Transfer ribonucleic acids (tRNAs) are extensively modified, especially their anticodon loops. Modifications at position 34 (wobble base) and 37 in these loops affect the tRNAs’ decoding ability, while modifications outside the anticodon loops, e.g. m1A58 of tRNAMeti, may be crucial for tRNA structure or stability. A number of gene products are required for the formation of modified nucleosides, e.g. at least 26 proteins (including Elongator complex) are needed for U34 modifications in yeast, and methyl transferase activity of the Trm6/61p complex is needed to form m1A58. The aim of the studies which this thesis is based upon was to investigate the functional aspects of tRNA modifications and regulation of the modifying enzymes’ activity. First, the hypothesis that ncm5U34, mcm5U34, or mcm5s2U34 modifications may be essential for reading frame maintenance was investigated. The results show that mcm5 and s2 group of mcm5s2U play a vital role in reading frame maintenance. Subsequent experiments showed that the +1 frameshifting event at Lys AAA codon occurs via peptidyl-tRNA slippage due to a slow entry of the hypomodified tRNA-Lys. Moreover, the hypothesis that Elp1p N-terminal truncation may regulate Elongator activity was investigated. Cleavage of Elp1p was found to occur between residue 203 (Lys) and 204 (Ala) and to depend on the vacuolar protease Prb1p. However, including trichloroacetic acid (TCA) during protein extraction abolished the appearance of truncated Elp1p, showing that its truncation is a preparation artifact. Finally, in glioma cell line C6, PKCα was found to interact with TRM61. RNA silencing of TRM6/61 causes a growth defect that can be partially suppressed by tRNAMeti overexpression. PKCα overexpression reduces the nuclear level of TRM61, likely resulting in reduced level of TRM6/61 complex in the nucleus. Furthermore, lower expression of PKCα in the highly aggressive GBM (relative to its expression in less aggressive Grade II/III glioblastomas) is accompanied by increased expression of TRM6/61 mRNAs and tRNAMeti, highlighting the clinical relevance of the studies.

Mes divers thèmes de recherche concernent la traduction qui, au sens large du terme, peut être considérée comme la production de biomolécules à rôle structural et/ou fonctionnel à partir de l'information contenue dans les gènes. Pour ma part je me suis intéressé tout d'abord à l'aminoacylation des ARNs de transfert (ARNt) ainsi qu'à la formation de certaines bases modifiées qui les composent. En effet, l'ARNt est un acteur clé de la synthèse protéique car c'est de l'exactitude de son aminoacylation que dépend grandement la fidélité de la synthèse protéique. <br />Chez la levure, il existe un complexe multi-synthétasique simplifié formé de la MetRS, la GluRS et Arc1p, une protéine dont le gène est en interaction non seulement avec la machinerie de transport nucléocytoplasmique mais également celle de la biosynthèse des ARNts. Ces découvertes m'ont amené à étudier le rôle de Mtr10p dans les échanges nucléocytoplasmiques et un possible lien avec le transport des ARNts. Les acquis lors de ces travaux auront été utiles dans l'étude du rôle d'Efl1p, une GTPase de type EF-2 impliquée dans une étape tardive (cytoplasmique) de la biogenèse de la grande sous-unité ribosomale 60S.<br />Grâce aux travaux antérieurs, j'ai pu me familiariser avec de nombreuses techniques biochimiques et génétiques qui s'avèreront précieuses pour mon projet d'étude de la localisation et de l'interactome des aminoacyl-ARNt synthétases (aaRS). En effet, il n'est pas concevable que le couple ARNt/aaRS évolue de façon autonome dans la cellule mais il doit interagir / communiquer d'une part avec les acteurs directs de la synthèse protéique et, d'autre part, avec des facteurs intervenant dans d'autres processus cellulaires. Trouver, comprendre et étudier les spécificités de divers organismes fournira peut-être de nouvelles pistes à but thérapeutique restées insoupçonnées jusqu'alors.<br />Un autre aspect de mon projet, toujours relié aux aaRS, traitera d'un système atypique de la machinerie traductionnelle qui est celui des voies de formation des aminoacyl-ARNts dans les systèmes asparagine et glutamine. En effet, un certain nombre d'organismes, pathogènes de l'homme, ne possèdent pas d'aaRS (AsnRS, GlnS) pour l'un et/ou l'autre de ces acides aminés et pallient à cette absence par l'utilisation d'une voie indirecte pour la formation de ces aminoacyl-ARNts. L'étude de ces voies est non seulement intéressante sur les plans évolutifs et relation structure-fonction ARN-protéine mais également d'un point de vue appliqué, certes à plus long terme, dans le but d'enrayer ces voies de biosynthèse amenant ainsi des cibles alternatives pour des molécules de type antibiotique. Cet aspect est d'autant plus important qu'à l'heure actuelle apparaissent des phénomènes de résistance aux antibiotiques posant de vrais problèmes de santé publique.

Depuis 1991, année de mon recrutement au CNRS, mon activité de recherche est centrée sur l'étude d'une famille d'enzymes intervenant dans la biosynthèse protéique : les aminoacyl-tRNA synthétases. Mon travail s'est articulé autour de l'étude de leur organisation fonctionnelle et de la compréhension des bases moléculaires de la reconnaissance spécifique entre ces enzymes et leurs substrats. Des approches multidisciplinaires (biologie moléculaire, biochimie, cristallographie aux rayons X, enzymologie et génétique) ont été mises en œuvre pour une exploration globale de ces problèmes. Nous avons pu progresser dans la connaissance de deux enzymes modèles : l'aspartyl-tRNA synthétase et l'arginyl-tRNA synthétase, deux enzymes appartenant respectivement à la classe II et à la classe I des aminoacyl-tRNA synthétases. Nous avons localisé les sites de fixation des différents substrats, proposé des mécanismes catalytiques et sélectionné in vivo des variants d'aminoacyl-tRNA synthétases et de tRNAs aux propriétés de reconnaissance modifiées.

In addition to its canonical role in aminoacylation, threonyl-tRNA synthetase (TARS) possesses pro-angiogenic activity that is susceptible to the TARS-specific antibiotic borrelidin. However, the therapeutic benefit of borrelidin is offset by its strong toxicity to living cells. The removal of a single methylene group from the parent borrelidin generates BC194, a modified compound with significantly reduced toxicity but comparable anti-angiogenic potential. Biochemical analyses revealed that the difference in toxicities was due to borrelidin's stimulation of amino acid starvation at ten-fold lower concentrations than BC194. However, both compounds were found to inhibit in vitro and in vivo models of angiogenesis at sub-toxic concentrations, suggesting a similar mechanism that is distinct from the toxic responses. Crystal structures of TARS in complex with each compound indicated that the decreased contacts in the BC194 structure may render it more susceptible to competition with the canonical substrates and permit sufficient aminoacylation activity over a wider concentration of inhibitor. Conversely, both borrelidin and BC194 induce identical conformational changes in TARS, providing a rationale for their comparable effects on angiogenesis. The mechanisms of TARS and borrelidin-based compounds on angiogenesis were subsequently tested using zebrafish and cell-based models. These data revealed ectopic branching, non-functional vessels, and increased cell-cell contracts following BC194-treatment or knockdown of TARS expression, suggesting a role for the enzyme in the maturation and guidance of nascent vasculature. Using various TARS constructs this function was found to be dependent on two interactions or activities associated with the TARS enzyme that are distinct from its canonical aminoacylation activity. Furthermore, observations that TARS may influence VEGF expression and purinergic signaling suggest the possibility for a receptor-mediated response. Taken together, the results presented here demonstrate a clear role for TARS in angiogenesis, independent of its primary function in translation. Although the exact molecular mechanisms through which TARS and borrelidin regulate this activity remain to be determined, these data provide a foundation for future investigations of TARS's function in vascular biology and its use as a target for angiogenesis-based therapy.

A regulação da localização de proteínas é um dos aspectos fundamentais na biologia celular vegetal. Os cloroplastos importam mais de 90% de suas proteínas do citosol, portanto, é importante caracterizar os fatores citosólicos que podem estar envolvidos no direcionamento de proteínas para as organelas. Um ensaio de duplohíbrido em leveduras com as proteínas cloroplastidiais HMPPK/TMPPase (TH1) e Glutamina Sintetase (GS) II usados como iscas revelou que a forma citosólica da glutamil-tRNA sintetase - GluRS (At5g26710) de Arabidopsis thaliana interagiu com ambas as proteínas. Estudos de Complementação da Fluorescência Bimolecular (BiFC) confirmaram tais interações in planta. Estudos com deleções na região Nterminal da GluRS mostraram que esta região é responsável pelas interações com HMPPK/TMPPase e GSII. Além disso, seis resíduos de aminoácidos parecem ser cruciais para a interação entre as proteínas. Curiosamente, foi mostrado que a GluRS está envolvida na localização de proteínas em leveduras. A fim de obter mais informações sobre o envolvimento da GluRS ns localização de proteínas nos cloroplastos, foram produzidos plantas de tabaco transgênicas expressando uma proteína quimérica, feita pela fusão do gene codificador da HMPPK/TMPPase, TH1- GFP, e GSII-GFP e posteriormente usados em ensaios de agroinfiltração com RNA de interferência (RNAi) para GluRS. Análises em microscópio confocal mostraram que TH1-GFP e GSII-GFP acumulam no citosol em vez de serem direcionados aos cloroplastos. Neste trabalho, mostramos pela primeira vez que a GluRS está envolvida na localização de proteínas cloroplastidiais em plantas e esse mecanismo é também conservado em Saccharomyces cerevisiae. / Regulation of protein localization is one of the key aspects in plant cell biology. Chloroplasts import more than 90% of their proteins from the cytosol, therefore, it is important to identify and characterize cytosolic factors that might be involved in protein delivery to the organelar envelope. A yeast two-hybrid screen with a chloroplastlocalized HMPPK/TMPPase protein and glutamine synthetase (GS), used as baits, revealed that the cytosolic form of the glutamyl-tRNA synthetase (GluRS) (At5g26710) from Arabidopsis thaliana interacted with both proteins. Bimolecular Fluorescence Complementation (BiFC) studies confirmed such interactions in planta. Deletion studies of GluRS showed that the N-terminal region of the protein is responsible for proteinprotein interactions (PPI) with TH1 and GS. In addition, six amino acid residues appeared to be crucial for PPI. Interestingly, GluRS has been also shown to be involved in regulating protein localization in yeast. In order to gain more information about the involvement of GluRS on protein localization in chloroplasts, we produced transgenic tobacco plants expressing a chimeric protein made by the fusion of TH1- GFP and GSIIGFP and agroinfiltrated with a RNA interference (RNAi) construct against GluRS. Confocal analysis showed that TH1-GFP and GSII-GFP accumulated in the cytosol instead of being targeted to chloroplasts. Here, we show for the same time that GluRS is involved in protein localization in plants and this mechanism is also conserved in Saccharomyces cerevisiae.

This thesis aims to address issues relating to genome architecture and base composition. The first part of this thesis addresses questions relating to codon usage. Initially I will investigate thousands of bacterial species using a detailed analysis of strengths of selection acting upon codons usage while also investigating patterns of optimal codon changes with respect to genomic base composition and tRNA abundance. I report that selection on codon usage increases throughout the length of highly expressed genes, in particular, the first quarter of genes have significantly lower selection. Further, it is clear that factors affecting genomic base composition can eventually lead to changes in optimal codons if the change in base composition is strong enough, however these patterns differ substantially between amino acids. The debate over translational efficiency vs. accuracy was addressed by comparing sites of differing conservation. Differing conservation were defined using a phylogenetic method, allowing sites to change in their extent of conservation throughout the tree. The results show that translational accuracy acts strongly on the top 10% of conserved sites, however is relatively weak when compared to the efficiency for other sites. Also detected is a reduction in apparent selection on codon usage on the bottom 10% of conserved sites which is likely to be caused by conflicting positive selection on amino acids. Finally, although differences in patterns are observed between amino acids, the general relationship to conservation is similar. As much of the variation in codon usage is determined by variation in base composition, this aspect of base composition is investigated in the second part of the thesis. The observed variation in intragenomic base composition in bacteria was found to be far higher than expected for GC-rich bacteria. The non-core part of the genome contributes to this variation to a greater extent than the core part, suggesting that processes such as AT-rich horizontal gene transfer may be involved. Secondly, base composition is modelled under Brownian motion and as an extension, the Ornstein- Uhlenbeck process, which allows for multiple optima throughout the tree. The model including optima fits the data better than standard Brownian motion or Brownian motion with multiple diffusion coefficients. Finally, I investigate a case where a previous codon usage analysis has been seriously confounded by an unusual genome architecture of abnormal regional base composition in two species of eukaryotic parasites in the genus Theileria. In both species, the background G+C content is 37% at most, out of the four syntenic chromosomes. In many orthologous regions however, T.annulata has a decreased G+C content of 28% while T.parva has an increased G+C content of 41%. Various factors coincide with this remarkable divergence: increased divergence at all types of site, recombination hot spots in T.parva, an increased frequency of tandem repeats and DNA sequence motifs in both species. The evolutionary origins of these unusual patterns will be discussed.

La traduction représente un processus central au sein de la cellule, elle assure le transfert de l’information génétique de l’ARNm vers les protéines. De nombreux acteurs y sont impliqués directement ou indirectement et parmi eux, chez les eucaryotes, la petite protéine Trm112. Celle-ci participe à la modification de plusieurs acteurs directs en interagissant et en activant quatre MTases. Le facteur de terminaison eRF1 est méthylé par le complexe Mtq2-Trm112, l’ARNr 18S par Bud23-Trm112 et certains ARNt par les complexes Trm9-Trm112 et Trm11-Trm112. Au cours de ce travail, les structures cristallographiques de Trm9-Trm112 et de Bud23-Trm112 de levure ont été résolues. L’étude comparative structurale de ces complexes et de la structure connue de Mtq2-Trm112, a permis de mettre en évidence que dans un même organisme, les séquences des trois protéines ont évolué de manière à conserver l’interaction avec Trm112. Même si les quatre partenaires présentent moins de 20% d’identité de séquence, les résidus clés pour l’interaction avec la petite protéine activatrice sont conservés ou partagent des caractéristiques identiques. En plus de l’analyse structurale, le complexe Trm9-Trm112 a fait l’objet d’une étude fonctionnelle chez S. cerevisiae ce qui a permis de cartographier le site actif de l’enzyme et de proposer un modèle de mécanisme d’action. Enfin, les premières études in vivo réalisées chez Haloferax volcanii suggèrent que cette plateforme serait également présente chez certains organismes procaryotes. / Protein synthesis is a central process in the cell; it ensures the transfer of genetic information from mRNA in to protein. A lot of actors are involved directly or indirectly in translation. In Eukaryotes, Trm112, a small protein, interacts with and activates four methyltransferases modifying direct actors of translation. The termination factor eRF1 is methylated by the Mtq2-Trm112 complex, the 18S rRNA by Bud23-Trm112 and some tRNA by the Trm9-Trm112 and Trm11-Trm112 complexes. During this work, the crystal structures of Trm9-Trm112 and Bud23-Trm112 complexes from yeast were solved. The comparative analysis of these two new structures with Mtq2-Trm112 structure highlights the structural plasticity allowing Trm112 to interact through a very similar mode with its partners although those share less than 20% sequence identity. In the same organism, the key residues for the interaction with Trm112 are conserved or share similar characteristics. In addition to the structural analysis, the function of the Trm9-Trm112 complex was studied in S. cerevisiae. This analysis allowed to map the active site of the enzyme and to propose a model of its mechanism of action. Finally, the first data obtained in vivo, with the Archaea Haloferax volcanii suggest that the Trm112 platform might also be present in some prokaryotic organisms.

Avec l'accroissement du nombre de génomes séquencés, l'organisation de ces données brutes et des données dérivées, l'extraction de l'information et des connaissances associées défie l'imagination. La notion de voisinage a été d'abord été introduite pour l'organisation des données dans des bases de données relationnelles. Pour extraire des informations pertinentes à partir de données massives, différents types de voisinages ont été étudiés ici. Tout d'abord, avec l'analysedes correspondances (CA) et en utilisant le regroupement supervisé ("model clustering" MBC), la proximité mutuelle des éléments formant deux entités biologiques centrales, les gènes (codant les protéines) et les acides aminés a été analysée. Nous montrons par exemple que les protéines de Psychromonas ingrahamii, bactérie psychrophile extrêmes, sont regroupées en six classes, et qu'il y a une forte opposition entre le comportement de l'asparagine (N) et des acides aminés sensibles à l'oxygène, ce que nous expliquons en terms de résistance au froid. Ensuite, nous avons analysé la répartition entre les îlots génomiques (GI) et le squelette du génome de base à partir d'une nouvelle méthode combinant composition en bases et en gènes, caractéristiques GI et de briser les synténies. L'application de cette approche à E. coli et B. subtilis a révélé que cette nouvelle méthode permet d'extraire certaines régions significative, non publiées auparavant.Enfin, pour illustrer un voisinage fin, la régulation de l'expression d'un gène et son évolution, nous avons étudié la relation entre les régions en amont du gène et la zone codante du gène thrS de façon approfondie. Nous avons constaté que ces deux régions associées à un gène, se sont comportés différemment dans l'histoire évolutive. Certaines des régions en amont porteuses de la fonction non-essentielle de régulation (qui contrôle l'expression de gène) ont évoluédifféremment de la région codante. / With more and more genomes being sequenced, the organization of those raw data and the derived data, the extraction of information and knowledge from these data has become a challenge. A key concept in this field is that of the neighborhood, especially with respect to the organization of data in relational databases. To extract information from bulk data, different kinds of neighborhoods were studied and each show interesting results in current study. .Firstly, through the Correspondence Analysis (CA) and later Model Based Clustering (MBC), two kinds of neighbors i.e. the genes (proteins) and amino acids were analyzed respectively, and it was found that proteins from Psychromonas ingrahamii are clustered into six classes, and there is strong opposition between asparagine (N) and the oxygen-sensitive amino acids. Secondly, the relationship between genomic islands and core genome (i.e. two closely linked neighbors withlarge range on the chromosome) was studied by a new method combining composition, GI features and synteny break. On applying to E. coli and B. subtilis it was revealed that this new method can extract some meaningful regions not published before. Thirdly, the relationship between upstream and coding regions of thrS gene (i.e. a case for two closely linked neighbors with small range on the chromosome) was studied extensively. It was found that these two regions associated to one gene, behaved differently in the evolutionary history.. Some of the upstream regions bearing non-essential function (i.e. regulation of gene expression) evolved more slowly than the coding region.

L’import d’ARN dans les mitochondries est un processus ubiquitaire chez les eucaryotes. Dans Saccharomyces cerevisiae un isoaccepteur cytosolique de l’ARNtLys, l’ARNtLys(CUU) (tRK1), est partiellement importé dans les mitochondries. L’adressage vers la surface mitochondriale a été étudié en détail mais l’étape de translocation dans les mitochondries reste toujours à démontrer. L’objectif principal de ce travail de thèse était l’identification et la caractérisation des protéines qui participent dans ce processus. Deux protéines de la membranes externe capables de former des canaux, Tom40 et VDAC1, ont été identifiées et leur rôle dans l’étape de translocation a été évalué in vitro et in vivo en utilisant des souches mutantes appropriées ou des agents capables de bloquer de manière spécifique les canaux formés par les protéines Tom40 et VDAC1. Ainsi il a été démontré que la délétion de VDAC1 ou l’inhibition du canal VDAC1 a conduit à une inhibition importante, néanmoins pas complète, de l’import de tRK1. Le blocage simultané des canaux Tom40 et VDAC1 par contre a causé un arrêt complet de l’import de tRK1 in vitro. Vu ces résultats, nous proposons que la translocation de tRK1 à travers la membrane mitochondriale externe puisse suivre deux chemins alternatifs. / RNA import into mitochondria is a ubiquitous process in eukaryotic cells. In Saccharomyces cerevisiae one cytosolic isoacceptor of tRNALys, tRNALys(CUU) (tRK1), is partially imported into mitochondria. Targeting of tRK1 to the mitochondrial surface is well described but the translocation of tRK1 into mitochondria is still poorly understood. This PhD work aimed to study this translocation step and the main objective was the identification and characterization of mitochondrial membrane proteins participating in this process. The two channel-forming proteins of the mitochondrial outer membrane, Tom40 and VDAC1, were identified and their role in tRK1 translocation further investigated with the help of in vitro and in vivo import studies using appropriate mutant strains or specific agents capable of blocking Tom40 and VDAC1. In this way, it could be demonstrated that deletion of the VDAC1 gene or inhibition of VDAC1 led to an important yet not complete inhibition of tRK1 import into mitochondria. Simultaneous blocking of the two channels formed by Tom40 and VDAC1, however, resulted in a complete inhibition of tRK1 import in vitro. Regarding these results we propose that tRK1 translocation through the mitochondrial outer membrane can use two alternative pathways.

<p>The stringent response plays a significant role in the survival of bacteria during different environmental conditions. It is activated by the binding of stringent factor (SF) to stalled ribosomes that have an unacylated tRNA in the ribosomal A-site which leads to the synthesis of (p)ppGpp. ppGpp binds to the RNA polymerase, resulting in a rapid down-regulation of rRNA and tRNA transcription and up-regulation of mRNAs coding for enzymes involved in amino acid biosynthesis. The importance of the A-site and unacylated tRNA in the activation of SF was confirmed by chemical modification and subsequent primer extension experiments (footprinting experiments) which showed that binding of SF to ribosomes resulted in the protection of regions in 23S rRNA, the A-loop and helix 89 that are involved in the binding of the A-site tRNA. An in vitro assay showed that the ribosomal protein L11 and its flexible N-terminal part was important in the activation of SF. Interestingly the N-terminal part of L11 was shown to activate SF on its own and this activation was dependent on both ribosomes and an unacylated tRNA in the A-site. The N-terminal part of L11 was suggested to mediate an interaction between ribosome-bound SF and the unacylated tRNA in the A-site or interact with SF and the unacylated tRNA independently of each other. Footprinting experiments showed that SF bound to the ribosome protected bases in the L11 binding domain of the ribosome that were not involved in an interaction with ribosomal protein L11. The sarcin/ricin loop, in close contact with the L11 binding domain on the ribosome and essential for the binding and activation of translation elongation factors was also found to be protected by the binding of SF. Altogether the presented results suggest that SF binds to the factor-binding stalk of the ribosome and that activation of SF is dependent on the flexible N-terminal domain of L11 and an interaction of SF with the unacylated tRNA in the A-site of the 50S subunit.</p>