spindle E/homeless

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).

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 or Deletion

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).

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).

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).

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date revised: 10 April 2008

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