To examine the distribution of endogenous Armi protein during oogenesis, polyclonal antibodies were generated against a C-terminal peptide and N-terminal fusion protein. On Western blots, the affinity-purified antibodies react with a polypeptide of the molecular weight expected for Armi, and both antibodies strongly label germline structures during oogenesis that are absent in armi mutants. These antibodies also produce diffuse labeling in the somatic follicle cells in wild-type and armi mutant egg chambers. This may be nonspecific labeling, because whole-mount in situ hybridization shows that armi transcripts are restricted to the germline and clonal analysis indicates that armi function is required in the germline for female fertility and axial patterning. The GFP-Armi fusion protein is incorporated into similar germline structures in living ovarioles, confirming that the antibody labeling is specific. Armi protein is first apparent early in oogenesis, in the cytoplasm of stem cells and mitotically dividing cystoblasts. In regions 2a and 2b of the germarium, Armi protein is most concentrated in the center of the germline cysts, where the pro-oocyte is located. In stage 1 and early stage 2 egg chambers, Armi accumulates at the anterior of the oocyte, near the ring canals. Armi also extends through the ring canals forming a branched structure that links the early oocyte with adjacent nurse cells. In stage 3 cysts, Armi accumulates at the posterior cortex and localizes to extensions that pass through the oocyte into the nurse cells (Cook, 2004).
The distribution of Armi in the germarium is reminiscent of the fusome, a vesiculated structure rich in membrane skeletal proteins that plays a critical role in orienting the cystoblast divisions. Double immunolabeling for Armi and the fusome marker Adducin demonstrate that the branched structure formed by Armi assembles as the fusome degenerates. During early oogenesis, Armi protein also accumulates in punctate structures around the nurse cell nuclei. This distribution is characteristic of nuage, amorphous perinuclear material implicated in RNA processing and transport. Through stages 4 to 7, Armi continues to be somewhat enriched at the posterior cortex of the oocyte, but at significantly lower levels. In stage 9 to 10 egg chambers, Armi is found throughout the cytoplasm of the oocyte and nurse cells, with slight enrichment at the oocyte cortex (Cook, 2004).
Armi protein and osk mRNA both accumulate in the oocyte during early oogenesis, when armi mutations lead to premature Osk protein expression. To define the spatial relationship between Armi protein and osk mRNA, wild-type egg chambers were immunostained for Armi and labeled for osk mRNA by FISH. In the germarium, osk mRNA and Armi protein are concentrated in similar regions, but the distributions do not precisely overlap. During stages 3 through 6, Armi protein and osk transcripts are concentrated near the posterior cortex of the oocyte in close proximity, but they assemble into distinct structures. These observations indicate that most of the osk mRNA in the oocyte is not physically associated with Armi protein (Cook, 2004).
The branched structure formed by Armi in early egg chambers also resembles the polarized microtubule cytoskeleton that directs asymmetric mRNA localization. Immunolabeling of wild-type ovaries for Armi and α-tubulin demonstrated that Armi protein is closely associated with the microtubule network in early egg chambers. Moreover, disruption of the microtubule network with colchicine disrupts the branched Armi network. However, Armi does not precisely colocalize with microtubules. In stage 1 egg chambers, for example, Armi accumulates near the MTOC and along microtubules that extend from the MTOC and pass through the ring canals. In single confocal optical sections, the most intense Armi labeling does not overlap with the MTOC or microtubule bundles, which appear to exclude Armi. Thus, most of the Armi protein does not directly associate with microtubules. Nonetheless, microtubules are required to establish the asymmetric distribution of Armi during early oogenesis (Cook, 2004).
To identify new genes involved in embryonic axis specification, a collection of P element transposon-induced maternal effect lethal mutations was screened for impaired localization of the pole plasm component Vasa and for disruption of D/V patterning as indicated by defects in the dorsal appendages on the eggshell. This screen identified a new mutation that produces variable appendage defects and significantly reduces Vasa accumulation at the posterior pole. Molecular and genetic analyses have demonstrated that the mutation was not in a previously identified gene. The locus was named armitage after the navigator on Robert Falcon Scott's failed Discovery expedition to the South Pole (Cook, 2004).
Excision of the P element in the original mutant, armi1, produced new armi alleles and revertant chromosomes that presumably restore gene function through precise transposon excision. Of the forty lines generated, 21 (52%) fully complemented armi1 and were homozygous fertile with normal embryonic patterning. The remaining lines failed to complement armi1, indicating that imprecise P element excision had generated new armi mutations. Most of these mutations were homozygous viable and female sterile, and the mutant females produced eggs with intermediate to weak defects in D/V patterning, suggesting that they were partial loss-of-function alleles. However, armi72.1 appears to be a strong loss-of-function allele. Homozygous armi1 females produce eggs where 67% showed strong D/V patterning defects as indicated by a complete lack of dorsal appendages. By contrast, 92% of eggs deposited by armi72.1 females completely lacked dorsal appendages, and 35% of these were also collapsed. armi72.1 over the deficiency Df(3L)E1 shows a similar range of defects, indicating that armi72.1 is a strong loss-of-function mutation. However, both armi1 and armi72.1 ovaries produce low levels of transcript, suggesting that neither allele is functionally null (Cook, 2004).
To further characterize armi patterning defects, fluorescent in situ hybridization (FISH) was performed for the three asymmetrically localized mRNAs that specify the anterior, posterior, and dorsal regions of the oocyte. In wild-type stage 9 to 11 oocytes, bcd mRNA accumulates along the anterior cortex and osk mRNA localizes to the posterior pole. In armi mutants, bcd mRNA showed a wild-type anterior distribution. However, osk RNA was either dispersed throughout the ooplasm or concentrated within the oocyte interior in 85% of armi1 egg chambers and 90% (n = 69) of armi72.1 egg chambers. The remaining egg chambers displayed weak posterior localization. osk mRNA consistently showed normal posterior localization in armirev39.2 revertants. During stages 2 through 6, osk mRNA accumulates at the posterior of wild-type oocytes. In armi mutants, osk mRNA was transported to the oocyte during these early stages, but it did not accumulate at the posterior cortex (Cook, 2004).
During stages 2 to 7, grk mRNA and protein also accumulate at the oocyte posterior. During these stages, grk mRNA is below the level of detection by FISH. However, Grk protein was detectable by antibody staining. In armi72.1 oocytes, Grk is dispersed throughout the cytoplasm. During mid-oogenesis, grk mRNA and Grk protein accumulate at the dorsal-anterior corner of the oocyte. In armi mutants, grk mRNA was undetectable by FISH. However, using a colorimetric detection method, grk mRNA was seen to form a weak ring near the anterior cortex, and immunostaining showed that Grk protein was dispersed throughout the oocyte. These observations indicate that the armi gene is required for axial polarization of the oocyte during early and mid-oogenesis (Cook, 2004).
Based on the SDE3 homology, it was postulated that armi might function in RNA silencing during oogenesis. Both osk and bcd mRNAs are produced through most of oogenesis but remain translationally silent until midoogenesis or egg activation, respectively. To determine whether armi mutations lead to premature translation of these mRNAs, egg chambers were immunolabeled for Bcd and Osk proteins. Bcd was undetectable during oogenesis in both wild-type and armi mutants, indicating that translational repression of bcd does not require armi. Wild-type embryos were immunolabeled for Bicoid as a positive control for bcd translation. In wild-type egg chambers, Osk protein is not produced until osk mRNA localizes to the posterior pole in mid-oogenesis. However, in armi72.1 mutants, Osk was produced in early oocytes before transcript localization. Osk started to accumulate as soon as transcript was apparent by FISH. Osk was also prematurely expressed in armi1 and armi72.1 hemizygous oocytes, but was not apparent in early armirev39.2 revertant oocytes (Cook, 2004).
Late stage egg chambers make up most of the ovary, and Osk is highly expressed during these stages in wild-type oocytes. It was therefore not possible to biochemically measure increased Osk protein expression in armi mutants by Western blotting. However, identical labeling and imaging procedures were used with all samples, allowing direct cytological comparison of Osk protein labeling in wild-type and mutant ovaries. Comparable levels of osk mRNA, as judged by FISH and Northern blotting, are present in armi mutants and in wild-type. armi mutants thus do not appear to affect osk transcript stability, but disrupt osk mRNA translational silencing (Cook, 2004).
In animals, miRNAs represses translation without affecting transcript stability, most likely through direct base pairing with target transcripts. miRNAs require components of the RNAi pathway for their maturation and to assemble into the RISC. To determine if components of the RNAi pathway are required for osk mRNA silencing, ovaries mutant for spindle-E (spn-E), aubergine (aub), and maelstrom (mael) were assayed for osk mRNA localization and Osk protein expression. aub and spn-E encode components of the Drosophila RNAi system, and mael is required for localization of a subset of RNAi pathway components in early Drosophila egg chambers. Mutations in all three genes lead to premature Osk protein expression without dramatically affecting the level of osk mRNA. The amount of Osk protein in aubHN2/aubQC42 oocytes was lower than in armi, spn-E, and mael mutants; however, neither aub mutation is a null allele, and the level of Osk expression is consistently above the background staining observed in wild-type controls. Significantly, mutations in aub, spn-E, and mael produce defects in osk mRNA localization during early and mid-oogenesis, as well as defects in D/V patterning that are strikingly similar to those produced by mutations in armi. Therefore, multiple components of the Drosophila RNAi system are required for osk mRNA silencing and embryonic axis specification (Cook, 2004).
To determine if armi is required for axial polarization of the microtubule cytoskeleton, armi egg chambers were immunolabeled with anti-tubulin antibodies. armi mutations do not affect initial organization of microtubules in stage 1 oocytes. During stages 2 to 7, microtubules are significantly more abundant in the oocyte than in the nurse cells and are organized by the posterior cortex of the oocyte. In armi mutants, a posterior MTOC was not apparent and microtubule levels in the oocyte and nurse cells were comparable. Armi is therefore essential for polarization of the oocyte cytoskeleton during early oogenesis. Significantly, spn-E, mael, and aub are also required for microtubule reorganization during early oogenesis (Cook, 2004).
This early polarized microtubule network is required for Grk-dependent differentiation of the posterior follicle cells, which in turn is required for triggering loss of cortical microtubules at the posterior pole during mid-oogenesis. Consistent with the observed defects in microtubule organization and Grk protein localization in early armi oocytes, cortical microtubules persist at the posterior cortex of armi mutants in mid-oogenesis. Loss of these posterior microtubules is thought to be essential for osk mRNA localization. These observations indicate that the RNAi system is required for initial anterior-posterior polarization of the microtubule cytoskeleton, which in turn is required for oskmRNA localization and posterior patterning during midoogenesis (Cook, 2004).
Mutations in armi and the other RNAi components lead to premature expression of Osk protein, raising the possibility that Osk misexpression directly or indirectly triggers the defects in microtubule organization. To test this, microtubule organization was analyzed in ovaries overexpressing Osk protein from a transgene. Microtubule organization in these ovaries was indistinguishable from wild-type controls. Moreover, armi1 osk54 and armi72.1 osk54 double mutant ovaries showed microtubule defects that were cytologically identical to the parental armi mutation. Therefore, the defects in osk mRNA silencing and microtubule polarization are genetically distinct consequences, suggesting that the RNAi pathway silences osk mRNA and additional transcripts encoding cytoskeletal regulators (Cook, 2004).
The RNA helicase Armitage is required to repress oskar translation in Drosophila oocytes; armi mutant females are sterile and armi mutations disrupt anteroposterior and dorsoventral patterning. armi has been shown to be required for RNAi. armi mutant male germ cells fail to silence Stellate, a gene regulated endogenously by RNAi, and lysates from armi mutant ovaries are defective for RNAi in vitro. Native gel analysis of protein-siRNA complexes in wild-type and armi mutant ovary lysates suggests that armi mutants support early steps in the RNAi pathway but are defective in the production of active RNA-induced silencing complex (RISC), which mediates target RNA destruction in RNAi. These results suggest that armi is required for RISC maturation (Tomari, 2004).
Silencing of the X-linked Ste gene by the highly homologous Y-linked Su(Ste) locus is an example of endogenous RNAi. In Drosophila testes, symmetrical transcription of Su(Ste) produces dsRNA, which is processed into siRNAs (Gvozdev, 2003). Su(Ste) siRNAs direct the degradation of Ste mRNA. Inappropriate expression of Ste protein in testes is diagnostic of disruption of the RNAi pathway. Both the Argonaute protein, aub, and the putative DEAD-box helicase, spn-E, have been shown to be required for RNAi in Drosophila oocytes. Both mutants fail to silence Ste, as evidenced by the accumulation of Ste protein crystals in the testes of aub and spn-E mutants. No Ste protein is detected in wild-type testes. Strikingly, Ste protein accumulates in testes of two different armi alleles, armi1 and armi72.1. Neither allele is expected to be a true null because armi1 is caused by a P element insertion 5' to the open reading frame, whereas armi72.1, which was created by an imprecise excision of the armi1 P element, corresponds to a deletion of sequences in the 5' untranslated region. Ste silencing is re-established in males homozygous for the revertant chromosome, armirev 39.2 (henceforth, armirev; Cook, 2004), which was generated by excision of the armi1 P element. These data suggest a role for Armi in Drosophila RNAi (Tomari, 2004).
Immunofluorescent detection of Ste protein in testes implicates both armi alleles in endogenous RNAi, but provides only a qualitative measure of allele strength. Since Ste protein in males reduces their fertility , the percent of embryos that hatch when mutant males are mated to wild-type (Oregon R) females provides a more quantitative measure of Ste dysregulation. Hatch rates were measured for the offspring of wild-type, armi1, armi72.1, and spn-E1 homozygous males mated to Oregon R females. For spn-E1 males, 82% of the progeny hatched. Seventy-five percent of the progeny of armi1 males hatched, but only 45% for armi72.1. In contrast, 97% of the offspring of wild-type males hatched. Thus, armi72.1 is a stronger allele than armi1, at least with respect to the requirement for armi in testes (Tomari, 2004).
In contrast to wild-type, lysates prepared from armi72.1 ovaries do not support siRNA-directed target cleavage in vitro: no cleavage product was observed in the armi72.1 lysate after 2 hr. This result was observed for more than ten independently prepared lysates. To determine if the RNAi defect was allele specific, ovaries from armi1 were tested. Phenotypically, this allele is weaker than armi72.1 in its effects on both male fertility and oogenesis. For armi72.1 females, 92% of the eggs lacked dorsal appendages, compared to 67% for armi1 eggs, and some armi1 eggs had wild-type or partially fused dorsal appendages. Consistent with its weaker phenotype, the armi1 allele showed a small amount of RNAi activity in vitro. The two alleles were analyzed together at least four times using independently prepared lysates. In all assays, total protein concentration was adjusted to be equal. Lysate from the revertant allele, armirev, which has wild-type dorsal appendages, showed robust RNAi, demonstrating that the RNAi defect in the mutants is caused by mutation of armi, not an unlinked gene (Tomari, 2004).
The rate of target cleavage was much slower for armi1 than for wild-type . Since the rate of target cleavage in this assay usually reflects the concentration of RISC, it was hypothesized that armi mutants are defective in RISC assembly. To test this hypothesis, a method to measure RISC was developed that requires less lysate than previously described techniques. Double-stranded siRNA was incubated with ovary lysate in a standard RNAi reaction. To detect RISC, a 5' 32P-radiolabled, 2'-O-methyl oligonucleotide complementary to the antisense strand of the siRNA was added. Like target RNAs, 2'-O-methyl oligonucleotides can bind to RISC containing a complementary siRNA, but unlike RNA targets, they cannot be cleaved and binding is essentially irreversible (Hutvagner, 2004). RISC/2'-O-methyl oligonucleotide complexes were then resolved by electrophoresis through an agarose gel (Tomari, 2004).
To validate the method, RISC formation was examined in embryo lysate. Four distinct complexes (C1, C2, C3, C4) were formed when siRNA was added to the reaction. Formation of these complexes required ATP and was disrupted by pre-treatment of the lysate with the alkylating agent N-ethylmaleimide (NEM), but it was refractory to NEM treatment after RISC assembly; these are all properties of RNAi itself. No complex was observed with an siRNA unrelated to the 2'-O-methyl oligonucleotide. The amount of complex formed by different siRNA sequences correlated well with their capacity to mediate cleavage. The four complexes were also detected in wild-type ovary lysate, suggesting that the same RNAi machinery is used during oogenesis and early embryogenesis. The lower amount of RISC formed in ovary compared to embryo lysates can be explained by the lower overall protein concentration of ovary lysates (Tomari, 2004).
The 2'-O-methyl oligonucleotide/native gel assay was used to analyze RISC assembly in armi mutant ovary lysates. armi mutants are deficient in RISC assembly. The extent of the deficiency correlated with allele strength: less C3/C4 complex formed in lysate from the strong armi72.1 allele than from armi1. Compared to the phenotypically wild-type armirev, >10-fold less RISC was produced in armi72.1 (Tomari, 2004).
The defect in RISC assembly in armi mutants is similar to that observed in lysates from aubHN2 ovaries. aub mutants do not support RNAi following egg activation and fail to silence the Ste locus in testes, and lysates from aubHN2 ovaries do not support RNAi in vitro. Aub is one of five Drosophila Argonaute proteins, core constituents of RISC. It is therefore not surprising that Aub is required for RISC assembly. Since RISC assembly in vitro was not detectable in aubHN2 lysates, the data suggest that Aub is the primary Argonaute protein recruited to exogenous siRNA in Drosophila ovaries. In contrast, ovaries from nanosBN, a maternal effect mutant not implicated in RNAi, are fully competent for both RISC assembly and siRNA-directed target RNA cleavage (Tomari, 2004).
Two intermediates have been identified in RISC assembly. Complex B forms rapidly upon incubation of siRNA in lysate, in the absence of ATP. The siRNA is then transferred to complex A, which contains the R2D2/Dcr-2 heterodimer. The siRNA is double stranded in both B and A. RISC is formed from complex A by a process that requires both siRNA unwinding and ATP. armi mutants are defective for the conversion of complex A to RISC. RISC does not form in ovary lysates from armi or aub mutants. However, both complexes B and A are readily detected in armi and aub mutants. Thus, armi and aub mutants are impaired in a step in RISC assembly after binding of the siRNA to the Dcr-2/R2D2 heterodimer (Tomari, 2004).
Armi might act after the formation of complex A to unwind siRNA duplexes prior to their assembly into RISC. To test this hypothesis, whether single-stranded siRNA circumvents the requirement for armi was tested. In vitro and in vivo, single-stranded siRNA triggers RNAi, albeit inefficiently. armi ovary lysates failed to support RNAi when the reactions were programmed with 5'-phosphorylated, single-stranded siRNA. The defect with single-stranded siRNA correlates with allele strength: some activity was seen in lysates from the weak allele, armi1, but none for the strong allele, armi72.1. The requirement for a putative ATPase -- Armi -- in RNAi triggered by single-stranded siRNA suggested the presence of an additional ATP-dependent step in the RISC assembly, after siRNA unwinding (Tomari, 2004).
To test if loading of single-stranded siRNA into RISC requires ATP, 5'-phosphorylated, single-stranded siRNA was added to embryo lysates depleted of ATP. After incubation for 2 hr, no cleavage product was detected, suggesting that there is at least one ATP-dependent step downstream of siRNA unwinding. The stability of single-stranded siRNA was not reduced by ATP depletion. In fact, single-stranded siRNA was slightly more stable in the absence of ATP. Thus differential stability cannot account for the requirement for ATP in RNAi triggered by single-stranded siRNA. In the RNAi pathway, there are at least three steps after siRNA unwinding: RISC assembly, target recognition, and target cleavage. To assess if either target recognition or cleavage was ATP dependent, single-stranded siRNA was incubated in a standard RNAi reaction with ATP to assemble RISC. Next, NEM was added to inactivate the ATP-regenerating enzyme, creatine kinase, and to block further RISC assembly. NEM was quenched with dithiothreitol (DTT), and hexokinase and glucose added to deplete ATP. Finally, mRNA target was added and the reaction incubated for 2 hr. Using this protocol, high ATP levels were maintained during RISC assembly, but less than 100 nM ATP was present during the encounter of RISC with the target RNA. Target recognition and cleavage did not require ATP when RISC was programmed with either double- or single-stranded siRNA, provided that ATP was supplied during RISC assembly (Tomari, 2004).
Four distinct RISC-like complexes common to embryo and ovary lysates have been detected. In ovaries, formation of these complexes is reduced >10-fold in armi mutants and is undetectable in aub mutants, which are RNAi defective. The requirement for Aub, an Argonaute protein, suggests that the complexes correspond to distinct RISC isoforms built on a common core of Aub and siRNA. These isoforms may play distinct regulatory roles (e.g., translational repression versus cleavage). Alternatively, the smallest, most abundant complexes may contain only the most stably associated protein constituents, whereas the larger, less abundant complexes may correspond to 'holo-RISC' that retain more weakly bound proteins. Clearly, a major challenge for the future is to define the protein constituents of each complex, their functional capacity, and their biological role. The development of a native gel assay that resolves distinct RISC complexes represents a step toward that goal (Tomari, 2004).
This study has identified two intermediates in RISC assembly. Complex B forms rapidly upon incubation of siRNA in lysate, in the absence of ATP. The siRNA is then transferred to complex A, which contains the previously identified R2D2/Dcr-2 heterodimer. The siRNA is double stranded in both B and A. RISC is formed from complex A by a process that requires both siRNA unwinding and ATP. Both aub and armi are required genetically for the production of RISC from complex A. The involvement of Armi, a putative RNA helicase protein, in the production of RISC from complex A and the finding that incorporation of single-stranded siRNA into RISC requires ATP suggest that Armi functions to incorporate single-stranded siRNA into RISC. However, the data cannot distinguish between direct and indirect roles for Armi in RISC assembly (Tomari, 2004). The Arabidopsis homolog of Armi, SDE3, together with the RNA-dependent RNA polymerase (RdRP) SDE1/SGS2, is required for PTGS triggered by transgenes that express single-stranded sense mRNA, but not silencing triggered by some RNA viruses (Dalmay, 2001). SDE3 has been proposed to facilitate the conversion of dsRNA into siRNA or the conversion of mRNA into complementary RNA by SDE1/SGS2 (Dalmay, 2001; Jorgensen, 2003). Recent studies show that SDE3 is not required for the production of siRNAs derived directly from a long dsRNA hairpin (Himber, 2003). Instead, SDE3 seems to play a role in the production of siRNAs generated by an RdRP-dependent amplification mechanism. The data from the Tamari study (2004) are not consistent with either of these functions for Armi. (1) Drosophila genomic, biochemical, and genetic data exclude a role for an RdRP in RNAi. (2) Armi is required for RISC assembly in Drosophila ovary lysates when RISC is programmed with siRNA, suggesting a role for Armi downstream of the conversion of dsRNA into siRNA, but upstream of target recognition by RISC. The apparently divergent functions of SDE3 and Armi could be reconciled if RISC is required for RdRP-mediated amplification of silencing. Alternatively, SDE3 and Armi may not have homologous functions (Tomari, 2004).
armi mRNA is abundant in oocytes and syncitial blastoderm embryos, but a low level can be detected throughout development, including in somatic tissues (Cook, 2004). While the requirement for armi in spermatogenesis and oogenesis makes Armi a good candidate for a component of the RNAi machinery in germ cells and early embryos, somatic functions for Armi are also possible. In this respect, armi is reminiscent of the maternally expressed Argonaute protein, piwi, which is also required during oogenesis. Although piwi mutants display no obvious somatic phenotype, Piwi is required in the soma both for posttranscriptional transgene silencing and for some types of transcriptional silencing. Whether Armi is likewise required for somatic transgene silencing remains to be tested (Tomari, 2004).
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).
Transposons and other selfish DNA elements can be found in all phyla, and mobilization of these elements can compromise genome integrity. The piRNA (PIWI-interacting RNA) pathway silences transposons in the germline, but it is unclear if this pathway has additional functions during development. This study shows that mutations in the Drosophila piRNA pathway genes, armi, aub, ago3, and rhi, lead to extensive fragmentation of the zygotic genome during the cleavage stage of embryonic divisions. Additionally, aub and armi show defects in telomere resolution during meiosis and the cleavage divisions; and mutations in ligase-IV, which disrupt non-homologous end joining, suppress these fusions. By contrast, lig-IV mutations enhance chromosome fragmentation. Chromatin immunoprecipitation studies show that aub and armi mutations disrupt telomere binding of HOAP, which is a component of the telomere protection complex, and reduce expression of a subpopulation of 19- to 22-nt telomere-specific piRNAs. Mutations in rhi and ago3, by contrast, do not block HOAP binding or production of these piRNAs. These findings uncover genetically separable functions for the Drosophila piRNA pathway. The aub, armi, rhi, and ago3 genes silence transposons and maintain chromosome integrity during cleavage-stage embryonic divisions. However, the aub and armi genes have an additional function in assembly of the telomere protection complex (Khurana, 2010).
Drosophila piRNAs have been implicated in transposon silencing and maintenance of genome integrity during female germline development. However, piRNA pathway mutations lead to complex developmental phenotypes, and piRNAs have been implicated in control of gene expression. Furthermore, the majority of piRNAs in other systems, including mouse testes, are not derived from repeated elements. The full extent of piRNA functions thus remains to be explored (Khurana, 2010).
Mutations in the majority of Drosophila piRNA pathway genes disrupt asymmetric localization of RNAs along the axes of the oocyte, and lead to maternal effect embryonic lethality. The axis specification defects linked to several of piRNA pathway mutations are dramatically suppressed by a null mutation in mnk, which encodes a Checkpoint kinase 2 (Chk2) homolog required for DNA damage signaling, indicating that the loss of asymmetric RNA localization is downstream of DNA damage. Oocyte patterning defects generally lead to embryonic lethality, but the mnk allele that suppresses the axis specification defects associated with piRNA mutations does not suppress embryonic lethality. piRNAs thus have an essential function during embryogenesis that is independent of Chk2 activation and DNA damage signaling. To gain insight into potential new functions for the piRNA pathway, the embryonic lethality associated with four piRNA pathway mutations was characterized. These studies reveal a novel function for a subset of piRNA genes in assembly of the telomere protection complex, and suggest that this process is directed by a subpopulation of 19-22 nt piRNAs (Khurana, 2010).
As wild type oocytes mature and initiate meiotic spindle assembly, the major chromosomes form a single mass at the spindle equator and the non-exchange 4th chromosomes move toward the poles. In OregonR, distinct 4th chromosomes were observed in 79% of stage 13 oocytes. In stage 13 aub and armi mutants, by contrast, distinct 4th chromosomes were observed in only 11% and 18% of stage 13 oocytes, respectively. However, a single primary mass of chromatin was always observed. These observations are consistent with γ-H2Av data suggesting that DNA breaks formed during early oogenesis are often repaired as the oocyte matures. In addition, both aub and armi mutations appear to inhibit separation of the small 4th chromosomes, although it is also possible that this small chromosome is fragmented and thus difficult to detect cytologically (Khurana, 2010).
Drosophila oocytes are activated as they pass through the oviduct, which triggers completion of the meiotic divisions. The first meiotic division is completed in the oviduct, but meiosis II can be observed in freshly laid eggs and is characterized by four well-separated meiotic products on tandem spindles. In aub and armi mutant embryos, the meiotic chromatin was either stretched across the paired meiotic spindles, or fragmented and spread over both spindles. No wild type meiotic figures were observed. Breaks thus appear to persist in some stage 14 oocytes, although this does not disrupt the karyosome organization during earlier stages. However, other oocytes appear to have intact chromosomes that fail to resolve during the meiotic divisions (Khurana, 2010).
Chromatin fragmentation could result from replication of broken chromosomes inherited from the female, or from post-fertilization fragmentation of the zygotic genome. To directly assay zygotic genome integrity, mutant females were mated to wild type males and dual-label FISH was used to monitor physically separate regions of the Y chromosome. In male embryos derived from wild type females, the two Y chromosome probes always co-segregated through anaphase and telophase. Mutant embryos showing chromatin fragmentation, by contrast, contained chromatin clusters that did not label for either Y chromosome probe, or that labeled for only one of the two probes. In mutant embryos that proceeded through cleavage stage mitotic cycles, the majority of segregating chromatids retained both Y chromosome markers, indicating that chromosome continuity had been maintained. Chromatids with only one of two markers were observed, however, indicating that breaks had separated regions on a Y chromosome arm from the centromere. The axial patterning defects associated with piRNA mutations are suppressed by mutations in mnk, but mnk did not suppress either the chromatin fragmentation or segregation defects linked to aub and armi. Mutations in aub and armi thus destabilize the genome of the zygote and disrupt chromosome resolution during the cleavage divisions through processes that are independent of DNA damage signaling (Khurana, 2010).
Mutations in the armi and aub genes disrupt piRNA production and transposon silencing, but have also been reported to inhibit homology dependent target cleavage by siRNAs. In addition, null mutations in argonaute2 (ago2), which block siRNA based silencing, have been reported to disrupt mitosis during the syncytial blastoderm stage. These observations raise the possibility that chromatin fragmentation and fusion in aub and armi mutants result from defects in the siRNA pathway. Therefore, cleavage was analyzed in embryos from females homozygous for null mutations in ago2 and dcr2, which block siRNA production and silencing. Consistent with previous studies, it was found that embryos from ago2 and dcr2 mutant females are viable. However, neither chromosome fragmentation nor a statistically significant increase in anaphase bridge formation was found relative to wild type controls. The loquacious (loqs) gene encodes a Dicer-1 binding protein required for miRNA production, and it was found that embryos from loqs mutant females also proceed through normal cleavage stage divisions. Chromosome segregation and maintenance of zygotic genome integrity during early embryogenesis thus appear to be independent of the siRNA and miRNA pathways, but require at least two components of the piRNA pathway (Khurana, 2010).
In S. pombe, mutations in ago1, dcr1 and rdp1 disrupt kinetochore assembly and thus lead to lagging mitotic chromosomes due to defects in centromere movement to the spindle poles. To determine if Drosophila piRNA mutations disrupt kinetochore assembly, dual label FISH was performed for centromeric dodeca-satellite sequences and the telomere-specific transposon HeT-A. In aub and armi mutants, centromeric sequences segregated to the spindle poles in essentially every anaphase figure, but telomere specific sequences were consistently present at the chromatin bridges. These observations indicate that armi and aub are not required for kinetochore assembly, but are needed for telomere resolution (Khurana, 2010).
Telomeres are protected from recognition as DNA double strand breaks by the telomere-protection complex (TPC), and defects in telomere protection thus lead to covalent ligation of chromosome ends by the non-homologous end-joining (NHEJ) pathway. DNA Ligase IV is required for NHEJ, and ligase IV mutations suppress fusions that result from covalent joining of unprotected chromosome ends. To determine if chromosome fusions in aub and armi are due to NHEJ, ligIV;aub and ligIV;armi double mutant females were generated and chromosome segregation was analyzed in the resulting embryos. In aub single mutant embryos, 50% of anaphase figures show bridges, but anaphase bridges are present in only 15% of ligIV;aub double mutants. By contrast, the fraction of embryos showing chromosome fragmentation increases in ligIV;aub double mutants. Chromosome fragmentation also increased in ligIV;armi mutant embryos, and as a result morphologically normal anaphase figures could not be observed. These findings strongly suggest that lagging chromosomes result from covalent ligation of chromosome ends by the NHEJ pathway, while chromatin fragmentation results from DNA breaks that are repaired by NHEJ. Mutations in armi and aub lead to significant over-expression of transposable elements, including DNA elements that are mobilized by a 'cut and paste' mechanism that directly produces double strand breaks. In addition, NHEJ pathway has been implicated in repair of gapped retroviral integration intermediates. Chromosome fragmentation may therefore result from transposon over-expression and mobilization, which induces breaks that overwhelm the NHEJ pathway. Telomere fusions, by contrast, appear to result from defects in telomere protection, which lead to chromosome end recognition by the NHEJ pathway (Khurana, 2010).
The Drosophila TPC includes HOAP and Modigliani (Moi), which may function only at chromosome ends, and HP1a and the MRN complex, which have additional roles in heterochromatic silencing and DNA repair. To directly assay for TPC recruitment, chromatin immunoprecipitation (ChIP) was used to measure HP1a and HOAP binding to the telomere specific transposon HeT-A. In wild type ovaries, HOAP and HP1a bind to multiple regions of HeT-A. In armi and aub mutants, by contrast, HOAP and HP1a binding to the Het-A 5'-UTR and ORF are significantly reduced. The 5' end of Het-A is oriented toward the chromosome end, and is therefore likely to lie at the telomere. Ovarian tissue consists of germ cells with a surrounding layer of somatic cells, which complicates interpretation of these biochemical studies. However, ChIP on 0-3 hour old embryos from aub and mnk,aub mutant females revealed significant reduction in HOAP binding at the HeT-A 5'-UTR. The aub and armi genes thus appear to be required for TPC recruitment, consistent with ligation of chromosome ends in mutant embryos (Khurana, 2010).
To determine if other piRNA pathway mutations disrupt telomere protection, the cleavage stage embryonic divisions was analyzed in ago3 and rhi mutants. The ago3 locus encodes a PIWI clade protein that primarily binds sense strand piRNAs, and rhi encodes a rapidly evolving HP1 homologue required for production of precursor RNAs from a subset of piRNA clusters. Essentially all of the rhi and ago3 mutant embryos showed chromatin fragmentation, as observed in the majority of aub and armi mutants. Therefore TPC assembly was analyzed in ovarian chromatin using ChIP for HOAP and HP1a. Surprisingly, neither ago3 nor rhi mutations disrupt HOAP or HP1a binding to Het-A, and rhi mutants show greater than wild type levels of HOAP binding to Het-A. By contrast, these rhi alleles reduce total piRNA production by 10 fold. The ago3 mutations appear to be null, and the rhi mutations are strong hypomorphc alleles. Assembly of the TPC in the ago3 and rhi mutants is therefore unlikely to be mediated by residual protein. Instead, these findings strongly suggest that aub and armi have a function in telomere protection that is not shared by ago3 or rhi (Khurana, 2010).
In Drosophila, chromosome breaks can be converted to stable telomeres, called terminal deletions, which accumulate additional copies of the telomeric elements HeT-A and TART. When terminal deletions are passaged in animals heterozygous for aub or the piRNA pathway gene spnE, the number of terminal TART repeats increase. The defects in TPC assembly in aub and armi could therefore be triggered by increased HeT-A and TART copy number, which could titrate TPC components. Therefore telomeric transposon copy number was assayed in aub and armi mutants, which show defects in TPC assembly, and in rhi and ago3 mutants, which do not. Telomeric transposon copy number and mitotic chromosome segregation was also analyzed in a wild-type variant, Gaiano, that has been reported to carry additional HeT-A repeats. Consistent with previous reports, it was found that Gaiano has 10 to 15 fold more HeT-A copies than OregonR controls. Despite the increase in telomere length, this stock is viable and fertile, and no telomere fusions or lagging chromosomes were observed during the cleavage stage embryonic divisions. In addition, it was found that aub mutants that show defects in TPC assembly do not accumulate additional copies of HeT-A or TART, while rhi and ago3 mutants that are wild type for TPC binding show an increase in telomere-specific transposon copy number. Assembly of the TPC is therefore independent of telomere specific transposon copy number (Khurana, 2010).
piRNAs are proposed to direct PIWI clade proteins to targets through sequence specific interactions. The current observations raised the possibility that armi and aub promote production of piRNAs that direct the telomere protection complex to transposons that make up chromosome ends. Published small RNA deep sequencing data was analyzed for species derived from a fourth chromosome cluster, defined by a high density of uniquely mapping piRNAs, containing multiple repeats of the telomeric transposons. This bioinformatic analysis showed that 70-80% of telomere specific piRNAs match this cluster. Length histograms for small RNAs from wt, rhi, ago3, aub and armi mutant ovaries map to this cluster. Significantly, aub and armi mutations lead to a preferential loss of shorter piRNAs mapping to the minus genomic strand. Loss of these shorter RNAs highlights the peak at 21 nt, which is retained in all of the mutants and likely represent endogenous siRNAs. The telomeric elements (HeT-A and TART) are almost exclusively on the minus genomic strand in this cluster, and the RNAs that are lost in aub and armi thus correspond to the sense strand of the target elements. Ovaries mutant for ago3 and rhi, by contrast, retain these shorter sense strand RNAs (Khurana, 2010).
The relative abundance of typical 23-29nt long piRNAs and the shorter 19-22nt species were quantified, excluding the 21nt endo-siRNA peak. All four mutations significantly reduce 23 to 29 nt piRNAs, although rhi mutants retain approximately 50% of wild type minus strand species. Loss of these piRNAs is consistent with over-expression of transposons matching this cluster in all four mutants. By contrast, the shorter minus strand RNAs are reduced by 3 to 10 fold in armi and aub, but are expressed at 80% to 95% of wild type levels in ago3 and rhi. In addition, short piRNA species from the telomeric cluster co-immunoprecipitate with Piwi protein, which localizes to the nucleus and is a likely effector of chromatin functions for the piRNA pathway. Binding of this subpopulation of piRNAs by Piwi is retained in ago3 mutants, which assemble the TPC, but significantly reduced in armi mutants, which block assembly of the TPC (Khurana, 2010).
Taken together, these observations suggest that the piRNA pathway has two genetically distinct functions during oogenesis and early embryogenesis. The pathway prevents DNA damage during oogenesis and maintains the integrity of the zygotic genome during the embryonic cleavage divisions, which likely reflects the established role for piRNAs in transposon silencing. This function requires aub, armi, rhi and ago3, which are also required for wild type piRNA production. In addition, these studies reveal a novel function for the piRNA genes aub and armi in telomere protection, whch may be mediated by a novel class of short RNAs that bind to Piwi. Consistent with this hypothesis, it has been reported that germline clones of piwi null alleles do not significantly disrupt oogenesis, but lead to maternal effect embryonic lethality and severe chromosome segregation defects during the cleavage division. A subpopulation of Piwi-bound piRNAs may therefore direct assembly of the TPC (Khurana, 2010).
PIWI-interacting RNAs (piRNAs) protect genome integrity from transposons. In Drosophila ovarian somas, primary piRNAs are produced and loaded onto Piwi. This study describes roles for the cytoplasmic Yb body components Armitage and Yb in somatic primary piRNA biogenesis. Armitage binds to Piwi and is required for localizing Piwi into Yb bodies. Without Armitage or Yb, Piwi is freed from the piRNAs and does not enter the nucleus. Thus, piRNA loading is required for Piwi nuclear entry. It is proposed that a functional Piwi-piRNA complex is formed and inspected in Yb bodies before its nuclear entry to exert transposon silencing (Saito, 2010).
In Drosophila, three sets of endogenous small RNAs have been identified so far: microRNAs (miRNAs), endogenous siRNAs (endo-siRNAs/esiRNAs), and PIWI-interacting RNAs (piRNAs). Of these, piRNAs are considered unique because of their germline-specific expression and specific interaction with germline-specific Argonaute proteins, PIWI proteins. The identification of the piRNAs associated with three PIWI proteins (Aubergine [Aub], Argonaute 3 [AGO3], and Piwi) has revealed distinct features of piRNAs associated with each PIWI and has led to two models for piRNA biogenesis: the primary processing pathway and the amplification loop pathway. In the amplification loop model, the Slicer (endonuclease) activity of Aub and AGO3 determines the formation of the 5' end of piRNAs. Zucchini (Zuc), a putative cytoplasmic nuclease, is involved in the primary processing pathway; however, its precise molecular function remains unclear. Furthermore, the factors other than zuc required for primary piRNA biogenesis are unknown (Saito, 2010).
The ovarian somatic cell (OSC) line consists of ovarian somas only. The expression of Aub and AGO3 is not detectable in OSCs because both proteins are germ cell-specific. This implies that the amplification loop does not operate in OSCs. However, OSCs express piRNAs and are loaded onto Piwi, indicating that the piRNAs in OSCs are generated specifically through the primary processing pathway. Thus, OSCs are an ideal tool to elucidate the molecular mechanisms of primary piRNA processing and Piwi function. Loss of zuc function drastically reduced the level of primary piRNAs in the ovaries. This was recapitulated in OSCs: Zuc depletion by RNAi caused a severe reduction in the piRNA level in OSCs. This result prompted a screen for other factors necessary for primary piRNA production using RNAi in OSCs (Saito, 2010).
To identify the genes required for somatic primary piRNA biogenesis, RNAi-based screening was performed in OSCs. The genes screened included armitage (armi), spindle-E (spn-E), and maelstrome (mael), all of which are implicated in piRNA biogenesis. However, their roles in somatic primary piRNA production remain unknown. Depletion of Armi reduced the piRNA levels to an extent very similar to that of Piwi and Zuc depletion, indicating that Armi is necessary for primary piRNA biogenesis in OSCs. Depletion of Mael and Spn-E showed little or no effect on piRNA accumulation in OSCs. Mutations in both genes have been shown to significantly reduce the piRNA levels in ovaries. Thus, spn-E and mael are factors functioning in the amplification loop. Depletion of Dicer1 and Dicer2 had little or no effect on the piRNA levels, confirming that neither protein is necessary for piRNA production (Saito, 2010).
Armi is the Drosophila ortholog of Arabidopsis Silencing-Defective 3 (SDE3) and mammalian Moloney leukemia virus 10 (MOV10). These orthologs contain a conserved ATP-dependent RNA helicase domain at their C termini and have been implicated in small RNA-mediated gene silencing. However, their precise functions remain unknown. To gain further insight into the function of Armi in somatic primary piRNA processing, a monoclonal antibody was produced against Armi. Western blotting showed a discrete band in both ovary and cultured Schneider2 (S2) cell lysates, indicating that Armi expression is not germline-specific. The ~150-kDa protein immunopurified from S2 cells with the anti-Armi antibody was confirmed to be Armi by mass spectrometry (Saito, 2010).
Immunostaining of OSCs and ovaries with the anti-Armi antibody confirmed an earlier observation that Armi is a cytoplasmic protein. The Armi signals were detected in both somatic and germ cells of ovaries. The somatic signal was considered a background signal because it did not disappear even in armi homozygous mutant egg chambers. In the present study, the cytoplasmic signal in OSCs mostly disappeared when Armi was depleted by RNAi. Thus, it is concluded that Armi is expressed in both somatic and germ cells in ovaries (Saito, 2010).
The subcellular localization of Armi in the armi trans-heterozygous mutants appeared very similar to that in the homozygous mutants. In addition, Western blotting revealed a band corresponding to Armi in the armi ovaries. By what mechanisms Armi is expressed in the mutant somas remains unclear. The simplest explanation is that the armi gene uses two distinct genomic elements as promoters in ovarian somas. In fact, armi homozygous mutants weakly express a shorter armi transcript than that expressed in the wild-type strain (Saito, 2010).
The Armi signal in germ cells was rather weak, and only a small proportion of Armi accumulated at, or near, the nuage, an electron-dense structure associated with nurse cell nuclei. Thus, Armi might not be a component of the nuage per se. This correlates well with the fact that armi mutations barely affected the ability of the ovaries to amplify endogenous piRNAs. In ovarian somas, Armi accumulated strongly at discrete cytoplasmic foci. Each somatic cell contained one or several foci. Interestingly, the Armi-positive foci were often located near the nucleus in both ovaries and OSCs (Saito, 2010)
Piwi is required for the silencing of transposons in gonads. In fact, Piwi depletion in OSCs caused derepression of transposons, as with Armi, Yb, and Zuc depletion. Under conditions where endogenous Piwi was depleted, expression of myc-Piwi-r, which was designed to be RNAi-insensitive, rescued transposon silencing. However, myc-Piwi-δN, which lacks 72 amino acids at the N terminus of Piwi and thus does not localize to the nucleus, did not rescue transposon silencing, although it does associate with piRNAs to the same extent as does the wild-type Piwi. myc-Piwi-δN13, which lacks 13 amino acids at the N terminus, behaved similarly. On the other hand, myc-Piwi-DDAA-r, a Slicer mutant of Piwi, could bind to mature piRNAs in OSCs, as does the wild-type Piwi, and rescued transposon silencing. These results might suggest that Piwi must be localized in the nucleus to silence the transposable elements, and that Piwi Slicer activity is unnecessary for its function. It is assumed that this system has evolved to prevent nascent Piwi, not loaded with piRNAs, from being imported into the nucleus. In other words, only the functional Piwi-piRNA complex (piRISC) formed at Yb bodies could be transported to the nucleus. At present, the mechanisms of this control system remain unclear. In the nongonadal somatic S2 cell line, where the expression of piRNAs is undetectable, transfected Piwi is localized to the nucleus, indicating that 'empty' Piwi can be transported to the nucleus. It seems that the machineries necessary for the nuclear transport of Piwi might recognize different features of Piwi in different cell types (Saito, 2010).
How is piRNA-free Piwi restrained in the cytoplasm in OSCs? One possibility is that some unknown protein binds the N-terminal end of Piwi, where its NLS (nuclear localization signal) resides, and interferes with the nuclear import machinery's ability to recognize Piwi as a cargo. The nuclear localization inhibitory factors may be retained on Piwi until a functional Piwi-piRNA complex is formed at Yb bodies. Once the complex is formed, a conformational change in Piwi would be induced, which would release the regulatory factors and reveal the Piwi NLS for recognition by the nuclear import machinery. It would be very interesting to determine the proteins that are associated with Piwi in OSCs under conditions of Armi or Zuc depletion, thus identifying the protein factors that restrain Piwi in the cytoplasm until it is loaded with mature piRNAs at Yb bodies (Saito, 2010).
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date revised: 15 October 2011
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