Interaction of yeast eIF4G with spliceosome components Implications in pre-mRNA processing events

International audienceAs evidenced from mammalian cells the eukaryotic translation initiation factor eIF4G has a putative role in nuclear RNA metabolism. Here we investigate whether this role is conserved in the yeast Saccharomyces cerevisiae. Using a combination of in vitro and in vivo methods, we show that, similar to mammalian eIF4G, yeast eIF4G homologues, Tif4631p and Tif4632p, are present both in the nucleus and the cytoplasm. We show that both eIF4G proteins interact efficiently in vitro with UsnRNP components of the splicing machinery. More specifically, Tif4631p and Tif4632p interact efficiently with U1 snRNA in vitro. In addition, Tif4631p and Tif4632p associate with protein components of the splicing machinery, namely Snu71p and Prp11p. To further delineate these interactions, we map the regions of Tif4631p and Tif4632p that are important for the interaction with Prp11p and Snu71p and we show that addition of these regions to splicing reactions in vitro has a dominant inhibitory effect. The observed interactions implicate eIF4G in aspects of pre-mRNA processing. In support of this hypothesis, deletion of one of the eIF4G isoforms results in accumulation of un-spliced precursors for a number of endogenous genes, in vivo. In conclusion these observations are suggestive of the involvement of yeast eIF4G in pre-mRNA metabolism

A functional network involved in the recycling of nucleocytoplasmic pre-60S factors

Eukaryotic pre-ribosomes go through cytoplasmic maturation steps before entering translation. The nucleocytoplasmic proteins participating in these late stages of maturation are reimported to the nucleus. In this study, we describe a functional network focused on Rei1/Ybr267w, a strictly cytoplasmic pre-60S factor indirectly involved in nuclear 27S pre-ribosomal RNA processing. In the absence of Rei1, the nuclear import of at least three other pre-60S factors is impaired. The accumulation in the cytoplasm of a small complex formed by the association of Arx1 with a novel factor, Alb1/Yjl122w, inhibits the release of the putative antiassociation factor Tif6 from the premature large ribosomal subunits and its recycling to the nucleus. We propose a model in which Rei1 is a key factor for the coordinated dissociation and recycling of the last pre-60S factors before newly synthesized large ribosomal subunits enter translation

60S ribosomal subunit assembly dynamics defined by semi-quantitative mass spectrometry of purified complexes

During the highly conserved process of eukaryotic ribosome formation, RNA follows a maturation path with well-defined, successive intermediates that dynamically associate with many pre-ribosomal proteins. A comprehensive description of the assembly process is still lacking. To obtain data on the timing and order of association of the different pre-ribosomal factors, a strategy consists in the use of pre-ribsomal particles isolated from mutants that block ribosome formation at different steps. Immunoblots, inherently limited to only a few factors, have been applied to evaluate the accumulation or decrease of pre-ribosomal intermediates under mutant conditions. For a global protein-level description of different 60S ribosomal subunit maturation intermediates in yeast, we have adapted a method of in vivo isotopic labelling and mass spectrometry to study pre-60S complexes isolated from strains in which rRNA processing was affected by individual depletion of five factors: Ebp2, Nog1, Nsa2, Nog2 or Pop3. We obtained quantitative data for 45 distinct pre-60S proteins and detected coordinated changes for over 30 pre-60S factors in the analysed mutants. These results led to the characterisation of the composition of early, intermediate and late pre-ribosomal complexes, specific for crucial maturation steps during 60S assembly in eukaryotes

mRNA Degradation and Decay

International audienceWhy is it important to understand mRNA degradation in a cell? First,RNA degradation has a clearing function and removes RNAs arising from tran-scription, splicing, export, or translation “accidents” to ensure robust geneexpression (see Chap. 8). Second, while regulation of gene expression has a veryimportant transcription component, mRNAs must be turned over rapidly for fastchanges in transcriptome composition. Coordinated destabilization of an entireclass of mRNAs can promote major physiological changes in a cell. Third, specificmechanisms of mRNA decay can serve to regulate gene expression throughfeedback control. Research on these topics has been frequently done first withyeasts and led to a better understanding of gene expression in eukaryotes. We startwith an overview of the methods for measuring mRNA decay on a large scale withan emphasis on how technical issues affect the current picture of global mRNAdecay in yeast. Next, we describe the importance of nuclear degradation in shapingthe stable transcriptome. Once in the cytoplasm, mRNAs are exposed to translationand we provide an overview of the complexes and individual enzymes that ensureprogressive deadenylation, mRNA decapping, and 5 ́ to 3 ́ or 3 ́ to 5 ́ exonucle-olytic RNA degradation. Finally, how organelle transcripts are degraded inmitochondria is briefly exposed

The ribosome-bound quality control complex: from aberrant peptide clearance to proteostasis maintenance

International audienceProteostasis in eukaryotes is maintained by compartment-specific quality control pathways, which enable the refolding or the degradation of defective polypeptides to prevent the toxicity that may arise from their aggregation. Among these processes, translational protein quality control is performed by the Ribosome-bound Quality Control complex (RQC), which recognizes nascent peptides translated from aberrant mRNAs, polyubiquitylates these aberrant peptides, extracts them from the stalled 60S subunit and finally escorts them to the proteasome for degradation. In this review, we focus on the mechanism of action of the RQC complex from stalled 60S binding to aberrant peptide delivery to the proteasome and describe the cellular consequences of a deficiency in the RQC pathway, such as aberrant protein aggregation. In addition, this review covers the recent discoveries concerning the role of cytosolic chaperones, as well as Tom1, to prevent the accumulation of aberrant protein aggregates in case of a deficiency in the RQC pathway

Dynamique des facteurs pré-ribosomiques au cours de la biogène de la grande sous-unité ribosomique chez S. cerevisiae

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Etude du rôle de facteurs pré-60S dans la biogenèse des ribosomes chez Saccharomyces cerevisiae

Chez Saccharomyces cerevisiae, environ 200 facteurs pré-ribosomiques sont impliqués dans la maturation du ribosome. La fonction de la plupart d entre eux n est pas encore caractérisée. Pendant mon travail de thèse, j ai mis en évidence un nouveau facteur, Ecm1, impliqué dans l export des précurseurs de la grande sous-unité 60S. ECM1 n est pas un gène essentiel. Cependant, il devient essentiel à la croissance cellulaire quand les complexes des pores nucléaires (NPC) ou l un des trois récepteurs d export Crm1/Nmd3, Mex67/Mtr2 ou Arx1 sont affectés. Ce défaut de croissance est dû au défaut d export des pré-60S qui provoque ainsi un déficit en sous-unité 60S mature. Il accompagne les particules précurseurs du noyau vers le cytoplasme et est ensuite réimporté par la karyophérine Kap123. Un crible double-hybride réalisé avec Ecm1 comme appât a permis de mettre en évidence un autre nouveau facteur pré-60S, Ypl009c. Bien qu Ypl009c s associe aux particules pré-60S dans le cytoplasme, son absence ne provoque pas de défaut de biogenèse de la sous-unité 60S. Par ailleurs, des tests de sensibilité aux antibiotiques effectués sur la souche ypl009c suggèrent un lien avec la traduction. Le défaut de traduction est exacerbé dans le double mutant ypl009c ski2 . Le lien fonctionnel entre Ypl009c et le complexe SKI avait été identifié dans un crible génétique. Le lien entre la biogenèse des ribosomes, la traduction et la dégradation des ARNm nous a conduit à tester si l absence d Ypl009c ne pouvait pas provoquer un défaut dans la dégradation des ARNm en cours de traduction. De manière surprenante, une augmentation des protéines synthétisées à partir d ARNm sans codon stop est observée en absence d Ypl009c, alors que les ARNm aberrants ne sont pas affectés. L ensemble de nos résultats suggère qu Ypl009c pourrait être un facteur important pour la reconnaissance des protéines aberrantes par le protéasomePARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

18 RNA Gene Analysis

International audienceThis chapter describes basic techniques to characterize the primary structure of RNA gene products and the proteins that associate with these RNA to form ribonucleoprotein particles. In yeast, the identification of protein coding genes is relatively straightforward. In its most unsophisticated form, it relies on the identification of open reading frames (ORFs) longer than a statistically defined threshold refined with the search of features, such as putative splice sites. Recently, protein coding gene determination was made considerably more accurate by comparative genomic studies in which protein sequence conservations allowed a reliable annotation of the genome for protein coding gene. The most widely used technique so far to characterize transcripts is Northern-blot hybridization. New technologies, such as those based on DNA microarrays, are now available and are powerful tools for genome-wide transcript analysis, but aside from the new tiling-array technology that is emerging or specialized arrays, it is essentially useful for relative quantifications but will not be able to detect any structural variations or heterogeneities

La chaperone Hsp40 Jjj1p et son rôle dans la biogenèse des ribosomes chez les Eucaryotes

Chez les Eucaryotes, deux réseaux de chaperones ont été décrits comme dédiés à la synthèse protéique et à la protection du protéome contre le stress. A coté de ces chaperonnes généralistes, d'autres sont spécifiquement impliquées dans des transitions structurales de protéines sur de larges complexes macromoléculaires. Au cours de cette thèse, nous avons identifié et caractérisé le rôle spécifique d'une chaperone Hsp40 dans la biogenèse des ribosomes. Jjj1p se lie tardivement à la particule pré-60S et permet la dissociation du récepteur d'export Arx1p. En absence de Jjj1p, le facteur pré-ribosomique Arx1p s'accumule anormalement sur les particules pré-60S cytoplasmiques, ce qui semble toxique pour la cellule. Nous avons montré que la fonction de Jjj1p dans la biogenèse des ribosomes est conservée dans l'évolution chez l'Homme et le poisson. Enfin, un crible de létalité synthétique nous a permis de mettre en évidence un lien entre Jjjp1 et la machinerie de dégradationThrough their action in protein folding, degradation, translocation across the membrane, and disassembly of complexes, molecular chaperones are very important for different cellular processes. Hsp70 and their Hsp40 partners are crucial in these processes. In eukaryotes, two chaperone networks have been described: a stress inducible network that protects the cellular proteome from stress and a stress-repressed chaperone network that is dedicated to protein biogenesis. Generally Hsp40/70 used to prevent polypeptide misfolding. But up to now, no chaperone has been shown to act specifically in ribosome biogenesis. We have shown that Jjj1p is required for the dissociation of the shuttling export receptor Arx1p from the late cytoplasmic pre-60S particles, relies on its chaperone activity. In jjj1D, Arx1p is abnormally accumulated in the cytoplasm on the pre-60S particle, which is toxic for the cell. The function of an Hsp40 chaperone in such a specific process is conserved throughout the evolutionMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF