La majorité des Légumineuses sont capables d’établir une symbiose avec des bactéries du sol nommées Rhizobia. Lors de cette interaction symbiotique, un nouvel organe est formé, la nodosité. Dans cet organe, les bactéries fixent l’azote atmosphérique au profit de la plante hôte. Pendant la symbiose Rhizobia-Légumineuse, deux grands changements transcriptômiques ont été observés par différentes technologies, comme le RNASeq (Maunoury et al., 2010) ou les expériences de microarrays (Benedito et al., 2008). Ces grands changements interviennent aux différentes étapes de développements des nodosités et sont médiés par différents régulateurs de l’expression génique comme certains FTs clés et des petits ARN. Ces petits ARN régulateurs sont produits après le clivage de précurseurs de long ARN double brin ou d’ARN en épingle à cheveux par des enzymes particulières de la famille des ribonucléases de type III (RNase III), nommées DICER-LIKE (DCL). De plus, des gènes codant des RNases III additionnelles sont présents dans le génome de plantes et leurs rôles restent encore à être déterminés.Dans cette étude, nous avons caractérisés la famille des RNases III chez Medicago truncatula mais aussi chez d’autres espèces de légumineuse. Nous avons également recherchés l’implication de MtRTL1b, une RNase III, lors du développement des nodosités.Cette RNase III est un orthologue spécifique des nodosités d’AtRTL1, un répresseur de silencing chez Arabidopsis thaliana. Tout d'abord, nous avons montré que l’expression de ce gène est activée juste avant la différenciation et est principalement restreinte à l’interzone, là où les bactéroïdes deviennent totalement différenciés dans les cellules hôtes, et dans la zone de fixation de la nodosité. La répression de l’expression de MtRTL1b, par ARN interférence dans des racines transgéniques, affecte le développement de la nodosité, la fixation de l’azote et la viabilité des bactéroïdes. Un phénotype opposé est observé lorsque MtRTL1b est exprimé de façon ectopique dans la racine. Les analyses des données de séquençage nous ont permis de mettre en évidence que le RNAi conduit à la sous-expression de 1038 gènes, incluant plus de 109 gènes codant des NCRs qui sont des peptides intervenant dans le développement des bactéroïdes et/ou pour leur viabilité dans les nodosités indéterminées. De plus,des gènes impliqués dans les voies métaboliques et la régulation de l’état d'oxydo-réduction mais aussi dans le processus symbiotique, comme la leghémoglobine, sont également sous-exprimés. Des données de séquençage de petits ARN et d’ARN double brins sont en cours d’analyse afin de caractériser les changements dans les populations de petit ARN et identifier les substrats ARN double brin de cette RNase III lors du développement des nodosités. / Almost all Legumes are able to establish symbiosis with soil bacteria called Rhizobia. During this interaction, a new organ is formed, the nodule. In this organ, bacteria fix the atmospheric nitrogen for the host plant. During Rhizobia-Legumes symbiosis twotranscriptomic changes were observed by different technologies like RNAseq (Maunoury et al., 2010) or microarrays experiment (Benedito et al., 2008). These dramatic changes occur at the different steps of nodule development and are mediated by various gene expression regulators including several keys transcription factors and small RNAs. These small regulatory RNAs are produced after cleavage of long double-stranded or hairpin RNA precursors by particular enzymes of the ribonuclease III (RNase III) family, called DICERLIKEproteins (DCL). However, additional RNase III encoding genes are present in plant genomes, whose roles remain to be fully determined.In this work, we characterized the RNAse III family in the model M. truncatula, as well as other legumes species. We also investigated the involvement in nodule development of MtRTL1b, one RNAse III, a nodule-specific orthologue of AtRTL1, a putative silencing repressor in Arabidopsis thaliana. First, we showed that the expression of this gene is activated just before differentiation and is mainly restricted in the interzone, where bacteroid become fully differentiated into the host cells and in the nitrogen fixation zone of the nodule. Repression of MtRTL1b expression, by RNA interference in transgenic roots, affected nodule development, nitrogen fixation and bacteroid viability while an opposite phenotype was observed in roots with ectopic expression of this gene. Then, RNASeq analyses showed that the RNAileads to the down-regulation of 1038 genes, including more than 109 NCRs, encoding peptides involved in bacteroid development and/or viability in indeterminate nodules. Moreover, genes involved in metabolic pathways and redox regulations as well as other genes involved in symbiosis, like leghemoglobins, are also down-regulated. RNAseq of small RNAs and double strand RNAs are under analysis to characterize changes in sRNA populations and identify dsRNA substrates of this RNAse III during nodule development.

RNA editing in trypanosomes is the post-transcriptional insertion or deletion of uridylates at specific sites in mitochondrial mRNAs. This process is catalyzed by a multienzyme, multisubunit complex through a series of enzymatic cycles directed by small, trans-acting RNA molecules. Despite impressive progress in our understanding of the mechanism of RNA editing and the composition of the editing complex, fundamental questions regarding RNP assembly and the regulation of catalysis remain. This dissertation presents studies of RNA-protein interactions between RNA editing complexes and substrate RNAs and the determination of substrate secondary structural determinants that govern them. Our results suggest that substrate association, cleavage and full-round editing by RNA editing complexes in vitro obey hierarchical determinants that increase in complexity as editing progresses and we propose a model for substrate recognition by RNA editing complexes. In addition, this dissertation also presents the characterization of a novel mitochondrial RNA helicase, named REH2 and its macromolecular interactions. Our data suggest that REH2 is intimately involved in interactions with macromolecular complexes that integrate diverse processes mediating mitochondrial gene expression. These results have implications for the mechanism of substrate RNA recognition by RNA editing complexes as well as for the integration of RNA editing to other facets of mitochondrial RNA metabolism.

Le staphylocoque doré (S. aureus) est un pathogène responsable d’infections diverses chez l’homme et l’animal. Il est responsable d’infections suppuratives (furoncles, endocardites, méningites…) ou non suppuratives (toxi-infections alimentaires…). L’émergence de souches bactériennes multi-résistantes aux antibiotiques augmente la gravité des infections et constitue un réel problème de santé publique. Depuis plusieurs années, les chercheurs tentent d’identifier des cibles pour de nouveaux antibiotiques. Parmi elles, les acides ribonucléiques (ARN) dits « régulateurs » constituent un modèle de choix. Environ 200 ARN régulateurs ont été identifiés chez S. aureus, mais jusqu'à maintenant, dans la plupart des cas, leurs fonctions ne sont pas connues. Pour identifier la fonction des ARN régulateurs chez S. aureus, nous avons utilisé une stratégie consistant à mettre en compétition 39 souches différentes dans chacune desquelles un ARN régulateur a été remplacé par une étiquette spécifique identifiable par séquençage. Cette expérience de compétition a été réalisée dans douze conditions de cultures différentes (conditions de stress différents) ainsi que chez un modèle souris. Les résultats de ces expériences ont été obtenus par analyse de séquençage massif haut débit. Un phénotype a été mis en évidence pour 11 des ARN régulateurs étudiés dans les conditions choisies. Nous avons aussi développé une méthode fiable pour identifier les cibles des ARN régulateurs. Cette méthode consistait à utiliser in vitro, ces ARN régulateurs comme appât pour piéger leurs cibles parmi un mélange d’ARN total. Les ARN cibles piégés ont ensuite été séquencés par ARN-seq haut débit. Cette stratégie a été appliquée pour déterminer les cibles de quatre ARN régulateurs chez S. aureus : RsaA, RsaE, RsaH et RNAIII. Plusieurs cibles putatives ont été identifiées et utilisées pour prédire le motif qui serait impliqué dans les interactions entre un ARN régulateur et sa cible. Le dernier chapitre de ce manuscrit concerne l’étude du mécanisme d’action des ARN régulateurs. En général, la liaison de l’ARN régulateur à son ARN messager cible va affecter la stabilité de ce dernier et engendrer sa dégradation par la ribonucléase III (RNase III). Chez S. aureus, RNase III est l’enzyme principalement impliquée dans la dégradation des complexes ARN régulateur-ARN messager (ARN double brin). RNase III a été décrite comme étant non essentielle chez S. aureus. Cependant, nous avons montré que cette ribonucléase est essentielle dans les souches de S. aureus contenant des prophages portant un système toxine/antitoxine (TA) de type I. Dans ces souches, la présence de RNase III est essentielle pour diminuer l’expression de ce système TA et contrer sa toxicité. / Staphylococcus aureus is an opportunistic Gram-positive pathogen bacterium and a leading cause of hospital- and community-acquired infections worldwide. In S. aureus, regulatory RNAs are key mediators in controlling bacterial virulence, viability and adaptability under various environmental conditions. About 200 regulatory RNAs were identified in S. aureus but up to date, their functions in most cases are unknown. To address the function of S. aureus regulatory RNAs, we used a strategy in which competitive fitness experiments were performed with mixed cultures comprising thirty-nine sRNA mutants. A bacterial population made of these mixed mutants was grown in twelve stress conditions and in a mouse model. The results were obtained by deep sequencing analysis. Eleven sRNA gene mutants were found to have an altered fitness in the tested conditions; three of them being requires for solely one studied condition, the others being modified by multiple stress conditions. The absence of sRNA genes generated negative fitness adaptation in all conditions, but one of the mutants had a strong growth defective phenotype at low-temperature. Current investigations indicate that the deleted sequence affects the 3’ end of a mRNA. This sequence is required for an optimum growth in cold conditions and contributes to the stability or translation of the associated mRNA. We also developed a robust procedure to identify reliably sRNA targets based on synthetic sRNAs that were used in vitro as bait to trap their corresponding targets which were subsequently identified by deep sequencing. Using this approach, we found new targets for RsaA, RsaE, RsaH and RNAIII. The binding of sRNAs to targeted mRNAs usually affect their stability by recruiting Ribonucleases (RNases). In S. aureus, RNase III encoded by rnc gene, is a major RNase involved in the degradation of sRNA-mRNA duplexes. RNase III was reported as nonessential in S. aureus. However, we report that the rnc gene is essential in some S. aureus strains due to the presence of prophages carrying type I toxin/antitoxin (TA) system. RNase III in these strains is essential to reduce the expression of TA systems to prevent their toxicity

Les endoribonucléases III sont conservées chez tous les eucaryotes. Ils jouent un rôle important dans le cycle de vie des acides ribonucléiques (ARN) que ce soit au niveau de leur maturation, leur régulation ou leur dégradation. Cependant, seul un petit nombre d'ARN ciblés par les ribonucléases (RNases) III sont connus et les motifs reconnus par l'enzyme sont encore mal caractérisés. Actuellement, la découverte de nouvelles cibles repose principalement sur la validation in vitro de gènes individuels. Il est important d'avoir une vue d'ensemble des cibles des RNases III pour comprendre le rôle de la dégradation spécifique des ARN dans le métabolisme cellulaire. Ainsi, cette thèse a comme objectif de développer des approches haut débit pour permettre une identification plus rapide des cibles tout en minimisant l'utilisation des ressources expérimentales. Elle présente l'utilisation combinée d'approches bio-informatiques, d'étude génétique de l'expression et de traitement in vitro dans le but d'avoir un portrait global des cibles de l'endoribonucléase III. Elle a aussi comme but d'identifier les motifs d'ARN non codants qui guident la reconnaissance par l'endoribonucléase III. Les principaux accomplissements de cette recherche sont : le développement de nouveaux algorithmes de prédiction des cibles de la RNase III, la détection de deux nouvelles classes de transcrits dont l'expression est dépendante de la RNase III, l'identification de quelques centaines de nouvelles cibles de la RNase III, la production d'un catalogue plus complet des motifs coupés par la RNase III et l'identification de nouvelles catégories de motifs coupées par la RNase III. Le tout procure un portrait global de l'impact de la RNase III sur le transcriptome et le cycle de vie des ARN. De plus, ce travail montre comment une approche intégrée incluant la recherche in silico, in vivo et in vitro permet de mieux comprendre le rôle d'un enzyme dans la cellule et comment chaque approche peut pallier les déficiences des autres approches et fournir globalement des résultats plus complets.

Gene regulation allows cells to control their metabolism, growth, division and adaptation to environmental changes. Every step of the genetic information transmission chain is regulated. One of the targets of gene regulation is the mRNA that acts as an intermediate between DNA and proteins. Regulation of the mRNA can happen at the level of its synthesis, processing and degradation. The RNase III of Saccharomyces cerevisiae , Rnt1p, is a nuclear enzyme implicated in the processing of many non-coding RNAs. Rnt1p binds and cleaves double-strand RNA structures capped by NGNN tetra-loops. Such structures can also be found in many mRNAs. Cleavage within the coding sequence of a mRNA results in its degradation and thus contributes to negatively regulate the expression of its gene. The work presented in this thesis characterized the effects of the loss of Rnt1p in yeast. A first publication highlighted the relationship between the localization of this enzyme and cell cycle progression. A change in localization of Rnt1p from the nucleolus to the nucleoplasm contributes to passage through G2/M. A second publication focused on the interaction between Rnt1p and the rRNA transcription machinery. Rnt1p was found to interact with RNA pol I and to be implicated in its transcription termination. We also used Rnt1p as a tool to uncover mRNAs that are posttranscriptionaly regulated in the nucleus. The case studied in this work shows a role for this nuclear degradation mechanism towards promoting the robustness of the cell wall stress response.

<p>The Escherichia coli small RNA (sRNA) MicA was identified recently in a genomewide search for sRNAs. It is encoded between the genes <i>gshA</i> and <i>luxS</i> in E. coli and its close relatives. The function of sRNAs in bacteria is generally believed to be in maintenance of homeostasis via stress-induced modulation of gene expression. Our studies on MicA have been aimed at attributing function(s) to this molecule.</p><p>We carried out high throughput assays aimed at identifying genes that are differentially regulated upon knocking out or overexpressing MicA. Among the protein candidates identified was the outer membrane protein, OmpA. Subsequent analysis allowed us to show this regulation to be antisense in nature with MicA binding within the translation initiation region of <i>ompA</i> mRNA. Furthermore, blocking the ribosome from loading caused a translational decoupling that instigates degradation of the mRNA. The regulation was apparent in early stationary phase and seen to be dependent on the RNA chaperone Hfq. </p><p>We went on to characterize the regulation of MicA, looking at its own transcription. Testing various stress conditions, we were able to identify putative promoter elements that we confirmed using transcriptional fusions. The results showed MicA to be dependent on the extracytoplasmic function ECF sigma E (σ^E) and could not detect MicA in mutants deleted for this factor.</p><p>Lastly, we identified an additional target for MicA being the adjacently encoded <i>luxS</i> mRNA. The LuxS protein is essential for the synthesis of the quorum sensing AI-2 molecule. Transcription of the <i>luxS </i>mRNA is commences within the <i>gshA</i> gene, on the other side of MicA coding region. We were able to show that MicA interacts with <i>luxS </i>mRNA and is recognized by RNase III which processes this complex leading to a shorter <i>luxS</i> mRNA isoform. The significance of this processing event is as yet undetermined. Our data elucidated a new promoter driving transcription of <i>luxS,</i> and we demonstrated this promoter to be stationary phase responsive.</p><p>In summary, the work presented here characterizes the sRNA MicA as a dual regulatory sRNA molecule, moonlighting between its cis-encoded target and its trans-encoded target. .</p>

The Escherichia coli small RNA (sRNA) MicA was identified recently in a genomewide search for sRNAs. It is encoded between the genes gshA and luxS in E. coli and its close relatives. The function of sRNAs in bacteria is generally believed to be in maintenance of homeostasis via stress-induced modulation of gene expression. Our studies on MicA have been aimed at attributing function(s) to this molecule. We carried out high throughput assays aimed at identifying genes that are differentially regulated upon knocking out or overexpressing MicA. Among the protein candidates identified was the outer membrane protein, OmpA. Subsequent analysis allowed us to show this regulation to be antisense in nature with MicA binding within the translation initiation region of ompA mRNA. Furthermore, blocking the ribosome from loading caused a translational decoupling that instigates degradation of the mRNA. The regulation was apparent in early stationary phase and seen to be dependent on the RNA chaperone Hfq. We went on to characterize the regulation of MicA, looking at its own transcription. Testing various stress conditions, we were able to identify putative promoter elements that we confirmed using transcriptional fusions. The results showed MicA to be dependent on the extracytoplasmic function ECF sigma E (σE) and could not detect MicA in mutants deleted for this factor. Lastly, we identified an additional target for MicA being the adjacently encoded luxS mRNA. The LuxS protein is essential for the synthesis of the quorum sensing AI-2 molecule. Transcription of the luxS mRNA is commences within the gshA gene, on the other side of MicA coding region. We were able to show that MicA interacts with luxS mRNA and is recognized by RNase III which processes this complex leading to a shorter luxS mRNA isoform. The significance of this processing event is as yet undetermined. Our data elucidated a new promoter driving transcription of luxS, and we demonstrated this promoter to be stationary phase responsive. In summary, the work presented here characterizes the sRNA MicA as a dual regulatory sRNA molecule, moonlighting between its cis-encoded target and its trans-encoded target. .

The site-specific cleavage of double-stranded (ds) RNA is a conserved early step in bacterial ribosomal RNA (rRNA) maturation that is carried out by ribonuclease III. Studies on the RNase III mechanism of dsRNA cleavage have focused mainly on the enzymes from mesophiles such as Escherichia coli. In contrast, little is known of the RNA processing pathways and the functions of associated ribonucleases in the hyperthermophiles. Therefore, structural and biochemical studies of proteins from hyperthermophilic bacteria are providing essential insight on the sources of biomolecular thermostability, and how enzymes function at high temperatures. The biochemical behavior of RNase III of the hyperthermophilic bacterium Thermotoga maritima is analyzed using purified recombinant enzyme and the cognate pre-ribosomal RNAs as substrates. The T. maritima genome encodes a ~5,000 nucleotide (nt) transcript, expressed from the single ribosomal RNA (rRNA) operon. RNase III processing sites are expected to form through base-pairing of complementary sequences that flank the 16S and 23S rRNAs. The Thermotoga pre-16S and pre-23S processing stems are synthesized in the form of small hairpins, and are efficiently and site-specifically cleaved by Tm-RNase III at sites consistent with an in vivo role of the enzyme in producing the immediate precursors to the mature rRNAs. T. maritima (Tm)-RNase III activity is dependent upon divalent metal ion, with Mg^2+ as the preferred species, at concentrations &gt;= 1 mM. Mn^2+, Co^2+ and Ni^2+ also support activity, but with reduced efficiency. The enzyme activity is also supported by salt (Na^+, K^+, or NH4^+) in the 50-80 mM range, with an optimal pH of ~8. Catalytic activity exhibits a broad temperature maximum of ~40-70 deg C, with significant activity retained at 95 deg C. Comparison of the Charged-versus-Polar (C-vP) bias of the protein side chains indicates that Tm-RNase III thermostability is due to large C-vP bias. Analysis of pre-23S substrate variants reveals a dependence of reactivity on the base-pair (bp) sequence in the proximal box (pb), a site of protein contact that functions as a positive determinant of recognition of E. coli (Ec)-RNase III substrates. The pb sequence dependence of reactivity is similar to that observed with the Ec-RNase III pb. Moreover, Tm-RNase III cleaves an Ec-RNase III substrate with identical specificity, and is inhibited by pb antideterminants that also inhibit Ec-Rnase III. These studies reveal the conservation acrosss a broad phylogenetic distance of substrate reactivity epitopes, both the positive and negative determinants, among bacterial RNase III substrates. / Chemistry

Gene expression pathways exhibit many “twists and turns,” with theoretically numerous ways in which the pathways can be regulated by both negative and positive feedback mechanisms. A key step in gene expression is RNA maturation (RNA processing), which in the bacterial cell can be accomplished through RNA binding and enzymatic cleavages. The well-characterized bacterial protein Ribonuclease III (RNase III), is a conserved, double-stranded(ds)-specific ribonuclease. In the gram-negative bacterium Escherichia coli, RNase III catalytic activity is subject to both positive and negative regulation. A recent study has indicated that an E. coli protein, YmdB, may negatively regulate RNase III catalytic activity. It has been proposed that YmdB inhibition of RNase III may be part of an adaptive, post-transcriptional physiological response to cellular stress. In E. coli, the model organism in this study, YmdB protein is encoded by the single ymdB gene, and has a predicted molecular mass of ~18.8 kDa. YmdB has been classified as a macrodomain protein, as it exhibits a characteristic fold that specifically provides an ADP-ribose (ADPR) binding site. While YmdB can bind ADPR with good affinity, there may be additional ligands for the binding site. Thus, YmdB protein may interact with other components in the cell, which in turn could modulate the interaction of YmdB with RNase III. In previous research conducted within the Nicholson laboratory at Temple University, affinity-purified Escherchia coli(Ec) YmdB and Aquifex aeolicus (Aa) YmdB were found to exhibit ribonucleolytic activity. This observation initiated the long-term goal of learning how YmdB regulates RNase III, and how the ribonucleolytic activity of YmdB may be involved in this process. The specific goal of this thesis project was to further characterize the ribonucleolytic activity of Ec-YmdB through site-specific mutational analysis. Mutations were introduced into a proposed adenine-binding pocket previously identified by crystallography and by molecular modeling. The adenine-binding pocket is a region within the macrodomain fold where ADP-ribose could bind. The mutations were examined for their effect on Ec-YmdB cleavage of a model RNA, R1.1. The results of this study will contribute to the development of a model describing how the ribonucleolytic activity of YmdB is regulated. / Biology

L’expression des gènes est un ensemble hautement régulé de mécanismes ayant pour objectifs de synthétiser les protéines fonctionnelles dont la cellule a besoin à partir des codes inscrits dans l’ADN. Pour contrôler la quantité de signal utilisé et pour que ce message puisse physiquement traverser la cellule, celle-ci utilise la transcription des ARN messagers comme intermédiaire. Pour assurer la qualité de ce signal et pour contrôler son niveau d’expression, plusieurs mécanismes de dégradation des ARNm se coordonnent dans les organismes en fonction de leurs spécificités propres. La littérature a depuis longtemps démontré les liens entre les machineries de synthèse des ARNm et celles de leur dégradation en identifiant comment ceux-ci travaillent ensemble pour assurer une bonne régulation génique. Un de ces mécanismes induit une terminaison dans la région non-codante en 3’ de certains gènes à partir d’un clivage par la ribonucléase III. Dans ce mémoire, nous voulons démontrer qu’un mécanisme similaire dépendant de la ribonucléase III peut induire une terminaison prémature de la transcription à l’intérieur même de séquences codantes. Ce mécanisme semble être indépendant du promoteur et du terminateur des gènes, préférant réguler sa sélectivité à partir de la structure liée au clivage de l’ARNm. Plusieurs séquences et structures sous forme de tige-boucles d’ARNm peuvent être reconnues par la ribonucléase III. Cependant, il existe des différences fonctionnelles entre les différentes tige-boucles et toutes n’induisent pas un mécanisme de terminaison prématurée. Comme ce type de mécanisme doit être inductible et/ou permissible afin de ne pas empêcher complètement l’expression des gènes cibles, nous pouvons potentiellement faire affaire à un nouveau modèle de régulation génique. / Abstract : Gene expression is a highly regulated coordination of processes with the objective of producing functional proteins from the DNA code of the cell. To control the amount of proteins produced, cells use messenger RNAs as an intermediate to permit genomic information to move across the cell. To assure the quality of this signal and to control the level of gene expression, many RNA degradation mechanisms coordinate together according to their own specificities. Scientific literature has demonstrated long ago the existing interactions between RNA synthesis and RNA degradation pathways and how they closely work together to achieve viable gene expression regulation. One of those mechanisms induces transcription termination in the 3’ untranscribed region of coding genes initiated with a RNA cleavage from a ribonuclease III. In this master thesis, we show that a similar RNase III dependent mechanism can induce premature transcription termination inside the coding sequence. This mechanism seems promotor and terminator independent and depends mostly on the sequence coding for a stem-loop structure in the mRNA. Different sequences can induce a stem-loop structure recognizable by RNase III. However, there are some functional differences between stem-loop structures and not all of them can induce premature transcription termination. Since this mechanism must not happen every time and somehow must be inducible to permit gene expression when needed, this could possibly lead to a new gene regulation model.