Cellular coexistence of two high molecular subsets of eEF1B complex

AbstractThe elongation factor eEF1B involved in protein translation was found to contain two isoforms of the eEF1Bδ subunit in sea urchin eggs. The eEF1Bδ2 isoform differs from eEF1Bδ1 by a specific insert of 26 amino acids. Both isoforms are co-expressed in the cell and likely originate from a unique gene. The feature appears universal in metazoans as judged from in silico analysis in EST-databanks. The eEF1B components were co-immunoprecipitated by specific eEF1Bδ2 antibodies. Quantification of the proteins in immunoprecipitates and on immunoblots demonstrates that eEF1Bδ1 and eEF1Bδ2 proteins are present in two subsets of eEF1B complex. We discuss and propose a model for the different subsets of eEF1B complex concomitantly present in the cell

EIF4E/4E-BP dissociation and 4E-BP degradation in the first mitotic division of the sea urchin embryo

AbstractThe mRNA’s cap-binding protein eukaryotic translation initiation factor (eIF)4E is a major target for the regulation of translation initiation. eIF4E activity is controlled by a family of translation inhibitors, the eIF4E-binding proteins (4E-BPs). We have previously shown that a rapid dissociation of 4E-BP from eIF4E is related with the dramatic rise in protein synthesis that occurs following sea urchin fertilization. Here, we demonstrate that 4E-BP is destroyed shortly following fertilization and that 4E-BP degradation is sensitive to rapamycin, suggesting that proteolysis could be a novel means of regulating 4E-BP function. We also show that eIF4E/4E-BP dissociation following fertilization is sensitive to rapamycin. Furthermore, while rapamycin modestly affects global translation rates, the drug strongly inhibits cyclin B de novo synthesis and, consequently, precludes the completion of the first mitotic cleavage. These results demonstrate that, following sea urchin fertilization, cyclin B translation, and thus the onset of mitosis, are regulated by a rapamycin-sensitive pathway. These processes are effected at least in part through eIF4E/4E-BP complex dissociation and 4E-BP degradation

mRNP3 and mRNP4 are phosphorylatable by casein kinase II in Xenopus oocytes, but phosphorylation does not modify RNA-binding affinity

AbstractmRNP3 and mRNP4 (also called FRGY2) are two mRNA-binding proteins which are major constituents of the maternal RNA storage particles of Xenopus laevis oocytes. The phosphorylation of mRNP3–4 has been implicated in the regulation of mRNA masking. In this study, we have investigated their phosphorylation by casein kinase II and its consequence on their affinity for RNA. Comparison of the phosphopeptide map of mRNP3–4 phosphorylated in vivo with that obtained after phosphorylation in vitro by purified Xenopus laevis casein kinase II strongly suggests that casein kinase II is responsible for the in vivo phosphorylation of mRNP3–4 in oocytes. The phosphorylation occurs on a serine residue in a central domain of the proteins. The affinity of mRNP3–4 for RNA substrates remained unchanged after the treatment with casein kinase II or calf intestine phosphatase in vitro. This suggests that phosphorylation of these proteins does not regulate their interaction with RNA but rather controls their interactions with other proteins

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

The genome of the sea urchin Strongylocentrotus purpuratus

We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus, a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes

Effets toxiques d'herbicides à base de glyphosate sur la régulation du cycle cellulaire et le développement précoce en utilisant l'embryon d'oursin

L'utilisation du développement précoce de l'oursin nous a permis de mettre en évidence des dysfonctionnements spécifiques du cycle cellulaire et du développement précoce. Nous montrons que le roundup provoque un délai dans l'apparition du premier clivage mitotique ; il retarde l'activation du complexe régulateur de l'entrée en phase M, le complexe CDK/cycline B. La cible moléculaire initiale du roundup est l'activité de synthèse d'ADN. Le mécanisme de surveillance de la transition G2M détecte l'anomalie et provoque le retard du cycle cellulaire. Parallèlement, nous avons mis en évidence que le roundup provoque des dysfonctionnements du développement embryonnaire précoce en affectant le mécanisme de transcription. Les deux effets décrits en présence de roundup sont asociés à des pathologies et leur existence pose problème en terme de santé humaineRENNES1-BU Sciences Philo (352382102) / SudocROSCOFF-Observ.Océanol. (292393008) / SudocSudocFranceF

Le facteur d'élongation de la traduction eEF1B (structure macromoléculaire et relation avec le cycle cellulaire)

La synthèse protéique est une étape cruciale dans la régulation de l'expression des gènes. Elle implique l'orchestration de différents facteurs autour de trois phases, l'initiation, l'élongation, et la terminaison, permettant l'expression des protéines de manière ciblée dans le temps et l'espace. Le facteur d'élongation eEF1 regroupe deux éléments : une protéine G nommée eEF1A et un complexe d'échange de GDP/GDP nommé eEF1B dont le rôle fondamentalement reconnu est de réactiver eEF1A. Nos résultats concernent notamment la structure du complexe eEF1B avec la mise en évidence de différentes sous-populations co-exprimées dans la cellule. Par ailleurs, nos résultats mettent en évidence une relocalisation de ces complexes dépendante du cycle cellulaire. Des analyses de séquences nous permettent de suggérer que le complexe est une cible universelle de la kinase CDK1/Cycline B. Ces résultats montrent l'importance des régulations du complexe eEF1B et plus généralement de l'élongation au cours du cycle cellulaire.RENNES1-BU Sciences Philo (352382102) / SudocROSCOFF-Observ.Océanol. (292393008) / SudocSudocFranceF