The role of PIWI and the miRNA machinery in Drosophila germline determination; increasing the maternal piwi dose increases Osk and Vasa levels

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

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

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

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

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

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

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

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

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

The HP1 homolog Rhino is required for transposon silencing and affects chromosome structure and egg polarity

Piwi-interacting RNAs (piRNAs) silence transposons and maintain genome integrity during germline development. In Drosophila, transposon-rich heterochromatic clusters encode piRNAs either on both genomic strands (dual-strand clusters) or predominantly one genomic strand (uni-strand clusters). Primary piRNAs derived from these clusters are proposed to drive a ping-pong amplification cycle catalyzed by proteins that localize to the perinuclear nuage. This study shows that the HP1 homolog Rhino is required for nuage organization, transposon silencing, and ping-pong amplification of piRNAs. rhi mutations virtually eliminate piRNAs from the dual-strand clusters and block production of putative precursor RNAs from both strands of the major 42AB dual-strand cluster, but not of transcripts or piRNAs from the uni-strand clusters. Furthermore, Rhino protein associates with the 42AB dual-strand cluster, but does not bind to uni-strand cluster 2 or flamenco. Rhino thus appears to promote transcription of dual-strand clusters, leading to production of piRNAs that drive the ping-pong amplification cycle (Klattenhoff, 2009).

Drosophila piRNA pathway mutations lead to germline DNA damage and disrupt axis specification through activation of Chk2 and ATR kinases, which function in DNA damage signaling. Mutations in the rhi locus lead to very similar patterning defects (Volpe, 2001). The mei-41 and mnk genes encode ATR and Chk2, respectively. To determine whether the axis specification defects associated with rhi result from damage signaling, double mutants were generated with mnk and mei-41 and axis specification was quantified by scoring for assembly of dorsal appendages, which are egg shell structures that form in response to dorsal signaling during oocyte development. Only 17% of embryos from rhiKG/rhi2 females had two wild-type appendages. However, 80% of embryos from mnk;rhiKG/rhi2 double-mutant females had two appendages. In addition, 33% of embryos from mei-41;rhiKG/rhi2 double-mutant females had two appendages. Consistent with these observations, rhi mutations disrupt dorsal localization of Gurken and posterior localization of Vasa in the oocyte, and localization of both proteins is restored in mnk; rhiKG/rhi2 double mutants (Klattenhoff, 2009).

Targets of Activity

The oskar gene directs germ plasm assembly and controls the number of germ cell precursors formed at the posterior pole of the Drosophila embryo. Mislocalization of Oskar RNA to the anterior pole leads to induction of germ cells at the anterior. Of the eight genes necessary for germ cell formation at the posterior, only three are essential at an ectopic site: oskar, vasa and tudor (Ephrussi, 1992).

Maternally synthesized Hsp83 transcripts are localized to the posterior pole of the early Drosophila embryo by a novel mechanism involving a combination of generalized RNA degradation and local protection at the posterior. Hsp83 RNA is not protected at the posterior pole of embryos produced by females carrying maternal mutations that disrupt the posterior polar plasm and the polar granules (Ding, 1993).

Localization of Vasa to pole plasm

Vasa (Vas), a key protein in establishing the specialized translational activity of the Drosophila pole plasm, accumulates at the posterior pole of the developing oocyte. Mutation in gustavus (gus), a gene that encodes a protein that interacts with Vas, blocks posterior localization of Vas, as does deletion of a segment of Vas containing the GUS binding site. Like Vas, Gus is present in cytoplasmic ribonucleoprotein particles. Heterozygotes for gus or a deletion including gus produce embryos with fewer pole cells and posterior patterning defects. Therefore, Gus is essential for the posterior localization of Vas. However, gus is not required for the posterior localization of oskar (osk). Apparent gus orthologs are present in mammalian genomes (Styhler, 2002).

To identify proteins that may interact with Vas during oogenesis, a yeast two-hybrid screen was performed using full-length Vas as bait and a Drosophila ovarian cDNA library as prey. Out of 145 independent positive clones analyzed, 23 encode a novel Vas-interacting protein that has been named Gustavus (Gus). The interaction between Vas and Gus was verified through GST pulldown assays and His-tagged Gus was found to bind purified GST-Vas. This experiment indicates that Vas and Gus can bind directly. In addition, immunoprecipitations were performed on ovary extracts using affinity purified anti-Gus and Vas could be detected in the anti-Gus immunoprecipitate, indicating that Vas and Gus are associated in vivo (Styhler, 2002).

gus is cytologically situated at 41E6, and corresponds to CG2944. Genome annotation predicts six potential gus transcripts, but only a single 1.6 kb gus transcript was detected on Northern blots of RNA prepared from ovaries, early embryos, and fertile adult females, and not in males or sterile females, indicating that gus is primarily expressed in ovaries. This transcript corresponds best in size to CG2944-RB, which encodes a 281 aa polypeptide, although a second in-frame initiator methionine six nucleotides downstream is in a better translational start sequence context. A predicted longer alternative splice form of gus, CG2944-RA, encodes a 349 aa polypeptide. Only a 32 kDa protein is detected on immunoblots, consistent with the size of the 281 aa polypeptide, suggesting that the shorter polypeptide is the major product of the gus gene. A peptide antibody was raised against a unique N-terminal fragment of the 349 aa form and no immunoreactivity was observed in Drosophila extracts. Based on this evidence, it is concluded that the major gus transcript corresponds to CG2944-RB, and Gus is primarily expressed as a 281 or 279 aa polypeptide (Styhler, 2002).

Gus contains a B30.2-like domain, a SPRY domain, and a SOCS box. SPRY domains are a subclass of the B30.2-like domain, and were first identified in ryanodine receptors, which mediate Ca2+ release from the sarcoplasmic reticulum. The SOCS box is a conserved domain that was initially discovered in SH2 domain-containing proteins of the suppressor of cytokine signaling (SOCS) family; the SOCS box is present in a number of proteins, including other SPRY domain-containing proteins. In mammalian cells, a portion of the SOCS box can bind the elongin BC complex, an association proposed to target proteins to the ubiquitination or proteasomal compartments (Styhler, 2002).

Gus is very closely related to proteins in a wide range of animal species. The hypothetical human proteins SSB-1 (GenPept accession number XP 045247.2; 70% identity over 277 amino acids, BLAST p < e-114) and SSB-4 (GenPept accession number XP 044664.1; 68% identity over 277 amino acids, BLAST p < e-106) are highly similar to Gus. SSB-1 and SSB-4 orthologs also exist in mouse. Apparent gus orthologs are also present in Ciona intestinalis (Ci META3; 59% identity over 243 amino acids, BLAST p < 2e-87) and C. elegans (GenPept accession number NP_497321.1; 54% identity over 218 amino acids, BLAST p < 7e-69). The 70 N-terminal amino acids of the predicted 349 aa Gus isoform have no significant similarity to any known protein. Two other Drosophila proteins, CG4643 and SP555, contain the same protein motifs as Gus, but are not as closely related as the probable orthologs described above (Styhler, 2002).

A gus mutation causes female sterility, and pole plasm assembly is blocked specifically at the step of posterior localization of Vas. Furthermore, a short region of Vas essential for binding Gus is also essential for posterior localization of Vas. Taken together, these results indicate that gus is necessary for Vas deployment and that the mechanism of Vas localization most likely involves a direct interaction between Vas and Gus (Styhler, 2002).

Like Vas and several other proteins involved in oocyte patterning, Gus is a component of cytoplasmic RNPs. In the Drosophila ovary, three classes of such RNPs have been distinguished based on morphological criteria: nuage particles, sponge bodies, and polar granules. Nuage particles are Vas positive and are concentrated around the outer periphery of the nurse cell nuclei; polar granules are related, larger, Vas-positive structures that are present in the pole plasm of late-stage oocytes and syncytial embryos; and sponge bodies are Exu and ME31B positive and present throughout the cytoplasm of the nurse cells and, to a lesser degree, the oocyte. The localization pattern of Gus would, by these criteria, indicate that it is a component of both nuage particles and sponge bodies. However, double-labeling experiments with Gus and Vas, Gus and Exu, or Gus and ME31B indicate overlapping but not entirely coincident distributions. From these results, it appears likely that what are termed nuage particles or sponge bodies are in fact heterogeneous collections of related RNPs that differ somewhat in their molecular composition and may individually be dynamic in their makeup. The observation that in gus mutant ovaries the density of cytoplasmic ME31B-positive particles is reduced but that such particles are not totally eliminated could be explained if gus activity is required for the assembly of some cytoplasmic RNPs (presumably a set that contains Gus) but other similarly distributed RNPs assemble independently of gus (Styhler, 2002).

At least through its initial stages, pole plasm assembly is a stepwise process, taking place in several stages that can be distinguished temporally. Completion of each particular stage is essential for subsequent stages of the process to occur. A great deal is known about the first step of pole plasm assembly, accumulation of osk RNA, which requires a polarized microtubule network, the plus-end-directed microtubule motor kinesin I, and at least seven gene activities in the oocyte: cappuccino, spire, Par-1, mago nashi, tsunagi, barentsz, and stau. Localization of osk RNA is intimately linked to control of its translation, since unlocalized osk is translationally repressed by Bruno, ME31B, Ypsilon Schachtel, and other factors, while that repression is alleviated at the posterior pole by factors including Aubergine (Aub) and Orb. Accumulation of the short isoform of OSK in the pole plasm sets off a cascade of events that results in the recruitment of all of the other factors necessary for the establishment of a functional germline, most immediately Vas. Vas arrives at the posterior pole through a mechanism distinct from that of osk, and Vas localization proceeds through four steps: (1) assembly of Vas into nuage particles that surround the nurse cell nuclei; (2) movement of nuage particles into the nurse cell cytoplasm; (3) transfer of nuage particles through the ring canals into the oocyte, and (4) localization of nuage particles at the posterior pole (Styhler, 2002).

Studies of posterior deployment of Vas, the final step of its localization, have been complicated by the fact that this step depends upon osk, so treatments such as depolymerization of the microtubule cytoskeleton that affect osk localization, indirectly affect Vas. Some potential insight into the mechanism of Vas localization has been provided by video-enhanced contrast microscopy to study the flow of unlabeled cytoplasmic particles in living stage 6-10a wild-type follicles. Unidirectional streaming of these particles is observed through the ring canals as early as stage 7, well before the bulk streaming of the nurse cell cytoplasm into the oocyte that commences at stage 10b. A circular cytoplasmic streaming of particles is observed within the oocyte beginning at stage 6-7. If these particles correspond, as is likely at least in part, to Vas-containing nuage particles, then these observations suggest the movement through the ring canals could correspond to the third step of Vas localization mentioned above, and the circular cytoplasmic streaming could contribute to posterior localization of Vas. Drug inhibition experiments have led to the conclusion that cytoskeletal actin or actin-associated cytoskeletal components, operating in concert with cytoplasmic myosin, are involved in the movement of particles through the ring canals, and that the microtubule cytoskeleton is required for circular streaming of the oocyte cytoplasm (Styhler, 2002 and references therein).

The effects of the gusZ409 mutation on pole plasm assembly can mostly be explained through the failure of Vas to be deployed to the posterior pole plasm in this mutant. An exception, however, is the striking reduction of Osk level at the posterior of stage 10 gusZ409 oocytes, since vas alleles have little if any effect on Osk accumulation at this developmental stage. This observation can be explained if gus also affects the spatial distribution of Aub, a translational activator of osk that is a component of polar granules and probably of nuage particles. Posterior accumulation of Aub is vas dependent and therefore is presumably dependent on gus, but Aub-mediated translational activation of osk does not require it to be posteriorly deployed. However, if Aub is a component of nuage particles like Vas, which is suggested by the observation that vas mutations largely abolish perinuclear accumulation of Aub-containing particles in nurse cells, then the gusZ409 mutation could delay or block the movement of Aub through the ring canals and into the oocyte cytoplasm and render it less able to activate translation of osk, thus explaining the observation of decreased Osk at the posterior (Styhler, 2002).

Vas is required for normal levels of Grk to be attained both in early oocytes and at the anterodorsal corner of stage 8-10 oocytes . In early gusZ409 oocytes, accumulation of Grk is clearly reduced, although by stage 10 the distribution and level of Grk is similar to that in wild-type oocytes. On the basis of these observations and the effects on osk translation, the gus mutant phenotypes are best explained by a delay in or reduced efficiency of movement of Vas-containing nuage particles from the nurse cells into the oocyte, followed by a complete failure of those particles to accumulate at the posterior pole. Minor effects on Grk accumulation, mediated through effects on the movement of Vas into the oocyte, could explain the dorsal appendage defects observed in the eggs produced by gusZ409 heterozygotes and homozygotes, although the aberrant positioning of the oocyte nucleus also must contribute to this phenotype, at least in gusZ409 homozygous ovaries (Styhler, 2002).

While a major role for Gus must be the targeting of Vas, the gus mutant phenotypes predict that Gus is involved in other processes as well, such as oocyte growth and chorion deposition. Interaction screens with gus should provide insight on additional protein partners that may be involved in Vas localization and these other developmental processes (Styhler, 2002).

Oskar interacts directly with Vasa and Staufen, in a yeast two-hybrid assay. These interactions also occur in vitro and are affected by mutations in oskar that abolish pole plasm formation in vivo. In the pole plasm, Oskar protein, like Vasa and Tudor, is a component of polar granules, the germ-line-specific RNP structures. Thus the Oskar-Vasa interaction constitutes an initial step in polar granule assembly. The biological significance of the direct interaction between OSK and STAU is unclear. A complex containing OSK could provide a scaffold for OSK transcript until structures containing Vasa as a component are established. It appears that OSK is a component of different complexes at different developmental stages (Breitwieser, 1996).

Vasa protein is required twice during oogenesis. First it is needed to assemble the perinucleus zone of oocytes and nurse cells; later it is required to assemble the pole plasm.

Posterior localization of Vasa protein depends upon the functions of four genes: cappuccino, spire, oskar and staufen. Localization of Vasa to the perinuclear nuage (fibrous bodies making electron-dense clumps on the cytoplasmic face of the nuclear envelop of germ line and nurse cells) is abolished in most vas alleles, but is unaffected by mutations in the four genes required upstream for Vasa's pole plasm localization. Thus localization of Vasa to the nuage particles in the perinuclear region of the oocyte is independent of the pole plasm assembly pathway. Proteins from two mutant alleles that retain the capacity to localize to the posterior pole of the oocyte are both severely reduced in RNA-binding and -unwinding activity as compared to the wild-type protein on a variety of RNA substrates, including in vitro synthesized pole plasm RNAs. Thus the RNA helicase function is not required for localization to the pole plasm. Initial recruitment of Vasa to the pole plasm must consequently depend upon protein-protein interactions but once localized Vasa must bind to RNA to mediate germ cell formation (Liang, 1994).

In oskar and staufen mutant females, vasa synthesis appears normal, but the Vasa protein is not localized. In tudor and valois mutant females, vasa is localized to the posterior pole of oocytes, but this localization is lost following egg activation. In addition to the posterior localized Vasa, there is a low level of Vasa distributed throughout the embryo. A function for this distributed Vasa is postulated based on the observation that embryos from Bicaudal-D mothers, in which abdominal determinants are incorrectly localized to the anterior pole, do not show any ectopic Vasa localization, though abdomen development at the anterior end depends on the amount of Vasa protein in the embryo (Hay, 1990).

pipsqueak acts after the establishment of the Oskar posterior anchor but before the localization of Vasa protein during oogenesis. Characterization of multiple alleles at the pipsqueak locus shows that pipsqueak, like vasa, is required for early stages of oogenesis, including but not limited to formation of the egg chamber and progression through Stage 6 of oogenesis. Genetic interaction studies suggest that pipsqueak acts at least partially through vasa; molecular studies indicate that pipsqueak affects vasa level in the ovary (Siegel, 1993).

Unlike other bicaudal mutants, Oskar mRNA is localized correctly to the posterior pole of the oocyte in bullwinkle mutants at stage 10. However, by early embryogenesis some Oskar mRNA is mislocalized to the anterior pole. Consistent with the mislocalization of Oskar mRNA, a fraction of the Vasa protein and Nanos mRNA are also mislocalized to the anterior pole of bullwinkle embryos. Mislocalization of Nanos mRNA to the anterior is dependent on functional Vasa protein. It seems that Bullwinkle interacts with the cytoskeleton and extracellular matrix and is necessary for gene product localization and cell migration during oogenesis after stage 10a (Rittenhouse, 1995).

licorne codes for a MAP kinase kinase exciting the p38 pathway in Drosophila. licorne mutant embryos are defined, for the purpose of this study, as hemipterous;licorne double mutants engineered to express a hemipterous transgene (see Licorne Effects of Mutation for more information about this genotype). lic mutant embryos show a segmentation phenotype that is reminiscent of the one produced by mutations in the maternal posterior-group genes, including oskar, vasa, and nanos. Most of the posterior-group genes can provoke both abdominal segmentation defects and a loss of germ cells, a dual defect that is due to the common localization of the posterior and germ cell determinants in the posterior germ plasm. Like several posterior-group genes, lic embryos lack or have a strongly reduced number of pole cells, as shown using Vasa and Nanos as markers. In most lic mutant embryos, Vasa protein fails to be accumulated at the posterior pole, although in some cases weak staining is observed. It is concluded that lic has a role in abdominal segmentation, proper Vasa protein and Nanos mRNA localization at the posterior pole, and formation of the pole cells. These results suggest that lic also has a role in germ plasm assembly (Suzanne, 1999).

The assembly of the germ plasm takes place during oogenesis and proceeds in several steps leading to the successive posterior localization of many different components (for review, see Rongo,1996). A pivotal step in this process is the localization of the OSK mRNA to the posterior pole of the oocyte in stage 8-9 egg chambers, which is the basis for the recruitment and assembly of downstream components like Vasa and Nanos. In lic germ-line clones, both OSK mRNA expression and early posterior localization appear normal until stage 8 of oogenesis. However, in stage 9 and older egg chambers, the OSK mRNA is mislocalized, diffusing in the whole oocyte in a gradient from the posterior to the anterior pole. In later stages, OSK transcripts are barely detectable, indicating that diffusion proceeds continuously in mutant egg chambers. A similar phenotype is observed in some osk missense mutants, suggesting a role for Osk protein in the anchoring of its own mRNA at the posterior pole. Staining of lic mutant egg chambers using an anti-Osk antibody did not allowed detection of any reduction in Osk protein accumulation, indicating that lic affects OSK mRNA localization independent of Osk translation (Suzanne, 1999).

In some lic egg chambers, the mislocalized OSK mRNAs also seem to partly accumulate in a more central position, reminiscent of the position of OSK transcripts in mutants that have not reorganized the microtubules, as in EGFR pathway mutants. This result suggests that lic oocytes are not completely repolarized. However, no defect in the positioning of the nucleus, or in the localization of a kinesin-lacZ microtubule-associated motor protein fusion is observed, suggesting that OSK mRNA mislocalization is a more sensitive assay and lic defects are weak. The correct localization of osk RNA at stage 8 and its later diffusion indicate that lic affects the maintenance of OSK mRNA asymmetric localization in the oocyte (anchoring) rather than the mechanism of localization per se, most likely as a result of incomplete polarization along the AP axis (Suzanne, 1999).

The translational repressor Cup is required for germ cell development in Drosophila

In Drosophila, germ cell formation depends on inherited maternal factors localized in the posterior pole region of oocytes and early embryos, known as germ plasm. This study reports that heterozygous cup mutant ovaries and embryos have reduced levels of Staufen (Stau), Oskar (Osk) and Vasa (Vas) proteins at the posterior pole. Moreover, Cup interacts with Osk and Vas to ensure anchoring and/or maintenance of germ plasm particles at the posterior pole of oocytes and early embryos. Homozygous cup mutant embryos have a reduced number of germ cells, compared to heterozygous cup mutants, which, in turn, have fewer germ cells than wild-type embryos. In addition, cup and osk interact genetically, because reducing cup copy number further decreases the total number of germ cells observed in heterozygous osk mutant embryos. Finally, cup mRNA and protein were detected within both early and late embryonic germ cells, suggesting a novel role of Cup during germ cell development in Drosophila (Ottone, 2012).

Germ plasm assembly is a stepwise process occurring during oogenesis. Accumulation of osk mRNA at the posterior of egg chambers is necessary for correct germ plasm assembly, which requires a polarized microtubule network, the plus-end motor kinesin I, and the activity of several genes (cappuccino, spire, par-1. mago nashi, barentz, stau, tsunagi, rab11, and valois). Localization of osk mRNA is strictly linked to the control of its translation, as unlocalized osk mRNA is silent. Upon localization at the posterior pole, the relieve of osk translational repression involves several factors, including Orb, Stau, and Aubergine. Localized Osk protein, in turn, triggers a cascade of events that result in the recruitment of all factors, such as Vas, Tud, and Stau proteins and nanos, germ less mRNAs, necessary for the establishment of functional germline structures (Ottone, 2012).

Posterior anchoring of Osk requires the functions of Vas, as well as Osk itself, to direct proper germ plasm assembly. Misexpression of Osk at the anterior pole of oocytes causes ectopic pole plasm formation, indicating that Osk is the key organizer of pole plasm assembly. Moreover, it has been demonstrated that endocytic pathways acting downstream of Osk regulate F-actin dynamics, which in turn are necessary to attach pole plasm components to the oocyte cortex. As far as Cup is concerned, it has been demonstrated that Cup is engaged in translational repression of unlocalized mRNAs, such as osk, gurken, and cyclinA, during early oogenesis (Ottone, 2012).

The current results establish that Cup is also a novel germ plasm component. First, Cup colocalizes with Osk, Stau, and Vas at the posterior pole of stage 10B oocytes. Second, biochemical evidence indicates that Cup interacts with Stau, Osk, and Vas. Vas localization occurs not through its association with localized RNAs, but rather through the interaction with the Osk protein, which represents an essential step in polar granule assembly (Ottone, 2012).

As a consequence of these interactions, Cup protein is mislocalized in osk and vas mutant stage 10 oocytes, demonstrating that Osk and Vas are essential to achieve a correct localization of Cup at the posterior cortex of stage 10 oocytes. This study suggests that the presence of Cup, Osk, Stau, and Vas are required for a correct germ plasm assembly. Moreover, several immuno-precipitation experiments, using anti- Tud and anti-Vas antibodies, identified numerous P-body related proteins, including Cup, as novel polar granule components (Ottone, 2012).

All the results suggest that Cup plays at least an additional role at stage 10 of oogenesis. Cup, besides repressing translation of unlocalized osk mRNA, is necessary to anchor and/or maintain Stau, Osk, and Vas at the posterior cortex. This novel function of Cup is supported by the findings that, when cup gene dosage is reduced, Stau, Osk, and Vas are partially anchored and/or maintained at the posterior pole, even if these proteins are not degraded. Consequently, pole plasm assembly is disturbed and cup mutant females lay embryos with a reduced number of germ cells. Since the role of Cup, a known multi-functional protein during the different stages of egg chamber development, cannot be easily studied in homozygous cup ovaries, it is not surprising that the involvement of Cup in pole plasm assembly remained undiscovered until now (Ottone, 2012).

During embryogenesis, Cup exerts similar functions. In particular, Osk, Stau, and Vas proteins and osk mRNA are not properly maintained and/or anchored at the posterior pole of embryos laid by heterozygous cup mutant mothers. Surprisingly, osk mRNA is increased in heterozygous cup mutant embryos. Since osk mRNA requires sufficient Osk protein to remain tightly linked at the posterior cortex, the reduced amount of Osk protein observed in heterozygous cup embryos, should be not sufficient to maintain all osk mRNA at the embryonic pole and could stimulate, by positive feedback, de novo osk mRNA synthesis. Also, a direct/indirect involvement of Cup in osk mRNA degradation and/or deadenylation cannot be excluded. The findings that Cup has been found together with Osk, when Osk is ectopically localized to the anterior pole of the embryos, and that reducing cup copy number further decreases the total number of germ cells, observed in heterozygous osk mutant embryos, strengthen the idea that Cup is involved in germ cell formation and/or in maintenance of their identity (Ottone, 2012).

Unlike Osk protein, both cup mRNA and protein were detected within germ cells until the end of embryogenesis. These observations suggest that zygotic cup functions, during germ cell formation and maintenance, are not limited to those carried out in combination with Osk. The finding that homozygous cup mutant embryos display a further decrease of germ cell number, in comparison with heterozygous embryos, supports this hypothesis. Whether or not cup zygotic function is involved in the translational repression of specific mRNAs, different from osk, remains to be explored (Ottone, 2012).

Signaling upstream of Vasa

The genes okra and spindle-B act during meiosis in Drosophila to repair double-stranded DNA breaks (DSBs) associated with meiotic recombination. Unexpectedly, mutations in these genes cause dorsoventral patterning defects during oogenesis. In addition to these patterning defects, mutations in genes of the spindle class also cause defects in the appearance of the oocyte nucleus. DNA within the germinal vesicle of wild-type egg chambers assumes a highly compacted spherical morphology by stage 3 of oogenesis, whereas the DNA within spindle-class-mutant oocyte nuclei is often fragmented or thread-like in appearance. Because mutations in vasa, which plays no part in DSB repair, produce a similar nuclear-morphology phenotype, it is unlikely that the fragmented appearance of the DNA in vasa and spindle-class mutants directly reflects unrepaired DSBs. It has been suggested that the appearance of the DNA in these mutants resembles the appearance of DNA in earlier stages of oogenesis, raising the possibility that the karyosome defect is indicative of delayed progression through meiosis (Ghabrial, 1999).

Patterning defects result from a failure to accumulate Gurken protein, which is required to initiate dorsoventral patterning during oogenesis. The block in Gurken accumulation in the oocyte cytoplasm reflects activation of a meiotic checkpoint in response to the persistence of DSBs in the nucleus. Vasa is a target of this meiotic checkpoint, and so may mediate the checkpoint-dependent translational regulation of Gurken (Ghabrial, 1999).

The mechanism through which the persistence of unrepaired DSBs during oogenesis causes patterning defects was explored. In S. cerevisiae, mutations in the DSB-repair genes RAD51 and DMC1 (the murine homolog of RAD51) cause cells to arrest in prophase of the first meiotic division. Meiotic arrest is also observed in Dmc1-deficient mice. In S. cerevisiae, this meiotic arrest is checkpoint dependent -- a failure to repair DSBs does not cause meiotic arrest directly; instead, recognition of the presence of DSBs triggers the activation of a pathway that halts the cell cycle until the damaged DNA is repaired. One of the checkpoint genes required to arrest meiosis in response to the presence of unrepaired DSBs is MEC1, which encodes a member of the ATM/ATR subfamily of phosphatidylinositol-3-OH-kinase-like proteins. In otherwise wild-type yeast, mec1 mutations lead to occasional premature meiosis I, as indicated by the persistence of foci of Rad51, which are believed to represent sites of DSB repair, on metaphase-I chromosomes. These results indicate that Mec1 may normally act to delay the cell cycle in the presence of repair intermediates. In Drosophila, mei-41 encodes a homolog of Mec1, and mei-41 mutants show meiotic non-disjunction as well as maternal-effect defects in the timing of mitotic cell cycles in the early embryo (Ghabrial, 1999).

To test whether the production of patterning defects by mutations in the spindle-class genes is due to the engagement of an analogous meiotic checkpoint, flies doubly mutant for okr, spn-B or spn-C and mei-41 were generated. Mutations in mei-41 are indeed able to suppress the dorsoventral patterning defects caused by mutations in the spindle-class genes. In double-mutant flies, a dramatic increase in the accumulation of Grk protein is observed, as indicated by whole-mount antibody staining and by restoration of anteroposterior and dorsoventral patterning in the eggshell. Significant suppression of the oocyte nuclear-morphology defect was also observed. Suppression of the spindle-class defects by mei-41 is not as complete as is that by mei-W68, raising the possibility that there may be some functional redundancy between mei-41 and a putative Drosophila ATM homolog, as appears to be the case for yeast MEC1 and TEL1. From these results, it is concluded that the patterning defects observed in mutants of the spindle class are caused by the activation of a mei-41-dependent checkpoint pathway in response to the persistence of unrepaired DSBs during meiosis (Ghabrial, 1999).

These results raise the question of how the mei-41-dependent checkpoint pathway affects accumulation of Grk. One candidate for a downstream target and effector of the mei-41-dependent pathway is the product of the vasa (vas) gene. vas encodes a protein similar to the translation-initiation factor eIF4A; it produces mutant phenotypes similar to those observed in okr, spn-B and spn-C mutants, and it has been implicated in the translational control of Grk and certain other oocyte-specific proteins. However, unlike okr, spn-B and spn-C mutations, mutations in vas are not suppressed by mutations in mei-41. The karyosome phenotype of vas mutants is also not suppressed by mei-41 mutations. This difference between vas and the spindle-class mutants analysed here indicates that Vas may act downstream of this mei-41-dependent meiotic checkpoint. To address this question more directly, Vas expression was studied in spindle-class-mutant backgrounds. At the level of whole-mount antibody staining, Vas does not appear to be affected. However, the mobility of Vas, as assessed by SDS polyacrylamide gel electrophoresis, is altered: Vas migration appears to be retarded in spn-B mutant ovaries as compared with wild-type ovaries or ovaries heterozygous for spn-B. These results indicate that Vas might be post-translationally regulated by the mei-41-dependent checkpoint pathway. In support of this interpretation, the mobility of Vas from ovarian lysates prepared from flies doubly mutant for spn-B and mei-W68 or mei-41 is restored to mobility levels observed in wild-type lysates. Taken together, these data support a model in which activation of the mei-41-dependent checkpoint pathway occurs in response to the presence of DSBs and leads to the modification of Vas, resulting in the downregulation of its activity and a consequent decrease in Grk translation (Ghabrial, 1999).

The COP9 signalosome (CSN) is linked to signaling pathways and ubiquitin-dependent protein degradation in yeast, plant and mammalian cells, but its roles in Drosophila development are just beginning to be understood. During oogenesis, one subunit of the CSN [COP9 complex homolog subunit 5 (CSN5/JAB1)], is required for meiotic progression and for establishment of both the AP and DV axes of the Drosophila oocyte. CSN5 mutations block the accumulation of the Egfr ligand Gurken in the oocyte, interfering with axis formation. CSN5 mutations also cause the modification of Vasa<, which is known to be required for Gurken translation. This CSN5 phenotype (defective axis formation, reduced Gurken accumulation and modification of Vasa) is very similar to the phenotype of the spindle-class genes that are required for the repair of meiotic recombination-induced DNA double-strand breaks. When these breaks are not repaired, a DNA damage checkpoint mediated by mei-41 is activated. Accordingly, the CSN5 phenotype is suppressed by mutations in mei-41 or by mutations in mei-W68, which is required for double strand break formation. These results suggest that, like the spindle-class genes, CSN5 regulates axis formation by checkpoint-dependent, translational control of Gurken. They also reveal a link between DNA repair, axis formation and the COP9 signalosome, a protein complex that acts in multiple signaling pathways by regulating protein stability (Doronkin, 2002).

vasa mutants show similar effects on axis determination and Grk protein accumulation as do spindle mutants and CSN5 germ-line clones. However, the vasa phenotypes are not suppressed by mei-41 or mei-W68 mutations, indicating that Vasa acts downstream of the meiotic checkpoint. Indeed, Vasa is one of the targets of Mei-41 activity since Vasa electrophoretic mobility is changed in spn-B mutants but restored in mei-41 spn-B double mutants (Ghabrial, 1999). Since Vasa protein binds to grk mRNA and is required for both its localization in the oocyte and its translation, it seems likely that the checkpoint effects on Grk accumulation are directly mediated by Vasa, although other Mei-41 targets cannot be excluded. The results of this study show effects of CSN5 mutants on Vasa mobility and are entirely consistent with the previous spn-B results, as would be expected if both types of mutants activate the same checkpoint (Doronkin, 2002).

Translational activation mediated by VASA

A discrete translation control element within the Nanos 3' untranslated region acts independently of the localization signal to mediate translational repression of unlocalized Nanos mRNA. This region, designated as nos translational control element (TCE) is evolutionarily conserved and is predicted to form a dual stem-loop structure. It is possible that TCE-mediated repression is achieved by blocking a stimulatory effect of a poly(A)-binding protein, or that a TCE-protein complex may itself block translational initiation or elongation. One component of the Nos localization machinery, VASAprotein, plays a role in overcoming TCE-mediated repression. RNAs bearing the Nos translational control element are completely inactive in embryos from vasa mutants. Thus, translation of Nos mRNA occurs by a specific derepression mechanism, requiring VAS protein (Gavis, 1996).

Bruno physically interacts with Vasa. Repression of translation by Bruno is alleviated once OSK mRNA is localized to the posterior pole of the oocyte. The mechanism of this process is unknown; however, it seems likely that the RNA helicase Vasa is involved, since it is localized to the posterior pole of the oocyte and is required for efficient activation of OSK translation. Immune sera, reactive to Bruno protein, cause a shift in the electrophoretic mobility of Osk protein (Webster, 1997).

Localization of specific mRNAs to distinct sites within the Drosophila oocyte is an early and key step in establishing the anterior-posterior and dorsal-ventral axes. A new function is described for the RNA helicase encoded by the "posterior" group gene vasa in the control of the localization of Gurken mRNA, a "dorsal-ventral" patterning gene. Two new ethyl methane sulfonate-induced, female sterile alleles of vas have been isolated. In these mutants, GRK mRNA fails to become localized properly and Grk protein is barely detectable. These mutants result in the ventralization of the eggshell, resembling defects of hypomorphic grk and Egfr mutants. Surprisingly, fs(1)K10, a recessive female sterile mutation that results in mislocalization of GRK mRNA to the anterior end of the oocyte, is epistatic to these vas alleles. In other words, fs(1)K10:vas double-mutants are dorsalized, just like fs(1)K10 alone. This result demonstrates that Grk protein levels sufficient to dorsalize the egg chamber can accumulate in vas mutants, if fs(1)K10 is also mutant. Taken together these results suggest that regulation of GRK mRNA localization normally occurs, directly or indirectly, through the Vas RNA-dependent RNA helicase and suggest that accumulation of Grk protein may normally depend on GRK mRNA localization (Tinker, 1998).

The Drosophila gene vasa (vas) encodes an RNA-binding protein required for embryonic patterning and germ cell specification. In vas mutants, translation of several germline mRNAs is reduced. Vas has been shown to interact directly with the Drosophila homolog of yeast translation initiation factor 2, encoded by a novel gene, dIF2. Embryos produced by vas/+;dIF2/+ females have pattern defects and fewer germline progenitor cells, indicating a functional interaction between endogenous vas and dIF2 activities. Mutations in other translation initiation factors do not enhance the vas phenotype, suggesting that dIF2 has a particular role in germ plasm function. It is concluded that Vas regulates translation of germline mRNAs by specific interaction with dIF2, an essential factor conserved from bacteria to humans (Carrera, 2000).

The 1144-amino acid dIF2 protein is 53% identical over 592 amino acids to yIF2. Still greater similarity (70% identity over 613 amino acids) exists between dIF2 and its human counterpart, hIF2, which can functionally rescue a yeast strain deficient for yIF2. dIF2 and yIF2 are more distantly related to archaean, mitochondrial, and eubacterial translation initiation factors 2 (IF2). Prokaryotic IF2 and eukaryotic yIF2 have been shown to deliver initiator methionine transfer RNA (Met-tRNAiMet) to the small ribosomal subunit, implying that the mechanism of translation initiation between prokaryotes and metazoans is more similar than had been anticipated. The Vas-interacting portion of one original dIF2 clone that was obtained in the two-hybrid screen contains the C-terminal half of dIF2 (amino acids 491-1144). Furthermore, the interaction between dIF2 and Vas depends on the presence of the domain common to DEAD box proteins in Vas, since an N-terminal truncated form of Vas containing only amino acids 1-310 fails to interact detectably (Carrera, 2000).

dIF2 mutants were used to determine whether vas and dIF2 interact functionally in vivo. vasPH165/+ females provide a sensitized background for gene dosage assays when cultured at 29°C, since 10%-30% of the embryos produced by such individuals fail to hatch and exhibit variable and minor patterning defects affecting both the anteroposterior and dorso-ventral axes. When one copy of dIF2 was removed from such females by introducing either the dIF2Delta1 or the Df(3L)G5 chromosome, the frequency of unhatched embryos increased from 10%-30% to as high as 89%. Many (30%-47%) of the unhatched embryos from vasPH165/+; dIF2/+ mothers exhibited severe segmentation defects, and an additional 26% showed a vas-like phenotype in which the fourth abdominal segment (A4) is completely or partially deleted. Segments A4 and A5 have been shown to be most sensitive to the activity of posterior group genes such as vas. Partial or total deletion of A4 was never observed among 62 embryos examined from control vasPH165/+; TM6B/+ females, nor among those from dIF2Delta1/+ females. In addition, and even more strikingly, evidence for the genetic interaction between vas and dIF2 was found by monitoring the number and position of germline precursor cells, termed pole cells. A marked reduction in the number of pole cells, formed in progeny of vasPH165/+; dIF2Delta1/+ trans-heterozygotes was observed, when compared to wild-type embryos or the progeny of females heterozygous for only vasPH165 or dIF2Delta1. In one set of experiments, almost all (90%) embryos produced by vasPH165/+; dIF2Delta1/+ trans-heterozygotes had fewer than 15 pole cells, as opposed to an average of 35-40 in wild-type embryos at the cellular blastoderm stage. Occasionally pole cells are completely absent in progeny of vasPH165/+; dIF2Delta1/+ trans-heterozygotes. Also, unlike pole cells in wild-type embryos, pole cells in progeny of vasPH165/+; dIF2Delta1/+ trans-heterozygotes are often interspersed with or positioned beneath somatic cells (Carrera, 2000).

This functional identification of a yIF2 homolog in Drosophila and its similarity to products predicted from mammalian genes indicates that the yIF2 translation initiation factor is highly conserved among metazoans. The physical interaction between Vas and dIF2 in vitro, the interaction between transfected vas and dIF2 in yeast cells, and that between the two endogenous genes in Drosophila all indicate that Vas may regulate translation of specific mRNAs through a direct link with the translation initiation machinery via dIF2. The failure to recover any other genes encoding known translation initiation factors from the interaction screen and the lack of a genetic interaction between vas and eIF4A or eIF4E mutations support this idea. Other DEAD box helicases related to Vas may function similarly; S. cerevisiae ded1 mutants exhibit defects in translation initiation. However, the precise step at which Vas exerts its function in the translation of a specific set of mRNAs is unclear. Since Vas is an RNA-binding protein that presumably interacts with mRNA, it appears likely that Vas acts at the level of mRNA recruitment to the ribosome. However, yIF2 has been implicated in promoting Met-tRNAiMet binding to ribosomes, which takes place prior to the recruitment of mRNA. Further analysis of VAS and dIF2 will provide additional insight into the specific functions of both of these molecules, which are conserved in mammals, and how they cooperate to initiate translation (Carrera, 2000).

Vasa promotes Drosophila germline stem cell differentiation by activating mei-P26 translation by directly interacting with a (U)-rich motif in its 3' UTR

Vasa (Vas) is a DEAD-box RNA-binding protein required in Drosophila at several steps of oogenesis and for primordial germ cell (PGC) specification. Vas associates with eukaryotic initiation factor 5B (eIF5B), and this interaction has been implicated in translational activation of gurken mRNA in the oocyte. Vas is expressed in all ovarian germline cells, and aspects of the vas-null phenotype suggest a function in regulating the balance between germline stem cells (GSCs) and their fate-restricted descendants. A biochemical approach was used to recover Vas-associated mRNAs. mei-P26, whose product represses microRNA activity and promotes GSC differentiation, was identified. vas and mei-P26 mutants interact, and mei-P26 translation is substantially reduced in vas mutant cells. In vitro, Vas protein bind specifically to a (U)-rich motif in the mei-P26 3' untranslated region (UTR), and Vas-dependent regulation of GFP-mei-P26 transgenes in vivo was dependent on the same (U)-rich 3' UTR domain. The ability of Vas to activate mei-P26 expression in vivo was abrogated by a mutation that greatly reduces its interaction with eIF5B. Taken together, these data support the conclusion that Vas promotes germ cell differentiation by directly activating mei-P26 translation in early-stage committed cells (Liu, 2009).

This study demonstrated that Vas regulates mei-P26 expression in vivo, and that a (U)-rich element in the mei-P26 3' UTR interacts with Vas in vitro and is required for Vas-mediated regulation in vivo. DEAD-box helicases such as Vas were not believed previously to be sequence-specific nucleic acid-binding proteins. This study has shown however, that Vas demonstrates specific binding, but that this requires domains that are distinct from the motifs shared by all DEAD-box proteins. Vas contains nine RGG repeats located between amino acids 17 and 165, within the region implicated in binding specificity that lies outside of the canonical DEAD-box segment. While the region N-terminal to the common DEAD-box motifs is highly variable in the sequences of Vas orthologs from different species, the presence of RGG repeats within that region is conserved; for example, zebrafish Vas contains nine such motifs, and human Vas (DDX4) contains four. Two RGG repeats are present in mammalian fragile X mental retardation protein (FMRP), and have been shown to specifically recognize a G quartet structure in semaphorin 3F RNA, indicating that this motif can discriminate among target RNAs. RGG repeats are often present in proteins that contain other RNA-binding domains; for example, Vas contains a DEAD-box signature, while FMRP contains two hnRNPK homology (KH) domains; thus it has been proposed that they serve an auxiliary role in RNA binding. It is suggested that the RGG repeats of Vas play such a role by conferring specificity to its association with RNA. RGG motifs are also targets for arginine methyltransferases, and arginine methylation has been linked to modulating the RNA-binding activity of heterogeneous nuclear RNP (hnRNP) A1. It is tempting to speculate that the RNA-binding activity of Vas might be similarly modulated, perhaps through the activity of the arginine methyltransferase Capsuléen, which like Vas is essential for germ cell specification (Liu, 2009).

A model has been proposed for Vas-mediated translational activation whereby Vas recruits eIF5B to target mRNAs. This study suggests that Vas can itself discriminate among potential mRNA targets, although it cannot be excluded that Vas may be recruited to other target mRNAs through indirect associations involving partner RNA-binding proteins. Precedents for regulation of translation at the step of subunit joining exist in several systems. For example, in early erythroid precursor cells, the mRNA encoding 15-lipoxygenase (r15-LOX) is translationally silenced at this step of translation initiation, dependent on a cytidine-rich 3' UTR element termed DICE (differentiation control element) and on two RNA-binding proteins: hnRNP K and hnRNP E1. Phosphorylation of a specific tyrosine residue of hnRNP K by c-Src reduces its affinity for DICE, thus activating translation. Another example is provided by the ASH1 mRNA in Saccharomyces cerevisiae, which is translationally repressed before localizing to the bud cortex by Puf6p, which binds the RNA and blocks subunit joining through an interaction with eIF5B. As for r15-LOX, repression is alleviated by phosphorylation of the RNA-binding protein. Vas differs from these other translational regulators in that it positively regulates its targets; like hnRNP K and Puf6p, however, post-translational modification of Vas has also been linked to a reduction of its activity (Liu, 2009).

Several phenotypes manifested in vas-null ovaries point toward a function for Vas in restricting cell fate during cystocyte divisions, and these could result from reduced mei-P26 expression. The relationship between Mei-P26 and Vas may be more complex, however, as both are linked to small RNA metabolism. Mei-P26 binds to AGO1, an RNase that is a core component of the RNA-induced silencing complex (RISC) that is involved in miRNA-mediated translational repression and RNA degradation pathways. Both Mei-P26 and AGO1 have been implicated in regulating GSC fate; Mei-P26 restricts growth and proliferation and promotes differentiation, while AGO1 does the reverse. Piwi, a key component of rasiRNA and piwi-interacting RNA (piRNA) pathways, has been implicated in stem cell self-renewal. Vas is associated with Piwi, Aubergine, and other components of the rasiRNA pathway, and has itself been linked to retrotransposon silencing. Therefore, Vas appears to be involved in both the AGO1 pathway, through its regulation of Mei-P26, and the rasiRNA pathway, potentially making it a key regulator of processes mediated by small RNAs in GSCs and early-stage committed cells (Liu, 2009).

A role for Vasa in regulating mitotic chromosome condensation in Drosophila

Vasa (Vas) is a conserved DEAD-box RNA helicase expressed in germline cells that localizes to a characteristic perinuclear structure called nuage. Previous studies have shown that Vas has diverse functions, with roles in regulating mRNA translation, germline differentiation, pole plasm assembly, and piwi-interacting RNA (piRNA)-mediated transposon silencing. Although vas has also been implicated in the regulation of germline proliferation in Drosophila and mice, little is known about whether Vas plays a role during the mitotic cell cycle. This study reports a translation-independent function of vas in regulating mitotic chromosome condensation in the Drosophila germline. During mitosis, Vas facilitates robust chromosomal localization of the condensin I components Barren (Barr) and CAP-D2. Vas specifically associates with Barr and CAP-D2, but not with CAP-D3 (a condensin II component). The mitotic function of Vas is mediated by the formation of perichromosomal Vas bodies during mitosis, which requires the piRNA pathway components aubergine and spindle-E. These results suggest that Vas functions during mitosis and may link the piRNA pathway to mitotic chromosome condensation in Drosophila (Pek, 2010).

In Drosophila, the association of Barr with mitotic chromosomes is highly dynamic, and Barr is loaded primarily at the centromeric regions before spreading distally. How such a dynamic event is regulated remains unclear. It is suggested that during mitosis, Vas forms bodies that are in close proximity to pericentromeric piRNA-generating loci, where they function to facilitate chromosomal recruitment of Barr to participate in the robust condensation of chromosomes. Alternatively, despite the localization to pericentromeres, Vas may promote the long-range stable association of Barr with entire mitotic chromosomes by a yet unknown mechanism. Barr appears to be a principal effector of Vas, because ectopic expression and enhanced localization of Barr to mitotic chromosomes suppresses, to a large extent, chromosomal defects in vas mutants. Furthermore, mitotic localization of Vas correlates with the timing of active chromosomal loading of Barr during prometaphase, and Vas interacts specifically with Barr and CAP-D2 (condensin I components). Interestingly, relocalization of Vas bodies to regions between segregating chromosomes were also observed during anaphase. In a small fraction of vas mutant germline cells (3.2%), segregation defects are not suppressed by ectopic expression of Barr, suggesting a condensin I-independent role for Vas during anaphase. It would be interesting to examine whether Vas also functions in a tether-based mechanism to facilitate the chromosome segregation seen in neuroblasts (Pek, 2010).

Studies have shown that loss of condensin function triggers the spindle assembly checkpoint (SAC). Consistent with the observation that vas promotes robust chromosomal localization of condensin I components, it was observed that the delay in mitotic progression in vas mutants is partially rescued by reducing a copy of the SAC genes bubR1 or zw10, suggesting that the SAC may be activated in vas mutants. However, it cannot be completely excluded that Vas may also regulate other factors that trigger or suppress the SAC (Pek, 2010).

Because Vas has a wide range of functions, vas mutants exhibit a pleiotropic phenotype, including showing a mild defect in stem cell maintenance and germline differentiation. In aub and spn-E mutants, in which the mitotic function of Vas is perturbed, less robust Barr localization was observed and a delay in chromosome condensation during prometaphase, but germline differentiation was unaffected. Similarly, cap-g (condensin I) mutants are female sterile and have impaired Barr localization and delayed chromosome condensation during prometaphase. In these mutants, although the dynamics of the synaptonemal complex during meiosis are perturbed, progression of oogenesis is unaffected. Moreover, introduction of the vasΔ617 or barr-GFP transgene into vas mutants restores mitotic defects of prometaphase delay and lagging chromosomes, but not the later-stage germline differentiation defects. Taken together, these data suggest that the function of vas in chromosome condensation is not required for progression of germline differentiation but is required to ensure the fidelity of chromosome segregation, which may contribute in part to the age-dependent atrophy, oocyte differentiation defect, and sterility in vas mutants. Analysis of a Vas variant that specifically abrogates the interaction of Vas with Barr would give insight into the molecular function of Vas in regulating Barr (Pek, 2010).

This study identifies a possible link between the piRNA pathway and chromosome condensation and segregation during mitosis. This is particularly intriguing because recent studies in C. elegans have shown that 22G-RNAs and several P granule components function to organize chromosomes with holocentric centromeres during mitosis. Together with this study in Drosophila, these studies raise an interesting possibility that components of the nuage and small RNA pathways may play a unique role in organizing chromosomes during the cell cycle in eukaryotes. It will be interesting to dissect the molecular involvement of piRNAs, siRNAs, and other small RNA pathway components in regulating mitotic chromosome condensation and segregation in germline cells (Pek, 2010).

It is proposed that Vas has a role in regulating chromosome condensation during mitosis of germline cells at least in part by facilitating robust chromosomal localization of Barr. This process is mediated by the formation of Vas perichromosomal bodies during mitosis and depends on aub and spn-E (Pek, 2010).

Dorso-ventral axis formation of the Drosophila oocyte requires Cyclin G

In general, cyclins control the cell cycle. Not so the atypical cyclins, which are required for diverse cellular functions such as for genome stability or for the regulation of transcription and translation. The atypical Cyclin G (CycG) gene of Drosophila has been involved in the epigenetic regulation of abdominal segmentation, cell proliferation and growth, based on overexpression and RNAi studies, but detailed analyses were hampered by the lack of a cycG mutant. For further investigations, the cycG locus was subjected to a detailed molecular analysis. Moreover, a cycG null mutant was studied that was recently established. The mutant flies are homozygous viable, however, the mutant females are sterile and produce ventralized eggs. This study shows that this egg phenotype is primarily a consequence of a defective Epidermal Growth Factor Receptor (EGFR) signalling pathway. By using different read outs, it was demonstrated that cycG loss is tantamount to lowered EGFR signalling. Inferred from epistasis experiments, it is concluded that CycG promotes the Grk signal in the oocyte. Abnormal accumulation but regular secretion of the Grk protein suggests defects of Grk translation in cycG mutants rather than transcriptional regulation. Accordingly, protein accumulation of Vasa, which acts as an oocyte specific translational regulator of Grk in the oocyte is abnormal. A role is proposed of cycG in processes that regulate translation of Grk and hence, influence EGFR-mediated patterning processes during oogenesis (Nigel, 2012).

This study has shown the ventralized phenotype of cycG mutant eggs results from a downregulation of the EGFR signalling pathway. CycG is required for the translational rather than the transcriptional regulation of Grk within the oocyte. One may think of several mechanisms through which CycG might influence grk mRNA translation. DroID, a comprehensive resource for gene interactions in Drosophila, identified several protein interaction partners of CycG that may relate to its role as translational regulator of grk mRNA. Notably, CycG was identified in vivo in protein complexes together with RNA binding proteins, several potential splice factors and translational regulators, for example Eukaryotic translation initiation factor 4AIII (eIF4AIII), Bicoid stability factor (Bsf), Barentz (Btz), Cap binding protein 80 (Cbp80), and SC35. Moreover, a large scale yeast-two hybrid screen picked Gustavus (Gus) as a partner of CycG. This interaction gives a direct link to dorso-ventral axis formation, since Gus is required for the correct localization of Vasa in the Drosophila egg. Most interestingly, Vasa protein levels appear reduced in cycGHR7 mutant ovaries. This may suggest that CycG is a cofactor of Gus, which acts on Vasa stability in the oocyte. In the absence of CycG, Vasa may be degraded more rapidly. Since Vasa is required for efficient translation of Grk, downregulation of Vasa could affect Grk accumulation and result in ventralized eggs. Alternatively, CycG may affect Grk translation indirectly. It was shown that a meiotic checkpoint induced by unrepaired double-strand breaks affects efficient translation of Grk, thereby causing a ventralized eggshell phenotype. A typical example are mutants in the spindle-A (spn-A) gene, which encodes a homologue of the Rad51 recombinase and which is required for double-strand break repair. Spn-A and CycG were found as molecular partners in yeast-two hybrid screens, and hence, CycG may in fact be involved in meiotic recombination repair. Finally, mutants affecting the rasi-RNA pathway cause similar ventralized eggs: in these mutants DNA breaks accumulate due to defects in transposon silencing, effecting the meiotic checkpoint as well. Because CycG has been involved in radiation sensitivity in both Drosophila and mammals, it is tempting to speculate that it may be involved in double-strand break repair during meiosis, as well. Hence, in the absence of CycG, meiotic double-strand breaks would accumulate, thereby activating the meiotic checkpoint and indirectly affecting grk mRNA translation and axis formation of the oocyte (Nigel, 2012).

Protein Interactions

Fat facets interacts with Vasa in the Drosophila pole plasm and protects it from degradation

Anterior-posterior patterning and germ cell specification in Drosophila requires the establishment, during oogenesis, of a specialized cytoplasmic region termed the pole plasm. Numerous RNAs and proteins accumulate to the pole plasm and assemble in polar granules. Translation of some of these RNAs is generally repressed and active only in pole plasm. Vasa (Vas) protein, an RNA helicase and a component of polar granules, is essential maternally for posterior patterning and germ cell specification, and Vas is a candidate translational activator in the pole plasm. Vas is stabilized within the pole plasm in that it is initially present throughout the entire embryo but strictly limited to the pole cells by the cellular blastoderm stage. hsp83 mRNA, which accumulates in the pole plasm through a stabilization-degradation mechanism, is another example. A biochemical approach has been used to identify proteins that copurify with Vas in crosslinked extracts. Prominent among these proteins was the ubiquitin-specific protease Fat facets (Faf), a pole plasm component, but one whose roles in posterior patterning and germ line specification have remained unclear. Evidence suggests that Faf interacts with Vas physically and reverses Vas ubiquitination, thereby stabilizing Vas in the pole plasm (Liu, 2003).

Vas and Faf can be copurified from chemically crosslinked embryonic and ovarian extracts; in faf mutants, the ubiquitination of Vas is increased and its levels are decreased in ovaries and more strikingly in progeny embryos. The simplest interpretation of these data is that Vas is a specific substrate for the deubiquinating enzyme Faf. It is believed the reduction of localized Osk observed in faf mutant ovaries is an indirect result of the reduced stability of Vas, since vas function is required for the stable accumulation of Osk in the pole plasm (Liu, 2003).

Heterozygotes for a vas null mutation produce embryos with a 20%-25% reduction in pole cell number, yet such embryos, unlike progeny of faf mutant mothers, show prominent posterior Vas staining. The severe phenotype of embryos produced by homozygous faf mothers renders difficult a direct analysis of its requirement in the pole plasm. However, based on the vas heterozygous phenotype, the obvious reduction in posterior Vas accumulation in faf mutants should affect development, and therefore Faf-mediated stabilization of Vas must contribute to pole plasm function. Faf-mediated protection of Vas is mostly restricted to the pole plasm, perhaps because during oogenesis, Faf itself becomes localized to the posterior pole and is incorporated into pole cells. As is the case for other pole plasm components, Faf localization depends on osk function, and a role for Faf in germ cell differentiation and development has been proposed. Interestingly, Usp9x, a bona fide mouse ortholog of Faf, is predominantly expressed in both germ cell and supporting cell lineages during gonadal development, suggesting a conserved role for Faf in mammalian germ line development (Liu, 2003).

Drosophila valois encodes a divergent WD protein that is required for Vasa localization and Oskar protein accumulation

valois (vls) was identified as a posterior group gene in the initial screens for Drosophila maternal-effect lethal mutations. Despite its early genetic identification, it has not been characterized at the molecular level until now. vls encodes a divergent WD domain protein and the three available EMS-induced point mutations cause premature stop codons in the vls ORF. A null allele was identified that has a stronger phenotype than the EMS mutants. The vlsnull mutant shows that vls+ is required for high levels of Oskar protein to accumulate during oogenesis, for normal posterior localization of Oskar in later stages of oogenesis and for posterior localization of the Vasa protein during the entire process of pole plasm assembly. There is no evidence for vls being dependent on an upstream factor of the posterior pathway, suggesting that Valois protein (Vls) instead acts as a co-factor in the process. Based on the structure of Vls, the function of similar proteins in different systems and phenotypic analysis, it seems likely that vls may promote posterior patterning by facilitating interactions between different molecules (Cavey, 2005).

Because all aspects of the vls mutant phenotype observed in embryos, including abdominal segment deletions, lack of pole cells, gastrulation defects and weak ventralization are rescued completely by a vls transgene and not even partially by a chk2 transgene, it is concluded that vls alone has a developmental requirement. Furthermore, chk2 function is only clearly required upon activation of cell cycle checkpoints. The vls phenotypes are reminiscent of a collapse of pole plasm assembly that seems to occur around stage 10 of oogenesis in vlsnull mutants. vas is crucial for the pole plasm to assemble properly and recruit the mRNAs and proteins required for pole cell specification and abdominal patterning. Genetic evidence implicates vas in the translational activation of several targets during oogenesis, including osk, grk and, in particular, nos at the posterior pole of the embryo. Vasa levels directly correlate with pole plasm activity, pole cell formation being more vulnerable to decreased Vasa levels than is abdominal patterning. Immunostaining for Vasa has been reported to show indistinguishable Vasa accumulation at the posterior pole of vls mutant and wild-type oocytes, and young embryos. These studies, performed with the homo- and hemi-zygous EMS mutants, showed a loss of posterior localization in the embryos from vls mothers sometime between fertilization and pole cell formation. The current study used vas-eGFP transgenes to assess the posterior localization of Vasa in vlsnull and hemizygous EMS alleles in detail. Maximal localization was still very weak and was found in oocytes and embryos from vlsEMS mothers. In vlsnull mutants a nearly complete failure to localize Vas-eGFP at the posterior pole was observed. This failure coincides with the collapse of the pole plasm and is probably the cause for the various embryonic phenotypes mentioned above. Consistent with this, the observed Vasa localization defects parallel the severity of the phenotypes of these vls alleles. The weak accumulation of Vasa at the posterior of vlsPG65 hemizygous oocytes gives rise to a grandchildless phenotype, whereas the almost complete absence of Vasa from the posterior of vlsnull oocytes results in a fully penetrant maternal-effect lethal phenotype (Cavey, 2005).

vls is thus required during oogenesis for the localization (transport or anchoring) of Vasa to the posterior cortex of the oocyte. The fact that Vls is not specifically enriched at the posterior may suggest that it acts to modify or transport pole plasm components before they reach the posterior pole. Preliminary experiments also failed to produce evidence that Vls and Vasa are part of the same protein complex. This suggests that the mode of action of vls on Vasa localization is transient or indirect. The fact that osk mRNA and protein are initially correctly localized implies that oocyte polarity is normal in vls mutants and that vls is not required for osk mRNA localization. Levels of Osk protein isoforms are then reduced in later stages and Western analysis reveals a much more drastic decrease of overall Osk levels than immunostaining does for both types of vls alleles. This suggests that most of the drop in Osk levels occurs during the late stages of oogenesis, when the vitelline membrane prevents antibody staining for oocyte Osk. Therefore, it seems that shortly after initiating pole plasm assembly, Osk fails to be maintained at the posterior of vls mutants and progressively disappears, concurrent with a complete collapse of the pole plasm (Cavey, 2005).

Several lines of evidence implicate the Short Osk isoform in directly anchoring Vas. Short Osk interacts strongly with Vasa in the two-hybrid system and recruits Vasa when ectopically localized in the oocyte. Because Vas-eGFP mis-localization patterns in stage 10 oocytes are indistinguishable in vls and osk54 mutants, vls could act directly at the level of Osk accumulation (e.g. in stimulating translation of osk), which is necessary for anchoring Vasa at the posterior pole. In contrast, it is also possible that vls acts primarily on Vasa protein localization. Because Vasa also seems to act in a positive feedback loop back on Osk protein accumulation, the lack of Vasa localization in vls mutants would then also preclude maintenance of posterior accumulation of Osk protein. In vls mutants, Osk levels appear to decrease just slightly after Vasa should have localized to the posterior pole, thus it appears that the failure to localize Vasa could be the cause of the pole plasm collapse in vls mutants. To investigate these issues further, Osk levels in vas and tud mutants were compared with those in vls mutants by Western analysis where a more significant drop was detected than by immunostaining. This analysis revealed generally stronger phenotypes for vls than for vas and tud mutants. A comparable decrease of Short Osk levels was observed on Western blots of vls, vas and tud mutant extracts, but with slight differences in the extent of reduction of the hyper- and hypo-phosphorylated forms, both of which are more severely affected in vls mutants. In addition, a clear reduction of Long Osk levels was observed in vls, a minor reduction in tud, but none in vas mutant extracts. However, this analysis is complicated by the fact that the vas and tud alleles that are useful and available, respectively, for these experiments, are not nulls. Their residual activity may therefore maintain Osk at the posterior for a longer period of time. These data are thus consistent with the idea that vls acts on either pathway target, Vasa or Osk, in a process which could involve additional intermediates that remain to be identified (Cavey, 2005).

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

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

vasa: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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