Control of oskar mRNA translation by Bruno in a novel cell-free system from Drosophila ovaries

The coupled regulation of oskar mRNA localization and translation in time and space is critical for correct anteroposterior patterning of the Drosophila embryo. Localization-dependent translation of oskar mRNA, a mechanism whereby oskar RNA localized at the posterior of the oocyte is selectively translated and the unlocalized RNA remains in a translationally repressed state, ensures that Oskar activity is present exclusively at the posterior pole. Genetic experiments indicate that translational repression involves the binding of Bruno protein to multiple sites, the Bruno Response Elements (BRE), in the 3' untranslated region (UTR) of oskar mRNA. We have established a cell-free translation system derived from Drosophila ovaries, which faithfully reproduces critical features of mRNA translation in vivo, namely cap structure and poly(A) tail dependence. We show that this ovary extract, containing endogenous Bruno, is able to recapitulate oskar mRNA regulation in a BRE-dependent way. Thus, the assembly of a ribonucleoprotein (RNP) complex leading to the translationally repressed state occurs in vitro. Moreover, we show that a Drosophila embryo extract lacking Bruno efficiently translates oskar mRNA. Addition of recombinant Bruno to this extract establishes the repressed state in a BRE-dependent manner, providing a direct biochemical demonstration of the critical role of Bruno in oskar mRNA translation. The approach that we describe opens new avenues to investigate translational regulation in Drosophila oogenesis at a biochemical level.     

Drosophila cup is an eIF4E binding protein that associates with Bruno and regulates oskar mRNA translation in oogenesis

Translational control is a critical process in the spatio-temporal restriction of protein production. In Drosophila oogenesis, translational repression of oskar (osk) RNA during its localization to the posterior pole of the oocyte is essential for embryonic patterning and germ cell formation. This repression is mediated by the osk 3' UTR binding protein Bruno (Bru), but the underlying mechanism has remained elusive. Here, we report that an ovarian protein, Cup, is required to repress precocious osk translation. Cup binds the 5'-cap binding translation initiation factor eIF4E through a sequence conserved among eIF4E binding proteins. A mutant Cup protein lacking this sequence fails to repress osk translation in vivo. Furthermore, Cup interacts with Bru in a yeast two-hybrid assay, and the Cup-eIF4E complex associates with Bru in an RNA-independent manner. These results suggest that translational repression of osk RNA is achieved through a 5'/3' interaction mediated by an eIF4E-Cup-Bru complex.     

Translational control of dosage compensation in Drosophila by Sex-lethal: cooperative silencing via the 5' and 3' UTRs of msl-2 mRNA is independent of the poly(A) tail.

Translational repression of male-specific-lethal 2 (msl-2) mRNA by Sex-lethal (SXL) controls dosage compensation in Drosophila. In vivo regulation involves cooperativity between SXL-binding sites in the 5' and 3' untranslated regions (UTRs). To investigate the mechanism of msl-2 translational control, we have developed a novel cell-free translation system from Drosophila embryos that recapitulates the critical features of mRNA translation in eukaryotes: cap and poly(A) tail dependence. Importantly, tight regulation of msl-2 translation in this system requires cooperation between the SXL-binding sites in both the 5' and 3' UTRs, as seen in vivo. However, in contrast to numerous other developmentally regulated mRNAs, the regulation of msl-2 mRNA occurs by a poly(A) tail-independent mechanism. The approach described here allows mechanistic analysis of translational control in early Drosophila development and has revealed insights into the regulation of dosage compensation by SXL.     

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.     

Drosophila sex-lethal inhibits the stable association of the 40S ribosomal subunit with msl-2 mRNA

The inhibition of male-specific lethal-2 (msl-2) mRNA translation in female flies is essential for X chromosome dosage compensation in Drosophila melanogaster. Translational repression of msl-2 requires sex-lethal (SXL) binding to uridine-rich sequences in both the 5' and 3' untranslated regions (UTRs) of the message. We delineate the msl-2 mRNA sequence elements that are important for regulation by SXL and identify functionally critical sequences adjacent to regulatory SXL binding sites. We demonstrate that SXL inhibits translation initiation and prevents the stable association of the 40S ribosomal subunit with the mRNA in a manner that does not require the presence of a cap structure at the 5' end of the mRNA. These results elucidate a critical regulatory step for dosage compensation in Drosophila melanogaster.     

A late phase of Oskar accumulation is crucial for posterior patterning of the Drosophila embryo, and is blocked by ectopic expression of Bruno

In Drosophila, posterior embryonic body patterning and germ cell formation rely on Oskar, a protein that is concentrated at the posterior pole of the oocyte. A program of mRNA localization and translational regulation ensures that Oskar is only expressed at the proper location. One key regulatory factor is Bruno, which represses translation of oskar mRNA before its localization. Ectopic expression of a bruno cDNA prolongs repression, even after oskar mRNA is localized, and posterior body patterning is efficiently and selectively blocked. Surprisingly, the initial accumulation of Oskar, while frequently reduced, is not eliminated, arguing that levels of Oskar previously thought to be sufficient for patterning do not suffice, or that Bruno acts at a downstream step in patterning. Expression of the bruno cDNA does not inhibit posterior patterning when Oskar is expressed independent of Bruno-mediated regulation, ruling out a downstream requirement for Bruno. Notably, an Oskar::GFP reporter protein reveals continual accumulation during the late phases of oogenesis. Taken together, these results strongly argue that a late phase in accumulation of Osk protein, typically not monitored because of imperviousness of late stage oocytes to antibodies, is crucial for body patterning.     

Proteomic analysis of reaper 5' untranslated region-interacting factors isolated by tobramycin affinity-selection reveals a role for La antigen in reaper mRNA translation

Translational control is a key step in gene expression regulation during apoptosis. To understand the mechanisms of mRNA translation of a pro-apoptotic gene, reaper (rpr), we adapted the tobramycin-aptamer technique described by Hartmuth et al. (Proc. Natl. Acad. Sci. USA 2002, 99, 16719-16724) for the analysis of proteins interacting with rpr 5' untranslated region (UTR). We assembled ribonucleoprotein complexes in vitro using translation extracts derived from Drosophila embryos and purified the RNA-protein complexes for mas spectrometry analysis. We identified the proteins bound to the 5' UTR of rpr. One of them, the La antigen, was validated by RNA-crosslinking experiments using recombinant protein and by the translation efficiency of reporter mRNAs in Drosophila cells after RNAinterference experiments. Our data provide evidence of the involvement of La antigen in the translation of rpr and set a protocol for purification of tagged-RNA-protein complexes from cytoplasmic extracts.     

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.     

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.     

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.     

Biochemical identification of Xenopus Pumilio as a sequence-specific cyclin B1 mRNA-binding protein that physically interacts with a Nanos homolog, Xcat-2, and a cytoplasmic polyadenylation element-binding protein

Translational activation of dormant cyclin B1 mRNA stored in oocytes is a prerequisite for the initiation or promotion of oocyte maturation in many vertebrates. Using a monoclonal antibody against the domain highly homologous to that of Drosophila Pumilio, we have shown for the first time in any vertebrate that a homolog of Pumilio is expressed in Xenopus oocytes. This 137-kDa protein binds to the region including the sequence UGUA at nucleotides 1335-1338 in the 3'-untranslated region of cyclin B1 mRNA, which is close to but does not overlap the cytoplasmic polyadenylation elements (CPEs). Physical in vitro association of Xenopus Pumilio with a Xenopus homolog of Nanos (Xcat-2) was demonstrated by a protein pull-down assay. The results of immunoprecipitation experiments showed in vivo interaction between Xenopus Pumilio and CPE-binding protein (CPEB), a key regulator of translational repression and activation of mRNAs stored in oocytes. This evidence provides a new insight into the mechanism of translational regulation through the 3'-end of mRNA during oocyte maturation. These results also suggest the generality of the function of Pumilio as a translational regulator of dormant mRNAs in both invertebrates and vertebrates.     

The 5' UTR of protein kinase C epsilon confers translational regulation in vitro and in vivo

We have examined translational regulation conferred by the 5' untranslated region (UTR) of PKCepsilon on expression of the luciferase reporter gene in vitro, using rabbit reticulocyte lysates and in vivo, in contact-inhibiting mouse Swiss 3T3 fibroblasts and non-contact-inhibiting Swiss 3T6 fibroblasts. In rabbit reticulocyte lysates, the 5' UTR of PKCepsilon significantly represses translation. In 3T3 and 3T6 cells, the 5' UTR of PKCepsilon reduces luciferase activity, but not to the same extent as it does in vitro. In rabbit reticulocyte lysate, the degree of repression mediated by different PKCepsilon 5' UTR-deletion constructs correlates with the free energy (DeltaG) of their predicted secondary structures. However, in cells, secondary structure is not the only determinant of repression; an internal region of the 5' UTR is both necessary and sufficient for repression. Mutation of an upstream AUG (uAUG) motif in this region partially relieves repression. We conclude that the 5' UTR of PKCepsilon can mediate translational regulation and that translation inhibition in vivo involves the uAUG motif. Our findings also suggest that there are factors present in fibroblasts, but not in rabbit reticulocyte lysates that substantially overcome the repressive qualities of the long, structured 5' UTR. Thus, we have identified a potential new level of regulation of PKC in mammalian cells.     

Dendritic BC1 RNA in translational control mechanisms

Translational control at the synapse is thought to be a key determinant of neuronal plasticity. How is such control implemented? We report that small untranslated BC1 RNA is a specific effector of translational control both in vitro and in vivo. BC1 RNA, expressed in neurons and germ cells, inhibits a rate-limiting step in the assembly of translation initiation complexes. A translational repression element is contained within the unique 3′ domain of BC1 RNA. Interactions of this domain with eukaryotic initiation factor 4A and poly(A) binding protein mediate repression, indicating that the 3′ BC1 domain targets a functional interaction between these factors. In contrast, interactions of BC1 RNA with the fragile X mental retardation protein could not be documented. Thus, BC1 RNA modulates translation-dependent processes in neurons and germs cells by directly interacting with translation initiation factors.     

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.