subunit of telomerase. The recently discovered TAHRE element shows extensive similarity to HeT-A, but contains a second ORF, which encodes a reverse transcriptase (Abad, 2004b). A HeT-A promoter located in the 3' region of the element directs synthesis of a downstream neighbor (Danilevskaya, 1997). The TART element was shown to be transcribed bidirectionally using a putative internal sense promoter and antisense one that was localized within the 1-kb region of the TART 3' end (Danilevskaya, 1999). Maintenance of Drosophila telomere length is mediated by HeT-A and TART transpositions to chromosome ends as well as by terminal recombination/gene conversion (Mikhailovsky, 1999; Kahn, 2000). Most of the observed spontaneous attachments to telomeres are HeT-A transpositions (Biessmann, 1992a; Kahn, 2000; Golubovsky, 2001), but TART attachments (Sheen, 1994) were also detected (Savitsky, 2006 and references therein).

The spn-E and aub genes, encoding an RNA helicase and a protein of Argonaute family, respectively, are involved in double-stranded RNA (dsRNA)-triggered RNA interference (RNAi) in embryos, in transcriptional silencing of transgenes, and in the control of Drosophila retrotransposon transcript abundance in the germline, especially in ovaries. No effects of RNAi gene mutations on HeT-A and TART expression and telomere structure were observed in somatic tissues (Perrini, 2004). This study shows that increased HeT-A and TART transcript abundance in ovaries, owing to RNAi mutations, is correlated with a high frequency of telomeric element attachments to broken chromosome ends. Addition of HeT-A or TART to a truncated X chromosome, with a break in the upstream regulatory region of yellow, activates yellow expression in aristae, which enables monitoring of the elongation events (Kahn, 2000; Savitsky, 2002). Using this genetic system, the effects of RNAi mutations were studied on the frequency and molecular nature of telomeric attachments. A high frequency of TART but not HeT-A attachments in heterozygous RNAi mutants suggests that TART may be the primary target of the RNAi-based silencing mechanism. These results highlight for the first time the importance of TART, but not the more abundant HeT-A element, in Drosophila telomere maintenance. The disappearance of short TART and HeT-A RNAs was found in spn-E mutant ovaries, strongly suggesting an RNAi-based pathway in the control of telomere maintenance in the Drosophila germline (Savitsky, 2006).

An RNAi-based mechanism has been proposed to evolve in order to immobilize transposable elements and was found to control expression of endogenous transposable elements and their mobility in different species. Drosophila telomeres are maintained by successive transpositions of specialized telomeric retroelements HeT-A and TART. This study shows that transposition of both telomeric elements is under the control of the spn-E and aub genes, known to be related to the RNAi machinery. Hence, an RNAi-based mechanism may be considered not only as a defense against retrotransposon expansion, but also as a regulatory system responsible for proper telomere length maintenance in Drosophila (Savitsky, 2006).

spn-E is required for appropriate localization of mRNA and proteins involved in the establishment of axis formation in the embryo and encodes a member of the DEAD/DE-H protein family possessing RNA-binding and RNA helicase activity. aub encodes a protein of the Argonaute family that was shown to be a component of the RNAi effector complex RISC. aub and spn-E mutations strongly diminished effects of the injected dsRNA into mature oocytes. Both genes are implicated in small interfering RNA (siRNA)-dependent silencing of testis-expressed Stellate genes. Thus, spn-E and aub are components of RNAi-based silencing pathways in Drosophila. Mutations in these genes result in the derepression of a wide spectrum of retrotransposons in the germline, including the HeT-A telomeric element (Aravin, 2001; Stapleton, 2001; Kogan, 2003). This study demonstrates that spn-E and aub mutations increase the frequency of telomeric element retrotranspositions to broken chromosome termini, suggesting that the RNAi machinery controls telomere length in Drosophila (Savitsky, 2006).

Both telomeric elements are shown to be the targets of RNAi. The present results emphasize the differences in the response of HeT-A and TART elements to RNAi mutations. Surprisingly, two different spn-E mutant alleles and an aub mutation in the heterozygous state increase considerably TART mobility, whereas attachments of HeT-A to broken chromosome ends were detected much more rarely in spn-E¹/+ ovaries and are not observed in ovaries of spn-E^hls3987/+ and aub^QC42/+ flies. One copy of a spn-E mutation is sufficient to increase TART transcript abundance. Strong accumulation of HeT-A transcripts is found only in homozygous mutants, correlating with a high frequency of HeT-A attachments to the broken chromosome ends in the developing oocytes. This observation argues that TART is a primary target of the RNAi machinery in ovaries (Savitsky, 2006).

TART and HeT-A, in spite of sharing the region of integration, are dissimilar in their structure and expression strategy. While both sense and antisense TART transcription has been demonstrated, antisense transcripts are more abundant. In situ RNA analysis detected sense and antisense TART transcripts in the cytoplasm of nurse cells in the late-stage egg chambers, suggesting a possibility of dsRNA formation. However, it was found that the level of antisense TART transcripts is not affected in RNAi mutants. Only sense HeT-A transcription was observed by Northern or by in situ RNA analyses. Nevertheless, HeT-A- and TART-specific siRNAs were revealed among the cloned short RNA species in Drosophila, and short RNAs corresponding to both HeT-A and TART elements are detected by Northern analysis. Antisense HeT-A RNA is probably transcribed at a low level from an unidentified promoter, possibly, from the HeT-A internal region. Actually, a low level of antisense activity of the HeT-A 3' end has been observed . While TART transcripts were observed only in the nurse cells, HeT-A transcripts were detected both in the growing oocyte and nurse cells. It is proposed that TART is a primary target of the RNAi controlling system, since one dose of an RNAi mutation causes preferential TART, but not HeT-A, attachments to broken chromosome ends in ovaries. In contrast, one dose of a mutant Su(var)205 gene (HP1) considerably increasess the frequency of HeT-A rather than TART attachments to the chromosome ends (Savitsky, 2002). Thus, a specific effect of RNAi components on telomeric element expression is observed . Although TART copies are much less abundant in the genome than HeT-A and no TART elements are detected in some telomeres, TART is a conserved component of telomeres in distant Drosophila species. TART was considered as a source of RT production, thus ensuring retrotranspositions of both TART and HeT-A elements. One may propose that TART supplies an RNAi-regulated template for RT production, thus providing telomere-specific transpositions of both elements (Savitsky, 2006).

Drosophila telomeres contain a multisubunit protein complex forming a chromosome cap protecting chromosomes from DNA repair and end-to-end fusions. However, no HeT-A or TART sequences were detected at the stably maintained broken chromosome end, which is protected from telomere fusions. Thus, a sequence-independent system performs telomere capping functions. The capping complex contains HP1, HOAP (HP1/ORC associated protein), as well as ATM-kinase and DNA repair MRN complex and the Ku70/Ku80 heterodimer. HP1 and the Ku heterodimer act also as negative regulators of telomere elongation by retrotransposition of telomeric elements. Deficiencies that remove either the Ku70 or the Ku80 gene increase the transposition rate of HeT-A and TART elements but exert no effect on the HeT-A expression, suggesting that Ku proteins control the accessibility of the telomere to transposition events. At the same time, mutations in the Su(var)205 gene increase both transcript abundance of HeT-A and TART and the frequency of their attachments to chromosome ends. RNAi affects both telomeric retrotransposon expression and the rate of transposition to the telomere. Probably, this effect is mediated through HP1 recruitment and silencing of HeT-A and/or TART chromatin (Savitsky, 2006).

siRNAs produced from telomeric elements TART and HeT-A belong to the long size class (25-29 nt) in contrast to 21-22-nt RNAs guiding post-transcriptional RNAi. In plants, long siRNAs are associated with RNA-directed DNA methylation and play an essential role in the transcriptional retrotransposon silencing. dsRNA and proteins of the RNAi machinery can direct chromatin alteration to homologous DNA sequences and induce transcriptional silencing. RNAi mutations cause delocalization of HP1 in yeast and Drosophila. Actually, the increase in accessibility of HeT-A chromatin and its enrichment in K9-acetylated H3 histone were revealed in ovaries of spn-E mutants. It is also possible that TART and/or HeT-A short RNAs can be targeted to telomeric repeats in a transcriptional silencing complex (Savitsky, 2006).

RNAi disruption affects neither HeT-A and TART expression, nor telomere fusions in somatic cells. No effect was observed of spn-E mutations on HeT-A expression, even in actively dividing cells of imaginal discs, where HeT-A expression was found. The data indicate a crucial role of the RNAi machinery in the regulation of telomere elongation in germinal cells. The appearance of a cluster of individuals with identical retroelement attachments indicates that dsRNA-mediated control of terminal elongation may occur at premeiotic stages of oogenesis (Savitsky, 2006).

This study has demonstrated that expression and retrotransposition of specific telomeric repeats is under control of an RNAi-based system in the Drosophila germline. In this case, the telomerase-dependent mechanism of telomere stability is substituted by retrotranspositions. Interestingly, telomerase-dependent telomere functioning during meiosis in the yeasts Schizosaccharomyces pombe and Tetrahymena is also under the control of RNAi machinery. These observations and the current data indicate that dsRNA-mediated regulation of telomere dynamics in the germline may be a general phenomenon independent of a mode of telomere maintenance (Savitsky, 2006).

REGULATION

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

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

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

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

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

The Tudor domain protein Tapas, a homolog of the vertebrate Tdrd7, functions in piRNA pathway to regulate retrotransposons in germline of Drosophila melanogaster

Piwi-associated RNAs (piRNAs) are a special class of small RNAs that provide defense against transposable elements (TEs) in animal germline cells. In Drosophila, germline piRNAs are thought to be processed at a unique perinuclear structure, nuage, which houses piRNA pathway proteins including the Piwi clade of Argonaute family proteins, along with several Tudor domain proteins, RNA helicases and nucleases. Tudor domain protein Tejas (Tej), an ortholog of vertebrate Tdrd5, is an important component of the piRNA pathway. The current study identified the paralog of Drosophila tej gene, tapas (tap), which is an ortholog of vertebrate Tdrd7. Like Tej, Tap is localized at the perinuclear structure in germline cells called nuage. The tap loss alone leads to a mild increase in transposon expression and decrease in piRNAs targeting transposons expressed in the germline. tap genetically interacts with other piRNA pathway genes, and Tap physically interacts with piRNA pathway components, such as Piwi family proteins Aubergine (Aub) and Argonaute3 (Ago3) and the RNA helicases Vasa (Vas) and Spindle-E (SpnE). tap together with tej is required for survival of germline cells during early stages and for polarity formation. It was further observed that loss of tej and tap together results in more severe defects in piRNA pathway in germline cells compared to single mutants: the double mutant ovaries exhibit mislocalization of piRNA pathway components and significantly greater reduction of piRNAs against transposons predominantly expressed in germline compared to single mutants. The single or double mutants did not have any reduction in piRNAs mapping to transposons predominantly expressed in gonadal somatic cells and those derived from unidirectional clusters such as flamenco. Consistently, the loss of both tej and tap function results in mislocalization of Piwi in germline cells, while Piwi remains localized to the nucleus in somatic cells. These data suggest that Tej and Tap work together for germline maintenance and piRNA production in germline cells. These observations suggest that tej and tap work together for the germline maintenance. tej and tap also function in a synergistic manner to maintain examined piRNA components at the perinuclear nuage and for piRNA production in Drosophila germline (Patil, 2014).

DEVELOPMENTAL BIOLOGY

Adult

In the germarium, spn-E transcript appears to be limited to the germline cells. Expression decreases through S4/S5, increases during S8, and is strongest in the nurse cells at S10. No transcript is detected in the oocyte until S11, when the nurse cell contents are deposited in the oocyte. The spn-E transcript is distributed uniformly in the early embryo (Gillespie, 1995).

EFFECTS OF MUTATION

The spindle E gene of Drosophila is required for anteroposterior and dorsoventral axis formation during oogenesis. At a low frequency, females homozygous for mutations in spn-E generate early egg chambers in which the oocyte is positioned incorrectly within the cyst. At a high frequency, late-stage egg chambers exhibit a ventralized chorion. To analyze Spn-E function, RNA localization patterns were determined for seven different transcripts in spn-E mutant ovaries. Previtellogenic transport to the oocyte is unaffected for all transcripts examined. Transport and localization of Bicoid and Oskar messages during vitellogenic stages are strongly disrupted, and the distribution and/or quantity of Gurken, Orb, and fs(1)K10 mRNAs are also affected, but to a lesser degree. In contrast, Hu-li tai shao and Bicaudal-D transcripts are transported and localized normally in spn-E mutants. In addition, Kinesin heavy chain:beta-Galactosidase fusion protein fails to localize correctly to the posterior of the oocyte in vitellogenic egg chambers. Examination of the microtubule structure with anti-alpha-Tubulin antibodies reveals aberrant microtubule organizing center movement and an abnormally dense cytoplasmic microtubule meshwork (Gillespie, 1995).

spindle E was initially detected in a P element insertion screen: a female sterile line was obtained in which the insertion mapped at 80A5-6. spindle E mutants contain mislocalized oocytes in a small percentage of vitellogenic egg chambers. Ovaries dissected from mutants contain a range of late-stage phenotypes. A wild-type egg chamber at stage 14 of oogenesis possesses two dorsal eggshell respiratory appendages, just lateral to the dorsal midline. Ninety to ninety-five percent of the egg chambers show aberrant appendage formation; the majority possess only one appendage or fused appendages emerging from one base on the dorsal midline (Gillespie, 1995).

Bicaudal-D (Bic-D) is essential for the establishment of oocyte fate and subsequently for polarity formation within the developing Drosophila oocyte. To find out where in the germ cells Bic-D performs its various functions, transgenic flies were made expressing a chimeric Bic-D::GFP fusion protein. Once Bic-D::GFP preferentially accumulates in the oocyte, it shows an initial anterior localization in germarial region 2. In the subsequent egg chamber stages 1-6 Bic-D::GFP preferentially accumulates between the oocyte nucleus and the posterior cortex in a focus that is consistently aligned with a crater-like indentation in the oocyte nucleus. After stage 6 Bic-D::GFP fluorescent signal is predominantly found between the oocyte nucleus and the dorso-anterior cortex. During the different phases several genes have been found to be required for the establishment of the new Bic-D::GFP distribution patterns. Dynein heavy chain (Dhc), spindle (spn) genes and maelstrom (mael) are required for the re-localization of the Bic-D::GFP focus from its anterior to its posterior oocyte position. Genes predicted to encode proteins that interact with RNA (egalitarian and orb) are required for the normal subcellular distribution of Bic-D::GFP in the germarium, and another potential RNA binding protein, spn-E, is required for proper transport of Bic-D::GFP from the nurse cells to the oocyte in later oogenesis stages. The results indicate that Bic-D requires the activity of mRNA binding proteins and a negative-end directed microtubule motor to localize to the appropriate cellular domains. Asymmetric subcellular accumulation of Bic-D and the polarization of the oocyte nucleus may reflect the function of this localization machinery in vectorial mRNA localization and in tethering of the oocyte nucleus. The subcellular polarity defined by the Bic-D focus and the nuclear polarity marks some of the first steps in antero-posterior and subsequently in dorso-ventral polarity formation (Pare, 2000).

The homeless gene of Drosophila encodes a member of the DE-H family of ATPase and RNA helicase proteins. Loss-of-function homeless mutations cause female sterility with numerous defects in oogenesis, including improper formation of both the anterior-posterior and dorsal-ventral axes and failure to transport and localize key RNAs required for axis formation. One homeless mutation was also found to affect male meiosis, causing elevated X-Y nondisjunction. This study analyzes the role of homeless in male meiosis. homeless mutations cause a variety of defects in male meiosis including nondisjunction of the X-Y and 2-2 pair, Y chromosome marker loss, meiotic drive, chromosome fragmentation, chromatin bridges at anaphase, and tripolar meiosis. In addition, homeless mutations interact with an X chromosomal factor to cause complete male sterility. These phenotypes are similar to those caused by deletion of the Suppressor of Stellate [Su(Ste)] locus. Like Su(Ste) deficiencies, homeless mutants also exhibit crystals in primary spermatocytes and derepression of the X-linked Stellate locus. To determine whether the regulatory role of hls is specific for Stellate or includes other repeated sequences as well, testis RNA levels were compared for nine transposable elements; all but one, copia, are expressed at the same levels in hls mutants and wild type. Copia, however, is strongly derepressed in hls mutant males. It is concluded that hls functions along with Su(Ste) and other recently described genes to repress the Stellate locus in spermatocytes, and that it may also play a role in repressing certain other repeated sequences (Stapleton, 2001).

The Y chromosome is known to be essential for male fertility in Drosophila melanogaster. Many aspects of the phenotype of flies lacking a Y chromosome (X0) reflect an unusual negative regulatory interaction that normally occurs between the X chromosome-linked Stellate (Ste) locus and the Y chromosome-linked Suppressor of Stellate [Su(Ste)] locus. That is, the Ste and Su(Ste) are normally silent. Deficiencies of Su(Ste) led to the derepression of the Ste elements in the male germ line and led to the mutant phenotype. Males lacking the Y linked Su(Ste) locus exhibit needle or star-shaped crystalline aggregates in the nuclei and the cytoplasm of primary spermatocytes and several meiotic defects, such as an undercondensation of meiotic chromosomes and an altered distribution of mitochrondria, leading, in many cases, to complete sterility. Both the formation and shape of crystals and the strength of the other meiotic abnormalities depend on the allelic state of the X-linked Ste locus (Aravin, 2001 and references therein).

aubergine (aub) and spindle-E mutations cause a relief of Stellate and sense Su(Ste) silencing. Stellate derepression in the presence of the intact Su(Ste) locus has been observed as a result of aubergine and spindle-E (spn-E) mutations, also known as sting and homeless, respectively. The Aubergine protein has homologs involved in PTGS and RNAi in plants, fungi, and animals. The spn-E gene encodes a putative RNA helicase that is also proposed as a participant in dsRNA-mediated silencing (Aravin, 2001).

A relief of Stellate silencing occurs as a result of the spn-E¹ mutation: this was confirmed by studying the expression of the Ste-lacZ reporter construct in the spn-E¹/+ and spn-E¹/spn-E¹ males. The expression of ß-galactosidase in testes is greatly enhanced in spn-E¹/spn-E¹ males as compared to the heterozygous ones. The effects of the aub^sting-1 and spn-E¹ mutations on the level of sense and antisense Su(Ste) transcripts were assessed. Both mutations, when homozygous, have no effect on the level of antisense Su(Ste) transcripts, but they do increase the level of sense Su(Ste) RNA. Thus, a common mechanism, assisted by the Aubergine and Spindle-E proteins is operated in Su(Ste) dsRNA-mediated silencing of Stellate and sense Su(Ste) expression. The effect of the spn-E¹ mutation is restricted to the germline, since no increase in the level of sense Su(Ste) transcripts in the heads of homozygous flies was observed (Aravin, 2001).

Maelstrom, a Drosophila spindle-class gene, encodes a protein that colocalizes with Vasa and RDE1/AGO1 homolog, Aubergine, in nuage

A hallmark of germline cells across the animal kingdom is the presence of perinuclear, electron-dense granules called nuage. In many species examined, Vasa, a DEAD-box RNA helicase, is found in these morphologically distinct particles. Despite its evolutionary conservation, the function of nuage remains obscure. A null allele of maelstrom (mael) has been characterized. Maelstrom protein is localized to nuage in a Vasa-dependent manner. By phenotypic characterization, maelstrom has been defined as a spindle-class gene that affects Vasa modification. In a nuclear transport assay, it has been determined that Maelstrom shuttles between the nucleus and cytoplasm, which may indicate a nuclear origin for nuage components. Interestingly, Maelstrom, but not Vasa, depends on two genes involved in RNAi phenomena for its nuage localization: aubergine and spindle-E (spn-E). Furthermore, maelstrom mutant ovaries show mislocalization of two proteins involved in the microRNA and/or RNAi pathways, Dicer and Argonaute2, suggesting a potential connection between nuage and the microRNA-pathway (Findley, 2003).

How germline status is established and maintained in sexually reproducing organisms is a fundamental question in developmental biology. A conserved feature of germ cells in species across the animal kingdom is the presence of a distinct morphological element called nuage. Ultrastructurally, nuage appears as electron-dense granules that are localized to the cytoplasmic face of the nuclear envelope. Despite the breadth of nuage in the animal kingdom, there is currently a lack of depth in understanding its function. In animals ranging from the nematode to vertebrates, the Vasa protein has been detected in these granules. Both nuage and Vasa thus offer potential clues as to what makes a germ cell unique (Findley, 2003).

One system with high potential for understanding the role of nuage is Drosophila. In females, Vasa-positive germline granules are continuously present throughout the life cycle, taking one of two forms, nuage or pole plasm. Pole plasm, which contains polar granules, is a determinant that is both necessary and sufficient to induce formation of the germ lineage in early embryogenesis. In Drosophila, nuage is first detectable when primordial germ cells are formed; it persists through adulthood, where it is present in all germ cell types of the ovary (Findley, 2003).

A null allele of the maelstrom gene, which encodes a novel protein with a human homolog, has been identified and characterized. The mutant displays each of the defects in oocyte development common to the spindle-class. Maelstrom localizes to nuage in a Vasa-dependent manner and maelstrom is required for proper modification of Vasa. Through mutant analysis, this study begins to unravel genetic dependencies of nuage particle assembly (Findley, 2003).

Spn-E encodes a putative Dex/hD-box RNA helicase, required for proper localization of several oocyte-destined RNAs and proteins over the course of oogenesis. While the localization of Spindle-E in the ovary has not been determined, its involvement in both RNAi and oogenesis, like Aubergine, prompted its inclusion in this analysis. As with aubergine mutants, the concentration of Maelstrom in perinuclear particles is lost in strong spn-E allelic combinations, spn-E^{616/hlsDelta125} and spn-E^{hls3987/hlsDelta125}. Vasa retains a perinuclear concentration in spn-E ovaries, but as in aubergine, the normal particulate appearance of nuage is less pronounced. Localization analysis has been extended to include the remaining members of the better characterized spn-class mutants, spn-A, spn-B, spn-C, spn-D and okr. Of particular interest was spn-B, which has been shown to modify Vasa as a consequence of meiotic checkpoint activation. The dependency of Maelstrom on Vasa for its localization could, in principle, be affected if Vasa is aberrant. However, in multiple allelic combinations of well-characterized spn genes (spn-B, spn-D and okr) and uncloned spn genes (spn-A and spn-C), colocalization of Vasa and Maelstrom in nuage particles was unperturbed at all stages of oogenesis (Findley, 2003).

The dissociation of Maelstrom from nuage particles in aubergine and spn-E backgrounds was intriguing in light of their requirement in RNAi in Drosophila spermatogenesis and late oogenesis. Importantly, proteins (or homologs) of RNAi pathway components also act in micro RNA (miRNA) processing. Since miRNAs have been shown to regulate RNA translation, it is conceivable that miRNAs are assembled in RNP particles formed in nuage. In this setting, nuage could represent a step in the generation of specificity in translational control in the germline. To explore this potential relationship between nuage and RNAi/miRNA processing pathways, the localization of additional RNAi components was examined in wild-type and maelstrom ovaries. Argonaute1 and Argonaute2 are RDE1/AGO1 homologs required for RNAi in Drosophila. Dicer is the core RNase of RNAi in Drosophila; it is also required for production of the small RNA effectors of the RNAi and miRNA pathways in C. elegans. In vertebrate cell lines, Dicer is primarily cytoplasmic. In wild-type Drosophila ovarioles, Dicer and AGO1 appear uniform and cytoplasmic in nurse cell cytoplasm; AGO2 appears cytoplasmic but relatively more granular. In maelstrom ovaries, AGO1 distribution is relatively unperturbed. However, AGO2 and Dicer are both dramatically mislocalized in maelstrom ovarioles. Beginning around stage 3, Dicer aggregates in discrete, often perinuclear foci in nurse cells. AGO2 is observed in perinuclear regions of nurse cells, which, by contrast, can colocalize with Vasa in nuage (Findley, 2003).

The failure of maelstrom oocytes to proceed to the karyosome stage, to establish cytoplasmic polarity and to accumulate Gurken qualifies the inclusion of maelstrom in the spindle class. Maelstrom is a component of Drosophila nuage and is required for proper modification (or processing) of a key nuage component, Vasa. Maelstrom is also present within the nucleus and cytoplasm of all germline cells, and can shuttle between these compartments in a CRM1-dependent manner. Of the known nuage-localizing proteins, Vasa appears to be a pivotal organizer or nucleator of nuage, whereas Maelstrom can be dissociated from nuage particles in aubergine and spn-E mutants. Furthermore, Dicer and AGO2 are mislocalized in the maelstrom background (Findley, 2003).

The characterized spn genes currently fall into two general classes: those that encode proteins that are likely to be directly involved in meiotic recombinational repair, such as okr, spn-B and spn-C; and those, such as maelstrom and vasa, whose mutant meiotic phenotype, protein sequence and/or localization suggest indirect roles. Work presented in this study suggests that the spn mutants can be sorted by an additional criterion: those that are also required for nuage assembly (vasa, aubergine, maelstrom and spn-E) and those that are not (spn-A, spn-B, spn-C, spn-D and okra). Taken together, these data suggest that the Vasa-like group of spn genes are essential in general 'nuage activities' in all cells of the germline. The activity of the spn-B-class genes, which are involved in recombination or meiotic checkpoint, could represent one avenue through which to use or modulate existing nuage functions that are operative within the germline cyst as a whole. Such nuage-related processes, if inactivated or defective, might culminate in polarity and translational defects within the oocyte (Findley, 2003).

Live imaging of nuage and polar granules: evidence against a precursor-product relationship and a novel role for Oskar in stabilization of polar granule components

Nuage, a germ line specific organelle, is remarkably conserved between species, suggesting that it has an important germline cell function. Very little is known about the specific role of this organelle, but in Drosophila three nuage components have been identified, the Vasa, Tudor and Aubergine proteins. Each of these components is also present in polar granules, structures that are assembled in the oocyte and specify the formation of embryonic germ cells. GFP-tagged versions of Vasa and Aubergine were used to characterize and track nuage particles and polar granules in live preparations of ovaries and embryos. Perinuclear nuage is a stable structure that maintains size, seldom detaches from the nuclear envelope and exchanges protein components with the cytoplasm. Cytoplasmic nuage particles move rapidly in nurse cell cytoplasm and passage into the oocyte where their movements parallel that of the bulk cytoplasm. These particles do not appear to be anchored at the posterior or incorporated into polar granules, which argues for a model where nuage particles do not serve as the precursors of polar granules. Instead, Oskar protein nucleates the formation of polar granules from cytoplasmic pools of the components shared with nuage. Surprisingly, Oskar also appears to stabilize at least one shared component, Aubergine, and this property probably contributes to the Oskar-dependent formation of polar granules. Bruno, a translational control protein, is associated with nuage, which is consistent with a model in which nuage facilitates post transcriptional regulation by promoting the formation or reorganization of RNA-protein complexes (Snee, 2004).

Perinuclear nuage contains, in addition to Vas and Aub, the Maelstrom (Mael), and Gustavus (Gus) proteins. Another component, Bruno (Bru), is a protein that acts in translational repression of osk and gurken (grk) mRNAs. By immunolocalization and expression of a GFP-tagged version of this protein, it was found that Bru is concentrated in perinuclear clusters, similar to the distribution of known nuage components. Double labelling experiments with GFPAub confirmed that Bru colocalizes with nuage. However, Bru is also present at high levels in the cytoplasm, raising the question of whether the colocalization reveals an association with nuage or simply reflects random overlap of an abundant protein with the more narrowly distributed nuage. Evidence that Bru is specifically associated with nuage comes from analysis of Bru distribution in vas mutants: as for other nuage components, the perinuclear clusters of Bru are strongly reduced. Given this identification of Bru as a nuage-associated protein, arrest (aret) mutants (the aret gene encodes Bru) were included in a genetic analysis of nuage. The other genes tested were vas, tud, aub and spindle E (spnE), each of which encodes a nuage component or has been shown to be required for nuage formation, or both (Snee, 2004).

Live imaging was used to better characterize the perinuclear nuage defects seen in static images and to extend the analysis to include cytoplasmic nuage particles. GFPAub was used as the nuage marker to test the role of vas, aret and tud, and VasGFP was used to test the roles of aub and spnE. The live imaging confirmed, for the most part, the basic observations from analysis of fixed samples. In vas mutants perinuclear nuage is almost completely absent, with only a few nuage clusters visible. Loss of spnE activity has a less extreme effect: the perinuclear nuage clusters are largely missing, but a perinuclear zone of VasGFP remains. Consistent with the results by using fixed samples, the persistent perinuclear zone of VasGFP is qualitatively different from wild type, appearing almost completely uniform and lacking any visible discontinuities. Similar results were obtained with the aub mutant, except that the VasGFP perinuclear clusters remain present up to stage 8 of oogenesis, after which they disappear. In aret and tud mutants no significant alteration of perinuclear nuage was detected (Snee, 2004).

In mutants whose perinuclear VasGFP is uniform (spnE^- and later stage aub^-), the protein undergoes rapid exchange with cytoplasmic pools, just as for VasGFP in perinuclear clusters of wild-type egg chambers. In photobleaching experiments the fluorescence-recovery half-time is 50 seconds in aub^- and 48.5 seconds in spnE^-, similar to the t_1/2=59 seconds for wild type (Snee, 2004).

Cytoplasmic nuage particles are affected differently in the vas, aub and spnE mutants. The vas and spnE mutants have few or no cytoplasmic nuage particles. By contrast, aub mutants have no dramatic reduction in the abundance of cytoplasmic nuage particles, even at times well after the disappearance of perinuclear nuage clusters at stage 8, and the particles have a fairly typical size distribution. These particles do not simply represent the default appearance of VasGFP; they are absent in the spnE mutant. Thus, it seems unlikely that perinuclear nuage clusters are required for the formation of cytoplasmic nuage particles, a conclusion consistent with the observation that cytoplasmic particles are produced only infrequently by detachment of perinuclear nuage clusters (Snee, 2004).

The consequences of loss of vas activity were examined in the male germ line. Just as in nurse cells, Vas appears to be concentrated in nuage in spermatocytes. Given the crucial role for Vas in the nuage of other cell types, either male nuage must differ in this requirement or nuage is not essential in the male germ line for fertility. To distinguish between these possibilities vas^AS spermatocytes were tested for the presence of nuage, using GFPAub as a marker. Although GFPAub was present in the cytoplasm, there were no visible perinuclear nuage clusters, indicating that nuage does not form in the vas mutant and is therefore not required for spermatocyte function. An alternate and less probable interpretation is that a rudimentary form of nuage, lacking Aub, is present and is sufficient to provide a minimal requirement for nuage in males (Snee, 2004).

In Drosophila, two types of function, not mutually exclusive, have been proposed for nuage. In one model nuage has been suggested to serve as a precursor to polar granules, a view initially based on ultrastructural similarities of the two organelles and supported by the identification of shared components. Another possible role for nuage is based on its position at the periphery of the nucleus, at or near nuclear pores. Specifically, nuage might act in some aspect of remodelling RNPs when RNAs are exported from the nucleus. Analysis of the movements and genesis of nuage particles provides two arguments against the first model: (1) the rate of release of perinuclear nuage clusters in the nurse cells is very low, much lower than expected if the clusters form polar granules; (2) no nuage particles arriving at the posterior pole of the oocyte and becoming incorporated into polar granules were detected. An additional observation that argues against a model where nuage is a precursor for polar granules, is the presence of cytoplasmic nuage particles in aub mutants, despite the fact that these mutants do not assemble polar granules. However, this evidence does not exclude the first model, because the nuage particles in the mutant might not be fully functional. A third argument is provided by the evidence that Osk cannot interact with nuage, leaving de novo assembly of polar granules as the only reasonable option. Overall, the results strongly suggest that nuage is not the precursor to polar granules, and it is believed that the shared features are simply indicative of similar biochemical activities, rather than a precursor-product relationship (Snee, 2004).

The data do not directly test the model that nuage might function as a transition zone in the movements of mRNAs from the nucleus to the cytoplasm, where RNP components might be exchanged or otherwise modified. However, new properties of nuage, and these relate to possible functions, have been identified. (1) It was found that Bruno, an RNA binding protein that acts as a translational repressor of osk and grk mRNAs, is associated with nuage. This extends the correlation of nuage components with factors that act in some aspect on mRNA localization or translational control. Of the previously identified nuage components, Vas and Gus are involved in the regulation of grk mRNA localization and translation, Aub is required for efficient translation of osk mRNA and has also been implicated in RNAi, and mael mutants display defects in the early stages of mRNA localization. Moreover, spnE, which is necessary for normal nuage formation, is required for the localization of multiple mRNAs and acts in RNAi. Thus, every known nuage component has a role in one or more types of post-transcriptional control of gene expression (Snee, 2004).

(2) The second property of nuage reported here, is the remarkably dynamic composition of perinuclear nuage clusters, despite their relatively fixed positions around the nucleus. This is in contrast to studies showing that general protein exchange is slow in mouse nuage. The rapid exchange of both Vas and Aub, the two proteins tested, suggests that the clusters are staging sites where these, and presumably additional proteins, become associated with other molecules and move off into the cytoplasm. Much like shuttling-proteins that escort RNAs in their travels from the nucleus to the cytoplasm, there might be a class of proteins that interact in nuage with newly exported RNAs and then facilitate post-transcriptional control events that occur in the cytoplasm. By this model nuage could be an organelle that concentrates and thus potentiates the activity factors normally present in all cells, but that must be especially active in germline cells because of their intensive reliance on post-transcriptional controls of gene expression (Snee, 2004).

It has been argued that nuage from the nurse cells is not used for polar granule assembly in the oocyte, yet these two subcellular structures clearly share components and may well have similar activities. One feature that clearly distinguishes polar granules from nuage is the presence of Osk protein. Under normal circumstances Osk is never in contact with nuage, because an elaborate set of post-transcriptional control mechanisms serves to prevent Osk accumulation in the nurse cells and to restrict the distribution of Osk protein within the oocyte to the posterior pole. The presence of Osk at this single location provides the cue for the assembly of polar granules, and misdirection of Osk to other sites in the oocyte leads to ectopic polar granule formation. Thus Osk is generally viewed as an anchor for the recruitment of the factors that form polar granules. Given the finding that polar granules are significantly more stable that perinuclear nuage clusters, it might be that Osk not only recruits other factors, but also strengthens their interactions. A further and unanticipated property of Osk was revealed in studies in which Osk was expressed precociously throughout the oocyte. Under these conditions GFPAub levels are substantially elevated in the oocyte. Two general explanations are possible. (1) Osk might stimulate the rate of transfer of GFPAub from the nurse cells to the oocyte. Such a model is not supported by any known property of Osk, and no increase in the rate at which GFPAub particles move into the oocyte was detected. Furthermore, GFPAub levels in the oocyte are enhanced even before the onset of known nurse cell to oocyte movements in the cytoplasm, and so Osk would have to dramatically alter the properties of the egg chamber under this model. (2) Osk could stabilize a normally labile pool of GFPAub in the oocyte. In the simplest form of this model, stabilization would occur as a consequence of the assembly into complexes, which could include factors other than Osk and GFPAub. This model appears to be most compatible with the data. In addition, such a model provides a possible explanation for the curious association of the Fat facets (Faf) protein, a deubiquitinating enzyme, with pole plasm. The role of Faf could be to stabilize one or more polar granule components, thereby enhancing the growth of polar granules (Snee, 2004).

The restriction of Osk protein to the posterior pole of the oocyte is known to be important for limiting the spatial distribution of posterior body patterning activity. By analogy, this restriction might also be important for allowing normal assembly and function of nuage in nurse cells, if Osk can compete with nuage for their shared components. To evaluate this possibility, ovaries were examined in which Osk was allowed to accumulate in the nurse cells as well as the oocyte. Osk does indeed nucleate the formation of large bodies in the nurse cell cytoplasm, but the presence of these bodies does not appear to limit the amount of perinuclear nuage. Notably, no Osk was observed in association with perinuclear nuage, which appears not to be affected by the ectopic Osk. The Osk protein can interact directly with Vas in the two-hybrid assay in yeast, and so its failure to associate with perinuclear nuage -- the regions of greatest Vas concentration -- in nurse cells is notable. One interpretation is that the site of Vas binding to Osk is blocked when it is in nuage. This fits with the model in which Osk protein nucleates polar granule formation not from nuage particles themselves, but from individual nuage components or subassemblies (Snee, 2004).

Does Spindle-E function in RNAi?

Gene silencing by double-stranded RNA is a widespread phenomenon called RNAi, involving homology-dependent degradation of mRNAs. RNAi is established in the Drosophila female germ line. mRNA transcripts are translationally quiescent at the arrested oocyte stage and are insensitive to RNAi. Upon oocyte maturation, transcripts that are translated become sensitive to degradation while untranslated transcripts remain resistant. Mutations in aubergine and spindleE, members of the PIWI/PAZ and DE-H helicase gene families, respectively, block RNAi activation during egg maturation and perturb translation control during oogenesis, supporting a connection between gene silencing and translation in the oocyte (Kennerdell, 2002).

To analyze the effects of dsRNA on mRNA stability in Drosophila oocytes, dsRNAs corresponding to the maternally expressed genes bicoid and hunchback were used. These genes were chosen because their mRNAs are synthesized, processed, and localized to the cytoplasm of oocytes during mid- to late oogenesis. To test the sensitivity of bicoid and hunchback to RNAi, fertilized eggs were initially injected with dsRNA. bicoid dsRNA reduces the expression of Bicoid protein and induces a bicoid loss-of-function phenotype in which embryos have partial transformation of anterior structures to posterior identities. The effect is robust enough that dsRNA-coated gold particles randomly introduced into fertilized eggs by a gene gun generate mutant phenotypes. hunchback dsRNA induces phenotypes in which embryos are missing thoracic and head segments. These phenotypes resemble mutant embryos generated when maternal and zygotic hunchback gene activity is reduced. To determine if dsRNA injection causes mRNA degradation, endogenous mRNA levels were measured using a semiquantitative RT-PCR assay. The level of bicoid mRNA was reduced about fourfold 40 min after injection of bicoid dsRNA. Likewise, injection of hunchback dsRNA resulted in a reduction of hunchback mRNA levels. Coinjection of a pan-specific ribonuclease inhibitor, vanadyl-ribonucleoside, with bicoid dsRNA results in no reduction of bicoid mRNA, indicating the effect requires a ribonuclease activity (Kennerdell, 2002).

Whether and when transcripts become sensitive to dsRNAs during oogenesis was determined. dsRNA was injected into staged oocytes and their consequent levels of bicoid and hunchback mRNAs were examined. Although oocytes earlier than stage 14 could not be injected, stage 14 oocytes could be examined for RNAi activity. Levels of bicoid and hunchback mRNAs were unchanged in stage 14 oocytes after injection of dsRNA, indicating that oocytes at this stage are unable to carry out RNAi (Kennerdell, 2002).

Oocytes of most animals arrest at species-specific stages of meiosis while differentiation of the oocytes occurs. Drosophila oocytes arrest transiently in prophase I while the oocytes are loaded with RNAs and proteins. Some of these molecules are differentially localized within the oocyte, imparting positional information to be used for embryonic axis formation. When Drosophila oocytes reach stage 14, they undergo meiotic arrest once more, this time at metaphase I. These arrested oocytes remain translationally quiescent in the ovary, potentially for weeks. Arrest is relieved as in most animal eggs by the process of maturation or activation that precedes fertilization. In the case of Drosophila, it appears that ovulation triggers activation of the oocyte to resume meiosis. When oocytes are activated, meiosis is completed and translation of maternal RNAs is dramatically elevated. Shortly thereafter, the oocyte is fertilized as it passes into the uterus (Kennerdell, 2002).

RNAi-like effects are not detected in arrested stage 14 oocytes injected with dsRNA. Was this a general feature of the female germ line? To explore this issue, dsRNA was injected into mature activated oocytes. Injection of dsRNA causes reduction in bicoid and hunchback mRNA levels comparable to those seen in embryos. To confirm that mRNA sensitivity to dsRNA is strictly coincident with oocyte maturation, arrested stage 14 oocytes were isolated from dissected ovaries and the oocytes were activated in vitro. This maturation procedure reactivates meiosis, mRNA translation, and vitelline membrane cross-linking. After maturation, oocytes were injected with bicoid dsRNA and assayed for bicoid mRNA levels. These oocytes showed a decrease in bicoid mRNA. Thus, immature Drosophila oocytes that are coordinately blocked for meiosis and translation are resistant to RNAi, and the block to these processes can be released by maturation or activation of oocytes (Kennerdell, 2002).

There are several possible ways in which RNAi might be blocked in arrested oocytes. One possibility is that an essential component of the RNAi machinery might be missing at this stage. Oocyte maturation would then involve synthesis of the component. To address if synthesis of a missing component is responsible, oocytes were activated in the presence of the protein synthesis inhibitor cycloheximide. Arrested stage 14 oocytes were preincubated with cycloheximide and then activated in vitro in the presence of cycloheximide. This treatment inhibits >95% of the protein synthesis that occurs during maturation. These oocytes were injected with bicoid dsRNA and, strikingly, they showed a decrease in bicoid mRNA levels that was comparable to that of normal mature oocytes. RNAi is established during oocyte maturation even when protein synthesis is blocked. Thus, RNAi establishment during oocyte maturation does not likely occur by synthesis of an essential protein component of the RNAi machinery (Kennerdell, 2002).

The stage 14 oocyte is coordinately blocked in both translation and RNAi. The two processes are released near simultaneously from this block, suggesting perhaps that a shared mechanism links their regulation. To test this possibility, the effectiveness of dsRNA was examined against a transcript that is present but not translated after oocyte maturation. The alphaTubulin67C gene encodes one of three alpha-tubulin proteins synthesized during oogenesis and embryogenesis. Transcript accumulates and is actively translated in early immature oocytes. However, after oocyte maturation, no translation of alphaTubulin67C mRNA occurs, even though transcripts at this stage are associated with ribosomes and are competent to drive translation in vitro. The stable pool of alphaTubulin67C mRNA is comparable to levels of bicoid and hunchback mRNA in mature oocytes. When two nonoverlapping dsRNAs against alphaTubulin67C transcript were independently injected into mature activated oocytes, no destruction of mRNA was detected. This suggests that the ability of dsRNAs to destroy transcripts during oogenesis is coupled to the translation activity of the transcript. Successful translation of transcripts is perhaps necessary to link a transcript to dsRNA-triggered degradation (Kennerdell, 2002).

Several Drosophila genes have been identified that affect translation of maternal mRNAs during oogenesis. One of these genes, aubergine (aub), encodes a protein with a PIWI and PAZ domain. To determine whether Aub has any role for RNAi in oocytes, the effect of aub mutations on RNAi activity was examined. bicoid and hunchback dsRNAs were injected into aub mutant oocytes that were activated in vitro. Degradation of bicoid and hunchback mRNAs was not observed in aub mutants, indicating that Aub is necessary for germ-line RNAi. Two independent aub alleles in heteroallelic combination produced the same result, indicating that the effect was not due to the influence of linked modifiers (Kennerdell, 2002).

The aub gene is a member of a family of genes implicated in RNAi and PTGS. Indeed, aub has been implicated in PTGS regulation of the Stellate repeats and Su(Ste) genes on X and Y chromosomes. Another member of the family, piwi, has been implicated in PTGS within somatic cells. A third family member, Ago2, is a subunit of the mRNA-cleaving complex that mediates RNAi in Drosophila embryonic cells. Thus, several members of this gene family in Drosophila have been implicated in RNAi and PTGS at various steps (Kennerdell, 2002).

It was of interest to determine if other translational regulatory genes are involved in RNAi. To test this possibility, two genes that possibly act through interactions with RNA were examined. vasa and spindle-E (spn-E) encode DexH-box RNA helicases. When activated spn-E mutant oocytes were injected with bicoid or hunchback dsRNAs, no reduction in cognate mRNA levels occurred. In contrast, activated vasa mutant oocytes injected with bicoid dsRNA were found to show transcript degradation comparable to wild type. It is concluded that activation of RNAi in oocytes is dependent on the activity of Spn-E but not Vasa (Kennerdell, 2002).

Arrested Drosophila oocytes are unable to generate RNAi silencing of endogenous maternal mRNAs, but selectively establish this capability upon egg maturation. How is RNAi activated by egg maturation? It is argued that RNAi is linked in some way to translation of maternal mRNAs, which is also specifically activated by egg maturation. Establishment of RNAi is probably not caused by translation of a missing RNAi component. Rather, the complete RNAi apparatus may be present and poised for action but is unable to target homologous substrate mRNAs until egg maturation. Translational masking of mRNAs, a mechanism that operates on maternal Drosophila gene expression, may conceivably be one way in which mRNA is blocked from RNAi attack. Alternatively, targeting of mRNA might require transcripts be assembled onto active polysomes. This may be the case, because siRNA-containing RISC complexes physically fractionate with polysomes, and siRNAs associate with polysomes in Trypanosoma brucei. There is no evidence to indicate that dsRNA-targeting requires ribosome translocation on transcripts, because it is found that cycloheximide inhibition of ribosome translocation does not block RNAi activity in activated mature oocytes (Kennerdell, 2002).

Coupling RNAi to translated mRNA might facilitate base-pairing interactions between siRNAs and an unfolded mRNA target, or it might simply be a means to mark RNAs to be scanned for destruction. The key evidence suggesting that transcript translation is linked to transcript degradation by RNAi comes from experiments in which dsRNA against the alphaTubulin67C message was tested. dsRNA is ineffective against the untranslated alphaTubulin67C transcript in mature activated oocytes, which are nevertheless competent to carry out RNAi against translated bicoid and hunchback transcripts. Thus, there is a correlation between the ability of a transcript to be translated and its ability to be destroyed by dsRNA (Kennerdell, 2002).

Aub and Spn-E are required for RNAi in Drosophila oocytes. They also regulate several features of germ-cell development, including translation of certain maternal mRNAs. Germ-line and stem-cell functions have been reported for orthologs of piwi and ago1 in a variety of species. The ego-1 gene of C. elegans is required for both germ-line development and germ-line PTGS. Finally, mutations in dcr-1, the C. elegans gene encoding Dicer, disrupt oogenesis in an unspecified manner. The developmental defects associated with mutations in these genes, including aub and spn-E, might reflect a loss of gene silencing important for oocyte development. Thus, Aub and Spn-E might play a specific role in gene silencing mechanisms, including RNAi, that nevertheless have a widespread impact on many features of development. Alternatively, Aub and Spn-E could be required for RNAi because they activate translation of germ-line transcripts including those for bicoid and hunchback. Although there is no evidence for translational control of bicoid mRNA in aub mutants, these mutants may perturb steps in the translation of transcripts that are essential for triggering RNAi. Future experiments should define the specific roles for Aub and Spn-E in dsRNA-mediated destruction and its relationship to translation control (Kennerdell, 2002).

Double-stranded RNA-mediated silencing of genomic tandem repeats and transposable elements in the D. melanogaster germline

To date, few natural cases of RNA-silencing-mediated regulation have been described. Repression has been analyzed of testis-expressed Stellate genes by the homologous Suppressors of Stellate [Su(Ste)] repeats that produce sense and antisense short RNAs. The Stellate promoter is dispensable for suppression, but local disturbance of complementarity between the Stellate transcript and the Su(Ste) repeats impairs silencing. Using in situ RNA hybridization, temporal control was found of the expression and spatial distribution of sense and antisense Stellate and Su(Ste) transcripts in germinal cells. Antisense Su(Ste) transcripts accumulate in the nuclei of early spermatocytes before the appearance of sense transcripts. The sense and antisense transcripts are colocalized in the nuclei of mature spermatocytes, placing the initial step of silencing in the nucleus and suggesting formation of double-stranded RNA. Mutations in the aubergine and spindle-E genes, members of the Argonaute and RNA helicase gene families, respectively, impair silencing by eliminating the short Su(Ste) RNA, but have no effect on microRNA production. Thus, different small RNA-containing complexes operate in the male germ line (Aravin, 2004).

A strong correlation is observed between Stellate silencing and the presence in testes of sense and antisense 25- to 27-nt RNAs homologous to Stellate and Su(Ste) sequences. The short RNAs are absent when Stellate genes are derepressed as a consequence of either a Su(Ste) locus deletion or mutations in the aub and spn-E genes. The cloning of short RNA from D. melanogaster testes also demonstrates the presence of short RNAs that are derived from Su(Ste) and are highly homologous to Stellate. A rigid size restriction of 21 to 23 nt has, however, been observed for siRNA in various in vitro studies of D. melanogaster RNAi. Examination of Dicer activity with different dsRNAs suggests a strong specificity of processing to 21- to 23-nt fragments in both Drosophila embryo extracts and cell culture. Furthermore, investigation of the functional anatomy of chemically synthesized siRNAs in embryo extracts defined the optimal length of siRNAs as 21 to 23 nt, while RNAs longer than 24 nt have practically no cognate-mRNA cleavage activity. It has been proposed that only RNAs that meet this size requirement can be loaded into the RISC. However, examples of the existence of two size classes of short RNAs (21 or 22 nt and 24 to 26 nt) involved in silencing have also been reported. Two different size variants of short RNAs were observed during artificial silencing in plants, with the short variant responsible for posttranscriptional gene silencing and the long one most likely participating in DNA methylation and spreading of the silencing signal. Furthermore, only RNAs from the long class have been detected that correspond to endogenous plant transposable elements. Two size classes of short RNAs are produced from dsRNA in plant extracts, and the activity of different Dicer proteins was shown to be responsible for producing each class. Cloning of endogenous short RNAs from D. melanogaster has also identified two size classes of short RNAs, with the short class (21 to 23 nt) including microRNAs and the long class (24 to 26 nt) comprising sequences derived from transcripts of transposable elements and other repetitive heterochromatic sequences (Aravin, 2004).

The larger size of the short Su(Ste) RNA may be explained by specific sequences affecting dsRNA processing by Dicer or by the presence in testes of specific factors that change the cleavage interval of dsRNA. However, exogenous Su(Ste) dsRNA is cleaved into 21- to 23-nt siRNA in testis extracts, most likely reflecting the activity of the same Dicer protein that acts in somatic tissues. The hypothesis is favored that the 25- to 27-nt Su(Ste) RNAs detected in vivo are produced by a mechanism at least partially different from conventional siRNA production. A clue to the origin of the short Su(Ste) RNAs comes from the finding that Su(Ste) dsRNA formation occurs in the nucleus, unlike that of artificial RNAi, in which dsRNA is believed to be processed in the cytoplasm. Both conventional-size siRNA and a longer short RNA have been observed during viroid replication in the plant nucleus. Two size classes of short RNAs may be produced in D. melanogaster by different Dicer proteins, as has been demonstrated in plants. Alternatively, specific nuclear factors may affect how a single Dicer protein processes dsRNA in the nucleus (Aravin, 2004).

Mutations in the aub and spn-E genes lead to elimination of short Su(Ste) RNA in testes. However, neither mutation affects processing of exogenously provided dsRNA to 21- to 23-nt siRNA in testis extracts. It has been observed that both aub and spn-E mutations block RNAi in oocytes produced by injected dsRNA. It has been proposed that both proteins affect RNAi because of their involvement in translational control, but the results suggest that Aub and Spn-E may be involved in the production and/or stabilization of siRNA. Similarly, the rde-1 and mut-7 genes of Caenorhabditis elegans are required for the production of siRNA in vivo but are dispensable for dsRNA processing in vitro. The corresponding proteins are required for long-term stabilization of siRNA rather than for dsRNA processing (Aravin, 2004).

The aub and spn-E mutations eliminate the short Su(Ste) RNA without affecting the abundance of two different microRNAs in testes. It is proposed that distinct protein complexes mediate production and/or stabilization of short Su(Ste) RNA and microRNAs in testes. Similarly, different members of the Argonaute family participate in artificial RNAi and in microRNA processing in C. elegans and plants, despite the central role of Dicer in both processes (Aravin, 2004).

A distinct small RNA pathway silences selfish genetic elements in the germline requires Spn-E

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Drosophila rasiRNA pathway mutations disrupt embryonic axis specification through activation of an ATR/Chk2 DNA damage response

Small repeat-associated siRNAs (rasiRNAs) mediate silencing of retrotransposons and the Stellate locus. Mutations in the Drosophila rasiRNA pathway genes armitage and aubergine disrupt embryonic axis specification, triggering defects in microtubule polarization as well as asymmetric localization of mRNA and protein determinants in the developing oocyte. Mutations in the ATR/Chk2 DNA damage signal transduction pathway dramatically suppress these axis specification defects, but do not restore retrotransposon or Stellate silencing. Furthermore, rasiRNA pathway mutations lead to germline-specific accumulation of γ-H2Av foci characteristic of DNA damage. It is concluded that rasiRNA-based gene silencing is not required for axis specification, and that the critical developmental function for this pathway is to suppress DNA damage signaling in the germline (Klattenhoff, 2007).

Mutations in the Drosophila armi, aub, and spn-E genes disrupt oocyte microtubule organization and asymmetric localization of mRNAs and proteins that specify the posterior apole and dorsal-ventral axis of the oocyte and embryo. Mutations in these genes block homology-dependent RNA cleavage and RISC assembly in ovary lysates, RNAi-based gene silencing during early embryogenesis, rasiRNA production, and retrotransposon and Stellate silencing. Mutations in dcr-2 and ago-2 genes, by contrast, block siRNA function, but they do not disrupt the rasiRNA pathway or embryonic axis specification. The rasiRNA pathway thus appears to be required for embryonic axis specification. However, the function of rasiRNAs in the axis specification pathway has not been previously established (Klattenhoff, 2007).

Cytoskeletal polarization, morphogen localization, and eggshell patterning defects associated with armi and aub are efficiently suppressed by mnk and mei-41, which respectively encode Chk2 and ATR kinase components of the DNA damage signaling pathway. In addition, armi and aub mutants accumulate γ-H2Av foci characteristic of DNA DSBs and trigger Chk2-dependent phosphorylation of Vas, an RNA helicase required for posterior and dorsal-ventral specification. Mutations in spn-E also disrupt the rasiRNA pathway, trigger axis specification defects, and lead to germline-specific accumulation of γ-H2Av foci. Significantly, the mnk and mei-41 mutations do not suppress Stellate or HeT-A overexpression, indicating that axis specification does not directly require rasiRNA-dependent gene silencing. Based on these findings, it is concluded that the rasiRNA pathway suppresses DNA damage signaling in the female germline, and that mutations in this pathway disrupt axis specification by activating an ATR/Chk2 kinase pathway that blocks microtubule polarization and morphogen localization in the oocyte (Klattenhoff, 2007).

The cause of DNA damage signaling in armi, aub, and spn-E mutants remains to be established. In wild-type ovaries, γ-H2Av foci begin to accumulate in region 2 of the germarium, when the Spo11 nuclease (encoded by the mei-W68 gene) initiates meiotic breaks. The axis specification defects associated with DNA DSB repair mutations are efficiently suppressed by mei-W68 mutations, indicating that meiotic breaks are the source of DNA damage in these mutants. The axis specification defects and γ-H2Av focus formation associated with armi, by contrast, are not suppressed by mei-W68. mei-W68 double mutants with aub or spn-E have not been analyzed, but this observation strongly suggests that meiotic DSBs are not the source of DNA damage in rasiRNA pathway mutations. Retrotransposon silencing is disrupted in armi, aub, and spn-E mutants, and transcription of LINE retrotransposons in mammalian cells leads to DNA damage and DNA damage signaling. Loss of retrotransposon silencing could therefore directly induce the DSBs in rasiRNA pathway mutants. However, DNA damage can also lead to loss of retrotransposon silencing. Mutations in the rasiRNA pathway could therefore disrupt DNA repair and thus induce DNA damage, which, in turn, induces loss of retrotransposon silencing. Finally, the HeT-A retrotransposon is associated with telomeres, and overexpression of this element could reflect a loss of telomere protection and could damage signaling by chromosome ends in the rasiRNA pathway mutants. The available data do not distinguish between these alternatives (Klattenhoff, 2007).

In mouse, the piwi-related Argonauts Miwi and Mili bind piRNAs, 30 nt RNAs derived primarily from a single strand that appear to be related to rasiRNAs. Mutations in these genes disrupt spermatogenesis and lead to germline apoptosis, which can be induced by DNA damage signaling. Mammalian piRNAs and Drosophila rasiRNAs may therefore serve similar functions in suppressing a germline-specific DNA damage response (Klattenhoff, 2007).

RNA helicase Belle/DDX3 regulates transgene expression in Drosophila

Belle (Bel), the Drosophila homolog of the yeast DEAD-box RNA helicase DED1 and human DDX3, has been shown to be required for oogenesis and female fertility. This study reports a novel role of Bel in regulating the expression of transgenes. Abrogation of Bel by mutations or RNAi induces silencing of a variety of P-element-derived transgenes. This silencing effect depends on downregulation of their RNA levels. Genetic studies have revealed that the RNA helicase Spindle-E (Spn-E), a nuage RNA helicase that plays a crucial role in regulating RNA processing and PIWI-interacting RNA (piRNA) biogenesis in germline cells, is required for loss-of-bel-induced transgene silencing. Conversely, Bel abrogation alleviates the nuage-protein mislocalization phenotype in spn-E mutants, suggesting a competitive relationship between these two RNA helicases. Additionally, disruption of the chromatin remodeling factor Mod(mdg4) or the microRNA biogenesis enzyme Dicer-1 (Dcr-1) also alleviates the transgene-silencing phenotypes in bel mutants, suggesting the involvement of chromatin remodeling and microRNA biogenesis in loss-of-bel-induced transgene silencing. Finally genetic inhibition of Bel function was shown to lead to de novo generation of piRNAs from the transgene region inserted in the genome, suggesting a potential piRNA-dependent mechanism that may mediate transgene silencing as Bel function is inhibited (Lo, 2016).

Transgene silencing refers to the activity of various host defense responses that ordinarily act on natural foreign or parasitic sequences such as transposable elements (TEs), viroids, RNA and DNA viruses, and bacterial DNA. Since transgenes or their transcripts can resemble these cellular invaders in a number of ways, they naturally become the targets of host protective reactions. There are at least two distinct host defense systems responsible for silencing transgenes. One performs its effect via de novo DNA methylation at the genome level. The second defense system operates post-transcriptionally to silence transgenes, which involves sequence-specific RNA degradation in the cytoplasm. Therefore, transgene silencing involves complex cell immune systems including epigenetic and RNA silencing mechanisms. Although many factors involved in transgene silencing have been identified, and several mechanisms have been proposed, there remains much to understand regarding this vital aspect of the cell immune system (Lo, 2016).

Drosophila oogenesis, which involves the generation of the female gamete (oocyte), nurse cells, and follicle cells, is an excellent system for the study of TE and transgene silencing. The egg chamber, the developmental unit of oogenesis, contains the germline cells (one oocyte and 15 nurse cells) and a layer of surrounding somatically derived epithelial follicle cells. Both the germline cells and follicle cells can produce small RNAs to silence TE expression. The nuage, a perinuclear structure within Drosophila nurse cells, is an RNA-rich organelle unique to the germline. The nuage is required for the processing and localization of germline mRNAs and for the biogenesis of PIWI-interacting RNAs (piRNAs), a class of small non-coding RNAs that function as the cell immune system for silencing TEs. In D. melanogaster, most primary piRNAs are produced from discrete pericentromeric and telomeric heterochromatic loci (called piRNA clusters) containing damaged repeated TE sequences. In fly germline cells, an additional step of piRNA biogenesis, the 'ping-pong cycle' mechanism, is employed to generate the secondary piRNAs. Multiple factors localized in the nuage of germline cells have been discovered to be essential for secondary piRNA biogenesis, including Aub, AGO3, Spindle-E (Spn-E), and Vasa. In follicle cells, piRNAs are only produced from piRNA clusters (e.g., flamenco ) via PIWI and other related nuclear factors, and there is no secondary piRNA biogenesis involved. Intriguingly, besides piRNA clusters, euchromatic transposon insertion sites have been identified as another origin to produce piRNAs and endo-siRNAs. This mechanism provides another layer of defense to suppress TE activity and can also serve as a way to affect expression of coding genes and microRNA (miRNA) genes adjacent to inserted TEs (Lo, 2016).

Vasa and Spn-E belong to a family of DEAD-box proteins defined by multiple distinct conserved motifs including the D-E-A-D (Asp-Glu-Ala-Asp) motif. Among the identified DEAD-box proteins, one subfamily is highly conserved from yeast to human, which includes orthologs in yeast (DED1), Drosophila (Belle (Bel)), Xenopus (An3), mice (PL10), and humans (DDX3). These DEAD-box subfamily proteins possess the ATP-dependent RNA helicase activity to unwind double-stranded RNA and remodel RNA-protein interactions. Yeast DED1 is a multifunctional protein that functions to regulate multiple stages of RNA processing and translation. DED1 has also been shown to play a specific role in cell-cycle control. DDX3, the human homolog of DED1, is known to be involved in modulating multiple biological processes, including antiviral innate immunity, mitotic chromosome segregation in somatic cells, the suppression of spermatogenesis, G1-S transition of the cell cycle, epithelial-mesenchymal transition (EMT), a bona fide component of the RNAi pathway, TNF-related apoptosis, and WNT signaling (Lo, 2016 and references therein).

Vasa, a paralog of Bel, is required for the formation and function of nuage to suppress TE expression by being involved in the production of piRNAs. Most recently, it has been reported that Vasa is a key component in the piRNA amplifier complex in the nuage and serves as a protein platform to recruit PIWI proteins, the Tudor protein Qin/Kumo and antisense piRNA guides in an ATP-dependent manner for the ping-pong-loop amplification of secondary piRNAs. Bel colocalizes with Vasa in the nuage and at the oocyte posterior during oogenesis, and is required for female fertility. Recent findings have shown that loss of bel delays activation of Notch signaling in follicle cells, which in turn leads to delayed cell differentiation and defects in the switch from the mitotic cycle to the endocycle. However, unlike Vasa, the specific roles of Bel in the nuage of germline cells, and whether it is involved in piRNA biogenesis, remain unknown (Lo, 2016).

From previous studies, it was unexpectedly found that the Gal4-driven expression of a UASp-Bel:GFP transgene was silenced in bel mutant germline cells. This silencing effect was not specific to bel-based transgenes because 13 out of 22 different transgenic lines tested could be silenced in either germline or somatic bel mutant cells, or both. Subsequently the RNA helicase Spn-E, the epigenetic regulator Modifier of mdg4 [Mod(mdg4)] and the miRNA biogenesis enzyme Dcr-1 was identified as crucial factors for this bel-related transgene silencing. Their abrogation could either partially or completely rescue the transgene-silencing phenotype induced by loss of bel. Importantly, small RNA deep sequencing analysis suggests that a piRNA-mediated mechanism is potentially involved in Bel-inactivation-induced transgene silencing. Together, these studies genetically link the function of Bel to Spn-E, Mod(mdg4), and Dcr-1, and suggest that transgene silencing induced by Bel inactivation may involve RNA processing, piRNA, miRNA, and epigenetic mechanisms (Lo, 2016).

This article reports that loss-of-bel function triggers transgene silencing, which occurs through reduction in transgene RNA levels. Furthermore, genetic studies indicate that this transgene silencing effect induced by bel abrogation requires the RNA helicase Spn-E, the insulator modulator Mod(mdg4) and/or the miRNA biosynthesis enzyme Dcr-1. Based on the functional roles of these three molecules, the data suggest that this transgene silencing effect may involve RNA processing, chromatin remodeling and/or miRNA biogenesis. This transgene silencing event occurring under various bel mutant backgrounds implies that Bel may regulate these three molecular mechanisms to sustain transgene expression in the normal physiological condition. Intriguingly, these studies also identified additional complexity in the relationship between Bel and Spn-E because the mislocalization of nuage components in spn-E mutants requires Bel. Therefore, these findings, taken together, provide new insight into Bel function and expand the molecular interaction network radiating from Bel (Lo, 2016).

These studies show that loss of bel gave rise to transgene silencing via decreased transgene RNA levels. This phenomenon could be attributable to either transcriptional suppression or increased RNA degradation. Some support for an RNA degradation/targeting mechanism comes from the finding that Spn-E is required for transgene silencing induced by loss of bel. In germline nurse cells, Spn-E, which is located in the cytoplasmic nuage, is crucial for properly maintaining the subcellular localization of piRNA-related protein factors in the nuage, the ping-pong reaction of piRNA biogenesis, and silencing of TEs. Spn-E is also required for the proper localization of RNA transcripts (e.g., Bicoid and Oskar) during oogenesis, which might be related to its role in organizing a cytoskeletal framework. Therefore, it is plausible that the Spn-E-dependent RNA processing activity and/or Spn-E-generated piRNAs mediate transgene RNA degradation. In addition, it is possible that piRNAs can also elicit transcriptional silencing of transgenes based on their nuclear epigenetic role in TE silencing. Another striking finding from these studies shows that Bel is involved in disrupting the subcellular localization of nuage components when Spn-E is abrogated. In contrast, loss of Bel alone had no impact on the localization of piRNA-related nuage protein components. These findings suggest that there could be a competitive relationship between Bel and Spn-E, where these two molecules negatively regulate each other. This hypothesis is also supported by observations that Spn-E is required for transgene silencing when Bel is abrogated. According to these findings, a model is envisioned that Bel may function as a negative regulator for ping-pong-cycle-mediated piRNA biogenesis via disrupting the nuage localization of piRNA-related proteins as Spn-E function is abrogated, whereas Spn-E may be aberrantly activated by loss of Bel, in turn leading to transgene silencing. Whether piRNAs generated from the Spn-E-mediated ping-pong cycle are involved in transgene silencing is currently unknown. However, a recent study has shown that piRNAs can be generated directly from the transposon-derived transgene insertion area located in the euchromatic genome, which have been proposed to be involved in transgene silencing. This finding links piRNAs to transgene silencing. Preliminary small RNA deep sequencing analysis showed that the viable trans-heterozygous bel74407/neo30 mutant ovaries, which manifest a partial transgene silencing phenotype, displayed no significant defect in overall piRNA biogenesis from piRNA clusters and the ping-pong cycle, indicating that unlike Vasa, Bel is not involved in regulating piRNA generation. This finding is consistent with the result that homozygous bel mutants had no defect in nuage protein localization, which is different from other piRNA-related nuage proteins whose defects can significantly disturb the localization of other nuage protein components. Nevertheless, deep sequencing analysis also identified de novo piRNAs generated in bel74407/neo30 mutant ovaries (but not in wild-type ovaries) that could be mapped to the integrated P-element-derived transgene sequence area (P[LacW]). This result is in line with previous findings and indicates that the de novo generation of piRNAs from the inserted transgene region in the genome occurs under the bel mutant background. Given that Bel is a paralog of Vasa and the current genetic findings also suggest a competitive relationship between Bel and Spn-E, loss of Bel may disrupt its normal regulation of some small RNA-related helicases and co-factors, which in turn aberrantly activates the small RNA pathway(s). Therefore, it is possible that loss of Bel may promote de novo piRNA biogenesis from the transgene insertion sites by freeing these small RNA regulators and provoking the activation of their related small RNA pathway(s), which is one possible mechanism leading to transgene silencing. Since it was not possible create viable mutant progeny bearing mutations at both bel and spn-E gene loci, it is still uncertain whether Spn-E is involved in regulating de novo piRNA biogenesis from transgene insertion sites. Although it was not possible to elucidate the detailed mechanism due to technical hurdles, exploring the regulatory roles of Bel and Spn-E in this new type of piRNA biogenesis will be key, interesting research for understanding molecular mechanisms underlying transgene silencing (Lo, 2016).

Another unexpected finding is that the spn-E mutant rescue of transgene silencing associated with loss of bel also occurs in somatic follicle cells. This indicates that in addition to its well-known function in germline cells, Spn-E may have a somatic function. As Spn-E is not implicated in somatic piRNA biogenesis (e.g., flamenco), it is uncertain how Spn-E mediates somatic transgene silencing and whether piRNAs participate in this event. Therefore, it will be important in the future to unravel whether the mechanism by which Spn-E facilitates transgene silencing in bel mutant somatic cells is the same as that in germline cells (Lo, 2016).

The common feature for transgenes silenced by Bel abrogation is their P-element-based integration into genomic DNA. The observation that some P-element-based transgenes were not silenced in bel mutant cells raises an interesting question about what factors can determine whether a transgene can be silenced or not. From genetic analysis of a series of transgenes, it was observed that six examined transgenes (ci-LacZ, dMyc-LacZ, dom-LacZ, C306-Gal4, ptc-Gal4, Tj-Gal4), which were generated by the insertion of two different P-element-derived vector sequences (P[LacW] and P[GawB]), were silenced under the bel mutant background. In contrast, two transgenes (histone-GFP, histone-RFP) generated by the insertion of P[His2Av]-derived vector sequences were not silenced by Bel inactivation. Although these data are not a conclusive result, they imply that the inserted transgene sequence itself, not the insertion location in the genome, may be a critical determinant for Bel-dependent transgene silencing since this silencing phenotype seems to be transgene-specific and a change in the transgene insertion location in the genome has no influence on whether this transgene can be silenced or not when Bel function is inhibited. It is possible that the transgene sequence determines a local chromosomal conformation and whether the transgene can be silenced under the bel mutant background is determined by whether its chromosomal structure can be recognized by epigenetic regulators involved in transgene silencing. Although further investigations are still needed to verify this hypothesis due to limited cases in this study, it raises the possibility that epigenetic regulation at the chromatin level may be involved in Bel-dependent transgene silencing. Indeed, besides Spn-E, the genetic study identified Mod(mdg4) as another crucial factor required for transgene silencing in both germline and somatic cells when Bel is abrogated. The mod(mdg4) gene encodes multiple nuclear factors through trans-splicing and this protein family is functionally involved in the modification of the properties of insulators, which are genomic elements that regulate gene expression. Mod(mdg4) proteins can function as chromatin modulators engaged in the organization of highly ordered chromatin domains. The involvement of Mod(mdg4) in transgene silencing suggests that nuclear epigenetic events are also crucial for induction of transgene silencing when Bel is inactivated. However, the role of Mod(mdg4) in transgene silencing might also be indirect, such as through its regulation of other genes that could contribute to silencing. Nevertheless, the findings suggest that the co-ordination between nuclear and cytoplasmic events mediated by Mod(mdg4) and Spn-E, respectively, is mandatory for induction of transgene silencing when Bel is functionally inhibited (Lo, 2016).

The miRNA biogenesis enzyme Dcr-1 is the third factor identified from these studies that is crucial for transgene silencing in the absence of Bel. Interestingly, the block in transgene silencing in this case (double mutant for bel and Dcr-1) only occurred in germline cells, but not in somatic cells. This discovery raises the possibility that there are additional miRNA-targeted proteins present in germline cells, but not in somatic cells, and they can interact with factors essential for germline transgene silencing. If this is a case, aberrantly elevated levels of these miRNA-targeted proteins in Dcr-1 mutant germline cells might interfere with transgene silencing. Besides this possible indirect role, another possibility is that the Dcr-1-dependent miRNA pathway may play a direct role in transgene silencing in germline cells as Bel is abrogated. The miRNA pathway has been shown to be implicated in transgene silencing in Drosophila S2 cells. Although the silencing mechanism is unclear, this finding raises a possible direct role of the Dcr-1-dependent miRNA pathway in bel-mutant transgene silencing. A study from Zhou has shown that Bel proteins were cofractionated with the miRNA-dependent RNA-induced silencing complexes (miRISCs) and co-immunoprecipitated with Ago1, the protein component of miRISCs. This finding suggests a compelling possibility that Bel may be directly involved in the miRNA pathway to regulate miRISC-dependent RNA silencing and Bel inactivation may result in the aberrant functionality of miRISC and its related RNA silencing. Since miRNAs can target mRNAs via their short seed sequences, a possibility which cannot be ruled out is that some miRNAs may directly target transgene RNAs to regulate their levels. Future investigation is needed to reveal which possibility is more relevant (Lo, 2016).

In conclusion, these findings provide novel insights into the regulatory role of Bel in the expression of transgenes in Drosophila and its functional linkage to crucial factors implicated in RNA processing, chromatin remodeling and miRNA biogenesis. These findings further advance understanding of the complex cellular functions of Bel. These studies of the role for Bel in transgene expression may have important, future implications for understanding the regulation of expression of newly invaded or transposed TEs and virus-retrotransposon DNA chimeras generated from viral infection as they may share the similar scenario as transgene integration (Lo, 2016).

EVOLUTIONARY HOMOLOGS

Drosophila helicases

Vasa protein is essential for the assembly of the pole plasm, a special cytoplasm found in the posterior portion of the egg and early embryo. Vasa is an RNA binding protein with an RNA dependent helicase. Vasa has been associated with two developmental processes. The first involves assembling the perinuclear region of the oocyte. Perinuclear cytoplasm is the precursor of the pole plasm. The helicase function of Vasa is required later, in a second process, for the assembly of pole plasm. Posterior localization of Vasa depends most likely on an interaction with Oskar. Oskar can successfully localize to the posterior pole without Vasa, but Oskar by itself cannot assemble the pole plasm. Both Oskar and Vasa activity are necessary for Nanos mRNA localization at the posterior pole (Liang, 1994). The function of Vasa is to overcome the repressive effect of the Nanos translational control element, an evolutionarily conserved dual stem-loop structure in the 3' untranslated region which acts independently of the localization signal to repress translation of Nanos mRNA (Gavis, 1996).

In Drosophila melanogaster, position-effect variegation of the white gene has provided a useful phenomenon by which to study chromosome structure and the genes that modify it. A new enhancer of variegation locus, Helicase at 25E, has been discovered in Drosophila. Deletion of the mutation Helicase at 25E enhances white variegation; this can be reversed by a transformed copy of Helicase at 25E+. In the presence of two endogenous copies, the transformed Helicase at 25E+ behaves as a suppressor of variegation. Helicase at 25E is an essential gene and functions both maternally and zygotically. The Helicase at 25E protein is similar to known RNA helicases, but contains an unusual variant (DECD) of the DEAD motif common to these proteins. Potential Helicase at 25E homologs have been found in mammals, yeast and worms. Helicase at 25E protein associates with salivary gland chromosomes and locates to nuclei of embryos and ovaries, but disappears in mitotic domains of embryos as chromosomes condense. It is proposed that the Helicase at 25E protein promotes an open chromatin structure that favors transcription during development by regulating the spread of heterochromatin, and that Helicase at 25E is regulated by, and may have a role in, the mitotic cell cycle during embryogenesis (Eberl, 1997).

Yeast helicases: PRP2, PRP8 and PRP22

In addition to small nuclear RNAs and spliceosomal proteins, ATP hydrolysis is needed for nuclear pre-mRNA splicing. A number of RNA-dependent ATPases that are involved in several distinct ATP-dependent steps in splicing have been identified in Saccharomyces cerevisiae and mammals. These so-called DEAD/H ATPases contain conserved RNA helicase motifs, although RNA unwinding activity has not been demonstrated in purified proteins. One such DEAH protein, PRP2 of S. cerevisiae plays a role in spliceosome activation. PRP2 binds to a precatalytic spliceosome prior to the first step of splicing. By blocking the activity of HP, a novel splicing factor(s) that is involved in a post-PRP2 step, it was found that PRP2 hydrolyzes ATP to cause a change in the spliceosome without the occurrence of splicing. The change is quite dramatic and could account for the previously reported differences between the precatalytic, pre-mRNA-containing spliceosome and the "active," intermediate-containing spliceosome. The post-PRP2-ATP spliceosome was further isolated and found able to carry out the subsequent reaction apparently in the absence of PRP2 and ATP. It is hypothesized that PRP2 functions as a molecular motor, similar to some DExH ATPases in transcription, in the activation of the precatalytic spliceosome for the transesterification reaction (Kim, 1996).

The RNA helicase-like splicing factor PRP2 interacts only transiently with spliceosomes. To facilitate analysis of PRP2 interactions with spliceosomal components, PRP2 protein was stalled in splicing complexes using two different methods. A dominant negative mutant form of PRP2 protein, which associates stably with spliceosomes, was found to interact directly with pre-mRNAs, as demonstrated by UV-crosslinking experiments. The use of various mutant and truncated pre-mRNAs revealed that this interaction requires a spliceable pre-mRNA and an assembled spliceosome; a 3' splice site is not required. To extend these observations to the wild-type PRP2 protein, spliceosomes were depleted of ATP; PRP2 protein interacts with pre-mRNA in these spliceosomes in an ATP-independent fashion. Comparison of RNA binding by PRP2 protein in the presence of ATP or gamma S-ATP shows that ATP hydrolysis rather than mere ATP binding is required to release PRP2 protein from pre-mRNA. Since PRP2 is an RNA-stimulated ATPase, these experiments strongly suggest that the pre-mRNA is the native co-factor stimulating ATP hydrolysis by PRP2 protein in spliceosomes. Since PRP2 is a putative RNA helicase, it is proposed that the pre-mRNA is the target of RNA displacement activity of PRP2 protein, promoting the first step of splicing (Teigelkamp, 1994).

To characterize sequences in the RNA helicase-like PRP2 protein of Saccharomyces cerevisiae that are essential for its function in pre-mRNA splicing, a pool of random PRP2 mutants was generated. A dominant negative allele was isolated which, when overexpressed in a wild-type yeast strain, inhibits cell growth by causing a defect in pre-mRNA splicing. This defect is partially alleviated by simultaneous co-overexpression of wild-type PRP2. The dominant negative PRP2 protein inhibits splicing in vitro and causes the accumulation of stalled splicing complexes. Immunoprecipitation with anti-PRP2 antibodies confirms that dominant negative PRP2 protein competes with its wild-type counterpart for interaction with spliceosomes, with which the mutant protein remains associated. The PRP2-dn1 mutation leads to a single amino acid change within the conserved SAT motif that in the prototype helicase eIF-4A is required for RNA unwinding. Purified dominant negative PRP2 protein has approximately 40% of the wild-type level of RNA-stimulated ATPase activity. Because ATPase activity is reduced only slightly, but splicing activity is abolished, it is proposed that the dominant negative phenotype is due primarily to a defect in the putative RNA helicase activity of PRP2 protein (Plumpton, 1994).

Five small nuclear RNAs (snRNAs) are required for nuclear pre-messenger RNA splicing: U1, U2, U4, U5 and U6. The yeast U1 and U2 snRNAs base-pair (respectively) to the 5' splice site and branch-point sequences of introns. The role of the U5 and U4/U6 small nuclear ribonucleoprotein particles (snRNPs) in splicing is not clear, though a catalytic role for the U6 snRNA has been proposed. Less is known about yeast splicing factors, but the availability of genetic techniques in Saccharomyces cerevisiae has led to the identification of mutants deficient in nuclear pre-mRNA splicing (prp2-prp27). Several PRP genes have now been cloned and their protein products characterized. The PRP8 protein is a component of the U5 snRNP and associates with the U4/U6 snRNAs/snRNP to form a multi-snRNP particle believed to be important for spliceosome assembly. Extragenic suppressors of the prp8-1 mutation of S. cerevisiae have been isolated. This study presents the preliminary characterization of one of these suppressors, spp81 is presented. The predicted amino-acid sequence of the SPP81 protein shows extensive similarity to a recently identified family of proteins thought to possess ATP-dependent RNA helicase activity. The possible role of this putative helicase in nuclear pre-mRNA splicing is discussed (Jamieson, 1991).

The product of the yeast PRP22 gene acts late in the splicing of yeast pre-messenger RNA, mediating the release of the spliced mRNA from the spliceosome. The predicted PRP22 protein sequence shares extensive homology with that of PRP2 and PRP16 proteins, which are also involved in nuclear pre-mRNA splicing. The homologous region contains sequence elements characteristic of several demonstrated or putative ATP-dependent RNA helicases. A putative RNA-binding motif originally identified in bacterial ribosomal protein S1 and Escherichia coli polynucleotide phosphorylase has also been found in PRP22 (Company, 1991).

Yeast helicases: Other DEAD-box helicases

The translation initiation factor eIF4E mediates the binding of the small ribosomal subunit to the cap structure at the 5' end of the mRNA. In Saccharomyces cerevisiae, the cap-binding protein eIF4E is mainly associated with eIF4G, forming the cap-binding complex eIF4F. Other proteins are detected upon purification of the complex on cap-affinity columns. Among them is p20, a protein of unknown function encoded by the CAF20 gene. p20 has a negative regulatory role in translational initiation. Deletion of CAF20 partially suppresses mutations in translation initiation factors. Overexpression of the p20 protein results in a synthetic enhancement of translation mutation phenotypes. Similar effects are observed for mutations in the DED1 gene, which has been isolated as a multicopy suppressor of a temperature-sensitive eIF4E mutation. The DED1 gene encodes a putative RNA helicase of the DEAD-box family. The analyses of its suppressor activity, of polysome profiles of ded1 mutant strains, and of synthetic lethal interactions with different translation mutants all indicate that the Ded1 protein has a role in translation initiation in S. cerevisiae (de la Cruz, 1997).

The DED1 gene, which encodes a putative RNA helicase, has been implicated in nuclear pre-messenger RNA splicing in the yeast Saccharomyces cerevisiae. Translation, rather than splicing, is severely impaired in two newly isolated ded1 conditional mutants. Preliminary evidence suggests that the protein Ded1p may be required for the initiation step of translation, as is the distinct DEAD-box protein, eukaryotic initiation factor 4A (eIF4A). The DED1 gene could be functionally replaced by a mouse homolog, PL10, which suggests that the function of Ded1p in translation is evolutionarily conserved (Chuang, 1997).

In Saccharomyces cerevisiae, ribosomal biogenesis takes place primarily in the nucleolus, in which a single 35S precursor rRNA (pre-rRNA) is first transcribed and sequentially processed into 25S, 5.8S, and 18S mature rRNAs, leading to the formation of the 40S and 60S ribosomal subunits. Although many components involved in this process have been identified, an understanding of this important cellular process remains limited. One of the evolutionarily conserved DEAD-box protein genes in yeast, DBP3, is required for optimal ribosomal biogenesis. DBP3 encodes a putative RNA helicase, Dbp3p (523 amino acids in length), which bears a highly charged amino terminus consisting of 10 tandem lysine-lysine-X (KKX) repeats. Disruption of DBP3 is not lethal but yields a slow-growth phenotype. This genetic depletion of Dbp3p results in a deficiency of 60S ribosomal subunits and a delayed synthesis of the mature 25S rRNA, which is caused by a prominent kinetic delay in pre-rRNA processing at site A3 and to a lesser extent at sites A2 and A0. These data suggest that Dbp3p may directly or indirectly facilitate RNase MRP cleavage at site A3. The direct involvement of Dbp3p in ribosomal biogenesis is supported by the finding that Dbp3p is localized predominantly in the nucleolus. The (KKX) repeats are dispensable for Dbp3p's function in ribosomal biogenesis but are required for its proper localization. The (KKX) repeats thus represent a novel signaling motif for nuclear localization and/or retention (Weaver, 1997).

The phylogenetically conserved U14 small nucleolar RNA is required for the processing of rRNA, and this function involves base pairing with conserved complementary sequences in 18S RNA. With a view to identifying other important U14 interactions, a stem-loop domain required for activity of Saccharomyces cerevisiae U14 RNAs (the Y domain) was first subjected to detailed mutational analysis. The mapping results show that most nucleotides of the Y domain can be replaced without affecting function, except for loop nucleotides conserved among five different yeast species. Defective variants were then used to identify both intragenic and extragenic suppressor mutations. All of the intragenic mutations map within six nucleotides of the primary mutation, suggesting that suppression involves a change in conformation and that the loop element is involved in an essential intermolecular interaction rather than intramolecular base pairing. A high-copy extragenic suppressor gene, designated DBP4 (DEAD box protein 4), encodes an essential, putative RNA helicase of the DEAD-DEXH box family. Suppression by DBP4 restores the level of 18S rRNA and is specific for the Y domain but is not allele specific. DBP4 is predicted to function either in the assembly of the U14 small nucleolar RNP or, more likely, in its interaction with other components of the rRNA processing apparatus. Mediating the interaction of U14 with precursor 18S RNA is an especially attractive possibility (Liang, 1997).

A new gene of S. cerevisiae, RRP3 (rRNA processing) is required for pre-rRNA processing. Rrp3 is a 60.9 kDa protein that is required for maturation of the 35S primary transcript of pre-rRNA and is required for cleavages leading to mature 18S RNA. RRP3 was identified in a PCR screen for DEAD box genes. DEAD box genes are part of a large family of proteins homologous to the eukaryotic transcription factor elF-4a. Most of these proteins are RNA-dependent ATPases and some of them have RNA helicase activity. This is the third yeast DEAD box protein that has been shown to be involved in rRNA assembly, but the only one required for the processing of 18S RNA. Mutants of the two other putative helicases, Spb4 and Drsl, both show processing defects in 25S rRNA maturation. In strains where Rrp3 is depleted, 35S precursor RNA is improperly processed. Rrp3 has been purified to homogeneity and has a weak RNA-dependent ATPase activity that is not specific for rRNA (O'Day, 1996).

Viral and Vertebrate helicases

Three distinct nucleic acid-dependent ATPases are packaged within infectious vaccinia virus particles; one of these enzymes (nucleoside triphosphate phosphohydrolase II or NPH-II) is activated by single-stranded RNA. Purified NPH-II is now shown to be an NTP-dependent RNA helicase. RNA unwinding requires a divalent cation and any one of the eight common ribo- or deoxyribonucleoside triphosphates. The enzyme acts catalytically to displace an estimated 10-fold molar excess of duplex RNA under in vitro reaction conditions. NPH-II binds to single-stranded RNA. Turnover of the bound enzyme is stimulated by and coupled to hydrolysis of NTP. Photocrosslinking of radiolabeled RNA to NPH-II results in label transfer to a single 73-kDa polypeptide. The sedimentation properties of the helicase are consistent with NPH-II being a monomer. Immunoblotting experiments identify NPH-II as the product of the vaccinia virus I8 gene. The I8-encoded protein displays extensive sequence similarity to members of the DE-H family of RNA-dependent NTPases. Mutations in the NPH-II gene define the vaccinia helicase as essential for virus replication in vivo. Encapsidation of NPH-II in the virus particle suggests a role for the enzyme in synthesis of early messenger RNAs by the virion-associated transcription machinery (Shuman, 1992).

A human RNA helicase gene, DBP1, was cloned by PCR methods using degenerate oligonucleotide primers corresponding to highly conserved motifs among known members of the DEAH-box protein family. The full-length DBP1 contains 3028 nucleotides and codes for a protein of 813 amino acids with a calculated mol. wt. of 92723 daltons. The predicted amino acid sequence shares extensive homology with Prp2, Prp16, and Prp22 proteins, which are required to splice mRNA precursors in budding yeast. The protein encoded by DBP1 has RGD, RD, and HS(A/T) repeat motifs close to the N-terminus. Southern blot analysis suggests the presence of a homolog of the DBP1 genes in other species, and Northern blot analysis shows that DBP1 is expressed ubiquitously in the various human organs investigated. The DBP1 gene is found to be on chromosome 4p15.3 and encodes a putative nuclear ATP-dependent RNA helicase (Imamura, 1997).

To identify human homologs of the yeast helicase family, PCR primers were constructed that correspond to the highly conserved region of the DEAH box protein family; five cDNA fragments were successfully amplified, using HeLa poly(A)+ RNA as a substrate. One fragment, designated HRH1 (human RNA helicase 1), is highly homologous to Prp22, which is involved in the release of spliced mRNAs from the spliceosomes. Expression of HRH1 in an S. cerevisiae prp22 mutant can partially rescue its temperature-sensitive phenotype. These results strongly suggest that HRH1 is a functional human homolog of the yeast Prp22 protein. Interestingly, it is HRH1 but not Prp22 that contains an arginine- and serine-rich domain (RS domain), characteristic of some splicing factors such as members of the SR protein family. HRH1 can interact in vitro and in the yeast two-hybrid system with members of the SR protein family through its RS domain. It is speculated that HRH1 might be targeted to the spliceosome through this interaction (Ono, 1994).

The prototypic DEAD/DExH family member eIF-4A, has RNA-dependent NTPase activity as a monomer but functions as a helicase when dimerized with eIF-4B, which binds RNA through an RNP RNA-binding domain. It is currently thought that most DEAD/DExH proteins either require accessory proteins to function or else recognize a specific sequence in an unknown RNA substrate. One RNA binding domain, termed the double-stranded RNA-binding domain (dsRBD), was first identified as a 70 residue repeat motif in three proteins: dsRNA-dependent (DAI) protein kinase, Xenopus RNA-binding protein xlrbpa and Drosophila maternal effect protein Staufen. Searches with dsRNA-binding domain profiles detect two copies of the domain in each of RNA helicase A, Drosophila Maleless and C. elegans ORF T20G5-11 (of unknown function). RNA helicase A is unusual in being one of the few characterised DEAD/DExH helicases that are active as monomers. Other monomeric DEAD/DExH RNA helicases (p68, NPH-II) have domains that match another RNA-binding motif, the RGG repeat. The DEAD/DExH domain appears to be insufficient on its own to promote helicase activity and additional RNA-binding capacity must be supplied either as domains adjacent to the DEAD/DExH-box or by bound partners as in the eIF-4AB dimer. The presence or absence of extra RNA-binding domains should allow classification of DEAD/DExH proteins as monomeric or multimeric helicases (Gibson, 1994).

Translation initiation factor elF-4B is an RNA-binding protein that promotes the association of the mRNA to the eukaryotic 40S ribosomal subunit. One of its better characterized features is the ability to stimulate the activity of the DEAD box RNA helicase elF-4A. In addition to an RNA recognition motif (RRM) located near its amino-terminus, elF-4B contains an RNA-binding region in its carboxy-terminal half. The elF-4A helicase stimulatory activity resides in the carboxy-terminal half of elF-4B, and the RRM has little impact on this function. To better understand the role of the elF-4B RRM, attempts were made to identify its specific RNA target sequence. To this end, in vitro RNA selection/amplifications were performed using various portions of elF-4B. These experiments were designed to test the RNA recognition specificity of the two elF-4B regions implicated in RNA binding and to assess the influence of elF-4A on the RNA-binding specificity. The RRM was shown to bind with high affinity to an RNA stem-loop structure with conserved primary sequence elements. Discrete point mutations in an in vitro-selected RNA identified residues critical for RNA binding. Neither the carboxy-terminal RNA-interaction region, nor elF-4A, influence the structure of the high-affinity RNA ligands selected by elF-4B, and elF-4A by itself does not select any specific RNA target. Previous studies have demonstrated an interaction of elF-4B with ribosomes, and it has been suggested that this association is mediated through binding to ribosomal RNA. The RRM of elF-4B interacts directly with 18S rRNA and this interaction is inhibited by an excess of the elF-4B in vitro-selected RNA. ElF-4B can bind simultaneously to two different RNA molecules, supporting a model whereby elF-4B promotes ribosome binding to the 5' untranslated region of an mRNA by bridging it to 18S rRNA (Methot, 1996).

Nuclear DNA helicase II (NDH II) unwinds both DNA and RNA. NDHII cDNA is 4,528 bases in length, which corresponds well with a 4.5-4.7-kilobase-long mRNA as detected by Northern blot analysis. The open reading frame of NDH II cDNA predicts a polypeptide of 1287 amino acids and a calculated molecular mass of 141,854 daltons. NDH II is related to a group of nucleic acid helicases from the DEAD/H box family II, with the signature motif DEIH in domain II. Two further proteins of this family, i.e. human RNA helicase A and Drosophila Maleless (Mle) protein, are found to be highly homologous to NDH II. With RNA helicase A, there is 91.5% identity and 95.5% similarity between the amino acid residues; with Mle protein, a 50% identity and an 85% similarity is observed. Antibodies against human RNA helicase A cross-react with NDH II, further supporting evidence that NDH II is the bovine homolog of human RNA helicase A. Immunofluorescence studies reveal a mainly nuclear localization of NDH II. A role for NDH II in nuclear DNA and RNA metabolism is suggested (Zhang, 1995).

Human p68 RNA helicase is a nuclear RNA-dependent ATPase that belongs to a family of putative helicases known as the DEAD box proteins. These proteins have been implicated in aspects of RNA function including translation initiation, splicing, and ribosome assembly in a variety of organisms ranging from Escherichia coli to humans. While members of this family are believed to function in the manipulation of RNA secondary structure, little is known about the regulation of these enzymes. p68 possesses a region of sequence similarity to the conserved protein kinase C phosphorylation site and calmodulin binding domain (also known as the IQ domain) of the neural-specific proteins neuromodulin (GAP-43) and neurogranin (RC3). p68 is phosphorylated by protein kinase C in vitro and binds calmodulin in a Ca(2+)-dependent manner. Both phosphorylation and calmodulin binding inhibit p68 ATPase activity, suggesting that the RNA unwinding activity of p68 may be regulated by dual Ca2+ signal transduction pathways through its IQ domain (Buelt, 1994).

RNA helicase A is an abundant nuclear enzyme found in HeLa cells that unwinds double-stranded RNA in a 3' to 5' direction. A complementary DNA (cDNA) clone expressing RNA helicase A was isolated by screening a human cDNA library with polyclonal antibodies produced against the purified protein. The deduced amino acid sequence from this clone shows that RNA helicase A is a member of the DEAH family of proteins and thought to be helicases. Sequence comparison among all known proteins of the DEAH family reveals that the highest homology is between RNA helicase A and the Maleless protein (Mle) of Drosophila. There is 49% identity and 85% similarity throughout the overall primary sequences of both proteins, suggesting that RNA helicase A is the human counterpart of Drosophila Mle. Polyclonal antibodies against Drosophila Mle recognize RNA helicase A in crude nuclear extracts of HeLa cells as well as the purified protein. A recombinant RNA helicase A containing 6 histidine residues at the NH2 terminus was expressed in Sf9 cells using a baculovirus vector. The protein isolated from insect cells and the enzyme purified from HeLa cells both exhibit identical RNA helicase and RNA-dependent ATPase activities (Lee, 1993).

The coactivator CBP has been proposed to stimulate the expression of certain signal-dependent genes via its association with RNA polymerase II complexes. Complex formation between CBP and RNA polymerase II requires RNA helicase A (RHA), a nuclear DNA/RNA helicase that is related to the Drosophila male dosage compensation factor Mle. In transient transfection assays, RHA is found to cooperate with CBP in mediating target gene activation via the CAMP responsive factor CREB (see Drosophila dCREB2). Since a mutation in RHA that compromises its helicase activity correspondingly reduces CREB-dependent transcription, it is proposed that RHA may induce local changes in chromatin structure that promote engagement of the transcriptional apparatus on signal responsive promoters. The involvement of a DNA helicase such as RHA in signal-dependent transcription is intriguing because it suggests that recruitment of CBP complexes may promote local unwinding of promoter DNA via RHA and thereby permit engagement of the transcriptional apparatus (Nakajima, 1997).

A rat cDNA of 117.4 kDa contains RNA helicase consensus motifs, among them a "DEAD" box, has been called HEL117 (for helicase of 117.4 kDa). In addition to the helicase consensus motifs, HEL117 contains an arginine-serine (RS)-rich domain, which occurs in some proteins involved in RNA splicing. The COOH-terminal region of 78 residues of HEL117 is 38.5% identical and 59% similar to the COOH-terminal region of a yeast PRP5 protein that is involved in RNA splicing. Rabbit antibodies identify a single polypeptide in rat cells, in the cells of other mammals, and in the chicken. The antibodies reveal a finely punctate and speckled intranuclear staining in immunofluorescence microscopy. A monoclonal antibody against a human splicing factor containing an RS domain (SC35) shows (in double immunofluorescence microscopy) largely overlapping staining consistent with HEL117 being involved in RNA splicing (Sukegawa, 1995).

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