Glorund, a Drosophila hnRNP F/H homolog, is an ovarian repressor of nanos translation

Patterning of the anterior-posterior body axis of the Drosophila embryo requires production of Nanos protein selectively in the posterior. Spatially restricted Nanos synthesis is accomplished by translational repression of unlocalized nanos mRNA together with translational activation of posteriorly localized nanos. Repression of unlocalized nanos mRNA is mediated by a bipartite translational control element (TCE) in its 3' untranslated region. TCE stem-loop II functions during embryogenesis, through its interaction with the Smaug repressor. Stem-loop III represses unlocalized nanos mRNA during oogenesis, but trans-acting factors that carry out this function have remained elusive. Here we identify a Drosophila hnRNP, Glorund, that interacts specifically with stem-loop III. We establish that the ability of the TCE to repress translation in vivo reflects its ability to bind Glorund in vitro. These data, together with the analysis of a glorund null mutant, reveal a specific role for an hnRNP in repression of nanos translation during oogenesis.     

Oskar allows nanos mRNA translation in Drosophila embryos by preventing its deadenylation by Smaug/CCR4

Anteroposterior patterning of the Drosophila embryo depends on a gradient of Nanos protein arising from the posterior pole. This gradient results from both nanos mRNA translational repression in the bulk of the embryo and translational activation of nanos mRNA localized at the posterior pole. Two mechanisms of nanos translational repression have been described, at the initiation step and after this step. Here we identify a novel level of nanos translational control. We show that the Smaug protein bound to the nanos 3' UTR recruits the deadenylation complex CCR4-NOT, leading to rapid deadenylation and subsequent decay of nanos mRNA. Inhibition of deadenylation causes stabilization of nanos mRNA, ectopic synthesis of Nanos protein and head defects. Therefore, deadenylation is essential for both translational repression and decay of nanos mRNA. We further propose a mechanism for translational activation at the posterior pole. Translation of nanos mRNA at the posterior pole depends on oskar function. We show that Oskar prevents the rapid deadenylation of nanos mRNA by precluding its binding to Smaug, thus leading to its stabilization and translation. This study provides insights into molecular mechanisms of regulated deadenylation by specific proteins and demonstrates its importance in development.     

A CCHC metal-binding domain in Nanos is essential for translational regulation.

The Drosophila Nanos protein is a localized repressor of hunchback mRNA translation in the early embryo, and is required for the establishment of the anterior-posterior body axis. 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. Using absorption spectroscopy and NMR we demonstrate that the CCHC motifs each bind one equivalent of zinc with high affinity. nanos mutations disrupting metal binding at either of these two sites in vitro abolish Nanos translational repression activity in vivo. We show that full-length and C-terminal Nanos proteins bind to RNA in vitro with high affinity, but with little sequence specificity. Mutations affecting the hunchback mRNA target sites for Nanos-dependent translational repression were found to disrupt translational repression in vivo, but had little effect on Nanos RNA binding in vitro. Thus, the Nanos zinc domain does not specifically recognize target hunchback RNA sequences, but might interact with RNA in the context of a larger ribonucleoprotein complex.     

Smaug, a novel and conserved protein, contributes to repression of nanos mRNA translation in vitro

Proper deployment of Nanos protein at the posterior of the Drosophila embryo, where it directs posterior development, requires a combination of RNA localization and translational controls. These controls ensure that only the posteriorly-localized nanos mRNA is translated, whereas unlocalized nanos mRNA is translationally repressed. Here we describe cloning of the gene encoding Smaug, an RNA-binding protein that interacts with the sequences, SREs, in the nanos mRNA that mediate translational repression. Using an in vitro translation assay, we demonstrate that SRE-dependent repression occurs in extracts from early stage embryos. Immunodepletion of Smaug from the extracts eliminates repression, consistent with the notion that Smaug is involved. Smaug is a novel gene and the existence of potential mammalian Smaug homologs raises the possibility that Smaug represents a new class of conserved translational repressor.     

The nanos translational control element represses translation in somatic cells by a Bearded box-like motif

Developmental control of translation is frequently mediated by regulatory elements that reside within 3' untranslated regions (3' UTRs). Two stem-loops within the nanos 3' UTR translational control element (TCE) act independently to direct translational repression of maternal nanos mRNA in the ovary or embryo. We have previously shown that the nanos TCE can also function in select somatic sites. Using an ectopic expression screen, we now identify a new site of TCE function, the dorsal pouch epithelium. Analysis of TCE mutants reveals that TCE activity in the dorsal pouch does not depend on either of the stem-loops required for maternal TCE function, but instead requires a third feature-a sequence that closely matches the Bearded box, a regulatory motif found in the 3' UTRs of several Notch pathway genes. In addition, we identify pleiohomeotic mRNA as an endogenous candidate for regulation by Bearded box-like motifs in the dorsal pouch. Together, these results suggest that the TCE has appropriated a conserved regulatory motif to expand its function to somatic tissues.     

The Drosophila hnRNP M homolog Rumpelstiltskin regulates nanos mRNA localization

Anterior-posterior axis patterning of the Drosophila embryo requires Nanos activity selectively in the posterior. This spatial asymmetry of Nanos is generated by the localization of nanos mRNA to the posterior pole of the embryo, where it is subsequently translated. Posterior localization of nanos is mediated by a complex cis-acting localization signal in its 3' untranslated region comprising several partially redundant localization elements. This localization signal redundancy has hampered the identification of trans-acting factors that act specifically to effect posterior localization of nanos. Here, we have used a biochemical approach to identify Rumpelstiltskin, a Drosophila heterogeneous nuclear ribonucleoprotein (hnRNP) M homolog, which binds directly to an individual nanos localization element. Rumpelstiltskin associates with nanos mRNA in vitro and in vivo, and binding by Rumpelstiltskin correlates with localization element function in vivo. Through analysis of a rumpelstiltskin null mutation by genetic strategies that circumvent redundancy, we demonstrate that Rumpelstiltskin regulates anterior-posterior axis patterning by functioning as a direct-acting nanos mRNA localization factor.     

Translational repression restricts expression of the C. elegans Nanos homolog NOS-2 to the embryonic germline

Members of the nanos gene family are evolutionarily conserved regulators of germ cell development. In several organisms, Nanos protein expression is restricted to the primordial germ cells (PGCs) during early embryogenesis. Here, we investigate the regulation of the Caenorhabditis elegans nanos homolog nos-2. We find that the nos-2 RNA is translationally repressed. In the adult germline, translation of the nos-2 RNA is inhibited in growing oocytes, and this inhibition depends on a short stem loop in the nos-2 3'UTR. In embryos, nos-2 translation is repressed in early blastomeres, and this inhibition depends on a second region in the nos-2 3'UTR. nos-2 RNA is also degraded in somatic blastomeres by a process that is independent of translational repression and requires the CCCH finger proteins MEX-5 and MEX-6. Finally, the germ plasm component POS-1 activates nos-2 translation in the PGCs. A combination of translational repression, RNA degradation, and activation by germ plasm has also been implicated in the regulation of nanos homologs in Drosophila and zebrafish, suggesting the existence of conserved mechanisms to restrict Nanos expression to the germline.     

Smaug, a novel RNA-binding protein that operates a translational switch in Drosophila.

During Drosophila embryogenesis, a gradient of Nanos protein emanating from the posterior pole organizes abdominal segmentation. This gradient arises from translational regulation of nanos mRNA, which is activated in the specialized cytoplasm at the posterior pole of the embryo and repressed elsewhere. Previously, we have defined cis-acting elements in the mRNA that mediate this translational switch. In this report, we identify a factor named Smaug that binds to these elements and represses translation in the bulk cytoplasm. Smaug interacts gentically and biochemically with Oskar, a key component of the pole plasm for activation of nanos mRNA and specification of the germline precursors. These observations suggest that Smaug operates a translational switch that governs the distribution of Nanos protein.     

A selective screen reveals discrete functional domains in Drosophila Nanos

The Drosophila protein Nanos encodes an evolutionarily conserved protein with two zinc finger motifs. In the embryo, Nanos protein function is required for establishment of the anterior-posterior body pattern and for the migration of primordial germ cells. During oogenesis, Nanos protein is involved in the establishment and maintenance of germ-line stem cells and the differentiation of oocyte precursor cells. To establish proper embryonic patterning, Nanos acts as a translational regulator of hunchback RNA. Nanos' targets for germ cell migration and development are not known. Here, we describe a selective genetic screen aimed at isolating new nanos alleles. The molecular and genetic analysis of 68 new alleles has allowed us to identify amino acids critical for nanos function. This analysis shows that the CCHC motifs, which coordinate two metal ions, are essential for all known functions of Nanos protein. Furthermore, a region C-terminal to the zinc fingers seems to constitute a novel functional domain within the Nanos protein. This "tail region" of Nanos is required for abdomen formation and germ cell migration, but not for oogenesis.     

Temporal complexity within a translational control element in the nanos mRNA

Translational control of gene expression plays a fundamental role in the early development of many organisms. In Drosophila, selective translation of nanos mRNA localized to the germ plasm at the posterior of the embryo, together with translational repression of nanos in the bulk cytoplasm, is essential for development of the anteroposterior body pattern. We show that both components to spatial control of nanos translation initiate during oogenesis and that translational repression is initially independent of Smaug, an embryonic repressor of nanos. Repression during oogenesis and embryogenesis are mediated by distinct stem loops within the nanos 3' untranslated region; the Smaug-binding stem-loop acts strictly in the embryo, whereas a second stem-loop functions in the oocyte. Thus, independent regulatory modules with temporally distinct activities contribute to spatial regulation of nanos translation. We propose that nanos evolved to exploit two different stage-specific translational regulatory mechanisms.     

Translational control of maternal Cyclin B mRNA by Nanos in the Drosophila germline

In the Drosophila embryo, Nanos and Pumilio collaborate to repress the translation of hunchback mRNA in the somatic cytoplasm. Both proteins are also required for repression of maternal Cyclin B mRNA in the germline; it has not been clear whether they act directly on Cyclin B mRNA, and if so, whether regulation in the presumptive somatic and germline cytoplasm proceeds by similar or fundamentally different mechanisms. In this report, we show that Pumilio and Nanos bind to an element in the 3' UTR to repress Cyclin B mRNA. Regulation of Cyclin B and hunchback differ in two significant respects. First, Pumilio is dispensable for repression of Cyclin B (but not hunchback) if Nanos is tethered via an exogenous RNA-binding domain. Nanos probably acts, at least in part, by recruiting the CCR4-Pop2-NOT deadenylase complex, interacting directly with the NOT4 subunit. Second, although Nanos is the sole spatially limiting factor for regulation of hunchback, regulation of Cyclin B requires another Oskar-dependent factor in addition to Nanos. Ectopic repression of Cyclin B in the presumptive somatic cytoplasm causes lethal nuclear division defects. We suggest that a requirement for two spatially restricted factors is a mechanism for ensuring that Cyclin B regulation is strictly limited to the germline.     

Multiple mechanisms collaborate to repress nanos translation in the Drosophila ovary and embryo

Translational control of gene expression is essential for development in organisms that rely on maternal mRNAs. In Drosophila, translation of maternal nanos (nos) mRNA must be restricted to the posterior of the early embryo for proper patterning of the anterior-posterior axis. Spatial control of nos translation is coordinated through the localization of a small subset of nos mRNA to the posterior pole late in oogenesis, activation of this localized mRNA, and repression of the remaining unlocalized nos mRNA throughout the bulk cytoplasm. Translational repression is mediated by the interaction of a cis-acting element in the nos 3' untranslated region with two proteins, Glorund (Glo) and Smaug (Smg), that function in the oocyte and embryo, respectively. The mechanism of Glo-dependent repression is unknown. Previous work suggests that Smg represses translation initiation but this model is not easily reconciled with evidence for polysome association of repressed nos mRNA. Using an in vitro translation system, we have decoupled translational repression of nos imposed during oogenesis from repression during embryogenesis. Our results suggest that both Glo and Smg regulate translation initiation, but by different mechanisms. Furthermore, we show that, during late oogenesis, nos translation is also repressed post-initiation and provide evidence that Glo mediates this event. This post-initiation block is maintained into embryogenesis during the transition to Smg-dependent regulation. We propose that the use of multiple modes of repression ensures inactivation of nos RNA that is translated at earlier stages of oogenesis and maintenance of this inactivate state throughout late oogenesis into embryogenesis.     

Spatial regulation of nanos is required for its function in dendrite morphogenesis

Spatial control of mRNA translation can generate cellular asymmetries and functional specialization of polarized cells like neurons. A requirement for the translational repressor Nanos (Nos) in the Drosophila larval peripheral nervous system (PNS) implicates translational control in dendrite morphogenesis [1]. Nos was first identified by its requirement in the posterior of the early embryo for abdomen formation [2]. Nos synthesis is targeted to the posterior pole of the oocyte and early embryo through translational repression of unlocalized nos mRNA coupled with translational activation of nos mRNA localized at the posterior pole [3, 4]. Abolishment of nos localization prevents abdominal development, whereas translational derepression of unlocalized nos mRNA suppresses head/thorax development, emphasizing the importance of spatial regulation of nos mRNA [3, 5]. Loss and overexpression of Nos affect dendrite branching complexity in class IV dendritic arborization (da) neurons, suggesting that nos also might be regulated in these larval sensory neurons [1]. Here, we show that localization and translational control of nos mRNA are essential for da neuron morphogenesis. RNA-protein interactions that regulate nos translation in the oocyte and early embryo also regulate nos in the PNS. Live imaging of nos mRNA shows that the cis-acting signal responsible for posterior localization in the oocyte/embryo mediates localization to the processes of class IV da neurons but suggests a different transport mechanism. Targeting of nos mRNA to the processes of da neurons may reflect a local requirement for Nos protein in dendritic translational control.     

Novel modes of localization and function of nanos in the wasp Nasonia

Abdominal patterning in Drosophila requires the function of nanos (nos) to prevent translation of hunchback (hb) mRNA in the posterior of the embryo. nos function is restricted to the posterior by the translational repression of mRNA that is not incorporated into the posteriorly localized germ plasm during oogenesis. The wasp Nasonia vitripennis (Nv) undergoes a long germ mode of development very similar to Drosophila, although the molecular patterning mechanisms employed in these two organisms have diverged significantly, reflecting the independent evolution of this mode of development. Here, we report that although Nv nanos (Nv-nos) has a conserved function in embryonic patterning through translational repression of hb, the timing and mechanisms of this repression are significantly delayed in the wasp compared with the fly. This delay in Nv-nos function appears to be related to the dynamic behavior of the germ plasm in Nasonia, as well as to the maternal provision of Nv-Hb protein during oogenesis. Unlike in flies, there appears to be two functional populations of Nv-nos mRNA: one that is concentrated in the oosome and is taken up into the pole cells before evidence of Nv-hb repression is observed; another that forms a gradient at the posterior and plays a role in Nv-hb translational repression. Altogether, our results show that, although the embryonic patterning function of nos orthologs is broadly conserved, the mechanisms employed to achieve this function are distinct.     

Live imaging of endogenous RNA reveals a diffusion and entrapment mechanism for nanos mRNA localization in Drosophila

BACKGROUND: Localization of nanos mRNA to the posterior pole of the Drosophila embryo directs local synthesis of Nanos protein that is essential for patterning of the anterior-posterior body axis and germ cell function. While nanos RNA is synthesized by the ovarian nurse cells and appears at the posterior pole of the ooctye late in oogenesis, the mechanism by which this RNA is translocated to and anchored at the oocyte posterior is unknown. RESULTS: By labeling endogenous nanos RNA with GFP, we have been able to follow the dynamic pathway of nanos localization in living oocytes. We demonstrate that nanos localization initiates immediately upon nurse cell dumping, whereby diffusion, enhanced by microtubule-dependent cytoplasmic movements, translocates nanos RNA from the nurse cells to the ooctye posterior. At the posterior, nanos is trapped by association, in particles, with the posteriorly localized germ plasm. Actin-dependent anchoring of nanos RNA complexed to the germ plasm at the posterior maintains localization in the face of rapid cytoplasmic movements. CONCLUSIONS: These results reveal a diffusion-based, late-acting posterior localization mechanism for long-range transport of nanos mRNA. This mechanism differs from directed transport-based localization mechanisms in its reliance on bulk movement of RNA.     

A common translational control mechanism functions in axial patterning and neuroendocrine signaling in Drosophila

Translational repression of maternal nanos (nos) mRNA by a cis-acting Translational Control Element (TCE) in the nos 3'UTR is critical for anterior-posterior patterning of the Drosophila embryo. We show, through ectopic expression experiments, that the nos TCE is capable of repressing gene expression at later stages of development in neuronal cells that regulate the molting cycle. Our results predict additional targets of TCE-mediated repression within the nervous system. They also suggest that mechanisms that regulate maternal mRNAs, like TCE-mediated repression, may function more widely during development to spatially or temporally control gene expression.     

Synthesis of the posterior determinant Nanos is spatially restricted by a novel cotranslational regulatory mechanism

Nanos (Nos) protein is required in the posterior of the Drosophila embryo to promote abdominal development, but must be excluded from the anterior to permit head and thorax development [1,2]. Spatial restriction of Nos is accomplished by selective translation of the 4% of nos mRNA localized to the posterior pole and translational repression of the remaining unlocalized mRNA [3-5]. Repression is mediated by a 90-nucleotide translational control element (TCE) in the nos 3' untranslated region (UTR) and the TCE-binding protein Smaug [4,6,7], but the molecular mechanism is unknown. We used sucrose density gradient sedimentation to ascertain whether unlocalized nos mRNA is excluded from polysomes and therefore repressed during translational initiation. Surprisingly, a significant percentage of nos mRNA was found to be associated with polysomes, even in mutants in which all nos mRNA is unlocalized and repressed. Using a regulated Drosophila cell-free translation system, we showed that ribosomes contained within these polysomes are capable of elongation in vitro, under conditions in which synthesis of Nos protein is repressed. Thus, synthesis of ectopic Nos protein is inhibited by a novel regulatory mechanism that does not involve a stable arrest of the translation cycle.     

RNA recognition via the SAM domain of Smaug

The Nanos protein gradient in Drosophila, required for proper abdominal segmentation, is generated in part via translational repression of its mRNA by Smaug. We report here the crystal structure of the Smaug RNA binding domain, which shows no sequence homology to any previously characterized RNA binding motif. The structure reveals an unusual makeup in which a SAM domain, a common protein-protein interaction module, is affixed to a pseudo-HEAT repeat analogous topology (PHAT) domain. Unexpectedly, we find through a combination of structural and genetic analysis that it is primarily the SAM domain that interacts specifically with the appropriate nanos mRNA regulatory sequence. Therefore, in addition to their previously characterized roles in protein-protein interactions, some SAM domains play crucial roles in RNA binding.