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.     

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.     

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.     

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.     

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.     

Functional domains of Drosophila UNR in translational control

Translational repression of male-specific-lethal 2 (msl-2) mRNA by Sex-lethal (SXL) is an essential regulatory step of X chromosome dosage compensation in Drosophila. Translation inhibition requires that SXL recruits the protein upstream of N-ras (UNR) to the 3' UTR of msl-2 mRNA. UNR is a conserved, ubiquitous protein that contains five cold-shock domains (CSDs). Here, we dissect the domains of UNR required for translational repression and complex formation with SXL and msl-2 mRNA. Using gel-mobility shift assays, the domain involved in interactions with SXL and msl-2 was mapped specifically to the first CSD (CSD1). Indeed, excess of a peptide containing this domain derepressed msl-2 translation in vitro. The CSD1 of human UNR can also form a complex with SXL and msl-2. Comparative analyses of the CSDs of the Drosophila and human proteins together with site-directed mutagenesis experiments revealed that three exposed residues within CSD1 are required for complex formation. Tethering assays showed that CSD1 is not sufficient for translational repression, indicating that UNR binding to SXL and msl-2 can be distinguished from translation inhibition. Repression by tethered UNR requires residues from both the amino-terminal Q-rich stretch and the two first CSDs, indicating that the translational effector domain of UNR resides within the first 397 amino acids of the protein. Our results identify domains and residues required for UNR function in translational control.     

Translational repression of gurken mRNA in the Drosophila oocyte requires the hnRNP Squid in the nurse cells

Establishment of the Drosophila dorsal-ventral axis depends upon the correct localization of gurken mRNA and protein within the oocyte. gurken mRNA becomes localized to the presumptive dorsal anterior region of the oocyte, but is synthesized in the adjoining nurse cells. Normal gurken localization requires the heterogeneous nuclear ribonucleoprotein Squid, which binds to the gurken 3′ untranslated region. However, whether Squid functions in the nurse cells or the oocyte is unknown. To address this question, we generated genetic mosaics in which half of the nurse cells attached to a given oocyte are unable to produce Squid. In these mosaics, gurken mRNA is localized normally but ectopically translated during the dorsal anterior localization process, even though the oocyte contains abundant Squid produced by the wild type nurse cells. These data indicate that translational repression of gurken mRNA requires Squid function in the nurse cells. We propose that Squid interacts with gurken mRNA in the nurse cell nuclei and, together with other factors, maintains gurken in a translationally silent state during its transport to the dorsal anterior region of the oocyte. This translational repression is not required for gurken mRNA localization, indicating that the information repressing translation is separable from that regulating localization.     

Requirement for both EDEN and AUUUA motifs in translational arrest of Mos mRNA upon fertilization of Xenopus eggs

Translational arrest of maternal Mos mRNA upon fertilization of Xenopus eggs is a prerequisite for the initiation of embryonic divisions. Recent studies suggest that an embryo deadenylation element (EDEN) present in the 3' untranslated region (3'UTR) is sufficient for deadenylation (and, hence, probably for translational arrest) of Mos mRNA after fertilization. By directly monitoring translation of numerous Mos mRNA constructs in Xenopus eggs, however, we show here that the EDEN is necessary but not sufficient for translational arrest of Mos mRNA. We demonstrate that two AUUUA motifs, each located solitarily and distantly from the EDEN, are also required for the translational arrest of Mos mRNA after fertilization. Significantly, translational arrest of Eg2 mRNA, another EDEN-containing maternal mRNA, also requires a single AUUUA motif located far from the EDEN. Analysis of the poly(A) tails of various Mos mRNA constructs indicates that the EDEN alone confers only partial deadenylation on Mos mRNA, and that the AUUUA motifs act to enhance EDEN-directed deadenylation in a position-dependent manner. Finally, introduction of an excess of the EDEN, but not the AUUUA motifs, into eggs can restore translation of endogenous Mos mRNA. These results suggest that the EDEN, only together with appropriately positioned AUUUA motifs and a trans-acting factor(s), can efficiently deadenylate and hence translationally arrest Mos (as well as Eg2) mRNA after fertilization.     

Control of translation by the 5'- and 3'-terminal regions of the dengue virus genome

The genomic RNAs of flaviviruses such as dengue virus (DEN) have a 5' m7GpppN cap like those of cellular mRNAs but lack a 3' poly(A) tail. We have studied the contributions to translational expression of 5'- and 3'-terminal regions of the DEN serotype 2 genome by using luciferase reporter mRNAs transfected into Vero cells. DCLD RNA contained the entire DEN 5' and 3' untranslated regions (UTRs), as well as the first 36 codons of the capsid coding region fused to the luciferase reporter gene. Capped DCLD RNA was as efficiently translated in Vero cells as capped GLGpA RNA, a reporter with UTRs from the highly expressed alpha-globin mRNA and a 72-residue poly(A) tail. Analogous reporter RNAs with regulatory sequences from West Nile and Sindbis viruses were also strongly expressed. Although capped DCLD RNA was expressed much more efficiently than its uncapped form, uncapped DCLD RNA was translated 6 to 12 times more efficiently than uncapped RNAs with UTRs from globin mRNA. The 5' cap and DEN 3' UTR were the main sources of the translational efficiency of DCLD RNA, and they acted synergistically in enhancing translation. The DEN 3' UTR increased mRNA stability, although this effect was considerably weaker than the enhancement of translational efficiency. The DEN 3' UTR thus has translational regulatory properties similar to those of a poly(A) tail. Its translation-enhancing effect was observed for RNAs with globin or DEN 5' sequences, indicating no codependency between viral 5' and 3' sequences. Deletion studies showed that translational enhancement provided by the DEN 3' UTR is attributable to the cumulative contributions of several conserved elements, as well as a nonconserved domain adjacent to the stop codon. One of the conserved elements was the conserved sequence (CS) CS1 that is complementary to cCS1 present in the 5' end of the DEN polyprotein open reading frame. Complementarity between CS1 and cCS1 was not required for efficient translation.