La methionyl-ARNt synthétase (MetRS) de Saccharomyces cerevisiae aminoacyle les ARNt méthionine initiateur et élongateur (ARNtiMet et ARNteMet), mais possède également des fonctions atypiques. Nous avons montré que la MetRS rejoint le noyau durant la transition diauxique afin de réguler la transcription des gènes nucléaires des complexes III et V de la chaîne respiratoire mitochondriale. Pour ce faire, la MetRS possède au moins deux signaux de localisation nucléaire (NLS) dans sa séquence, l’un se situant dans les 55 premiers acides aminés (aa) et le second, au delà de la partie N-terminale lui permettant de recruter les sous-unités Rpb4 et Rpb7 de l’ARN pol II. Nous avons montré qu’en fermentation, la MetRS est clivée entre le 114ème et le 132ème aa et que cette forme clivée est essentielle à la viabilité des cellules, puisqu’un variant non clivé (MetRSK11A) ne permet pas la croissance. Nous avons surproduit et purifié un mutant de la MetRS clivée (MetRSΔ142) et montré que ce variant est plus efficace pour l’aminoacylation de l’ARNtiMet que la forme entière de MetRS. Ainsi, notre étude suggère que chez S. cerevisiae, la forme longue de MetRS cytoplasmique permet l’aminoacylation de l’ARNteMet, la forme longue de MetRS nucléaire régule la transcription, et la forme clivée de MetRS nucléaire et cytoplasmique permet l’aminoacylation de l’ARNtiMet / Methionyl-tRNA synthetase (MetRS) is the enzyme in charge of aminocylation of tRNA methionine initiator and elongator (tRNAiMet et tRNAeMet), but also displays atypical functions in Saccharomyces cerevisiae. In the present work, we showed that MetRS is imported to the nucleus during the diauxic shift in order to regulate transcription of genes coding for the complexes III and V subunits of the mitochondrial respiratory chain. To do so, MetRS harbors at least two nuclear localization signals (NLS), located within the 55 first aminoacids (NLS1) and beyond the N-terminal part (NLS2). The N- terminal part is responsible for the recruitment of RNA pol II subunits Rpb4 and Rpb7. We also showed that MetRS is cleaved through the 114th and the 132nd aminoacid during fermentation and that the proteolysed form is essential for the viability of the cell, since a mutant of MetRS which is not cleaved (MetRSK11A) did not allows the growth. We showed that an overproduced and purified a mutant representative of the cleaved form (MetRSΔ142) is more efficient for tRNAiMet aminoacylation than the full length MetRS. Thus, our study suggests that in S. cerevisiae, the cytoplasmic full length MetRS aminoacylates tRNAeMet, the nuclear full length MetRS regulates genes transcription, and the cytoplasmic and nuclear cleaved MetRS aminoacylates the tRNAiMet.

Les complexes multisynthétasiques (MSC) sont des complexes multi-protéiques identifiés dans un grand nombre d’organismes pro- et eucaryotes. Ils impliquent des protéines d’assemblages et des aminoacyl-ARNt synthétases (aaRSs), responsables de l’aminoacylation de leurs ARNts homologues au cours de la traduction. La taille et la composition des MSC varient selon les organismes, et le rôle de ces complexes n’est pas encore totalement compris. Il semblerait néanmoins que chez les eucaryotes, l’accrétion en complexe soit une stratégie mise en oeuvre par les cellules pour empêcher les aaRSs d’assurer des fonctions additionnelles. Chez S.cerevisiae,nous montrons que la dynamique du complexe AME, composé de la méthionyl- et de la glutamyl-ARNt synthétase (MRS et ERS) ainsi que de la protéine d’ancrage Arc1p, est dépendante du métabolisme de la levure. En respiration la MRS joue le rôle de facteur de transcription et régule l’expression des gènes nucléaires du complexe III et V de la chaîne respiratoire, tandis que l’ERS active la traduction mitochondriale. Cette étude montre que la relocalisation synchrone est primordiale pour l’adaptation des cellules au métabolisme respiratoire. / Multisynthetase complexes (MSC) are complexes made of several proteins and were identified in a wide variety of organisms from pro- to eukaryotes. They are usually made of assembly factors and aminoacyl-tRNA synthetases (aaRSs), which are responsible for the aminoacylation of their corresponding tRNAs during translation. Depending on the organisms, size and composition of these complexes differ greatly and their role is not fully understood yet. Although it seems that in eukaryotes, accretions of aaRSs into MSC prevent aaRSs to perform their additional functions. In the yeast Saccharomyces cerevisiae, we show that the dynamic of the AME complex, made of the méthionyl- and glutamyl-tRNA synthetases (MRS and ERS) and the assembly protein Arc1p is linkedto yeast metabolism. In respiration, MRS is imported in the nucleus to act as a transcription factor and regulates the expression of nuclear genes belonging to complex III and V of the respiratory chain, while ERS is imported in mitochondria to activate translation. This study shows that synchronous relocation of both aaRSs is crucial for yeast cells to adapt to respiratory metabolism.

Les ARNt sont des molécules adaptatrices reliant l'information génétique de l’ARN messagers à la séquence d'acides aminés primaire des protéines. Les ARNt ont une structure typique, appelée "feuille de trèfle". Certains ARNt mitochondriaux montrent une forte dérivation de cette structure. Un cas extrême peut être observé dans les mitochondries du nématode R. culicivorax. Cette étude vise la caractérisation fonctionnelle de ces ARNt «bizarres» et de définir leurs propriétés structurales et leur fonctionnalité avec des protéines partenaires telles que les CCAses et les aminoacyl-ARNt synthetases. Ce travail révèle que les ARNt sans bras forment une structure secondaire en forme d'épingle à cheveux et que leurs structures 3D présentent une grande flexibilité intrinsèque. Les tests initiaux n’ont pas démontré l'activité d'aminoacylation. Cependant, les ARNt sans bras représentent des molécules fonctionnelles pour le CCAse, indiquant des adaptations de l’enzyme aux ARNt sans bras. / TRNAs are adapter molecules linking the genetic information of messenger RNAs with the primary amino acid sequence of proteins. tRNAs have a typical cloverleaf-like secondary structure. Some mitochondrial tRNAs show a high derivation from this canonical tRNA structure. An extreme case of structural truncations can be observed in mitochondria of the nematode R. culicivorax. This study aims the functional characterization of such “bizarre” tRNAs in defining their structural properties and their functionality with interacting partner proteins such as CCA-adding enzymes and aminoacyl-tRNA synthetases. This work reveals that armless tRNAs form a hairpin-shaped secondary structure. 3D structures exhibit a high intrinsic flexibility. Initial tests could not demonstrate aminoacylation activity. However, armless tRNAs represent functional molecules for CCA-incorporation, indicating adaptations of CCA-adding enzymes to armless tRNAs.

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.

Nos organismos eucariotos, aproximadamente 50% das proteínas traduzidas no citoplasma são transportadas para as organelas, onde irão desempenhar suas funções. Com isso, surgiu um intricado sistema de transporte intracelular de proteínas. Nas plantas, a presença de uma segunda organela endossimbionte, o plastídio, tornou este sistema mais complexo e gerou demanda adicional por transporte. Ainda, grande maioria das proteínas mitocondriais e plastidiais são codificadas por genes nucleares e importadas do citosol. O dogma uma proteína-uma localização foi associado ao conceito de um gene-uma proteína na biologia celular. Entretanto, proteínas individuais podem ter mais de uma função, e mais recentemente, proteínas codificadas por um único gene foram identificadas em mais de um compartimento subcelular, o que deu origem ao conceito de duplo direcionamento (DD). Um exemplo bem estudado de DD vem das proteínas da família das aminoacil-tRNA sintetases (aaRS), que participam da síntese protéica ao acoplar o aminoácido ao seu tRNA cognato. Dentre as aaRSs, a glutamil-tRNA sintetase citosólica (GluRS), através de sua extensão N-terminal, parece estar envolvida com outras funções além da tradução. Em Arabidopsis thaliana, há dois genes nucleares que codificam a GluRS, um para uma proteína de duplo direcionamento (DD) e outro para uma proteína citosólica. Resultados recentes em nosso laboratório mostraram que a GluRS citosólica pode estar relacionada ao controle da localização subcelular de proteínas organelares em Arabidopsis. Para verificar um eventual papel desta proteína na localização subcelular de outras proteínas, foram realizados ensaios de duplo-híbrido em levedura, os quais mostraram interação entre a GluRS e a glutamina sintetase (GS) de Arabidopsis thaliana, proteína de DD para mitocôndrias e cloroplastos Esta interação foi confirmada in planta, sendo a sequência da GluRS responsável pela interação localizada na região N-terminal, do resíduo 207 ao 316. Análises filogenéticas apontam que esta região encontra-se ausente nas bactérias e que originou-se provavelmente em Archea, entre 2,6 e 1,8 bilhões de anos. Além disso, observa-se que esta sequência é conservada em fungos, musgos e plantas vaculares, tendo originado-se em Arabidopsis há cerca de 2 bilhões de anos. / In eukaryotic organisms, about 50% of cytoplasmic translated proteins are transported to the organelles, where they can play their roles. Thus, a complex system for intracellular transport was established. In plants, the presence of a second endosymbiont organelle, the plastid, turned this system still more intricated and required an additional transport mechanism. Besides, most of organellar proteins are coded by nuclear genes and imported from the cytosol. The one protein-one localization was associated to the idea of one gene-one protein, which has long been established in molecular biology. However, individual proteins can show more than one function, and recently, proteins coded by one single gene were identified in more than one subcellular compartment, which has originated the concept of dual targeting. One of the most studied example of dual targeted proteins is the aminoacyl-tRNA synthetase (aaRS) family, which are related to protein synthesis by attaching the correct amino acid onto the cognate tRNA molecule. Among the aaRSs, cytosolic glutamyl-tRNA synthetase (GluRS), through its N-terminal extension, seems to be involved in other cellular role beyond translation. In Arabidopsis thaliana, there are two genes encoding GluRS, one for a dual-targeted protein and other for a cytosolic protein. Recent results in our laboratory showed that GluRS interacts with proteins destinated to other organelles, which suggest that this protein might have a role in interfering on protein localization in Arabidopsis. In order to gain some information on the role of this protein in subcellular localization, yeast two-hybrid assays were performed. These studies showed the interaction between GluRS and glutamine synthetase (GS), a mitochondrial and chloroplastic dual-targeted protein. This interaction was confirmed in planta. In addition, the GluRS sequence associated to protein interaction was localized at its N-terminal portion, between the residues 207 316. Phylogenetic analysis revealed that this region is absent in bacteria and it probably arose from Archea between 2.6 and 1.8 billion years ago. Also, this sequence is conserved in fungi, moss and all the green plants investigated. Finally, datation analysis showed that this sequence arose in Arabidopsis between 2 and 1.7 billion years ago.

La GlnRS de Deinococcus radiodurans se distingue des autres GlnRS par la présence d'un appendice additionnel en C-terminal (C-ter). Celui-ci adopterait le même repliement que la famille de protéines YqeY de fonction inconnue et une région de la sous-unité GatB de l'AdT. Son architecture atypique, trouvée dans 4 organismes, corresponds à la fusion de protéine de la voie directe et indirecte d'aminoacylation des ARNt. La structure cristallographique de la GlnRS-Dr n'a pas permis de résoudre la région C-ter, la maille étant suffisamment large pour l'accommoder. Des analyses en RMN du C-ter isolé ont confirmé la présence d'une région majoritairement structurée. D'autres structures ont été résolues en présence de petits substrats (glutamine, analogues d'adénylate) ainsi que la forme tronquée en C-ter. Dans 2 cas, une conformation verrouillée unique du site actif a été mise en évidence. Des analyses structurales et fonctionnelles et les propriétés de l'empilement cristallin sont exposées.

Ma thèse porte sur l'étude fonctionnelle et structurale des partenaires de la réaction d'aminoacylation spécifique de la tyrosine dans la mitochondrie humaine. Contrairement à ce qui a été observé pour les autres systèmes d'aminoacylation, les réactions de charges croisées entre bactéries et archaea/eucaryotes sont impossibles suite à la nature différente de la première paire de bases de l'ARNtTyr. La tyrosyl-ARNt synthétase (TyrRS) mitochondriale humaine est la première TyrRS connue à ce jour qui s'affranchisse de la barrière d'espèces et qui ne discrimine pas les ARNtTyr en fonction de la nature de leur première paire de bases. La TyrRS mitochondriale est un homodimère de forme allongée susceptible de fixer l'ARNtTyr à cheval sur ses deux monomères. Elle se distingue des autres TyrRS par la présence de deux insertions à sa surface, l'une potentiellement impliquée dans la reconnaissance de l'ARNtTyr et l'autre qui pourrait constituer une zone d'interaction avec un cofacteur.

Accurate translation of genetic information necessitates the tuned expression of a large group of genes. Amongst them, controlled expression of the enzymes catalyzing the aminoacylation of tRNAs, the aminoacyl-tRNA synthetases (aaRS), is essential to insure translational fidelity. Here, it is shown that expression of AspRS is regulated in Saccharomyces cerevisiae by a feedback mechanism, that necessitates the binding of AspRS to its messenger RNA. The correlation between AspRS expression and mRNAAspRS and tRNAAsp concentrations, as well as the presence of AspRS in the nucleus, suggest an original regulation mechanism. It is proposed that the surplus of AspRS, not sequestered by tRNAAsp, is imported in the nucleus where it binds to mRNAAspRS and thus inhibits its accumulation. <br />We have established the folding of the 300-nucleotides long 5' end of mRNAApRS and identified the structural signals involved in the regulation process. We propose that the mRNAAspRS fragment folds in two independent and symmetrically structured domains spaced by two single-stranded connectors. Domain I displays a tRNAAsp anticodon-like stem-loop structure that is restricted in domain II to a short double-stranded helix. The overall mRNA structure, based on enzymatic and chemical probing, support a model where each monomer of yeast AspRS binds one individual domain and recognizes the mRNA structure like it recognizes its cognate tRNAAsp.<br />Finally, the consequences of an increased concentration of AspRS in the cell have been tested. In vitro, high AspRS concentrations lead to mis-aspartylation of tRNAAsn and tRNAGlu. In vivo, the design of a reporter gene conferring an antibiotic resistance, dependent on mischarged tRNAs, did not allow to detect any cross aminoacylation. However, the proteomic analysis of yeasts overexpressing AspRS pointed out the conditions of AspRS accumulation in the cell by detecting the presence of an additional control mechanism at the post-translational level.

During protein synthesis, elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to the A-site of mRNA-programmed ribosomes in a GTP-dependent manner. To enable future studies on the functional and structural requirement of EF-Tu’s function, a Cysteine-free variant of EF-Tu was constructed suitable for subsequent labelling of the protein and use in kinetic studies. Here, the kinetic properties of three Cysteine-less EF-Tu variants are reported, demonstrating that only the variant with the Alanine substitution in position 81 retains wild-type activity with respect to the interaction with guanine nucleotides, aa-tRNA and the ribosome. To explore a possible tRNA independent pathway for the GTPase activation signal, three residues in domain II of EF-Tu (Arginine 279, Glycine 280, Arginine 283) were mutated; the activity of EF-Tu variants were analyzed. Results suggest that these residues are indeed required for efficient ribosome-dependent stimulation of the GTPase activity of EF-Tu. / x, 85 leaves : ill. (some col.) ; 29 cm