Smaug assembles an ATP-dependent stable complex repressing nanos mRNA translation at multiple levels

The nanos (nos) mRNA encodes the posterior determinant of the Drosophila embryo. Translation of the RNA is repressed throughout most of the embryo by the protein Smaug binding to Smaug recognition elements (SREs) in the 3' UTR. Translation is locally activated at the posterior pole by Oskar. This paper reports that the SREs govern the time- and ATP-dependent assembly of an exceedingly stable repressed ribonucleoprotein particle (RNP) in embryo extract. Repression can be virtually complete. Smaug and its co-repressor Cup as well as Trailer hitch and the DEAD box protein Me31B are part of the repressed RNP. The initiation factor eIF4G is specifically displaced, and 48S pre-initiation complex formation is inhibited. However, later steps in translation initiation are also sensitive to SRE-dependent inhibition. These data confirm several previously untested predictions of a current model for Cup-dependent repression but also suggest that the Cup model by itself is insufficient to explain translational repression of the nos RNA. In the embryo extract, recombinant Oskar relieves translational repression and deadenylation by preventing Smaug's binding to the SREs.     

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.     

microRNA‐independent recruitment of Argonaute 1 to nanos mRNA through the Smaug RNA‐binding protein

Argonaute (Ago) proteins are typically recruited to target messenger RNAs via an associated small RNA such as a microRNA (miRNA). Here, we describe a new mechanism of Ago recruitment through the Drosophila Smaug RNA‐binding protein. We show that Smaug interacts with the Ago1 protein, and that Ago1 interacts with and is required for the translational repression of the Smaug target, nanos mRNA. The Ago1/nanos mRNA interaction does not require a miRNA, but it does require Smaug. Taken together, our data suggest a model whereby Smaug directly recruits Ago1 to nanos mRNA in a miRNA‐independent manner, thereby repressing translation.     

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.     

Tab2 is a novel conserved RNA binding protein required for translation of the chloroplast psaB mRNA

The chloroplast psaB mRNA encodes one of the reaction centre polypeptides of photosystem I. Protein pulse-labelling profiles indicate that the mutant strain of Chlamydomonas reinhardtii, F14, affected at the nuclear locus TAB2, is deficient in the translation of psaB mRNA and thus deficient in photosystem I activity. Genetic studies reveal that the target site for Tab2 is situated within the psaB 5'UTR. We have used genomic complementation to isolate the nuclear Tab2 gene. The deduced amino acid sequence of Tab2 (358 residues) displays 31-46% sequence identity with several orthologues found only in eukaryotic and prokaryotic organisms performing oxygenic photosynthesis. Directed mutagenesis indicates the importance of a highly conserved C-terminal tripeptide in Tab2 for normal psaB translation. The Tab2 protein is localized in the chloroplast stroma where it is associated with a high molecular mass protein complex containing the psaB mRNA. Gel mobility shift assays reveal a direct and specific interaction between Tab2 and the psaB 5'UTR. We propose that Tab2 plays a key role in the initial steps of PsaB translation and photosystem I assembly.     

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.     

The conserved GTPase LepA contributes mainly to translation initiation in Escherichia coli

LepA is a paralog of EF-G found in all bacteria. Deletion of lepA confers no obvious growth defect in Escherichia coli, and the physiological role of LepA remains unknown. Here, we identify nine strains (ΔdksA, ΔmolR1, ΔrsgA, ΔtatB, ΔtonB, ΔtolR, ΔubiF, ΔubiG or ΔubiH) in which ΔlepA confers a synthetic growth phenotype. These strains are compromised for gene regulation, ribosome assembly, transport and/or respiration, indicating that LepA contributes to these functions in some way. We also use ribosome profiling to deduce the effects of LepA on translation. We find that loss of LepA alters the average ribosome density (ARD) for hundreds of mRNA coding regions in the cell, substantially reducing ARD in many cases. By contrast, only subtle and codon-specific changes in ribosome distribution along mRNA are seen. These data suggest that LepA contributes mainly to the initiation phase of translation. Consistent with this interpretation, the effect of LepA on ARD is related to the sequence of the Shine–Dalgarno region. Global perturbation of gene expression in the ΔlepA mutant likely explains most of its phenotypes.     

RNA-binding properties and translation repression in vitro by germ cell-specific MSY2 protein

The large amount of MSY2 protein, a mouse germ cell-specific Y-box protein, in oocytes and its degradation by the late two-cell stage suggest that MSY2 may stabilize and/or regulate the translation of maternal mRNAs. We report here the ability of bacterially expressed recombinant MSY2 protein to bind to mRNA and repress translation in vitro. Although MSY2 displays some sequence specificity in binding to short RNA sequences derived from the 3' untranslated region (UTR) of the protamine 1 (Prm1) mRNA, as determined by both gel shift and filter binding assays, essentially no sequence specificity is observed when full-length Prm1 mRNA is used. The binding of MSY2 is approximately 10-fold greater to the full-length Prm1 mRNA than to a 37-nucleotide sequence derived from the 3' UTR, and gel shift assays indicate that multiple MSY2 molecules bind to a single Prm1 mRNA. MSY2 binding to luciferase mRNA at ratios of protein to mRNA that are likely to exist in the oocyte also leads to a moderate inhibition of protein synthesis in vitro. Given the abundance of MSY2 in mouse oocytes (2% of total oocyte protein), these data suggest that MSY2 packages mRNAs in vivo with relatively little sequence specificity, which may lead to both stabilization and translation repression of maternal mRNAs.     

Isolation and in vitro translation of a mammalian protamine mRNA

mRNA was isolated from sexually mature rat, rabbit, and bovine testes. Poly(A+) and (A-) RNAs were prepared and hybridized to a rainbow-trout protamine probe. The bovine (A+) fraction showed significant hybridization compared to the other species and these related sequences were also found in total bovine DNA. Bovine mRNA programmed the in vitro synthesis of a basic protein that possessed protamine-like properties. The mRNA was fractionated by agarose-gel electro-phoresis and the fractions hybridized to the trout protamine probe. A significant hybridization signal was observed corresponding to a mRNA of approximately 400 nucleotides in length which coded for the protamine-like protein. The data support the view that we have isolated a mammalian (bovine) protamine mRNA.     

Structure and dynamics of the quaternary hunchback mRNA translation repression complex

A key regulatory process during Drosophila development is the localized suppression of the hunchback mRNA translation at the posterior, which gives rise to a hunchback gradient governing the formation of the anterior-posterior body axis. This suppression is achieved by a concerted action of Brain Tumour (Brat), Pumilio (Pum) and Nanos. Each protein is necessary for proper Drosophila development. The RNA contacts have been elucidated for the proteins individually in several atomic-resolution structures. However, the interplay of all three proteins during RNA suppression remains a long-standing open question. Here, we characterize the quaternary complex of the RNA-binding domains of Brat, Pum and Nanos with hunchback mRNA by combining NMR spectroscopy, SANS/SAXS, XL/MS with MD simulations and ITC assays. The quaternary hunchback mRNA suppression complex comprising the RNA binding domains is flexible with unoccupied nucleotides functioning as a flexible linker between the Brat and Pum-Nanos moieties of the complex. Moreover, the presence of the Pum-HD/Nanos-ZnF complex has no effect on the equilibrium RNA binding affinity of the Brat RNA binding domain. This is in accordance with previous studies, which showed that Brat can suppress mRNA independently and is distributed uniformly throughout the embryo.     

In vitro translation of rabbit lung Clara cell secretory protein mRNA

The major secretory product of Clara cells is a low molecular weight protein (CCSP) whose extracellular function, at this time, is not known. The primary translation product of its mRNA is a protein with molecular weight approximately 1 kD greater than that of the native secreted protein (6.0 kD). The primary translation product is not detected in incubated lung tissue, only the secretory protein is found. The primary translation product is trypsin sensitive whereas the secretory protein is not. Cell free translation of the mRNA in the presence of microsomes results in cleavage of the signal peptide and the appearance of the lower molecular weight trypsin-resistant secretory protein. These data indicate that the low molecular weight Clara cell secretory protein is synthesized as a larger, trypsin sensitive, protein. Passage of the protein into the cisternae of the endoplasmic reticulum results in loss of the signal peptide and alterations to the tertiary structure of the protein rendering it trypsin insensitive.     

The novel microtubule-associated protein MAP3 contributes to the in vitro assembly of brain microtubules

MAP3 is a novel microtubule-associated protein found in brain and a variety of other tissues (Huber, G., Alaimo-Beuret, D., and Matus, A. (1985) J. Cell Biol. 100, 496-507). In this study, monoclonal antibodies were used to assess its influence on the polymerization of brain tubulin. When added to unpolymerized brain microtubules, anti-MAP3 IgG produced a dose-related inhibition of subsequent assembly. Under the same circumstances, nonimmune mouse IgG did not influence either the rate or the extent of tubulin polymerization. We also used immobilized antibodies to deplete brain MAPs selectively in either MAP3 or MAP1. MAP3-depleted MAPs showed a reproducible decrease in activity compared to control preparations that had been exposed to immobilized nonimmune IgG. MAP1-depleted MAPs did not differ significantly in performance from the nonimmune treated controls. We conclude that MAP3 contributes to the net assembly of brain microtubules observed in vitro. This may be particularly relevant in neonatal animals where brain MAP3 is more abundant than in the adult.     

Aubergine is a component of a nanos mRNA localization complex

Localization of nanos (nos) mRNA to the posterior pole of the Drosophila oocyte is essential for abdominal segmentation and germline development during embryogenesis. Posterior localization is mediated by a complex cis-acting localization signal in the nos 3' untranslated region that comprises multiple partially redundant elements. Genetic analysis suggests that this signal is recognized by RNA-binding proteins and associated factors that package nos mRNA into a localization competent ribonucleoprotein complex. However, functional redundancy among localization elements has made the identification of individual localization factors difficult. Indeed, only a single direct-acting nos localization factor, Rumpelstiltskin (Rump), has been identified thus far. Through a sensitized genetic screen, we have now identified the Argonaute family member Aubergine (Aub) as a nos localization factor. Aub interacts with nos mRNA in vivo and co-purifies with Rump in an RNA-dependent manner. Our results support a role for Aub, independent of its function in RNA silencing, as a component of a nos mRNA localization complex.     

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.