A Variant Mimicking Hyperphosphorylated 4E-BP Inhibits Protein Synthesis in a Sea Urchin Cell-Free, Cap-Dependent Translation System

BACKGROUND: 4E-BP is a translational inhibitor that binds to eIF4E to repress cap-dependent translation initiation. This critical protein:protein interaction is regulated by the phosphorylation of 4E-BP. Hypophosphorylated 4E-BP binds to eIF4E and inhibits cap-dependent translation, whereas hyperphosphorylated forms do not. While three 4E-BP proteins exist in mammals, only one gene encoding for 4E-BP is present in the sea urchin genome. The protein product has a highly conserved core domain containing the eIF4E-binding domain motif (YxxxxLPhi) and four of the regulatory phosphorylation sites. METHODOLOGY/PRINCIPAL FINDINGS: Using a sea urchin cell-free cap-dependent translation system prepared from fertilized eggs, we provide the first direct evidence that the sea urchin 4E-BP inhibits cap-dependent translation. We show here that a sea urchin 4E-BP variant, mimicking phosphorylation on four core residues required to abrogate binding to eIF4E, surprisingly maintains physical association to eIF4E and inhibits protein synthesis. CONCLUSIONS/SIGNIFICANCE: Here, we examine the involvement of the evolutionarily conserved core domain and phosphorylation sites of sea urchin 4E-BP in the regulation of eIF4E-binding. These studies primarily demonstrate the conserved activity of the 4E-BP translational repressor and the importance of the eIF4E-binding domain in sea urchin. We also show that a variant mimicking hyperphosphorylation of the four regulatory phosphorylation sites common to sea urchin and human 4E-BP is not sufficient for release from eIF4E and translation promotion. Therefore, our results suggest that there are additional mechanisms to that of phosphorylation at the four critical sites of 4E-BP that are required to disrupt binding to eIF4E

The translational repressor 4E-BP called to order by eIF4E: new structural insights by SAXS

eIF4E binding protein (4E-BP) inhibits translation of capped mRNA by binding to the initiation factor eIF4E and is known to be mostly or completely unstructured in both free and bound states. Using small angle X-ray scattering (SAXS), we report here the analysis of 4E-BP structure in solution, which reveals that while 4E-BP is intrinsically disordered in the free state, it undergoes a dramatic compaction in the bound state. Our results demonstrate that 4E-BP and eIF4E form a ‘fuzzy complex’, challenging current visions of eIF4E/4E-BP complex regulation

Oligomerization of EDEN-BP is required for specific mRNA deadenylation and binding.

International audienceBACKGROUND INFORMATION: mRNA deadenylation [shortening of the poly(A) tail] is often triggered by specific sequence elements present within mRNA 3' untranslated regions and generally causes rapid degradation of the mRNA. In vertebrates, many of these deadenylation elements are called AREs (AU-rich elements). The EDEN (embryo deadenylation element) sequence is a Xenopus class III ARE. EDEN acts by binding a specific factor, EDEN-BP (EDEN-binding protein), which in turn stimulates deadenylation. RESULTS: We show here that EDEN-BP is able to oligomerize. A 27-amino-acid region of EDEN-BP was identified as a key domain for oligomerization. A mutant of EDEN-BP lacking this region was unable to oligomerize, and a peptide corresponding to this region competitively inhibited the oligomerization of full-length EDEN-BP. Impairing oligomerization by either of these two methods specifically abolished EDEN-dependent deadenylation. Furthermore, impairing oligomerization inhibited the binding of EDEN-BP to its target RNA, demonstrating a strong coupling between EDEN-BP oligomerization and RNA binding. CONCLUSIONS: These data, showing that the oligomerization of EDEN-BP is required for binding of the protein on its target RNA and for EDEN-dependent deadenylation in Xenopus embryos, will be important for the identification of cofactors required for the deadenylation process

Microevolution of bank voles (Myodes glareolus) at neutral and immune-related genes during multiannual dynamic cycles : Consequences for Puumala hantavirus epidemiology

Understanding howhost dynamics, including variations of population size and dispersal, may affect the epidemiology of infectious diseases through ecological and evolutionary processes is an active research area. Here we focus on a bank vole (Myodes glareolus) metapopulation surveyed in Finland between 2005 and 2009. Bank vole is the reservoir of Puumala hantavirus (PUUV), the agent of nephropathia epidemica (NE, a mild form of hemorrhagic fever with renal symptom) in humans. M. glareolus populations experience multiannual density fluctuations that may influence the level of genetic diversity maintained in bank voles, PUUV prevalence and NE occurrence. We examine bank vole metapopulation genetics at presumably neutral markers and immunerelated genes involved in susceptibility to PUUV (Tnf-promoter, Tlr4, Tlr7 and Mx2 gene) to investigate the links between population dynamics, microevolutionary processes and PUUV epidemiology. We show that genetic drift slightly and transiently affects neutral and adaptive genetic variability within the metapopulation. Gene flow seems to counterbalance its effects during the multiannual density fluctuations. The low abundance phase may therefore be too short to impact genetic variation in the host, and consequently viral genetic diversity. Environmental heterogeneity does not seem to affect vole gene flow, which might explain the absence of spatial structure previously detected in PUUV in this area. Besides, our results suggest the role of vole dispersal on PUUV circulation through sex-specific and density-dependent movements. We find little evidence of selection acting on immune-related genes within this metapopulation. Footprint of positive selection is detected at Tlr-4 gene in 2008 only. We observe marginally significant associations between Mx2 genotype and PUUV genogroups. These results show that neutral processes seem to be the main factors affecting the evolution of these immune-related genes at a contemporary scale, although the relative effects of neutral and adaptive forces could vary temporally with density fluctuations. Immune related gene polymorphism may in turn partly influence PUUV epidemiology in this metapopulation. (C) 2016 Published by Elsevier B.V.Peer reviewe

The genomic repertoire for cell cycle control and DNA metabolism in S. purpuratus

A search of the Strongylocentrotus purpuratus genome for genes associated with cell cycle control and DNA metabolism shows that the known repertoire of these genes is conserved in the sea urchin, although with fewer family members represented than in vertebrates, and with some cases of echinoderm-specific gene diversifications. For example, while homologues of the known cyclins are mostly encoded by single genes in S. purpuratus (unlike vertebrates, which have multiple isoforms), there are additional genes encoding novel cyclins of the B and K/L types. Almost all known cyclin-dependent kinases (CDKs) or CDK-like proteins have an orthologue in S. purpuratus; CDK3 is one exception, whereas CDK4 and 6 are represented by a single homologue, referred to as CDK4. While the complexity of the two families of mitotic kinases, Polo and Aurora, is close to that found in the nematode, the diversity of the NIMA-related kinases (NEK proteins) approaches that of vertebrates. Among the nine NEK proteins found in S. purpuratus, eight could be assigned orthologues in vertebrates, whereas the ninth is unique to sea urchins. Most known DNA replication, DNA repair and mitotic checkpoint genes are also present, as are homologues of the pRB (two) and p53 (one) tumor suppressors. Interestingly, the p21/p27 family of CDK inhibitors is represented by one homologue, whereas the INK4 and ARF families of tumor suppressors appear to be absent, suggesting that these evolved only in vertebrates. Our results suggest that, while the cell cycle control mechanisms known from other animals are generally conserved in sea urchin, parts of the machinery have diversified within the echinoderm lineage. The set of genes uncovered in this analysis of the S. purpuratus genome should enhance future research on cell cycle control and developmental regulation in this model

A specific role for the C-terminal region of the Poly(A)-binding protein in mRNA decay

mRNA poly(A) tails affect translation, mRNA export and mRNA stability, with translation initiation involving a direct interaction between eIF4G and the poly(A)-binding protein Pab1. The latter factor contains four RNA recognition motifs followed by a C-terminal region composed of a linker and a PABC domain. We show here that yeast mutants lacking the C-terminal domains of Pab1 display specific synthetic interactions with mutants in the 5′-3′ mRNA decay pathway. Moreover, these mutations impair mRNA decay in vivo without significantly affecting mRNA export or translation. Inhibition of mRNA decay occurs through slowed deadenylation. In vitro analyses demonstrate that removal of the Pab1 linker domain directly interferes with the ability of the Pop2–Ccr4 complex to deadenylate the Pab1-bound poly(A). Binding assays demonstrate that this results from a modulation of poly(A) packaging by the Pab1 linker region. Overall, our results demonstrate a direct involvement of Pab1 in mRNA decay and reveal the modular nature of this factor, with different domains affecting various cellular processes. These data suggest new models involving the modulation of poly(A) packaging by Pab1 to control mRNA decay

Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress

Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages

EuReCa ONE—27 Nations, ONE Europe, ONE Registry A prospective one month analysis of out-of-hospital cardiac arrest outcomes in 27 countries in Europe

AbstractIntroductionThe aim of the EuReCa ONE study was to determine the incidence, process, and outcome for out of hospital cardiac arrest (OHCA) throughout Europe.MethodsThis was an international, prospective, multi-centre one-month study. Patients who suffered an OHCA during October 2014 who were attended and/or treated by an Emergency Medical Service (EMS) were eligible for inclusion in the study. Data were extracted from national, regional or local registries.ResultsData on 10,682 confirmed OHCAs from 248 regions in 27 countries, covering an estimated population of 174 million. In 7146 (66%) cases, CPR was started by a bystander or by the EMS. The incidence of CPR attempts ranged from 19.0 to 104.0 per 100,000 population per year. 1735 had ROSC on arrival at hospital (25.2%), Overall, 662/6414 (10.3%) in all cases with CPR attempted survived for at least 30 days or to hospital discharge.ConclusionThe results of EuReCa ONE highlight that OHCA is still a major public health problem accounting for a substantial number of deaths in Europe.EuReCa ONE very clearly demonstrates marked differences in the processes for data collection and reported outcomes following OHCA all over Europe. Using these data and analyses, different countries, regions, systems, and concepts can benchmark themselves and may learn from each other to further improve survival following one of our major health care events

Le Facteur de terminaison de la traduction eRF3 et les protéines de liaison aux ARNm polyadénylés (liens structuraux et fonctionnels)

Du gène à la protéine, l'étude des régulations de l'expression des gènes n'a fait que mieux révéler la complexité du monde eucaryote. Les mécanismes post transcriptionnels ont un rôle fondamental indiscutable. Une queue poly(A) est mise en place à l'extrémité de presque tous les ARNm après transcription. Elle joue un rôle important dans le contrôle de la stabilité et de la traduction des ARNm. C'est par la famille des protéines de liaison à la queue poly(A) (PABP) que la queue poly(A) intervient dans l'expression génique. Nous avons montré chez la levure que la PABP n'intervient pas seulement dans l'initiation de la traduction mais aussi dans la dernière étape de la synthèse protéique via son interaction avec le facteur de terminaison de la traduction eRF3. La PABP interagit in vivo avec eRF3 chez les eucaryotes, de la levure à l'homme, suggérant que la fonction associée à cette interaction dans la terminaison de la traduction est conservée.Nous avons caractérisé une nouvelle PABP de classe I chez le Xénope, appelée ePABP pour PABP embryonnaire. ePABP est capable de substituer fonctionnellement le gène levure PAB1. L'analyse du profil d'expression des protéines PABP de Xenope (ePABP et PABP1) au cours du développement précoce et dans les tissus adultes révèle une expression différente pour ces deux protéines. Nous proposons différents rôles clefs que peuvent jouer ces protéines dans la régulation de l'expression des gènes au cours du développement.La surproduction du facteur de terminaison eRF3 seul dans des cellules humaines en culture est sans effet sur la terminaison, mais elle modifie l'abondance de certains ARNm. L'effet de la surexpression d'eRF3 est différent en fonction du promoteur, suggérant un rôle d'eRF3 via la transcription ou un mécanisme couplé. De plus, nos analyses de localisation d'eRF3 n'ont pas permis de détecter cette protéine dans le noyau, suggérant que l'effet d'eRF3 est cytoplasmique et donc probablement indirect.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

Flux géniques et dispersion chez un rongeur à démographie cyclique dans un paysage agricole intensif

La dispersion est un trait d'histoire de vie qui joue un rôle majeur dans le fonctionnement des populations naturelles. Comprendre ce phénomène et son évolution est aujourd'hui déterminant pour la gestion des populations dans des écosystèmes de plus en plus anthropisés. Cette étude s'est attachée à caractériser la dispersion et ses déterminants chez le campagnol des champs, Microtus avalis, dans un paysage agricole de l'Ouest de la France. L'instabilité spatio-temporelle des agro-écosystèmes et sa démographie cyclique font de ce petit rongeur colonial un modèle exceptionnel pour aborder cette problématique. La constitution d'une banque de marqueurs microsatellites nous a permis d'utiliser des approches de génétique des populations et de génétique paysagère. Une seule entité génétiquement homogène couvre les 500 km du site d'étude bien que les distances de dispersion inférée soient faibles. Le patron d'isolement par la distance qui caractérise cette population indique que seule la distance limite le flux génique dans ce paysage. La densité influence positivement le flux génique en faisant varier la balance entre dispersion efficace et dérive génétique au cours des cycles. Nous montrons que les patrons de dispersion sont différents entre mâles et femelles (dispersion biaisée vers les mâles) suggérant que la dispersion n'est pas déterminée par les mêmes causes évolutives selon le sexe. La colonisation par les femelles des habitats temporaire (les cultures annuelles), particulièrement massive lors des pullulations, permet la cohésion spatiale de la population. Sa cohésion génétique est assurée par les migrations répétées des mâles entre colonies pour la reproduction. Ce fonctionnement est rendu possible par l'extrême rapidité cycle de vie du campagnol des champs qui compense l'instabilité du paysage. L'intégration des différents résultats de cette étude dans un modèle de dispersion couplé à un modèle dynamique de paysage devrait permettre d'évaluer l'impact de l'évolution de l'agriculture sur cette espèce emblématique de la biodiversité de ces paysages.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF