Unmasking mRNA in Clam Oocytes: Role of Phosphorylation of a 3′ UTR Masking Element-Binding Protein at Fertilization

AbstractDuring meiotic maturation or after fertilization of invertebrate and vertebrate oocytes, many of the quiescent stored mRNAs are recruited into polysomes. In the clam,Spisula solidissima,such masked messages include the abundant mRNAs encoding cyclin A and the small subunit of ribonucleotide reductase. We have previously shown that mRNA-specific unmasking of these two messages can be achievedin vitro,in oocyte cell-free extracts, by the addition of antisense RNAs corresponding to a fairly short (130–140 nucleotides) segment in their cognate 3′ untranslated regions. We postulated that the antisense RNAs prevented the binding of a masking repressor protein (Standartet al.,1990). Here we report UV-crosslinking and gel retardation studies which show that the masking portions of the translationally regulated mRNAs bind an oocyte protein of 82 kDa (p82), which is phosphorylated after fertilization. This modification was accompanied by altered RNP complex formation in gel retardation assays. These changes presumably reflect the activation of translation of the masked mRNAs. The role of p82 phosphorylation in maternal mRNA unmasking was assessed in a novelin vitroactivation system developed from clam oocytes, based upon the natural rise in pH which accompanies fertilization. Concomitant with mRNA unmasking, several kinases, including cdc2 and MAP kinases were activated in this system, as was p82 phosphorylation. Inhibitors of serine/threonine kinases, including 6-DMAP, staurosporine, and H7 inhibited p82 phosphorylation, whereas inhibitors of tyrosine kinases, protein kinase C, cAMP-dependent protein kinase, and p70s6kdid not prevent this modification. A specific inhibitor of cdc2 kinase, p27Kip1, prevented p82 phosphorylation and translational activation, strongly suggesting that p82 modification is required for unmasking

CPEB and miR-15/16 Co-Regulate Translation of Cyclin E1 mRNA during Xenopus Oocyte Maturation.

Cell cycle transitions spanning meiotic maturation of the Xenopus oocyte and early embryogenesis are tightly regulated at the level of stored inactive maternal mRNA. We investigated here the translational control of cyclin E1, required for metaphase II arrest of the unfertilised egg and the initiation of S phase in the early embryo. We show that the cyclin E1 mRNA is regulated by both cytoplasmic polyadenylation elements (CPEs) and two miR-15/16 target sites within its 3'UTR. Moreover, we provide evidence that maternal miR-15/16 microRNAs co-immunoprecipitate with CPE-binding protein (CPEB), and that CPEB interacts with the RISC component Ago2. Experiments using competitor RNA and mutated cyclin E1 3'UTRs suggest cooperation of the regulatory elements to sustain repression of the cyclin E1 mRNA during early stages of maturation when CPEB becomes limiting and cytoplasmic polyadenylation of repressed mRNAs begins. Importantly, injection of anti-miR-15/16 LNA results in the early polyadenylation of endogenous cyclin E1 mRNA during meiotic maturation, and an acceleration of GVBD, altogether strongly suggesting that the proximal CPEB and miRNP complexes act to mutually stabilise each other. We conclude that miR-15/16 and CPEB co-regulate cyclin E1 mRNA. This is the first demonstration of the co-operation of these two pathways.This study was supported by the Biotechnology and Biological Sciences Research Council (BB/E016316/1). AG was funded by Cancer Research UK and the RATHER consortium, and JA was funded by the Cambridge Overseas Trust and the Parke Davis Bursary (Downing College).This is the final version of the article. It first appeared from PLOS via https://doi.org/10.1371/journal.pone.014679

The DDX6-4E-T interaction mediates translational repression and P-body assembly.

4E-Transporter binds eIF4E via its consensus sequence YXXXXLΦ, shared with eIF4G, and is a nucleocytoplasmic shuttling protein found enriched in P-(rocessing) bodies. 4E-T inhibits general protein synthesis by reducing available eIF4E levels. Recently, we showed that 4E-T bound to mRNA however represses its translation in an eIF4E-independent manner, and contributes to silencing of mRNAs targeted by miRNAs. Here, we address further the mechanism of translational repression by 4E-T by first identifying and delineating the interacting sites of its major partners by mass spectrometry and western blotting, including DDX6, UNR, unrip, PAT1B, LSM14A and CNOT4. Furthermore, we document novel binding between 4E-T partners including UNR-CNOT4 and unrip-LSM14A, altogether suggesting 4E-T nucleates a complex network of RNA-binding protein interactions. In functional assays, we demonstrate that joint deletion of two short conserved motifs that bind UNR and DDX6 relieves repression of 4E-T-bound mRNA, in part reliant on the 4E-T-DDX6-CNOT1 axis. We also show that the DDX6-4E-T interaction mediates miRNA-dependent translational repression and de novo P-body assembly, implying that translational repression and formation of new P-bodies are coupled processes. Altogether these findings considerably extend our understanding of the role of 4E-T in gene regulation, important in development and neurogenesis.BBSRC [BB/J00779X/1 to N.S.]; CNRS PICS (to D.W.); Agence Nationale pour la Recherche [ANR-14-CE09-0013-01ANR to D.W.]; Gates Cambridge Foundation (to A.K.); Fondation Wiener – Anspach of the Université Libre de Bruxelles and the Cambridge Newton Trust (C.V.). Funding for open access charge: BBSRC

Dual RNA processing roles of Pat1b via cytoplasmic Lsm1-7 and nuclear Lsm2-8 complexes

Pat1 RNA-binding proteins, enriched in P-bodies, are key players in cytoplasmic 5’ to 3’ mRNA decay, activating decapping of mRNA in complex with the Lsm1-7 heptamer. Using co-immunoprecipitation and immunofluorescence approaches coupled with RNAi, we provide evidence for a nuclear complex of Pat1b with the Lsm2-8 heptamer, which binds to the spliceosomal U6 snRNA. Furthermore, we establish the set of interactions connecting Pat1b/Lsm2-8/U6 snRNA/SART3 and additional U4/U6.U5 tri-snRNP components, in Cajal bodies, the site of snRNP biogenesis. RNAseq following Pat1b depletion revealed the preferential up-regulation of mRNAs normally found in P-bodies and enriched in 3’ UTR AU-rich elements. Changes in >180 alternative splicing events were also observed, characterized by skipping of regulated exons with weak donor sites. Our data demonstrate the unsuspected dual role of a decapping enhancer in pre-mRNA processing as well as in mRNA decay via distinct nuclear and cytoplasmic Lsm complexes.This work was funded by a fellowship to CV from the Fondation Wiener – Anspach, BBSRC (BB/J00779X/1) and the Newton Trust (University of Cambridge) to NS, and CNRS PICS and ANR (14- CE09-0013-01ANR) to DW. The CMMI is supported by the European Regional Development Fund and the Walloon Region

GC content shapes mRNA storage and decay in human cells.

mRNA translation and decay appear often intimately linked although the rules of this interplay are poorly understood. In this study, we combined our recent P-body transcriptome with transcriptomes obtained following silencing of broadly acting mRNA decay and repression factors, and with available CLIP and related data. This revealed the central role of GC content in mRNA fate, in terms of P-body localization, mRNA translation and mRNA stability: P-bodies contain mostly AU-rich mRNAs, which have a particular codon usage associated with a low protein yield; AU-rich and GC-rich transcripts tend to follow distinct decay pathways; and the targets of sequence-specific RBPs and miRNAs are also biased in terms of GC content. Altogether, these results suggest an integrated view of post-transcriptional control in human cells where most translation regulation is dedicated to inefficiently translated AU-rich mRNAs, whereas control at the level of 5' decay applies to optimally translated GC-rich mRNAs

The active form of Xp54 RNA helicase in translational repression is an RNA-mediated oligomer

Previously, we reported that in clam oocytes, cytoplasmic polyadenylation element-binding protein (CPEB) co-immunoprecipitates with p47, a member of the highly conserved RCK family of RNA helicases which includes Drosophila Me31B and Saccharomyces cerevisiae Dhh1. Xp54, the Xenopus homologue, with helicase activity, is a component of stored mRNP. In tethered function assays in Xenopus oocytes, we showed that MS2–Xp54 represses the translation of non-adenylated firefly luciferase mRNAs and that mutations in two core helicase motifs, DEAD and HRIGR, surprisingly, activated translation. Here we show that wild-type MS2–Xp54 tethered to the reporter mRNA 3′-untranslated region (UTR) represses translation in both oocytes and eggs in an RNA-dependent complex with endogenous Xp54. Injection of mutant helicases or adenylated reporter mRNA abrogates this association. Thus Xp54 oligomerization is a hallmark of translational repression. Xp54 complexes, which also contain CPEB and eIF4E in oocytes, change during meiotic maturation. In eggs, CPEB is degraded and, while eIF4E still interacts with Xp54, this interaction becomes RNA dependent. Supporting evidence for RNA-mediated oligomerization of endogenous Xp54, and RNA-independent association with CPEB and eIF4E in oocytes was obtained by gel filtration. Altogether, our data are consistent with a model in which the active form of the Xp54 RNA helicase is an oligomer in vivo which, when tethered, via either MS2 or CPEB to the 3′UTR, represses mRNA translation, possibly by sequestering eIF4E from the translational machinery

P-Bodies: Cytosolic Droplets for Coordinated mRNA Storage

International audienc

eIF4E-binding proteins: new factors, new locations, new roles

Abstract The cap-binding translation initiation factor eIF4E (eukaryotic initiation factor 4E) is central to protein synthesis in eukaryotes. As an integral component of eIF4F, a complex also containing the large bridging factor eIF4G and eIF4A RNA helicase, eIF4E enables the recruitment of the small ribosomal subunit to the 5 end of mRNAs. The interaction between eIF4E and eIF4G via a YXXXXLφ motif is regulated by small eIF4E-binding proteins, 4E-BPs, which use the same sequence to competitively bind eIF4E thereby inhibiting cap-dependent translation. Additional eIF4E-binding proteins have been identified in the last 10-15 years, characterized by the YXXXXLφ motif, and by interactions (many of which remain to be detailed) with RNAbinding proteins, or other factors in complexes that recognize the specific mRNAs. In the present article, we focus on the metazoan 4E-T (4E-transporter)/Cup family of eIF4E-binding proteins, and also discuss very recent examples in yeast, fruitflies and humans, some of which predictably inhibit translation, while others may result in mRNA decay or even enhance translation; altogether considerably expanding our understanding of the roles of eIF4E-binding proteins in gene expression regulation. eIF4E (eukaryotic initiation factor 4E) and 4E-BP (eIF4E-binding protein) eIF4E is central to translation initiation, enabling the recruitment of the small ribosomal subunit to mRNA by its recognition of the 5 cap structure and as a component of the eIF4F complex, also containing eIF4G and eIF4A. eIF4E is a small, approximately 25 kDa, protein, whose structure resembles a cupped hand formed by antiparallel β-sheets, backed by α-helices on its convex side. The eIF4E cavity specifically interacts with the m 7 G(5 )ppp(5 )N (where N is typically G or A) cap added to RNA polymerase II transcripts (reviewed i