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.     

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.     

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.     

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.     

Overlapping but distinct RNA elements control repression and activation of nanos translation

Spatially restricted synthesis of Nanos protein in the Drosophila embryo is essential for anterior-posterior patterning. Nanos translation is restricted to the posterior of the embryo by translational repression of nanos mRNA throughout the bulk cytoplasm and selective activation of posteriorly localized nanos mRNA. A 90-nucleotide translational control element (TCE) mediates translational repression. We show that TCE function requires formation of a bipartite secondary structure that is recognized by Smaug repressor and at least one additional factor. We also demonstrate that translational activation requires the interaction of localization factors with sequences that overlap TCE structural motifs. The identification of separate but overlapping recognition motifs for translational repressors and localization factors provides a molecular mechanism for the switch between translational repression and activation.     

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.     

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.     

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.     

Dual nature of translational control by regulatory BC RNAs

In higher eukaryotes, increasing evidence suggests, gene expression is to a large degree controlled by RNA. Regulatory RNAs have been implicated in the management of neuronal function and plasticity in mammalian brains. However, much of the molecular-mechanistic framework that enables neuronal regulatory RNAs to control gene expression remains poorly understood. Here, we establish molecular mechanisms that underlie the regulatory capacity of neuronal BC RNAs in the translational control of gene expression. We report that regulatory BC RNAs employ a two-pronged approach in translational control. One of two distinct repression mechanisms is mediated by C-loop motifs in BC RNA 3' stem-loop domains. These C-loops bind to eIF4B and prevent the factor's interaction with 18S rRNA of the small ribosomal subunit. In the second mechanism, the central A-rich domains of BC RNAs target eIF4A, specifically inhibiting its RNA helicase activity. Thus, BC RNAs repress translation initiation in a bimodal mechanistic approach. As BC RNA functionality has evolved independently in rodent and primate lineages, our data suggest that BC RNA translational control was necessitated and implemented during mammalian phylogenetic development of complex neural systems.     

The Xenopus ELAV protein ElrB represses Vg1 mRNA translation during oogenesis

Xenopus laevis Vg1 mRNA undergoes both localization and translational control during oogenesis. We previously characterized a 250-nucleotide AU-rich element, the Vg1 translation element (VTE), in the 3'-untranslated region (UTR) of this mRNA that is responsible for the translational repression. UV-cross-linking and immunoprecipitation experiments, described here, revealed that the known AU-rich element binding proteins, ElrA and ElrB, and TIA-1 and TIAR interact with the VTE. The levels of these proteins during oogenesis are most consistent with a possible role for ElrB in the translational control of Vg1 mRNA, and ElrB, in contrast to TIA-1 and TIAR, is present in large RNP complexes. Immunodepletion of TIA-1 and TIAR from Xenopus translation extract confirmed that these proteins are not involved in the translational repression. Mutagenesis of a potential ElrB binding site destroyed the ability of the VTE to bind ElrB and also abolished translational repression. Moreover, multiple copies of the consensus motif both bind ElrB and support translational control. Therefore, there is a direct correlation between ElrB binding and translational repression by the Vg1 3'-UTR. In agreement with the reporter data, injection of a monoclonal antibody against ElrB into Xenopus oocytes resulted in the production of Vg1 protein, arguing for a role for the ELAV proteins in the translational repression of Vg1 mRNA during early oogenesis.     

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.     

Localization of nanos RNA controls embryonic polarity.

Anterior-posterior polarity of the Drosophila embryo is initiated during oogenesis through differential maternal RNA localization. The RNA of the anterior morphogen bicoid is localized to the anterior pole of the embryo, where bicoid protein controls head and thorax development. The RNA of the posterior morphogen nanos is localized to the posterior pole, where nanos protein is required for abdomen formation. Here we show that the nanos 3' untranslated region, like that of the bicoid RNA, is sufficient for RNA localization. We have used the bicoid RNA localization signal to mislocalize nanos, producing embryos with two sources of nanos protein. Such embryos form two abdomens with mirror image symmetry. Embryos with nanos RNA localized only to the anterior have greater nanos gene activity than embryos with nanos RNA localized posteriorly. We propose a role for RNA localization in regulating nanos activity.     

The maternal gene nanos has a central role in posterior pattern formation of the Drosophila embryo

A group of maternal genes, the posterior group, is required for the development of the abdominal region in the Drosophila embryo. We have used genetic as well as cytoplasmic transfer experiments to order seven of the posterior group genes (nanos, pumilio, oskar, valois, vasa, staufen and tudor) into a functional pathway. An activity present in the posterior pole plasm of wild-type embryos can restore normal abdominal development in posterior group mutants. This activity is synthesized during oogenesis and the gene nanos most likely encodes this activity. The other posterior group genes have distinct accessory functions: pumilio acts downstream of nanos and is required for the distribution or stability of the nanos-dependent activity in the embryo. Staufen, oskar, vasa, valois and tudor act upstream of nanos. Embryos from females mutant for these genes lack the specialized posterior pole plasm and consequently fail to form germ-cell precursors. We suggest that the products of these genes provide the physical structure necessary for the localization of nanos-dependent activity and of germ line determinants.     

Determinants of RNA Binding and Translational Repression by the Bicaudal-C Regulatory Protein

Bicaudal-C (Bic-C) RNA binding proteins function as important translational repressors in multiple biological contexts within metazoans. However, their RNA binding sites are unknown. We recently demonstrated that Bic-C functions in spatially regulated translational repression of the xCR1 mRNA during Xenopus development. This repression contributes to normal development by confining the xCR1 protein, a regulator of key signaling pathways, to specific cells of the embryo. In this report, we combined biochemical approaches with in vivo mRNA reporter assays to define the minimal Bic-C target site within the xCR1 mRNA. This 32-nucleotide Bic-C target site is predicted to fold into a stem-loop secondary structure. Mutational analyses provided evidence that this stem-loop structure is important for Bic-C binding. The Bic-C target site was sufficient for Bic-C mediated repression in vivo. Thus, we describe the first RNA binding site for a Bic-C protein. This identification provides an important step toward understanding the mechanisms by which evolutionarily conserved Bic-C proteins control cellular function in metazoans.     

Translational repression of a C. elegans Notch mRNA by the STAR/KH domain protein GLD-1

In C. elegans, the Notch receptor GLP-1 is localized within the germline and early embryo by translational control of glp-1 mRNA. RNA elements in the glp-1 3'untranslated region (3' UTR) are necessary for repression of glp-1 translation in germ cells, and for localization of translation to anterior cells of the early embryo. The direct regulators of glp-1 mRNA are not known. Here, we show that a 34 nucleotide region of the glp-1 3' UTR contains two regulatory elements, an element that represses translation in germ cells and posterior cells of the early embryo, and an element that inhibits repressor activity to promote translation in the embryo. Furthermore, we show that the STAR/KH domain protein GLD-1 binds directly and specifically to the repressor element. Depletion of GLD-1 activity by RNA interference causes loss of endogenous glp-1 mRNA repression in early meiotic germ cells, and in posterior cells of the early embryo. Therefore, GLD-1 is a direct repressor of glp-1 translation at two developmental stages. These results suggest a new function for GLD-1 in regulating early embryonic asymmetry. Furthermore, these observations indicate that precise control of GLD-1 activity by other regulatory factors is important to localize this Notch receptor, and contributes to the spatial organization of Notch signaling.     

The Drosophila PNG kinase complex regulates the translation of cyclin B

The Drosophila PAN GU (PNG) kinase complex regulates the developmental translation of cyclin B. cyclin B mRNA becomes unmasked during oogenesis independent of PNG activity, but PNG is required for translation from egg activation. We find that although polyadenylation of cyclin B augments translation, it is not essential, and a fully elongated poly(A) is not required for translation to proceed. In fact, changes in poly(A) tail length are not sufficient to account for PNG-mediated control of cyclin B translation and of the early embryonic cell cycles. We present evidence that PNG functions instead as an antagonist of PUMILIO-dependent translational repression. Our data argue that changes in poly(A) tail length are not a universal mechanism governing embryonic cell cycles, and that PNG-mediated derepression of translation is an important alternative mechanism in Drosophila.