<p>Protein biosynthesis is a core process in all living organisms. Assembly of the protein chain from aminoacids is catalysed by the ribosome, ancient and extremely complex macromolecular machine. Several different classes of accessory molecules are involved in translation, and one set of them, called translational GTPases (trGTPases), was in the focus of this work. </p><p>In this thesis properties of two trGTPases– EF-G and eRF3 - were studied by means of direct biochemical experiments. EF-G is a bacterial trGTPase involved in two steps of translation: translocation and ribosomal recycling. Translocation is a process of the ribosomal movement along the mRNA, and recycling as the step when upon completion of the protein ribosome is released from the mRNA via splitting in two ribosomal subunits. We found that off the ribosome EF-G has similar affinities to GDP and GTP, and thus given the predominance of the latter in the cell, EF-G should be present mostly in the complex with GTP. However, binding to the ribosome increases factors affinity to GTP drastically, ensuring that it is in the GTP-bound state. GDP can not promote neither translocation, not recycling, and GDPNP can not promote recycling. It can, however, promote translocation, but in so doing it results in an intermediate ribosomal state and translocation process can be reversed by addition of GDP, which is not the case for the EF-G•GTP-catalyzed reaction.</p><p>The second trGTPase we investigated is eukaryotic termination factor eRF3. This protein together with another factor, eRF1, is involved translation termination, which is release of the synthesized protein from the ribosome. We demonstrateed, that eRF3 alone has basically no propensity to bind GTP and thus resides in the GDP-bound state. Complex formation between eRF1 and eRF3 promotes GTP binding by the latter, resulting in the formation of the ternary complex eRF1•eRF3•GTP, which in turn is catalyzing the termination event.</p><p>Experimental investigations of trGTPases where rationalized within a generalized thermodynamical framework, accommoding the existent experimental observations, both structural and biochemical. </p>

Protein biosynthesis is a core process in all living organisms. Assembly of the protein chain from aminoacids is catalysed by the ribosome, ancient and extremely complex macromolecular machine. Several different classes of accessory molecules are involved in translation, and one set of them, called translational GTPases (trGTPases), was in the focus of this work. In this thesis properties of two trGTPases– EF-G and eRF3 - were studied by means of direct biochemical experiments. EF-G is a bacterial trGTPase involved in two steps of translation: translocation and ribosomal recycling. Translocation is a process of the ribosomal movement along the mRNA, and recycling as the step when upon completion of the protein ribosome is released from the mRNA via splitting in two ribosomal subunits. We found that off the ribosome EF-G has similar affinities to GDP and GTP, and thus given the predominance of the latter in the cell, EF-G should be present mostly in the complex with GTP. However, binding to the ribosome increases factors affinity to GTP drastically, ensuring that it is in the GTP-bound state. GDP can not promote neither translocation, not recycling, and GDPNP can not promote recycling. It can, however, promote translocation, but in so doing it results in an intermediate ribosomal state and translocation process can be reversed by addition of GDP, which is not the case for the EF-G•GTP-catalyzed reaction. The second trGTPase we investigated is eukaryotic termination factor eRF3. This protein together with another factor, eRF1, is involved translation termination, which is release of the synthesized protein from the ribosome. We demonstrateed, that eRF3 alone has basically no propensity to bind GTP and thus resides in the GDP-bound state. Complex formation between eRF1 and eRF3 promotes GTP binding by the latter, resulting in the formation of the ternary complex eRF1•eRF3•GTP, which in turn is catalyzing the termination event. Experimental investigations of trGTPases where rationalized within a generalized thermodynamical framework, accommoding the existent experimental observations, both structural and biochemical.

La traduction est considérée comme une étape clé de l'expression des gènes permettant à la cellule de s'adapter aux variations de son environnement en réponse aux signaux internes ou externes. Des études bioinformatiques ont montrés que la moitié des ARN messagers chez l'homme portent, en amont de leur phase codante, des éléments régulateurs appelés uORF. Le laboratoire a montré qu'un défaut de terminaison de la traduction par déplétion du facteur de terminaison eRF3 modifie l'expression de gènes dont l'ARNm contient des uORF comme le gène ATF4. Cette modification se fait soit par un mécanisme de réinitiation après traduction de l'uORF soit par une augmentation de la stabilité de l'ARNm résultant d'un défaut de sa dégradation par la voie du "Nonsense-mediated mRNA Decay" (NMD). A travers leur association dans le même complexe et leur implication dans la terminaison de la traduction et la NMD, eRF3 et Upf1 contribuent à la régulation fine de l'expression des gènes. Cependant, on ne sait pas dans quelle mesure ces deux facteurs affectent la traduction et la stabilité des ARNm. Nous avons évalué la traduction par ribosome profiling et le taux de transcrits par RNA-seq dans les cellules humaines déplétées en eRF3 ou en Upf1. Ces analyses nous ont permis de dresser une carte des uORF traduites dans le transcriptome des cellules humaines HCT116. Nous avons également observé que peu de gènes cibles sont communs entre la déplétion en eRF3 ou en Upf1. Nos résultats appuient fortement l'hypothèse qu'il y a au moins deux classes de transcrits portant des uORF, l'une dont la régulation implique la terminaison de la traduction et l'autre dont la régulation implique la NMD. / Regulation of gene expression at the translational level is increasingly being recognized as a key mechanism by which cells can rapidly change their gene expression pattern in response to internal or external stimuli. Bioinformatic studies revealed that half of human transcripts present at least one expression regulatory element uORF in the 5’ leader sequence preceding the main ORF. We have previously shown that translation termination disruption caused by eRF3a depletion induces upregulation of the transcriptional activator ATF4 and its targeted genes partly by a translational control at uORFs, and partly in relation to a defect in Nonsense-mediated mRNA Decay activation, increasing ATF4 mRNA stability. Through their physical association and their involvement in translation termination and NMD, eRF3 and Upf1 are regulating the protein and mRNA levels of a significant number of genes and thus contribute to the fine-tuning of their expression. It is not known yet, in what extent both of these factors affect translational control and what is the subset of genes that are regulated by these factors. In this study, we evaluated translation by ribosome profiling and mRNA level by RNA-seq in human cells subjected to either eRF3a or Upf1 depletion. These analyses allowed us to draw a transcriptome-wide map of uORFs and obtained a list of functional uORFs in our reference HCT116 transcriptome. We also observe that only a small fraction of these are common targets for both eRF3a and Upf1. Our results provide strong support for the notion that different classes of transcripts bearing uORFs are regulated either by translational processes involving translation termination or by NMD.

La désadénylation des ARNm fait intervenir les complexes de désadénylation PAN2-PAN3 et CCR4-NOT-TOB mais aussi le complexe de terminaison de la traduction eRF1-eRF3. Ces trois complexes ont la capacité d'interagir avec la protéine PABP. Cependant, le rôle d'eRF3 n'est pas clairement établi. Il a été décrit que les facteurs eRF3, PAN3 et TOB sont en compétition pour l'interaction avec PABP et qu'il y a un couplage entre la terminaison de la traduction et la désadénylation assuré par eRF3. Chez l'homme, le gène eRF3/GSPT1 présente 5 formes alléliques qui diffèrent par le nombre de répétitions de codons GGC à l'extrémité 5' du cadre de lecture (7, 9, 10, 11 et 12-GGC). Une corrélation entre l'allèle 12-GGC et le risque de développement de cancer du sein et de l'estomac a été mis en évidence. Notre objectif est (i) d'améliorer notre compréhension du rôle d'eRF3 dans le processus de couplage traduction-dégradation des ARNm, (ii) de comprendre l'effet du polymorphisme de la région N-terminale d'eRF3 sur son interaction avec PABP. A travers la méthode de résonnance plasmonique de surface (SPR), nous montrons que l'affinité de la forme allélique 12-GGC est 10 fois plus faible que celle d'eRF3a (10-GGC). Cette différence est essentiellement due à la plus faible association de la forme 12-GGC avec PABP. La plus faible affinité de la forme 12-GGC d'eRF3 entrainerait une dérégulation de la désadénylation au moins pour certains ARNm et pourrait ainsi promouvoir la prolifération cellulaire et la carcinogenèse. La région N-terminale d'eRF3 contenant la répétition de glycine joue un rôle crucial dans l'interaction eRF3-PABP, dans la désadénylation et donc dans la stabilité de l'ARNm. / The mRNA deadenylation involves the deadenylation complexes PAN2-PAN3 and CCR4-NOT-TOB and the translation termination complex eRF1-eRF3. All three proteins, eRF3, PAN3 and TOB, interact with the PABP protein. However, the role of eRF3 is still unclear. It has been reported that eRF3, TOB and PAN3 compete for the binding to PABP. Recently, it has been suggested that eRF3 may regulate mRNA deadenylation in a translation termination-coupled manner. In human, the gene eRF3/GSPT1, contains a trinucleotide GGC repeat in its 5’ end which lead to 5 allelic forms of the gene. There are five known alleles of this gene (7, 9, 10, 11 and 12-GGC). A strong correlation between the longest allele (12-GGC) and gastric and breast cancer development has been reported. Our project was (i) to improve our understanding on the role of eRF3 in the coupling of mRNA deadenylation with translation termination, (ii) to understand whether the GGC repeat polymorphism of eRF3 influences eRF3-PABP interaction. The kinetic measurements of eRF3-PABP interaction obtained by Surface Plasmon Resonance (SPR) show that the affinity of the allelic 12-GGC form is 10 fold lower than that of eRF3a (10-GGC). This decrease is mostly due to difference in the association rate of the complex. The weaker affinity of the 12-GGC allelic form may result in a deregulation of deadenylation, at least for some mRNAs, and thus, could promote cell proliferation and carcinogenesis. In fine, we show that the N-terminal region of eRF3 containing the glycine expansion plays a key role in the eRF3-PABP interaction, in the deadenylation process, and hence, in mRNA stability.