nanos: Biological Overview | Evolutionary Homologs | Regulation | mRNA localization and post-transcriptional regulation | Developmental Biology | Effects of Mutation | References

Gene name - nanos

Synonyms -

Cytological map position - 91F13

Function - translational repressor - RNA binding protein

Keywords - posterior group - oocyte

Symbol - nos

FlyBase ID:FBgn0002962

Genetic map position - 3-66.2

Classification - Nanos RNA binding domain

Cellular location - cytoplasmic



NCBI links: | Entrez Gene
Recent literature
Little, S. C., Sinsimer, K. S., Lee, J. J., Wieschaus, E. F. and Gavis, E. R. (2015). Independent and coordinate trafficking of single Drosophila germ plasm mRNAs. Nat Cell Biol. PubMed ID: 25848747
Summary:
Messenger RNA localization is a conserved mechanism for spatial control of protein synthesis, with key roles in generating cellular and developmental asymmetry. Whereas different transcripts may be targeted to the same subcellular domain, the extent to which their localization is coordinated is unclear. Using quantitative single-molecule imaging, this study analysed the assembly of Drosophila germ plasm mRNA granules inherited by nascent germ cells. The germ-cell-destined transcripts nanos, cyclin B and polar granule component travel within the oocyte as ribonucleoprotein particles containing single mRNA molecules but co-assemble into multi-copy heterogeneous granules selectively at the posterior of the oocyte. The stoichiometry and dynamics of assembly indicate a defined stepwise sequence. The data suggest that co-packaging of these transcripts ensures their effective segregation to germ cells. In contrast, compartmentalization of the germline determinant oskar mRNA into different granules limits its entry into germ cells. This exclusion is required for proper germline development.
Weidmann, C.A., Qiu, C., Arvola, R.M., Lou, T.F., Killingsworth, J., Campbell, Z.T., Tanaka Hall, T.M. and Goldstrohm, A.C. (2016). Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio. Elife [Epub ahead of print]. PubMed ID: 27482653
Summary:
Collaboration among the multitude of RNA-binding proteins (RBPs) is ubiquitous, yet understanding of these key regulatory complexes has been limited to single RBPs. This study investigated combinatorial translational regulation by Drosophila Pumilio (Pum) and Nanos (Nos), which control development, fertility, and neuronal functions. The obtained results show how the specificity of one RBP (Pum) is modulated by cooperative RNA recognition with a second RBP (Nos) to synergistically repress mRNAs. Crystal structures of Nos-Pum-RNA complexes reveal that Nos embraces Pum and RNA, contributes sequence-specific contacts, and increases Pum RNA-binding affinity. Nos shifts the recognition sequence and promotes repression complex formation on mRNAs that are not stably bound by Pum alone, explaining the preponderance of sub-optimal Pum sites regulated in vivo. These results illuminate the molecular mechanism of a regulatory switch controlling crucial gene expression programs, and provide a framework for understanding how the partnering of RBPs evokes changes in binding specificity that underlie regulatory network dynamics.
Ji, Y. and Tulin, A. V. (2016). Poly(ADP-ribosyl)ation of hnRNP A1 protein controls translational repression in Drosophila. Mol Cell Biol [Epub ahead of print]. PubMed ID: 27402862
Summary:
Poly(ADP-ribosyl)ation of heterogeneous nuclear RNA binding proteins (hnRNP) regulates the post-transcriptional fate of RNA during development. Drosophila hnRNP A1, Hrp38, is required for germline stem cell maintenance and oocyte localization. The mRNA-targets regulated by Hrp38 are mostly unknown. This study identified 428 Hrp38-associated gene transcripts in the fly ovary, including mRNA of the translational repressor Nanos. Hrp38 binds to the 3' untranslated region (3' UTR) of Nanos mRNA, which contains a translation control element. Translation of the luciferase reporter bearing Nanos 3' UTR is enhanced by dsRNA-mediated Hrp38 knockdown as well as by mutating potential Hrp38-binding sites. These data show that poly(ADP-ribosyl)ation inhibits Hrp38 binding to Nanos 3' UTR, increasing the translation in vivo and in vitro Hrp38 and Parg null mutants showed an increased ectopic Nanos translation early in the embryo. It is concluded that Hrp38 represses Nanos translation, whereas its poly(ADP-ribosyl)ation relieves the repression effect, allowing restricted Nanos expression in the posterior germ plasm during oogenesis and early embryogenesis.
Ote, M., Ueyama, M. and Yamamoto, D. (2016). Wolbachia protein TomO targets nanos mRNA and restores germ stem cells in Drosophila Sex-lethal mutants. Curr Biol [Epub ahead of print]. PubMed ID: 27498563
Summary:
Wolbachia, endosymbiotic bacteria prevalent in invertebrates, manipulate their hosts in a variety of ways: they induce cytoplasmic incompatibility, male lethality, male-to-female transformation, and parthenogenesis. However, little is known about the molecular basis for host manipulation by these bacteria. In Drosophila melanogaster, Wolbachia infection makes otherwise sterile Sex-lethal (Sxl) mutant females capable of producing mature eggs. Through a functional genomic screen for Wolbachia genes with growth-inhibitory effects when expressed in cultured Drosophila cells, this study identified the Wolbachia gene WD1278 encoding a novel protein called toxic manipulator of oogenesis (TomO), which phenocopies some of the Wolbachia effects in Sxl mutant D. melanogaster females. TomO enhances the maintenance of germ stem cells (GSCs) by elevating Nanos (Nos) expression via its interaction with nos mRNA, ultimately leading to the restoration of germ cell production in Sxl mutant females that are otherwise without GSCs.
Tamayo, J. V., Teramoto, T., Chatterjee, S., Hall, T. M. and Gavis, E. R. (2017). The Drosophila hnRNP F/H homolog Glorund uses two distinct RNA-binding modes to diversify target recognition. Cell Rep 19(1): 150-161. PubMed ID: 28380354
Summary:
The Drosophila hnRNP F/H homolog, Glorund (Glo), regulates nanos mRNA translation by interacting with a structured UA-rich motif in the nanos 3' untranslated region. Glo regulates additional RNAs, however, and mammalian homologs bind G-tract sequences to regulate alternative splicing, suggesting that Glo also recognizes G-tract RNA. To gain insight into how Glo recognizes both structured UA-rich and G-tract RNAs, mutational analysis was used guided by crystal structures of Glo's RNA-binding domains, and two discrete RNA-binding surfaces were identified that allow Glo to recognize both RNA motifs. By engineering Glo variants that favor a single RNA-binding mode, it was shown that a subset of Glo's functions in vivo is mediated solely by the G-tract binding mode, whereas regulation of nanos requires both recognition modes. These findings suggest a molecular mechanism for the evolution of dual RNA motif recognition in Glo that may be applied to understanding the functional diversity of other RNA-binding proteins.
Gotze, M., Dufourt, J., Ihling, C., Rammelt, C., Pierson, S., Sambrani, N., Temme, C., Sinz, A., Simonelig, M. and Wahle, E. (2017). Translational repression of the Drosophila nanos mRNA involves the RNA helicase Belle and RNA coating by Me31B and Trailer hitch. RNA 23(10): 1552-1568. PubMed ID: 28701521
Summary:
Translational repression of maternal mRNAs is an essential regulatory mechanism during early embryonic development. Repression of the Drosophila nanos mRNA, required for the formation of the anterior-posterior body axis, depends on the protein Smaug binding to two Smaug recognition elements (SREs) in the nanos 3' UTR. In a comprehensive mass spectrometric analysis of the SRE-dependent repressor complex, Smaug, Cup, Me31B, Trailer hitch, eIF4E, and PABPC were identified, in agreement with earlier data. As a novel component, the RNA-dependent ATPase Belle (DDX3) was found, and its involvement in deadenylation and repression of nanos was confirmed in vivo. Smaug, Cup, and Belle bound stoichiometrically to the SREs, independently of RNA length. Binding of Me31B and Tral was also SRE-dependent, but their amounts were proportional to the length of the RNA and equimolar to each other. It is suggested that "coating" of the RNA by a Me31B*Tral complex may be at the core of repression.
Malik, S., Jang, W. and Kim, C. (2017). Protein interaction mapping of translational regulators affecting expression of the critical stem cell factor Nos. Dev Reprod 21(4): 449-456. PubMed ID: 29354790
Summary:
The germline stem cells of the Drosophila ovary continuously produce eggs throughout the life- span. Intricate regulation of stemness and differentiation is critical to this continuous production. The translational regulator Nos is an intrinsic factor that is required for maintenance of stemness in germline stem cells. Nos expression is reduced in differentiating cells at the post-transcriptional level by diverse translational regulators. However, molecular mechanisms underlying Nos repression are not completely understood. Through three distinct protein-protein interaction experiments, this study identified specific molecular interactions between translational regulators involved in Nos repression. The findings suggest a model in which protein complexes assemble on the 3' untranslated region of Nos mRNA in order to regulate Nos expression at the post-transcriptional level.
Sugimori, S., Kumata, Y. and Kobayashi, S. (2018). Maternal Nanos-dependent RNA stabilization in the primordial germ cells of Drosophila embryos. Dev Growth Differ 60(1): 63-75. PubMed ID: 29278271
Summary:
Nanos (Nos) is an evolutionary conserved protein expressed in the germline of various animal species. In Drosophila, maternal Nos protein is essential for germline development. In the germline progenitors, or the primordial germ cells (PGCs), Nos binds to the 3' UTR of target mRNAs to repress their translation. In contrast to this prevailing role of Nos, this study reports that the 3' UTR of CG32425 mRNA mediates Nos-dependent RNA stabilization in PGCs. The level of mRNA expressed from a reporter gene fused to the CG32425 3' UTR was significantly reduced in PGCs lacking maternal Nos (nos PGCs) as compared with normal PGCs. By deleting the CG32425 3' UTR, the region was identified required for mRNA stabilization, which includes Nos-binding sites. In normal embryos, CG32425 mRNA was maternally supplied into PGCs and remains in this cell type during embryogenesis. However, as expected from the reporter assay, the levels of CG32425 mRNA and its protein product expressed in nos PGCs were lower than in normal PGCs. Thus, it is proposed that Nos protein has dual functions in translational repression and stabilization of specific RNAs to ensure proper germline development.
Ote, M. and Yamamoto, D. (2018). Enhancing Nanos expression via the bacterial TomO protein is a conserved strategy used by the symbiont Wolbachia to fuel germ stem cell maintenance in infected Drosophila females. Arch Insect Biochem Physiol: e21471. PubMed ID: 29701280
Summary:
The toxic manipulator of oogenesis (TomO) protein has been identified in the wMel strain of Wolbachia that symbioses with the vinegar fly Drosophila melanogaster, as a protein that affects host reproduction. TomO protects germ stem cells (GSCs) from degeneration, which otherwise occurs in ovaries of host females that are mutant for the gene Sex-lethal (Sxl). TomO homologs were isolated from wPip, a Wolbachia strain from the mosquito Culex quinquefasciatus. One of the homologs, TomOwPip 1, exerted the GSC rescue activity in fly Sxl mutants when lacking its hydrophobic stretches. The GSC-rescuing action of the TomOwPip 1 variant was ascribable to its abilities to associate with Nanos (nos) mRNA and to enhance Nos protein expression. The analysis of structure-activity relationships with TomO homologs and TomO deletion variants revealed distinct modules in the protein that are each dedicated to different functions, i.e., subcellular localization, nos mRNA binding or Nos expression enhancement. It is proposed that modular reshuffling is the basis for structural and functional diversification of TomO protein members.
Kawaguchi, S., Ueki, M. and Kai, T. (2020). Drosophila MARF1 ensures proper oocyte maturation by regulating nanos expression. PLoS One 15(4): e0231114. PubMed ID: 32243476
Summary:
Meiosis and oocyte maturation are tightly regulated processes. The meiosis arrest female 1 (MARF1) gene is essential for meiotic progression in animals; however, its detailed function remains unclear. This study examined the molecular mechanism of dMarf1, a Drosophila homolog of MARF1 encoding an OST and RNA Recognition Motif (RRM) -containing protein for meiotic progression and oocyte maturation. Although oogenesis progressed in females carrying a dMarf1 loss-of-function allele, the dMarf1 mutant oocytes were found to contain arrested meiotic spindles or disrupted microtubule structures, indicating that the transition from meiosis I to II was compromised in these oocytes. The expression of the full-length dMarf1 transgene, but none of the variants lacking the OST and RRM motifs or the 47 conserved C-terminal residues among insect groups, rescued the meiotic defect in dMarf1 mutant oocytes. These results indicate that these conserved residues are important for dMarf1 function. Immunoprecipitation of Myc-dMarf1 revealed that several mRNAs are bound to dMarf1. Of those, the protein expression of nanos (nos), but not its mRNA, was affected in the absence of dMarf1. In the control, the expression of Nos protein became downregulated during the late stages of oogenesis, while it remained high in dMarf1 mutant oocytes. It is proposed that dMarf1 translationally represses nos by binding to its mRNA. Furthermore, the downregulation of Nos induces cycB expression, which in turn activates the CycB/Cdk1 complex at the onset of oocyte maturation.
BIOLOGICAL OVERVIEW

nanos is required maternally for two processes during oogenesis. nanos is expressed in the early germarium where it is needed for continued egg chamber production. In mature eggs and the developing zygote it is also required to specify posterior identity. Nanos mRNA is localized to the pole plasm, a specialized cytoplasm later incorporated into pole cells, the precursors of the fly's reproductive system (Wang, 1991, 1994).

For its role in specifying posterior identity, nos is required briefly but immediately after fertilization. It has two known targets: hunchback and bicoid. Translation of both targets is inhibited by the binding of NOS protein to the 3' untranslated region of their mRNAs. While Nanos itself does not bind to the "Nanos response elements" of HB mRNA, Pumilio does bind and appears to bring Nanos protein into the complex (Murata, 1995). Inhibition of Hunchback mRNA, its maternal transcript in particular, guarantees an anterior to posterior gradient of HB protein. The restriction of HB mRNA permits the ordered expression of genes specifying the abdominal region: Krüppel, knirps and giant. In the absence of nos, all abdominal segments are lost. It would seem that one of the requirements for NOS mRNA localization, involving proteins of the posterior group are imposed by the presence of the Nanos response elements in BCD and HB mRNAs (Wharton, 1991).

Eight genes are required for NOS mRNA localization. Among these are oskar, a gene necessary for organization of the germ plasm, pumilio, whose coded protein binds the 3'UTR of NOS mRNA, and vasa, coding for an ATP dependent RNA helicase. Translation of NOS mRNA requires the posterior localization. Consequently when the NOS mRNA is freed from its association with the posterior pole NOS translation does not proceed and embryos develop abdominal defects notwithstanding the stability of the unlocalized NOS mRNA within their systems. The translational silencing is mediated by NOS's 3'UTR sequence. Repression can be alleviated by either replacement of the 3'UTR with heterologous 3'UTR sequence or by posterior localization (Gavis, 1994).

Although it was previously thought that Nos functions primarily to allow abdomen formation, Nos is now also known to be required for embryonic germ cell migration. In many animal groups, factors required for germline formation are localized in germ plasm, a region of the egg cytoplasm. In Drosophila embryos, germ plasm is located in the posterior pole region and is inherited in pole cells, the germline progenitors. Transplantation experiments have demonstrated that germ plasm contains factors that can form germline, and germ plasm also directs abdomen formation. Genetic analysis has shown that a common mechanism directs the localization of the abdomen and germline-forming factors to the posterior pole. The critical factor for abdomen formation is Nanos. Nos is also essential for germline formation in Drosophila; pole cells lacking Nanos activity fail to migrate into the gonads, and therefore do not become functional germ cells. A function for Nos protein in Drosophila germline formation is compatible with observations of its association with germ plasm in other animals (Kobayashi, 1996). The product of nos is required at the posterior pole of the embryo for the differentiation of abdominal structures, but not for pole cell formation. Kobayashi reported that nos also controls the timing of the initiation of transcription of germline-specific genes. The experiments that led to this hypothesis have been repeated and further experiments are reported. Contrary to what had been earlier reported, germ cells of embryos deriving from nos females do not show premature gene expression. Germ cells of such embryos, however, often show artifactual lacZ staining even in the absence of a lacZ gene (Heller, 1998).

Experiments by another laboratory, however, show that Nos regulates certain germ cell markers. In order to probe the gene expression in pole cells, ten enhancer-trap lines that showed beta-gal expression in pole cells were screened. All of these enhancer-trap markers are fully activated in pole cells after their migration to the embryonic gonads. In the pole cells lacking Nos, the expression of nine out of ten enhancer-trap markers is affected. Among nine markers, five (Type-A) were prematurely expressed in the pole cells during the course of their migration. The expression of the other four markers (Type-B) initiates correctly after pole-cell migration, but their expression is significantly reduced. Thus, it is concluded that the maternal Nos plays a dual role in zygotic gene regulation in pole cells: to define the stages of expression for Type-A markers, and to enhance expression for Type-B markers. Contrary to the results presented here, Heller (1998) has recently reported that no premature expression of Type-A markers occurs in the pole cells of embryos derived from nos mutant females. This discrepancy is due to the difference in the nos mutant alleles used for these analyses. The current study used the much stronger allele, nosBN. pumilio mutations, like nanos, affect the expression of the enhancer-trap markers in pole cells (Asaoka, et al. in preparation). This suggests that Nos cooperates with Pum in pole cells to regulate the gene expression by means of a translational repression mechanism. This is likely to occur with a similar mechanism by which Pum, along with Nos, represses the translation of maternal Hunchback mRNA. The premature expression of Type-A markers in nos pole cells can be explained by a failure of Nos to repress translationally the production of regulatory factor(s) responsible for Type-A enhancer activation. These activator(s) may be stored in pole cells as mRNA(s) whose translation is repressed by Nos. Once maternal Nos protein disappears from pole cells at around zygotic stage 15, the activator mRNA(s) is translated to produce activator(s) for Type-A enhancers. In contrast, Nos could act as a permissive factor that would allow the expression of Type-B enhancers (Asaoka, 1998).

To study the mechanism of nanos-mediated translational repression, the mechanism by which maternal Hunchback mRNA is translationally activated was examined. In the oocyte from wild-type females, where no HB translation is detected, the mRNA has a poly(A) tail length of approximately 30 nucleotides. However, concomitant with translation of the mRNA at between 0.5 and 1.5 hours after egg deposition, the poly(A) tail is elongated to approximately 70 nt. In the absence of nanos activity, the poly(A) tail of Hunchback mRNA is elongated to approximately 100 nt concomitant with its translation, suggesting that cytoplasmic polyadenylation directs activation. However, in the presence of nanos the length of the Hunchback mRNA poly(A) tail is reduced via the nanos response element present in the HB 3'UTR. To determine if nanos activity represses translation by altering the polyadenylation state of Hunchback mRNA, various in vitro transcribed RNAs were injected into Drosophila embryos and changes in polyadenylation were determined. nanos activity reduces the polyadenylation status of injected Hunchback RNAs by accelerating their deadenylation. Pumilio activity, which is necessary to repress the translation of Hunchback mRNA, is also needed to alter polyadenylation. An examination of translation indicates a strong correlation between poly(A) shortening and suppression of translation. These data indicate that nanos and pumilio determine posterior morphology by promoting the de-adenylation of maternal Hunchback mRNA, thereby repressing its translation (Wreden, 1997).

nanos plays an important role during zygotic development in downregulating mitosis and transcription during the development of the Drosophila germline. Germ cells in embryos derived from nos mutant mothers do not migrate to the primitive gonad and prematurely express several germline-specific markers. These defects have been traced back to the syncytial blastoderm stage. Pole cells in nos minus embryos fail to establish/maintain transcriptional quiescence; the sex determination gene Sex-lethal (Sxl) and the segmentation genes fushi tarazu and even-skipped are ectopically activated in nos minus germ cells. nos minus germ cells are unable to attenuate the cell cycle and instead continue dividing. Unexpectedly, removal of the Sxl gene in the zygote mitigates both the migration and mitotic defects of nos minus germ cells. Supporting the conclusion that Sxl is an important target for nos repression, ectopic, premature expression of Sxl protein in germ cells disrupts migration and stimulates mitotic activity (Deshpande, 1999).

Soon after formation, wild-type pole cells in Drosophila downregulate RNA polymerase II transcription until they have been incorporated into the primitive gonad. The premature activation of these germline-specific genes is likely to reflect a more general defect in transcriptional regulation that arises early in embryogenesis, soon after the pole cells are formed. Instead of shutting off RNA polymerase II transcription, nos- pole cells inappropriately transcribe several somatic genes. Why do nos- germ cells fail to regulate RNA polymerase II transcription? The only known regulatory target for nos in the embryo is the hb transcription factor. Nos together with the Pumilio protein is thought to bind to maternally derived hb mRNA and block its translation. Since Hb protein is produced throughout much of the posterior in the absence of Nos, one possibility is that this gap gene protein activates transcription in the pole cells. However, this explanation does not seem likely. Although hb regulates eve and ftz in the soma, it is not clear that the ectopic expression of only the Hb protein would be sufficient to activate either of these genes in the absence of other factors. In addition, hb has no known role in controlling the activity of Sxl-Pe. In fact, ectopic expression of Hb in the soma seems to repress rather than activate Sxl-Pe. Finally, germ cells derived from nos-hb- germline clones exhibit a similar set of developmental defects as nos- germ cells. Conversely, these defects are not induced when hb is ectopically expressed in pole cells (Deshpande, 1999).

A more likely possibility is that nos- germ cells have a defect in the system responsible for attenuating RNA polymerase II activity. If this is true, there must be additional target(s) for nos regulation, in addition to maternal hb mRNA. In this context it is interesting to note that the failure to establish/maintain transcriptional quiescence in nos- pole cells is reminiscent of the defects seen in the germ cell lineage of C. elegans pie-1 mutants. In pie-1 mutants, the germ cells transcribe many genes that are normally expressed only in the soma, assuming an inappropriate identity. The Pie-1 protein contains two copies of a C3H zinc finger motif found in proteins implicated in pre-mRNA metabolism. Antibody staining indicates that Pie-1 is restricted to germ cells and is localized preferentially in the nucleus. A plausible explanation for why pie-1 mutants fail to repress transcription comes from studies on the phosphorylation of the RNA polymerase II large subunit carboxy-terminal domain (CTD). The CTD contains tandem repeats of a seven-amino acid sequence that contains two serine residues (2 and 5) that are targets for phosphorylation. Phosphorylation is thought to play an important role in polymerase elongation and in the recruitment of pre-mRNA modifying enzymes. In wild-type C. elegans, RNA polymerase II phosphorylated in the serine-2 residues of the CTD is detected in somatic cells but is not observed in the germline. In contrast, in pie-1 mutants, phosphorylated CTD serine-2 is detected in both cell types. The available evidence suggests that Pie-1 may directly inhibit CTD serine-2 phosphorylation, perhaps by interfering with a protein that recognizes the CTD (Deshpande, 1999 and references therein).

Like the C. elegans germline lineage, pole cells in Drosophila also have greatly reduced levels of phosphorylated CTD serine-2, which could be responsible for the inhibition of RNA polymerase II in the fly germline. However, it is not clear at this point whether the failure to establish/maintain transcriptional quiescence in nos- pole cells is due to a defect in the system that regulates serine-2 phosphorylation. The level of CTD serine-2 phosphorylation in nos- pole cells is not greatly different from that seen in wild type. It is possible that the changes in the level of CTD serine-2 phosphorylation in nos- pole cells were too small to detect. Alternatively, RNA polymerase II activity in the germline might be controlled not only by CTD phosphorylation, but also by some other unknown mechanism which is the target for the Nos protein. Supporting this later possibility is the finding that only a subset of the genes expressed in the soma of early embryos are ectopically activated in nos- pole cells. By contrast, changes in the status of CTD phosphorylation might be expected to have quite global effects on transcription. Further studies will be required to resolve this question (Deshpande, 1999 and references therein).

Another feature that distinguishes germline precursors from the soma is the cell cycle. Since hb has no known role in cell cycle regulation, the cause of the cell cycle defect was suspected to be a failure in the regulation of some other target gene. One possible candidate is cyclin B. However, no obvious abnormalities could be detected in cyclin B accumulation in nos- pole cells at earlier stages of embryogenesis. Since cell cycle defects are evident in nos- pole cells as early as the syncitial blastoderm stage, the reduction in cyclin B midway through embryogenesis might be simply a consequence of continued cycling rather than arresting the cycle in G2 at the blastoderm stage. Another possible cell cycle candidate is String. Germ cells are blocked in G2 because Cdc2 is inhibited by the phosphorylation of two amino acid residues, Thr-14 and Tyr-15. Since phosphorylated Cdc2 is normally reactivated by the string phosphatase, cdc25stg, it is possible that Nos prevents the translation of maternally derived stg mRNA in germ cells (Deshpande, 1999 and references therein).

In wild-type embryos, transcription factors, such as Runt, Sisterless-a, and Scute, are responsible for activating the Sxl establishment promoter, Sxl-Pe. The genes encoding these positive regulators are on the X chromosome, and they are expressed in the early precellular zygote in direct proportion to the number of gene copies. Sufficient quantities of the X-linked activators are produced by 2X/2A nuclei to activate Sxl-Pe, while quantities produced by 1X/2A nuclei are insufficient to activate Sxl-Pe. Pole cells differ from the surrounding soma in that these activators are not expressed at detectable levels in the germline precursors, and Sxl-Pe remains off in both sexes. The failure to express these activators most likely reflects the global downregulation of RNA polymerase II transcription in wild-type pole cells. Thus, one mechanism that might account for the inappropriate activation of Sxl-Pe in nos- pole cells would be a general derepression of somatically active genes. As a consequence, the genes encoding the X-linked activators would be expressed, and these in turn would activate Sxl-Pe. Although it seems reasonable to believe that the ectopic expression of the X-linked activators could contribute to the activation of Sxl-Pe in nos- pole cells, it does not readily explain why Sxl-Pe is turned on not only in 2X but also in 1X pole cells. Moreover, when Scute protein expression was examined in nos- embryos, the level of Scute protein in pole cells was less than that seen in 1X/2A somatic nuclei. For this reason, it is suspected that Sxl-Pe may be activated in nos- pole cells by a mechanism that, at least in part, bypasses the normal regulation of this promoter by the X/A counting system (Deshpande, 1999).

Although Nos protein is likely to control Sxl-Pe activity by an indirect mechanism, a number of lines of evidence indicate that Sxl is an important nos regulatory target. In wild-type, Sxl proteins are normally not expressed in the germline until after the formation of the primitive gonad, and at this stage expression is restricted to the female germline. As a consequence of the ectopic activation of Sxl-Pe, Sxl proteins are present in nos- pole cells at the blastoderm stage. It would appear that the premature appearance of Sxl proteins in the pole cells is an important contributing factor to the nos- phenotype. The migration and cell cycle defects of nos- germ cells can be alleviated by the elimination of the Sxl gene. Conversely, it is possible to induce both of these defects in wild-type germ cells by ectopically expressing Sxl protein. While the removal of Sxl mitigates some of the defects of nos- germ cells, it should be noted that these cells are still abnormal. They fail to establish/maintain transcriptional quiescence, and they cannot form a functional adult germline. This finding indicates that Sxl is not the only target for nos regulation (Deshpande, 1999).

Why does ectopic expression of Sxl protein (either in the absence of nos or in the presence of the Sxl transgene) disrupt germ cell migration and induce cell cycle defects? Sxl encodes an RNA-binding protein that functions in the soma as both a splicing and translational regulator. Since the Sxl protein is predominantly localized in the cytoplasm of nos- pole cells, it is imagined that Sxl also functions to regulate the translation of mRNAs encoding proteins critical to migration or cell cycle control. An important goal for future study will be the identification of these Sxl targets (Deshpande, 1999).

Dynein-Dependent Transport of nanos RNA in Drosophila Sensory Neurons Requires Rumpelstiltskin and the Germ Plasm Organizer Oskar

Intracellular mRNA localization is a conserved mechanism for spatially regulating protein production in polarized cells, such as neurons. The mRNA encoding the translational repressor Nanos (Nos) forms ribonucleoprotein (RNP) particles that are dendritically localized in Drosophila larval class IV dendritic arborization (da) neurons. In nos mutants, class IV da neurons exhibit reduced dendritic branching complexity. This study investigated the mechanism of dendritic nos mRNA localization by analyzing requirements for nos RNP particle motility in class IV da neuron dendrites. Dynein motor machinery components were shown to mediate transport of nos mRNA in proximal dendrites. Two factors, the RNA-binding protein Rumpelstiltskin and the germ plasm protein Oskar function in da neurons for formation and transport of nos RNP particles. nos was shown to regulate neuronal function, most likely independently of its dendritic localization and function in morphogenesis. These results reveal adaptability of localization factors for regulation of a target transcript in different cellular contexts (Xu, 2013).

This study has combined a method that allows live imaging of mRNA in intact Drosophila larvae with genetic analysis to investigate the mechanism underlying transport of nos mRNA in class IV da neurons. Live imaging over the short time periods allowed has provided a snapshot into the steady-state behavior of nos*RFP particles in the proximal dendrites of mature da neurons. The results indicate that anterograde transport of nos RNP particles into and within da neuron dendrites is mediated by dynein and is consistent with the minus-end out model for microtubule polarity in the proximal dendrites of da neurons (Zheng, 2008). This model predicts that bidirectional trafficking would be mediated by opposite polarity motors and the predominance of retrograde movement of nos*RFP particles when dynein function is partially compromised is consistent with this. Moreover, Rab-5 endosomes, whose accumulation in class IV da neuron dendrites is dynein-dependent, also exhibit bidirectional movement (Satoh, 2008), suggesting that different cargos may use similar dendritic transport strategies. Unfortunately, the severe defects caused by loss of kinesin have thus far hampered confirmation of a role for kinesin in these events (Xu, 2013).

The observed bidirectional movement of nos RNP particles resembles the constant bidirectional transport observed for dendritic mRNAs near synapses in hippocampal neurons. In contrast to da neurons, proximal dendrites of mammalian neurons have mixed microtubule polarity so that bidirectional trafficking could be mediated by a single motor that switches microtubules or by switching between the activities of plus-end and minus-end motors. The association of kinesin with neuronal RNP granule components and inhibition of CaMKIIα RNA transport by dominant-negative inhibition of kinesin has implicated kinesin as the primary motor for dendritic mRNA transport. However, a recent study showed that dynein mediates unidirectional transport of vesicle cargoes into dendrites of cultured hippocampal neurons as well as bidirectional transport within the dendrites. Whether dynein plays a role in RNP particle transport in mammalian dendrites as it does in Drosophila neurons remains to be determined (Xu, 2013).

Despite its prevalence, the role of bidirectional motility is not yet clear. A recently proposed 'sushi belt' model suggests that neuronal RNP particles traffic back and forth along the dendrite until they are recruited by an active synapse and disassembled for translation (Doyle, 2011). Although da neuron dendrites do not receive synaptic input, this continual motility may provide a reservoir of nos mRNA that can be rapidly mobilized for translation locally in response to external signals that regulate dendrite branching (Xu, 2013).

These studies have shown that nos mRNA can be adapted for different localization mechanisms depending on cellular context: diffusion/entrapment in late oocytes that lack a requisite polarized microtubule cytoskeleton and microtubule-based transport during germ cell formation in the embryo and in class IV da neurons. Surprisingly, Rump and Osk are specifically required for nos localization in both oocytes and da neurons, suggesting that they function in the assembly or recognition of a fundamental nos RNP that can be adapted to both means of localization. However, because it is not possible to distinguish individual particles within the cell body, the possibility cannot be ruled out that Rump and/or Osk mediate coupling of nos RNP particles to dynein motors rather than particle formation. Within the germ plasm, nos associates with Vasa (Vas), a DEAD-box helicase, and is transported together with Vas into germ cells. Although dendritic branching complexity is reduced in vas mutants, no effect on dendritic localization of nos RNP particles was detected, suggesting that only a subset of germ plasm components are shared by neuronal localization machinery. A role for osk in learning and memory was proposed based on the isolation of an enhancer trap insertion upstream of osk in a screen for mutants with defective long-term memory, but osk function in memory formation has not been directly tested. Notably, however, a recent study showed that the osk ortholog in the cricket Gryllus bimaculatus functions in development of the embryonic nervous system rather than in germ cell formation. Thus, the ancestral function of osk appears to be in neural development, whereas its role in germ plasm formation is a later adaptation in higher insects. The results showing that Osk protein function is not limited to Dipteran germ plasm organization but also plays an important role in neuronal development and function supports this idea (Xu, 2013).

The data indicate that the Nos/Pum complex is not only required for da neuron morphogenesis, but also for nociceptive function. However, nociception does not appear to require local function of Nos/Pum in the dendrites and reduced dendritic branching does not necessarily correlate with a deficit in nociception. These results suggest that morphogenesis and function are regulated separately and that Nos/Pum plays a second role in regulating the somatic translation of proteins required for the nociceptive response. Systematic identification of Nos/Pum targets will be essential to further investigate these different roles (Xu, 2013).

Maternal Nanos inhibits Importin-alpha2/Pendulin-dependent nuclear import to prevent somatic gene expression in the Drosophila germline

Repression of somatic gene expression in germline progenitors is one of the critical mechanisms involved in establishing the germ/soma dichotomy. In Drosophila, the maternal Nanos (Nos) and Polar granule component (Pgc) proteins are required for repression of somatic gene expression in the primordial germ cells, or pole cells. Pgc suppresses RNA polymerase II-dependent global transcription in pole cells, but it remains unclear how Nos represses somatic gene expression. This study shows that Nos represses somatic gene expression by inhibiting translation of maternal importin-alpha2 (impalpha2) mRNA. Mis-expression of Impalpha2 caused aberrant nuclear import of a transcriptional activator, Ftz-F1, which in turn activated a somatic gene, fushi tarazu (ftz), in pole cells when Pgc-dependent transcriptional repression was impaired. Because ftz expression was not fully activated in pole cells in the absence of either Nos or Pgc, it is proposed that Nos-dependent repression of nuclear import of transcriptional activator(s) and Pgc-dependent suppression of global transcription act as a 'double-lock' mechanism to inhibit somatic gene expression in germline progenitors (Asaoka, 2019).

How germ cell fate is established and maintained is a century-old question in developmental, cellular, and reproductive biology. Metazoan species have two distinct modes of germline specification. In some species, germline progenitors are characterized by inheritance of a specialized ooplasm, or the germ plasm, which contains maternal factors necessary and sufficient for germline development. In other species, germline progenitors are specified by inductive signals from surrounding tissues. Irrespective of the mode of germline specification, transcriptional repression of somatic genes is common in germline progenitors, implying that this phenomenon is critical for separation of the germline from the soma (Asaoka, 2019).

In Drosophila, the germ plasm is localized in the posterior pole of cleavage embryos (stage 1-2), and is partitioned into germline progenitors called pole cells (stage 3-4). In pole cells of blastoderm embryos (stage 4-5), the genes required for somatic differentiation are transcriptionally repressed by two maternal proteins in the germ plasm, Polar granule component (Pgc) and Nanos (Nos). Pgc is a Drosophila-specific peptide that suppresses RNA polymerase II-dependent transcription in pole cells by inhibiting the function of positive transcriptional elongation factor b (P-TEFb, a dimer of Cyclin dependent kinase 9 and Cyclin T). By contrast, Nos is an evolutionarily conserved protein that plays an essential role in germline development in various animals. For example, in Drosophila, pole cells lacking Nos (nos pole cells) can adopt a somatic, rather than a germline, fate. Furthermore, depletion of Nos is reported to show ectopic expression of somatic genes, such as fushi tarazu (ftz), even-skipped (eve), and the sex-determination gene Sex lethal (Sxl), in pole cells. Thus, maternal Nos is required in pole cells for repression of somatic genes and establishment of the germ/soma dichotomy. However, the mechanism by which Nos represses somatic gene expression remains unknown (Asaoka, 2019).

Nos acts as a translational repressor of mRNAs that harbor a discrete sequence motif called Nanos Response Element (NRE) in the 3' UTR. NRE contains an evolutionarily conserved Pumilio (Pum)-binding sequence, UGU trinucleotide. In abdominal patterning, Pum represses translation of maternal hunchback (hb) mRNA by binding to NREs in its 3' UTR and recruiting Nos to the RNA/protein complex. Deletion of the NREs from hb mRNA causes its ectopic translation in the posterior half of embryos, which in turn suppresses abdomen formation. Furthermore, deletion of NREs causes hb translation in pole cells, suggesting that NRE-dependent translational repression occurs in pole cells. Indeed, Nos represses translation of head involution defective (hid) mRNA in pole cells in an NRE-like-sequence-dependent manner. In addition, Nos and Pum repress Cyclin B translation in pole cells by binding to a discrete sequence containing two UGU trinucleotides (Cyclin B NRE) These findings led to a speculation that Nos, along with Pum, represses somatic gene expression in pole cells by suppressing translation of mRNAs containing NRE or UGU in their 3' UTRs (Asaoka, 2019).

This study reports that, in pole cells, Nos, along with Pum, represses translation of importin-α2 (impα2)/Pendulin/oho31/CG4799 mRNA, which contains an NRE-like sequence in its 3' UTR. The impα2 mRNA encodes a Drosophila Importin-α homologue that plays a critical role in nuclear import of karyophilic proteins. Nos inhibits expression of a somatic gene, ftz, in pole cells by repressing Impα2-dependent nuclear import of the transcriptional activator, Ftz-F1. Based on these observations, it is proposed that Nos-dependent inhibition of nuclear import of transcriptional activators and Pgc-dependent global transcriptional silencing act as a 'double-lock' mechanism to repress somatic gene expression in pole cells (Asaoka, 2019).

Maternally supplied impα2 mRNA is distributed throughout cleavage embryos. When embryos develop to the blastoderm stage, impα2 mRNA is degraded in the somatic region, but not in pole cells, resulting in enrichment of impα2 mRNA in pole cells. However, this study found that expression of Impα2 protein was at background levels in pole cells. Because impα2 mRNA contains a sequence very similar to the NRE (hereafter, NRE-like sequence) in its 3' UTR, it was assumed that impα2 mRNA is a target of Nos/Pum-dependent translational repression in pole cells. To investigate this possibility, the expression of the Impα2 protein was first monitored in pole cells of embryos lacking maternal Nos or Pum (nos or pum embryos, respectively). In these pole cells, expression of Impα2 protein was higher than in those of control (nos/+) embryos. Because neither nos nor pum mutation affected the impα2 mRNA level in pole cells, these observations show that Nos and Pum repress protein expression from the impα2 mRNA in pole cells (Asaoka, 2019).

Whether this repression is mediated by the NRE-like sequence in the impα2 3' UTR was investigated. To this end, impα2 mRNA, with or without the NRE-like sequence (impα2 WT and impα2 ΔNRE, respectively), was maternally supplied to embryos, and their protein expression was examined in pole cells at the blastoderm stage. Because a triple Myc tag sequence was inserted at the C-terminal end of the coding sequence, protein expression from these mRNAs could be monitored using an anti-Myc antibody. When impα2 WT mRNA was supplied to normal (y w) embryos, the tagged protein was expressed at low levels in the soma, but was barely detectable in pole cells. By contrast, the tagged protein from impα2 ΔNRE mRNA was detected in normal pole cells. Similar protein expression was observed in pole cells lacking Nos (nos pole cells), when impα2 WT mRNA was supplied, as well as when impα2 ΔNRE mRNA was supplied. Because the frequency of tagged protein expression from impα2 ΔNRE mRNA did not increase in cells lacking Nos, these results indicate that the NRE-like sequence mediates Nos-dependent repression of Impα2 protein expression in pole cells (Asaoka, 2019).

The NRE-like sequence of impα2 mRNA contains two UGU trinucleotides. The UGU trinucleotide is a core sequence of an RNA motif (Nos-Pum SEQRS motif: 5'-HWWDUGUR) that was highly enriched in a SEQRS (in vitro selection, high-throughput sequencing of RNA, and sequence specificity landscapes) analysis of the Nos-Pum-RNA ternary complex. Hence, it was asked whether Pum and Nos form a ternary complex with impα2 mRNA in an NRE-like sequence-dependent manner. To address this question, electrophoretic mobility shift assay (EMSA) was performed using the Pum RNA-binding domain and the Nos protein containing Zn finger motifs and C-terminal region, which are reported to form a Nos-Pum-target RNA ternary complex in vitro. Nos and Pum together, but neither alone, formed a complex with impα2 RNA containing an NRE-like sequence (WT), whereas alteration of the NRE-like sequence (mut) abolished this interaction. These results demonstrate that Nos and Pum are able to interact with the impα2 3' UTR in an NRE-like sequence-dependent manner. The observations described above led to a conclusion that Nos, along with Pum, directly represses impα2 translation in pole cells (Asaoka, 2019).

Impα2 is a Drosophila homologue of Importin-α that mediates nuclear import of karyophilic proteins with classical nuclear localization signal (NLS). It was predicted that ectopic production of Impα2 in nos pole cells would cause aberrant nuclear import of NLS-containing karyophilic proteins. To explore this possibility, this study focused on a transcriptional activator, Ftz-F1, which contains a classical NLS and is expressed throughout early embryos, including pole cells. In normal embryos, Ftz-F1 was enriched in the cytoplasm of pole cells, although it was in the nuclei of somatic cells. In the absence of maternal Nos, the percentage of embryos with Ftz-F1 signal accumulating in pole-cell nuclei was higher than in normal embryos. Furthermore, the nuclear/cytoplasmic ratio of Ftz-F1 signal intensities in nos pole cells was higher than in normal pole cells. To determine whether this aberrant concentration of Ftz-F1 was caused by mis-expression of Impα2, this study expressed Impα2 ectopically in pole cells of normal embryos. To this end, impα2 mRNA in which the 3' UTR was replaced with the nos 3' UTR, was maternally supplied under the control of the nos promoter; the mRNA was localized to the germ plasm and pole cells under the control of the nos 3' UTR. The percentage of these embryos (impα2-nos3'UTR embryos) with Ftz-F1 in pole-cell nuclei and the nuclear/cytoplasmic ratio of Ftz-F1 intensities in their pole cells were higher than those of normal pole cells. These observations suggest that mis-expression of Impα2 in pole cells caused by depletion of maternal Nos results in aberrant nuclear import of Ftz-F1 (Asaoka, 2019).

Depletion of maternal Nos results in ectopic expression of the somatic genes ftz, eve and Sxl in pole cells. Because Ftz-F1 is required for proper expression of ftz in the soma, it was asked whether mis-expression of Impα2 causes ectopic expression of ftz in pole cells. In normal embryos, ftz mRNA was expressed in seven stripes of somatic cells, but never expressed in pole cells. By contrast, in impα2-nos3'UTR embryos, ftz mRNA was rarely detectable in pole cells. It is assumed that this low frequency of ftz expression was due to Pgc-mediated silencing of global mRNA transcription. To test this idea, Impα2 was expressed in pole cells of embryos lacking maternal Pgc (pgc impα2-nos3'UTR embryos); the frequency of ftz expression was drastically increased, compared to those of impα2-nos3'UTR embryos and the embryos lacking Pgc (pgc embryos). A similar situation was observed in embryos lacking both Pgc and Nos activities (pgc nos embryos). The percentage of embryos expressing ftz in pole cells was 82.8%, an increase relative to 35.8% in pgc embryos. Furthermore, ectopic ftz expression in pgc nos pole cells was suppressed by injecting double-stranded RNA (dsRNA) against impα2. Therefore, it is concluded that ectopic expression of ftz in pole cells is cooperatively repressed by Nos-dependent suppression of Impα2 production and Pgc (Asaoka, 2019).

In addition to ftz expression, eve was expressed ectopically in pole cells of pgc impα2-nos3'UTR embryos. Ectopic eve mRNA and its protein expression were significantly higher in pgc impα2-nos3'UTR pole cells than pgc or impα2-nos3'UTR pole cells (S3 Fig). Expression of the sex-determination gene Sxl was examined in early pole cells, because Sxl is also repressed by nos in both male and female pole cells. In males, Sxl mRNA expression was rarely detectable in pole cells of nos, impα2-nos3'UTR, pgc, and pgc impα2-nos3'UTR embryos. By contrast, in females, the percentage of embryos expressing Sxl mRNA in pole cells was significantly higher in pgc impα2-nos3'UTR embryos than in impα2-nos3'UTR, and pgc embryos. These results indicate that eve and Sxl, like ftz, are cooperatively repressed in pole cells by Impα2 depletion and Pgc-dependent transcriptional silencing. Because there is no evidence for the involvement of Ftz-F1 in eve and Sxl expression, it is likely that Impα2 mediates nuclear import of other transcriptional activator(s) for eve and/or Sxl in pole cells (Asaoka, 2019).

Nos is required in pole cells for mitotic quiescence, repression of apoptosis, and proper migration to embryonic gonads. Hence, it was asked whether mis-expression of Impα2 causes defects in these processes. First, using an antibody against a phosphorylated form of histone H3 (PH3), a marker of mitosis, whether pole cells enter mitosis in stage 7-9 embryos was investigated. Premature mitosis was detected in pole cells of nos embryos, as described previously, but never in pole cells of impα2-nos3'UTR or pgc impα2-nos3'UTR embryos. Second, using an antibody against cleaved Caspase-3, a marker of apoptosis, whether pole cells enter apoptosis in stage 10-16 embryos was investigated. Pole cells never expressed the apoptotic marker in impα2-nos3'UTR embryos, whereas in pgc impα2-nos3'UTR embryos, 20.4% of pole cells expressed the apoptotic marker. The latter was statistically indistinguishable from pgc pole cells, which have been reported to enter apoptosis. These data indicate that mis-expression of Impα2 does not affect apoptosis of pole cells even in the absence of pgc function. Last, whether mis-expression of Impα2 affects pole cell migration was investigated. The ability of pole cells to migrate properly into the embryonic gonads was never impaired in impα2-nos3'UTR embryos, and the percentage of pole cells entering the gonads in pgc impα2-nos3'UTR embryos was statistically indistinguishable from that of pgc pole cells, which has been reported to exhibit migration defect. These observations indicate that mis-expression of Impα2 does not induce premature mitosis, apoptosis, or mis-migration of pole cells. This can be partly explained by the facts that Cyclin B and hid mRNAs are the targets for Nos-dependent translational repression regulating mitosis and apoptosis in pole cells, respectively (Asaoka, 2019).

During the course of the experiments described above, it was observed that impα2-nos3'UTR interacts genetically with the pgc mutation to cause dysgenic gametogenesis. Because almost all of the ovaries in females derived from pgc mothers mated with y w males were agametic, the effect of impα2-nos3'UTR in pgc/+ background was examined. The percentage of dysgenic ovaries in pgc/+ impα2-nos3'UTR females derived from pgc/+ impα2-nos3'UTR mothers mated with y w males was significantly higher than those in pgc/+ and impα2-nos3'UTR females. In the dysgenic ovaries, almost all of the egg chambers fail to complete the vitellogenic stage, and consequently only a few mature oocytes were present. Furthermore, the percentages of dysgenic and agametic testes in pgc impα2-nos3'UTR males derived from pgc impα2-nos3'UTR mothers mated with y w males were higher than those in pgc and impα2-nos3'UTR males. In these testes, the abundance of Vasa-positive germline cells was reduced (dysgenic) or absent (agametic). Because dysgenic and agametic gonads were barely detectable in females and males derived from reciprocal crosses, the data suggest that mis-expression of Impα2 from maternal transcript, concomitant with maternal pgc depletion in pole cells, causes defects in gametogenesis. However, it cannot be tested whether concomitant depletion of maternal Nos and Pgc causes a similar phenotype because nos pole cells degenerate before adulthood, even when apoptosis in these cells is genetically repressed (Asaoka, 2019).

Expression of Importin-α subtypes is spatio-temporally regulated in the soma during development in multiple animal species, including Drosophila, and they control nuclear transport of unique karyophilic proteins to activate different sets of somatic genes. Drosophila genome contains three Importin-α family genes: impα1, 2, and 3. impα1/Kap-α1/CG8548 mRNA is not detectable in pole cells during early embryogenesis, and its protein product is ubiquitously expressed at a very low level throughout embryogenesis. By contrast, maternal impα3/Kap-α3/CG9423 mRNA is detectable in germ plasm during pole cell formation, and production of Impα3 protein is upregulated during the blastoderm stage. Because Impα3 production was independent of maternal nos activity, it is likely that Nos-dependent repression of Impα2 production is solely responsible for suppression of somatic gene expression in pole cells. By contrast, pole cells become transcriptionally active during gastrulation, when Impα2 is undetectable in these pole cells. Thus, the onset of zygotic transcription in pole cells may require Impα3-dependent nuclear import of transcription factors, in addition to the disappearance of Pgc and the alteration in chromatin-based regulation. After gastrulation, maternal impα2 mRNA is rapidly degraded in pole cells, and neither impα2 mRNA nor protein is detectable in the germline before adulthood. This suggests that maternal impα2 is dispensable for germline development, and that maternal impα2 mRNA partitioned into early pole cells must be silenced by Nos and Pum in order to suppress mis-expression of somatic genes (Asaoka, 2019).

Depletion of maternal Nos activities caused mis-expression of ftz in pole cells. Although ftz expression was barely observed in pole cells lacking only maternal Nos, it was partially derepressed in pole cells in the absence of Pgc alone, probably because a trace amount of Ftz-F1 enters pole cell nuclei even in the absence of the impα2 translation. Therefore, it is proposed that a subset of somatic genes, including ftz and eve, are repressed in pole cells by two distinct mechanisms: Nos-dependent repression of nuclear import of transcriptional activators and Pgc-dependent silencing of mRNA transcription. Pgc inhibits P-TEFb-dependent phosphorylation of Ser2 residues in the heptad repeat of the C-terminal domain (CTD) of RNA polymerase II, a modification that is critical for transcriptional elongation; thus, mRNA transcription in pole cells is globally suppressed by Pgc. By contrast, Nos inhibits transcription of particular genes by repressing Impα2-dependent nuclear import of the corresponding transcriptional activators (Asaoka, 2019).

Nos is evolutionarily conserved and expressed in the germline progenitors of various animal species. In C. elegans, nos-1 and -2 are essential for rapid turnover of maternal lin-15B mRNA, which encodes a transcription factor that would otherwise cause inappropriate transcriptional activation in primordial germ cells. In the germline progenitors of Xenopus embryos, Nos-1, along with Pum, destabilizes maternal VegT mRNA and represses its translation to inhibit somatic (endodermal) gene expression, which is activated by VegT protein. Furthermore, in the germline progenitors (small micromeres) of sea urchin embryos, Nos silences maternal mRNA encoding a deadenylase, CNOT6, to stabilize other maternal mRNAs inherited into small micromeres. This study demonstrated that Nos inhibits translation of maternal impα2 mRNA in pole cells in order to suppress nuclear import of a transcriptional activator for somatic gene expression. Based on these observations, it is proposed that Nos silences maternal transcripts that are inherited into germline progenitors but deter the proper germline development. In addition to Nos-dependent silencing of maternal transcripts, transient suppression of RNA polymerase II elongation is observed during germline development of a wide range of animals, including Drosophila, C. elegans, Xenopus, and an ascidian, Halocynthia roretzi. Therefore, it is proposed that the 'double-lock' mechanism achieved by Nos and global suppression of RNA polymerase II activity plays an evolutionarily widespread role in germline development (Asaoka, 2019).

The PIWI protein Aubergine recruits eIF3 to activate translation in the germ plasm

Piwi-interacting RNAs (piRNAs) and PIWI proteins are essential in germ cells to repress transposons and regulate mRNAs. In Drosophila, piRNAs bound to the PIWI protein Aubergine (Aub) are transferred maternally to the embryo and regulate maternal mRNA stability through two opposite roles. They target mRNAs by incomplete base pairing, leading to their destabilization in the soma and stabilization in the germ plasm. This study reports a function of Aub in translation. Aub is required for translational activation of nanos mRNA, a key determinant of the germ plasm. Aub physically interacts with the poly(A)-binding protein (PABP) and the translation initiation factor eIF3. Polysome gradient profiling reveals the role of Aub at the initiation step of translation. In the germ plasm, PABP and eIF3d assemble in foci that surround Aub-containing germ granules, and Aub acts with eIF3d to promote nanos translation. These results identify translational activation as a new mode of mRNA regulation by Aub, highlighting the versatility of PIWI proteins in mRNA regulation (Ramat, 2020).

Translational control is a widespread mechanism to regulate gene expression in many biological contexts. This regulation has an essential role during early embryogenesis, before transcription of the zygotic genome has actually started. In Drosophila, embryonic patterning depends on the translational control of a small number of maternal mRNAs, among them, nanos (nos) mRNA encodes a key posterior determinant required for abdominal segmentation and development of the germline. nos mRNA is present in the whole embryo, but a small proportion accumulates at the posterior pole in the germ plasm, a specialized cytoplasm in which the germline develops. Localization and translational control of nos mRNA are linked, such that the pool of nos mRNA present in the bulk of the embryo is translationally repressed, whereas the pool of nos mRNA localized in the germ plasm is translationally activated to produce a Nos protein gradient from the posterior pole. Both repression of nos mRNA translation in the bulk of the embryo and activation in the germ plasm are required for embryonic development (Ramat, 2020).

The coupling between mRNA localization and translational control depends in part on the implication of the same factors in both processes. The Smaug (Smg) RNA binding protein specifically recognizes nos mRNA through binding to two Smaug recognition elements (SRE) in its 3'UTR. Smg is both a translational repressor of nos, and a localization factor through its role in mRNA deadenylation and decay in the bulk of the embryo, by recruitment of the CCR4-NOT deadenylation complex. Smg directly interacts with the Oskar (Osk) protein that is specifically synthesized at the posterior pole of oocytes and embryos and drives germ plasm assembly. Smg interaction with Osk prevents Smg binding to nos mRNA, thus contributing to relieving both Smg-dependent translational repression and mRNA decay in the germ plasm. Osk is therefore a key player in the switch of nos and other germ cell mRNA regulation between soma and germ plasm of the embryo (Ramat, 2020).

A role of Aubergine (Aub) in the localization of germ cell mRNAs to the germ plasm has been demonstrated. Aub is one of the three PIWI proteins in Drosophila. PIWI proteins belong to a specific clade of Argonaute proteins that bind 23-30 nucleotides (nt)-long small RNAs referred to as Piwi-interacting RNAs (piRNAs). piRNAs and PIWI proteins have an established role in the repression of transposable elements in the germline of animals. piRNAs target transposable element mRNAs through complementarity and guide interaction with PIWI proteins that, in turn, cleave targeted mRNAs through their endonucleolytic activity. In addition to this role, piRNAs have a conserved function in the regulation of cellular mRNAs in various biological contexts. In the Drosophila embryo, Aub loaded with piRNAs produced in the female germline is present both at low levels in the bulk of the embryo and at higher levels in the germ plasm. Aub binds maternal germ cell mRNAs through incomplete base pairing with piRNAs. Aub binding to these mRNAs induces their decay in the bulk of the embryo, either by direct cleavage or recruitment together with Smg of the CCR4-NOT deadenylation complex. In contrast, in the germ plasm Aub recruits Wispy, the germline-specific cytoplasmic poly(A) polymerase, leading to poly(A) tail elongation and stabilization of Aub-bound mRNAs. Thus, Aub and piRNAs play a central role in the localization of germ cell mRNAs through two opposite functions in mRNA stability: mRNA destabilization in the bulk of the embryo and stabilization in the germ plasm. The role of piRNAs and PIWI proteins in cellular mRNA regulation in other contexts, including mouse spermiogenesis and sex determination in Bombyx, also depends on their function in the regulation of mRNA stability (Ramat, 2020).

This study describes translational activation as a new mechanism of mRNA regulation by piRNAs and PIWI proteins. Using ectopic expression of Osk in the whole embryo to mimic the germ plasm, this study shows that Aub and piRNAs are required for nos mRNA translation. Mass spectrometry analysis of Aub interactors in early embryos identifies several components of the translation machinery, including translation initiation factors. This study finds that Aub physically interacts with the poly(A)-binding protein (PABP) and several subunits of the translation initiation complex eIF3. Furthermore, PABP and eIF3d accumulate in foci that assemble around and partially overlap with Aub-containing germ granules in the germ plasm. Polysome gradient analysis indicates that Aub activates translation at the initiation step. Finally, functional experiments involving the concomitant decrease of Aub and eIF3d show that both proteins act together in nos mRNA translation in the germ plasm. These results identify translational activation as a new level of mRNA regulation by PIWI proteins. Moreover, they expand the role of the general eIF3 translation initiation complex in translation regulatory mechanisms required for developmental processes (Ramat, 2020).

Several studies have reported the role of PIWI proteins in cellular mRNA regulation at the level of stability. piRNA-dependent binding of mRNAs by PIWI proteins leads to their decay in different biological systems. In addition, in Drosophila embryos, mRNA binding by the PIWI protein Aub also leads to their stabilization in a spatially regulated manner. This study reports a novel function of Aub in direct translational control of mRNAs. Using nos mRNA as a paradigm, it was shown that Aub is required for nos mRNA translation. Nos protein levels are also strongly reduced in armi mutant, in which piRNA biogenesis is massively affected, suggesting that Aub loading with piRNAs is necessary for its function in translational activation. Consistent with this, deletion of two piRNA target sites in nos mRNA decreases its translation. Importantly, Nos levels are not affected in a panx mutant background. Panx is a piRNA factor required for transcriptional repression of transposable elements, but has no function in piRNA biogenesis. In addition, as is the case for aub and armi mutants, panx mutant embryos do not develop. Finally, Nos levels are similar in unfertilized eggs and embryos overexpressing Osk, demonstrating that Nos protein synthesis is independent of embryonic development. Together, these results strongly argue for a direct role of Aub and piRNAs in nos mRNA translational control, independently of their role in transposable element regulation or developmental defects in piRNA pathway mutants (Ramat, 2020).

Mass spectrometry analysis of Aub interactors points to a strong link with the translation machinery. In addition, polysome gradient analyses reveal Aub association with actively translated mRNAs in polysomal fractions. A link has been reported previously between the PIWI proteins Miwi and Mili and the translation machinery in mouse testes, where Miwi and Mili were found to associate with the cap-binding complex. However, the role of Miwi and Mili in translational control has not been characterized. This study has deciphered the molecular mechanisms of Aub function in translational activation of germ cell mRNAs in the Drosophila embryo. This study demonstrates a physical interaction between Aub and the translation initiation factors PABP, eIF4E and subunits of the eIF3 complex. These interactions are in agreement with polysome gradient analyses in WT and aub mutant backgrounds that indicate a role of Aub in translation initiation (Ramat, 2020).

Recent data have identified specific roles of eIF3 in the regulation of translation. eIF3 is the most elaborate of translation initiation factors containing twelve subunits and an associated factor, eIF3j. This complex promotes all steps of translational initiation and does so in part through direct association with other translation initiation factors, contributing to their functional conformations on the small ribosomal subunit surface. In addition to this role in basal translation, the eIF3a, b, d and g subunits were shown to directly bind 5'UTR of specific mRNAs, leading to cap-dependent translation activation or repression. The eIF3d subunit that attaches to the edge of the complex appears to play an especially important role in various modes of eIF3-dependent translational control: (1) eIF3d is involved in the translational repression of Drosophila sex-lethal mRNA through binding to its 5'UTR. (2) eIF3d was reported to directly bind the cap structure of specific mRNAs in mammalian cells, thus bypassing the requirement of eIF4E binding to the cap for translation initiation. (3) In the same line, eIF3d was involved in cap-dependent translational activation of specific mRNAs for neuronal remodeling in Drosophila larvae, in a context where eIF4E is blocked by 4E-binding protein (4E-BP). Other studies have reported the role of eIF3 in promoting cap-independent translation, thus highlighting eIF3 functional versatility in the control of translation. eIF3 was shown to directly bind methylated adenosine m6A, in mRNA 5'UTRs to induce cap-independent translation under stress conditions. Furthermore, PABP bound to the poly(A) tail was also shown to cooperate with eIF3 for its binding to mRNA 5'UTR triggering cap-independent translation (Ramat, 2020).

This study describes a new mode of eIF3-dependent translational activation through its recruitment by the PIWI protein Aub. Based on previous information on the nos translation repressor complex and data presented in this study on translational activation, the following model is proposed. nos mRNA translation is repressed in the somatic part of the embryo by two mechanisms. First, the 4E-BP protein Cup in complex with Smg binds to eIF4E and prevents eIF4G recruitment and cap-dependent translation. The detailed mechanism of Cup recruitment to the repressor complex has not been clarified, but Cup was shown to directly associate with the Not1 subunit of the CCR4-NOT complex and this interaction might stabilize Cup association with eIF4E. CCR4-NOT itself is recruited to nos mRNA by Smg and Aub. Second, two translational repressors, the RNA helicase Me31B (Drosophila DDX6) and its partner Tral coat the length of nos mRNA and prevent translation through a cap-independent mechanism. Again the mode of Me31B/Tral specific recruitment to nos mRNA has not been determined, but the CCR4-NOT complex might also be involved since DDX6 directly binds the Not1 subunit of CCR4-NOT. Aub coprecipitation with components of the nos translational repressor complex is consistent with its association with the CCR4-NOT complex in the soma and suggests that Aub might be involved in translational repression, in addition to mRNA decay. In the germ plasm, Osk interaction with Smg prevents Smg binding to nos mRNA9 and this contributes to CCR4-NOT displacement from the mRNP complex. Consistent with this, CCR4 is depleted in the germ plasm. The lack of CCR4-NOT on nos mRNA might preclude the recruitment of Me31B/Tral and relieve the cap-independent mechanism of translational repression. This study finds that Aub physically interacts with PABP and several subunits of eIF3. It is proposed that these associations would lead to translational activation independently of eIF4E through binding of eIF3 to nos 5'UTR, followed by direct recruitment of the 40 S ribosome by eIF3 and PABP, as previously reported for translation of XIAP mRNA. Alternatively, eIF3 might act through direct binding of eIF3d to the cap structure; however, this hypothesis is not favored. Indeed, if eIF3d interaction with the cap was involved, overexpression of the point mutant eIF3dhelix11 that is unable to bind the cap, would be expected to induce negative dominant defects, due to the lack of translation mediated by this interaction. However, overexpression of eIF3dhelix11 with the nos-Gal4 driver did not induce any defects in embryonic development or Nos protein synthesis (Ramat, 2020).

Germ granules coordinate germ cell mRNA regulation with piRNA inheritance through the role of PIWI proteins in both processes. Recent studies in C. elegans have shown that piRNA/PRG1-dependent mRNA accumulation in germ granules prevent their silencing, strengthening the function of piRNAs in germ granules for mRNA storage and surveillance. In Drosophila, Aub mediates the link between piRNAs and mRNA regulation in germ granules since Aub localization to germ granules depends on its loading with piRNAs and Aub/piRNAs play a general role in the localization and stabilization of germ cell mRNAs in germ granules. How do germ granules accommodate translational control has remained more elusive. In Drosophila embryos, germ granules contain mRNAs that are translated sequentially. This study demonstrates a direct role of Aub in translational activation. Strikingly, PABP and eIF3d tend to colocalize with Aub at the periphery of germ granules. This is reminiscent of a study analyzing translational control in relation to RNA granules in Drosophila oocytes, in which translational repressors such as Me31B were found to concentrate in the granule core with repressed mRNAs, whereas the translational activator Orb was localized at the edge of the granules where mRNAs docked for translation. Similarly, germ granules in embryos might be partitioned into functional subdomains involved in various steps of mRNA regulation, including storage (in an internal region of granules) and translational activation (at the granule periphery). This work reveals the central role of Aub in activation of translation. Future studies will undoubtedly address the complexity of mRNA regulation by PIWI proteins in relation with germ granules (Ramat, 2020).

While this manuscript was under review, a role of Miwi and piRNAs in translational activation during mouse spermiogenesis has been demonstrated (Dai, 2019). Miwi was shown to be in complex with PABP and several subunits of eIF3 for its function in translational activation, which is required for spermatid development. This reveals a striking evolutionary conservation of PIWI protein function in translational control for key developmental processes (Ramat, 2020).


GENE STRUCTURE

Bases in 5' UTR - 261

Exons - three

Bases in 3' UTR - 874


PROTEIN STRUCTURE

Amino Acids - 400

Structural Domains

The C-terminal region of Nanos is homologous to Xcat-2, an RNA binding protein of Xenopus. Although NOS has a much higher molecular weight than X-cat2, the Nanos and X-cat2 shared domain forms a putative zinc finger, potentially able to bind to either RNA or protein (Mosquere, 1993).

Analysis of nanos mutants reveals that a small, evolutionarily conserved, C-terminal region is essential for Nanos function in vivo, while no other single portion of the Nanos protein is absolutely required. Within the C-terminal region are two unusual Cys-Cys-His-Cys (CCHC) motifs that are potential zinc-binding sites. One equivalent of zinc is bound with high affinity by each of the CCHC motifs. nanos mutations disrupting metal binding at either of these two sites in vitro abolish Nanos translational repression activity in vivo (Curtis, 1997).


nanos: Evolutionary Homologs | Regulation | mRNA localization and post-transcriptional regulation | Developmental Biology | Effects of Mutation | References

date revised: 20 August 2019

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