Les approches bioinformatiques développées au cours de cette thèse ont permis d’une part le développement de banques de données concernant les ARNt classiques ainsi que les ARNt mitochondriaux de métazoaires. Celles-ci sont basées sur de nouveaux outils pour la détection de gènes d’ARNt «bizarres» et des alignements de séquences basés sur les propriétés structurales préservées. Les analyses des séquences collectées ont conduit non seulement à une vision globale de la diversité des ARNt dans les génomes mitochondriaux couvrant l’ensemble des groupes taxonomiques des métazoaires, mais également une meilleure connaissance de l’organisation des génomes et d’en proposer des liens évolutifs. Elles ont également permis de confirmer et d’élargir l’existence d’ARNt les plus petits connus à ce jour et de poser les bases de compréhension des repliements tridimensionnaux des ARNt mitochondriaux. Ces travaux permettent de mieux appréhender la compréhension des relations structure/fonction des ARNt mitochondriaux humains, et en particulier les dysfonctionnements dans les pathologies mitochondriales. / The bioinformatic approaches presented in this thesis include the development of databases for classical tRNAs and the mitochondrial tRNAs of metazoans. They are based on new tools for the detection of "bizarre" tRNA genes and sequences, and for the calculation of alignments based on their structural features. The analysis of collected sequences have led to an global overview on the diversity of tRNAs in mitochondrial genomes covering all taxonomic groups of metazoans, but also to a better understanding of genome organization and their evolution. The present study revealed the existence of the smallest known tRNA so far and provides the basis for understanding the three-dimensional folding of mitochondrial tRNA. This work helps to better understand the structure/function relationships of human mitochondrial tRNAs and, in particular, the dysfunctions in mitochondrial pathologies.

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.

Parmi les petits ARN non codants, les fragments dérivant d’ARNt (tRF) ont été identifiés dans tous les embranchements de la vie. Cependant, très peu de donnée existe sur les tRF de plantes. Les populations de tRF issues de plusieurs banques de petits ARN (différents tissus, plantes soumises à des stress abiotiques, ou fractions immunoprécipitées avec la protéine ARGONAUTE1) ont été analysées. Les populations sont essentiellement constituées de tRF-5D ou des tRF-3T (clivage dans la boucle D ou T respectivement) et elles varient d’une banque à l’autre. Par une approche in silico suivie de tests de clivage in vitro, des RNases T2 d’A. thaliana (RNS) ont été identifiées comme étant capables de cliver les ARNt dans la région de l’anticodon, de la boucle D et de la boucle T. Lors de l’étude de l’expression des RNS, nous avons observé que deux d’entre elles sont fortement exprimées à un stade de maturation tardif des siliques. Ainsi, la population en tRF issue de stades de développement avancés des siliques a été analysée. Des expériences de carences en phosphate nous ont permis de démontrer l’implication d’une des RNS dans la genèse de tRF dans A. thaliana. Au final, nos données ouvrent de nouvelles perspectives quant à l’implication des RNS et des tRF comme des acteurs majeurs dans l’expression des gènes chez les plantes. / Among the small ncRNAs, tRNA-derived RNA fragments (tRFs) were identified in all domains of life. However, only few data report on plants tRFs. Short tRF were retrieved from A. thaliana small RNA libraries (various tissues, plants submitted to abiotic stress or argonaute immunoprecipitated fractions). Mainly tRF-5D or tRF-3T (cleavage in the D or T region respectively) were found, and fluctuations in the tRF population were observed.Using in vitro approaches, A. thaliana RNase T2 endoribonucleases (RNS) were shown to cleave tRNAs in the anticodon region but also in the D or T region. Through a whole study of RNS expression, we show that two RNS are also strongly expressed in the siliques at a late stage of development. Thus, we analyzed the tRF population of this particular developmental stage. Upon phosphate starvation, we demonstrate also the implication of one RNS in the production of tRFs in planta. Altogether, our data open new perspectives for RNS and tRFs as major actors of gene expression inplants.

The studies reported in this thesis describe the work done in the area of translation initiation where a previously unknown role of multiple copies of initiator tRNA in E. coli has been reported. Also the role of SsrA resume codon in resumption of translation, until not clearly known has been reported here. Chapter -1 discusses the relevant literature in understanding translation and initiator tRNA selection on the ribosome during initiation. It also discusses the literature pertaining to the aspect of release of stalled ribosomal complexes by SsrA. This is followed by the next chapter (chapter- 2) which discusses the materials and methods used throughout the study. Chapter- 3 describes the studies leading to the role of multiple copies of initiator tRNA in E. coli in governing the fidelity of initiator tRNA selection on the P site of the ribosome. This is followed by Chapter-4 which describes the role of the resume codon of the SsrA in governing the efficiency of trans-translation in releasing the stalled ribosomal complexes. The summaries of the chapters 3 and chapter 4 are briefly described below. i) Role of conserved 3GC base pairs of initiator tRNA in the initiator-elongator tRNA discrimination. Translation initiation is the first step in the very important and highly conserved biological process of protein biosynthesis. The process involves many steps, a wide array of protein factors at each specialized step and a large ribonucleoprotein particle; the ribosome to decode the information of the mRNA template into biologically active proteins. The process of initiation is still unclear largely due to fewer reports of available structural data. One of the very interesting questions that people have been trying to address is how the initiator tRNA is selected on the P- site of the ribosome and what is the importance of the conserved three GC base pairs in the anticodon stem of the initiator tRNA. Here in this study, I have studied this question by using the classical genetic technique of generating and characterizing the mutant initiator tRNA defective at the step of initiation. I have identified and analyzed the suppressors which are capable of rescuing this defect in initiation. The study involves two such E. coli suppressor strains (named D4 and D27). These suppressors can initiate translation from a reporter CAT mRNA with amber codon, independent of the presence of the three consecutive GC base pairs in the anticodon stem of initiator tRNAs. Mapping of the mutations revealed that the mutants are defective in expression of the tRNA1fMet (metZVW) gene locus which encodes the initiator tRNA. Both the suppressors (D4 and D27) also allow initiation with elongator tRNA species in E. coli. Taken together, the results show that E. coli when deficient in the initiator tRNA concentration can lead to initiation with elongator tRNA species. ii) The Role of SsrA/tmRNA in ribosome recycling and rescue. Occasionally during the process of translation, the ribosomes stall on the mRNA before the polypeptide synthesis is complete. This situation is detrimental to the organism because of the sequestration of the tRNAs as ‘peptidyl tRNAs’ and the ribosomes. In E. coli one of the pathways to rescue stalled ribosomes involves disassembly of these stalled complexes to release peptidyl tRNAs which are then recycled by peptidyl tRNA hydrolase (Pth), an essiential enzyme in E. coli. The other pathway which is not essential in E. coli but is conserved in all prokaryotes involves SsrA or tmRNA (transfer messenger RNA). The tmRNA is charged with alanine and recognizes the stalled ribosomal complexes and acts as tRNA to bind the A-site. It also functions as mRNA by adding a undecapeptide (which is actually a tag for degradation by cellular proteases) to the existing polypeptide and there is normal resumption of the translation. In most sequences of SsrA ORF, the first codon of the ORF, called as resume codon, is conserved. I wanted to understand the importance of the conservation of the resume codon. Towards this end I randomly mutated the resume codon and studied the effect of the altered resume codon in the rescue of stalled ribosomal complexes. The effect of over-expression of these mutants was investigated in the rescue of the Pthts defect since it is known that the overexpression of SsrA rescues the temperature sensitive phenotype of the Pthts strain and so causes less accumulation of peptidyl–tRNA in E. coli .The effect for these mutants has also been studied by the growth of hybrid λimmP22 phages. I also used AGA minigene system to study the effect of various mutants which has been shown to sequester tRNAArg (UCU) in the ribosomal P-site, translation of this minigene causes toxicity to E. coli. I have tried to study the effect of the SsrA mutants in rescue of toxicity caused by the minigene. Overall, the observations indicate that the conservation of the resume codon is important in E. coli and having mutated resume codon probably leads to deficient trans-translation during one or the other growth conditions.

Bacteriophages (hereafter referred as phages) can translate their mRNAs efficiently by maximizing the use of codons decoded by the most abundant tRNAs of their bacterial hosts. Translation efficiency directly influences phage fitness and evolution. Reengineered phages find application in controlling their host population in both health and industry. The objective of this thesis work is to examine the factors shaping codon choices of single stranded DNA (ssDNA) and double stranded DNA (dsDNA) Escherichia coli phages. In chapter two, we employed two indices, rRSCU (correlation in relative synonymous codon usage between phages and their hosts) and CAI (codon adaptation index) to measure codon adaptation in phages. None of the analyzed ssDNA phages encode tRNAs while some dsDNA phages encode their own tRNAs. Both rRSCU and CAI are negatively correlated with number of tRNA genes encoded by these dsDNA phages. We observed significantly greater rRSCU for dsDNA phages (without tRNAs) than ssDNA phages. In addition, we propose that ssDNA phages have evolved a novel codon adaptation strategy to overcome the disruptive effect of their high C→T mutation rates in codon adaptation with host. In chapter three, we formulated an index phi to measure selection by host translation machinery and to present explicit linear and nonlinear models to characterize the effect of C→T mutation and host-tRNA-mediated selection on phage codon usage. The effect of selection (phi) on codon usage is detectable in most dsDNA and ssDNA phage species. C→T mutations also interfere with nonsynonymous substitutions at second codon positions, especially in ssDNA phages. Strand asymmetry along with the accompanying local variation in mutation bias can significantly affect codon adaptation in both dsDNA and ssDNA phages.

Covalent nucleotide modifications in RNAs affect numerous biological processes, and novel functions are continually being revealed even for well-known modifications. Among all RNA species, transfer RNAs (tRNAs) are highly enriched with diverse modifications, which are known to play roles in decoding and tRNA stability, charging, and cellular trafficking. However, studies of tRNA modifications have been limited in a small scale and performed by groups with different methodologies. To systematically compare the functions of a large set of noncoding RNA modifications in translational regulation, I carried out ribosome profiling in 57 budding yeast mutants lacking nonessential genes involved in tRNA modifications. Deletion mutants with enzymes known to modify the anticodon loop or non-tRNA substrates such as rRNA exhibited the most dramatic translational perturbations, including altered dwell time of ribosomes on relevant codons, and altered ribosome density in protein-coding regions or untranslated regions of specific genes. Several mutants that result in loss of tRNA modifications in locations away from the anticodon loop also exhibited altered dwell time of ribosomes on relevant codons. Translational upregulation of the nutrient-responsive transcription factor Gcn4 was observed in roughly half of the mutants, consistent with the previous studies of Gcn4 in response to numerous tRNA perturbations. This work also discovered unexpected roles for tRNA modifying enzymes in rRNA 2’-O-methylation, and in transcriptional regulation of TY retroelements. Taken together, this work revealed the importance and novel functions of tRNA modifications, and provides a rich resource for discovery of additional links between tRNA modifications and gene regulation.

No description available.

In prokaryotes, protein synthesis and folding are often coupled, and the protein begins to fold from the N-terminus as it is being synthesized. It has been hypothesised that there could be kinetic coupling of the speed of translation and the folding, which means that an altered rate of synthesis can cause a possible misfolding of the protein. Testing this hypothesis will be impactful for protein misfolding diseases such as Alzheimer’s, Parkinson’s, Huntington’s etc., and also help in the study of the effect of synonymous, non-synonymous and rare codon changes on a protein. However, research works in this regard are far and few and none of them have been carried out in a homologous in vitro system. This project is an attempt to study the co-translational folding of Escherichia coli protein dihydro folate reductase (DHFR) using an E. coli reconstituted transcription/ translation system (RTTF) in vitro. The preparatory phase involves: preparation of UAG mutants of the DHFR DNA (for site-specific incorporation of fluorescent dyes), preparation of amber tRNAs which recognise the UAG codons, aminoacylation of the tRNAs and labelling the amino acids with fluorescent dyes. The experimental phase involves: incorporation of each of the fluorescent amino acids in the protein during in vitro synthesis in steady-state, observing incorporation of the same in stopped-flow spectrofluorimeter, attempting to observe fluorescent resonance energy transfer (FRET) between the two dyes due to co-translational folding. The preparatory and experimental phases were completed successfully, and it has been established that the amino acids with the fluorescent moieties can be incorporated site specifically in the mutant protein. The synthesis of the protein was observed using stopped-flow spectrometer for each of the fluorescent amino acids individually.  The synthesis of the mutants using two sets of dye pairs was also observed using a steady-state fluorimeter as well as stopped-flow spectrofluorimeter and the FRET between the two fluorophores was obtained. Although further experiments are required to fully validate and standardize this technique,  it will, even now,  aid in the study of the folding of proteins in a cell-free system.

Les ribozymes sont des ARN naturels ou artificiels possédant une activité catalytique. Les ribozymes artificiels ont été identifiés in vitro par la méthode SELEX, et plusieurs d'entre eux ont été caractérisés par des études cinétiques. Ces molécules sont impliquées dans des réactions de clivage, de ligation, de modification d'extrémités d'ARN, de polymérisation, de phosphorylation et d'activation de groupements acyl. Parce qu'elle est nécessaire à la traduction, l'aminoacylation des ARN joue un rôle évolutif important dans la transition du monde de l'ARN vers le monde moderne de l'ADN et des protéines, et elle est centrale à l'établissement du code génétique. Plusieurs ribozymes catalysant le transfert d'acides aminés à partir de cofacteurs activants ont pu être isolés et caractérisés depuis une vingtaine d'années, ce qui a documenté la possibilité d'aminoacylation d'ARNt en l'absence des aminoacyl ARNt synthétases. En développant un nouveau protocole SELEX basé sur l'oxydation au périodate, le but de notre travail est de découvrir de nouveau ribozymes d'une taille de l'ordre d'une vingtaine de nucléotides pouvant combiner la catalyse de l'activation des acides aminé et la transestérification. Bien que des molécules catalysant l'une ou l'autre des deux réactions ont été identifiées, aucun ribozyme n'existe à ce jour qui puisse utiliser des acides aminés libres et un cofacteur activant pour réaliser l'aminoacylation en 3' dans un même milieu réactionnel. La sélection de molécules actives dans une approche SELEX exige la présence de régions constantes sur les deux extrémités des séquences pools aléatoires initiaux. Ces régions sont nécessaires pour l'amplification par PCR, mais elles imposent des contraintes importantes pour l'identification de ribozymes car elles peuvent complètement inhiber leur activité par interférence structurelle. Nous présentons un protocole optimisé qui minimise la taille de ces régions constantes. D'autre part, notre nouveau design est très spécifique pour la sélection d'ARN aminoacylés sur l'extrémité 3'. Ce protocole a été utilisé pour réaliser 6 à 7 cycles de sélection avec différents pools, et un enrichissement en séquences spécifiques a pu être mis en évidence. Bien que certains tests avec les pools sélectionnés a révélé une activité possible, des essais avec des séquences spécifiques de ces pools n'ont pour l'instant pas pu confirmer l'activité catalytique recherchée. Un protocole basé sur le même principe de sélection a été utilisé dans une étude parallèle pour identifier les ARN aminoacylés présents dans l'ARN total d'Escherichia coli. Dans ce deuxième travail, note but est d'identifier tous les d'ARN aminoacylés par séquençage massif, avec à la clé la découverte possible de molécules autres que les ARNt et ARNtm. En utilisant les ARNt comme modèle, nous nous sommes aperçus qu'un protocole RNAseq standard n'était pas adapté à cause des bases modifiées présentes sur ces molécules. Nous avons développé et mis au point un nouveau protocole pour l'identification de n'importe quelle séquence aminoacylée en 3'. La nouvelle approche présentée devrait permette l'étude exhaustive de l'aminoacylation de toutes les séquences présentes dans l'ARN total. / Ribozymes are natural or in vitro selected RNA molecules possessing a catalytic activity. Artificial ribozymes have been extensively investigated by in vitro SELEX experiments, and characterized by kinetic assays. Ribozymes are involved in RNA cleavage, ligation, capping, polymerization, phosphorylation and acyl activation. Because it is required for translation, RNA aminoacylation plays an important role in the evolution from the late RNA world to the modern DNA and protein world, and is central to the genetic code. Several ribozymes catalyzing amino acid transfer from various activating groups have already been selected and characterized in the past two decades, documenting the possibility of tRNA aminoacylation in the absence of aminoacyl tRNA synthetase. With a newly designed SELEX protocol based on periodate oxydation, the aim of our investigation is to uncover small ribozymes of the order of 20 nucleotides that could catalyze both amino acid activation and transesterification. Although molecules catalyzing either reaction have been identified, no existing ribozyme could use free amino acids and activating cofactor(s) as substrates for 3' esterification in a single reactional context. The selection of active molecules in a SELEX procedure requires the presence of constant tracks on both ends of the sequences constituting the initial random pools. These tracks are required for PCR amplification, but they impose significant burden to the identification of ribozymes because they can prevent any activity through structural inhibition. We present an optimized protocol that significantly minimizes the size of these constant tracks. At the same time, our newly design protocol is very specific for the selection of 3'-end aminoacylated RNA. Working with this protocol, we performed 6 to 7 cycles of selection with different pools, and observed an enrichement with specific sequences. Although some experiments performed with entire pools did reveal a possible activity, no activity could be so far confirmed with specific sequences. A similar protocol was also applied in a parallel study to identify aminoacylated RNA from total RNA in Escherichia coli. In this other approach, our goal is to possibly identify new classes of aminoacylated RNA while using the deep sequencing technology. Using tRNA to validate our protocol, we realized that a standard RNAseq procedure could not work due to the presence of modified bases. We established a new method for bank preparation to identify any sequence aminoacylated at the 3' end. Ultimately, this new approach will allow us to study the level of aminoacylation of any sequence present in total RNA.

Mitochondria are organelles present in most eukaryotic cells. They generate most of the cells adenosine triphosphate (ATP) supply which make them essential for cell viability. It is assumed that they are derived from a proteobacterial ancestor as they retain their own, drastically small genome. The importance in studying mitochondrial genome evolution came from the discovery of a large number of human diseases that are caused by mitochondrial dysfunction (e.g., Parkinson and Alzheimer). Many of these diseases are a result of a mutation in one of the mitochondrial genes or a defective mitochondrial DNA (mtDNA) maintenance, mostly caused by genetic defects in proteins involved in mtDNA replication. In order to explore the diversity and understand the evolution of mitochondrial genomes (mitogenomes) in animals, multiple methods have been developed in this study to deal with two biological problems related to the mitochondrial genome evolution. A new method for identifying the mitochondrial origins of replication is presented. This method deals with the problem of determining the origins of replication, which despite many previous efforts has remained non-trivial even in the small genomes of animal mitochondria. The replication mechanism is of central interest to understand the evolution of mitochondrial genomes since it allows the duplication of the genetic information. The extensive work that has been done to study the replication of mitochondrial genomes has generated the assumption of the strand displacement model (SDM) also known as the standard model of replication that is known to leave the mitochondrial H-strand in a single stranded state exposing it to mutation and damage. Later on, other models of replication have been suggested such as the strand coupled bidirectional replication model, its refinement which assumes the bidirectional mode but with a unidirectional start, and the \"RNA incorporation throughout the lagging strand\" (RITOLS) model proposed as a refinement of the strand displacement model. Based on the observation that the GC-skew is correlated with the distance from the replication origins in the light of the strand displacement model of replication, a new computational method to infer the position of both the heavy strand and the light strand origins from nucleotide skew data has been developed. The method has been applied in a comprehensive survey of deuterostome mitochondria where conserved positions of the replication origins for the vast majority of vertebrates and cephalochordates have been inferred. Deviations from the consensus picture are presumably associated with genome rearrangements. Additionally, two methods for the identification of tRNA remolding events throughout Metazoa have been developed. Remolding changes the identity of a tRNA by a duplication and a point mutation(s) of the anticodon. This new tRNA takes the identity of another tRNA which is then lost. This can lead to artifacts in the annotation of mitogenomes and thus in studies of mitogenomic evolution. In this work, novel methods are developed to detect tRNA remolding in large-scale data sets. The first method represents an extension of the similarity-based approach to determine remolding candidates with high confidence. This approach uses an extended set of criteria based on both sequence and structural similarities of the tRNAs in conjunction with statistical tests. The second method is a novel phylogeny-based likelihood method which evaluates specific topologies of gene phylogenies of the two tRNA families relevant to a putative remolding event. Both methods have been applied to survey tRNA remolding throughout animal evolution. At least three novel remolding events are identified in addition to the ones previously mentioned in the literature. A detailed analysis of these remoldings showed that many of them are derived ancestral events.