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.

The activation of the innate immune system is the first line of host defence against infection. Nucleic acids can potently stimulate this response and trigger a series of signalling cascades leading to cytokine production and the establishment of an inflammatory state. Mutations in genes encoding nucleases have been identified in patients with autoimmune diseases, including Aicardi-Goutières syndrome (AGS). This rare childhood inflammatory disorder is characterised by the presence of high levels of the antiviral cytokine interferon-α in the cerebrospinal fluid and blood, which is thought to be produced as a consequence of the activation of the innate immunity by unprocessed self-nucleic acids. This thesis therefore aimed to define the role of one of the AGS nucleases, the Ribonuclease H2 (RNase H2) complex, in innate immunity, and to establish if nucleic acid substrates of this enzyme were able to induce type I interferon production in vitro. The AGS nucleases may function as components of the innate immune response to nucleic acids. Consistent with this hypothesis, RNase H2 was constitutively expressed in immune cells, however, its expression was not upregulated in response to type I interferons. RNase H2-deficient cells responded normally to a range of nucleic acid PAMPs, which implied that a role for RNase H2 as a negative regulator of the immune response was unlikely, in contrast to the reported cellular functions of two other AGS proteins, TREX1 and SAMHD1. Therefore, no clear evidence was found for the direct involvement of RNase H2 in the innate immune response to nucleic acids. An alternative model for the pathogenesis of disease hypothesises that decreased RNase H2 activity within the cell results in an accumulation of RNA:DNA hybrids. To investigate the immunostimulatory potential of such substrates, RNA:DNA hybrids with different physiochemical properties were designed and synthesised. Methods to purify the hybrids from other contaminating nucleic acid species were established and their capacity as activators of the innate immune response tested using a range of in vitro cellular systems. A GU-rich 60 bp RNA:DNA hybrid was shown to be an effective activator of a pro-inflammatory cytokine response exclusively in Flt3-L bone marrow cultures. This response was completely dependent on signalling involving MyD88 and/or Trif, however the specific receptor involved remains to be determined. Reduced cellular RNase H2 activity did not affect the ability of Flt3-L cultures to mount a cytokine response against the RNA:DNA hybrid. These in vitro studies suggested that RNA:DNA hybrids may be a novel nucleic acid PAMP. Taken together, the data in this thesis suggest that the cellular function of RNase H2 is in the suppression of substrate formation rather than as a component of the immune response pathways. Future studies to identify endogenous immunostimulatory RNA:DNA hybrids and the signalling pathways activated by them should provide a detailed understanding of the molecular mechanisms involved in the pathogenesis of AGS and related autoimmune diseases.

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.

L’auto-incompatibilité (AI) est la capacité génétiquement déterminée d’une plante fertile de rejeter son propre pollen. Chez les Solanacées l’AI dépend des éléments d’un locus fort complexe (locus S) multigénique. L’élément du locus-S exprimé dans le pistil est une ribonucléase (S-RNase) dont le rôle est de dégrader l’ARN chez le pollen self, tandis que l’élément du locus S exprimé dans le pollen est un ensemble de protéines du type F-box, qui sont normalement impliquées dans la dégradation des protéines. Cependant, comment les S-RNases self restent actives lors des croisements incompatibles et comment les S-RNases non-self sont inactivées lors des croisements compatibles ce n’est encore pas clair. Un modèle propose que les S-RNases non-self soient dégradées lors des croisements compatibles. Un autre modèle propose que toutes les S-RNases, self et non-self, soient d'abord séquestrées à l’intérieur d’une vacuole, et elles y resteraient lors des croisements compatibles. Lors de croisements incompatibles, par contre, elles seraient relâchées dans le cytoplasme, où elles pourront exercer leur action cytotoxique. Notre étude tente de répondre à ces questions. Notamment, nous cherchons à mettre en évidence la localisation vacuolaire et/ou cytoplasmique des S-RNases et leur concentration par immunolocalisation, en utilisant un anticorps ciblant la S11-RNase de Solanum chacoense et la microcopie électronique à transmission. Nos résultats montrent que la densité de marquage observée pour les S-RNases cytoplasmiques est significativement plus haute dans les tubes incompatibles que dans ceux compatibles ce qui nous indique que pour qu’un tube pollinique soit compatible il doit contenir une faible densité de S-RNase cytoplasmique. / Self-incompatibility (SI) is a widespread genetic device used by flowering plants to reject their own pollen, and thus to avoid inbreeding. This cell-cell recognition mechanism is mediated by molecular interactions between gene products expressed in the pollen and those expressed in specialized cells of the pistil. The genetic determinants of the system are produced from a highly complex multigenic S-locus with multiple S-haplotypes, although other genes outside the S-locus also contribute to the phenomenon in a non-allele specific manner. SI discriminates between self and non-self pollen, as the former will be rejected (incompatible cross), whereas the latter will be allowed to accomplish fertilization (compatible cross). In the Solanaceae (to which Solanum chacoense belongs) the pistillar determinant to SI is an extremely polymorphic stylar extracellular S-RNase, whereas the pollen determinant involves the collaborative action of several members of the F-box family (SLF or S-locus F-box). This has led to the hypothesis that during compatible crosses, ubiquitin-mediated degradation of non-self S-RNases takes place (degradation model). However, it has also been found that non-self S-RNases appear to be sequestered in the vacuole during compatible crosses (sequestration model). The objective of our study was to discriminate between these two models by using immunolocalization techniques and transmission electron microscopy. We have found that the concentration of S-RNases is significantly higher in incompatible pollen tubes than in compatible ones.

The HBV RNAse H has been cloned into the PET43a vector, which contains the NusA protein which works as a solubilizing fusion protein. The fusion NUS-RNAse H protein was cleaved by enterokinase; the cleaved RNAse H is about 17 Kda which remains soluble and active. A fluorescence assay utilizing a quenching mechanism was used to characterize the activity of NUS-RNAse H and cleaved RNAse H proteins. The beacon is a RNA:DNA hybrid oligonucleotide labeled with a 5'DABCYL and a 3'fluorescein, when RNAse H digests the RNA, DABCYL is released resulting in high fluorescence. The digestion of the RNA was also confirmed by gel analysis. The protein was identified by N-terminal amino acid sequence analysis of the fusion protein, SDS-PAGE, western blot utilizing HBV positive sera for primary antibodies, and enzyme immunoassay by peroxidase labeling of HBV RNAse H. Structural analysis of the protein was done by circular dichroism, tryptophan fluorescence, the generation of a model from HIV RNAse H and initial crystals which unfortunately did not diffract. The ability to produce good amounts soluble RNAse H, the development of a sensitive assay to test for activity and the solution of the crystal structure will help develop new anti-viral inhibitors.

<p>Mitotic genome instability can occur during the repair of double-strand breaks (DSBs) in DNA, which arise from endogenous and exogenous sources. Studying the mechanisms of DNA repair in the budding yeast, Saccharomyces cerevisiae has shown that Homologous Recombination (HR) is a vital repair mechanism for DSBs. HR can result in a crossover event, in which the broken molecule reciprocally exchanges information with a homologous repair template. The current model of double-strand break repair (DSBR) also allows for a tract of information to non-reciprocally transfer from the template molecule to the broken molecule. These “gene conversion” events can vary in size and can occur in conjunction with a crossover event or in isolation. The frequency and size of gene conversions in isolation and gene conversions associated with crossing over has been a source of debate due to the variation in systems used to detect gene conversions and the context in which the gene conversions are measured. </p><p>In Chapter 2, I use an unbiased system that measures the frequency and size of gene conversion events, as well as the association of gene conversion events with crossing over between homologs in diploid yeast. We show mitotic gene conversions occur at a rate of 1.3x10-6 per cell division, are either large (median 54.0kb) or small (median 6.4kb), and are associated with crossing over 43% of the time. </p><p>DSBs can arise from endogenous cellular processes such as replication and transcription. Two important RNA/DNA hybrids are involved in replication and transcription: R-loops, which form when an RNA transcript base pairs with the DNA template and displaces the non-template DNA strand, and ribonucleotides embedded into DNA (rNMPs), which arise when replicative polymerase errors insert ribonucleotide instead of deoxyribonucleotide triphosphates. RNaseH1 (encoded by RNH1) and RNaseH2 (whose catalytic subunit is encoded by RNH201) both recognize and degrade the RNA in within R-loops while RNaseH2 alone recognizes, nicks, and initiates removal of rNMPs embedded into DNA. Due to their redundant abilities to act on RNA:DNA hybrids, aberrant removal of rNMPs from DNA has been thought to lead to genome instability in an rnh201Δ background. </p><p> In Chapter 3, I characterize (1) non-selective genome-wide homologous recombination events and (2) crossing over on chromosome IV in mutants defective in RNaseH1, RNaseH2, or RNaseH1 and RNaseH2. Using a mutant DNA polymerase that incorporates 4-fold fewer rNMPs than wild type, I demonstrate that the primary recombinogenic lesion in the RNaseH2-defective genome is not rNMPs, but rather R-loops. This work suggests different in-vivo roles for RNaseH1 and RNaseH2 in resolving R-loops in yeast and is consistent with R-loops, not rNMPs, being the the likely source of pathology in Aicardi-Goutières Syndrome patients defective in RNaseH2.</p> / Dissertation

<p><i>Staphylococcus aureus</i> (<i>S. aureus</i>) is a Gram-positive pathogen that causes a wide range of infections in both hospitals and communities, of which the total mortality rate is higher than AIDS, tuberculosis, and viral hepatitis combined. The drug resistant <i>S. aureus </i>is a member of the “ESKAPE” pathogens that require immediate and sustained actions of novel method to combat. However, the current antimicrobial development against <i>S. aureus</i> is in stagnation, which underscores the urgent need for novel antimicrobial scaffolds and targets. <i>S. aureus</i> Ribonuclease P protein (RnpA) is an essential protein that plays important roles in both tRNA maturation and mRNA degradation pathways. The goal of this research was to evaluate RnpA as an antimicrobial target using biophysical methods. The crystal structures of wild-type RnpA in three different constructs were determined, among which the tag-free RnpA construct has a structural model of 2.0 Å resolution and R_crys/R_free= 0.214/0.234, and its crystals are reproducible. This crystal structure of tag-free <i>S. aureus </i>RnpA shows a globular representation with key structural motifs, including the “RNR” Ribonuclease P RNA binding region and a substrate binding central cleft, which shares high similarity to previously solved RnpA structures from other species despite of their low sequence identity. Meanwhile, in a screen of <i>S. aureus </i>RnpA mutants performed by our collaborator, RnpA^P89A was found lacking the mRNA degradation activity while retaining the tRNA maturation function, and causing defects in cell viability. We therefore studied this mutant using differential scanning fluorimetry, crystallography, and circular dichroism. It was shown that RnpA^P89A is thermally less stable than wild-type RnpA by ~2.0 ˚C, but no secondary structural or 3D conformational differences were found between the two proteins. Although the mutant RnpA^P89A requires further characterization, the results of the studies in this thesis have begun to shed light on the relatively new role of <i>S. aureus </i>RnpA in mRNA degradation, and will serve as useful tools in future structure-based drug discovery for multi-drug resistant <i>S. aureus </i>treatment. </p>

<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>

<p>RNA is the only biological molecule that can function both as a repository of information and as a catalyst. This, together with the ability to self-replicate, led to recognition of RNA as ‘prelude to life’.</p><p>My work highlights some of the important features of RNA as a catalyst, exemplified by RNase P. It addresses questions of evolutionary preservations of residues and structure, involvement of metal ions and finally structure evolution towards minimal catalytically competent RNA motifs.</p><p>RNase P is the only enzyme involved in 5’ end processing of all pre-tRNAs. Until recently, it was believed that the RNA moiety of RNase P is responsible for mediating catalysis only in Bacteria. However, my recent study conclusively demonstrated that eukaryotic RNase P RNA is catalytically competent in vitro in absence of proteins. These findings evidenced evolutionary preservation of RNA-mediated catalysis in RNase P.</p><p>RNase P RNA is a metalloeznyme. In my studies I analyzed the contributions of individual chemical groups at the cleavage site to catalysis. My findings suggested that the 2’OH of N_-1 and the exocyclic amine of G₊₁ are involved in positioning of functionally important metal ions. Additionally, data appointed the function of Pb²⁺ as both structural metal ion and important in generating the nucleophile. My studies further indicate a conformational change upon RNase P RNA -substrate complex formation in keeping with an induced fit mechanism. </p><p>Studying the effects of reducing the ribozyme size upon dissection of bacterial RNase P RNAs, we defined the smallest catalytically competent domain i.e. P15-loop. Derivatives of this autonomous metal ion binding domain, (the smallest being 31nt-s), are able to cleave both whole-length pre-tRNAs as well as hairpin substrates, though with severely reduced rates relative to their parent ribozymes. The study has inferred that partite ES interactions at the cleavage site prove sufficient for catalysis.</p>

myotubesProper muscle function depends upon the fine tuning of the different molecular components of the neuromuscular junction (NMJ). Synaptic acetylcholinesterase (AChE) is responsible for rapidly terminating neurotransmission. Neuroscientists in the field have elucidated many aspects of synaptic AChE structure, function, and localization during the last 75 years. Nevertheless, how the enzyme is regulated and targeted to the NMJ is not completely understood. In skeletal muscle the synaptic AChE form derives from two separate genes encoding the catalytic and the collagenic tail (ColQ) subunits respectively. ColQ-AChE expression is regulated by muscle activity; however, how this regulation takes place remains poorly understood. We found that over or down-regulation of ColQ is sufficient to change the levels of AChE activity by promoting assembly of higher order oligomeric forms including the collagen-tailed forms. Furthermore, when peptides containing the Proline Rich Attachment Domain (PRAD), the region of ColQ that interacts with the AChE, are fed to muscle cells or cell lines expressing AChE, they are taken up by the cells and retrogradely transported to the endoplasmic reticulum (ER)/Golgi network where they induce assembly of newly synthesize AChE into tetramers. This results in an increase, as a consequence, in total cell associated AChE activity and active tetramer secretion, making synthetic PRAD peptides potential candidates for the treatment of organophosphate pesticides and nerve gas poisoning. To study the developmental regulation of ColQ-AChE we determined the levels of ColQ and ColQ mRNA in primary quail muscle cells in culture and as a function of muscle activity. Surprisingly, we found dissociation between transcription and translation of ColQ from its assembly into ColQ-AChE indicating the importance of posttranslational controls in the regulation of AChE folding and assembly. Furthermore, we found that the vast majority of the ColQ molecules in QMCs are not assembled into ColQ-AChE, suggesting that they can have alternative function(s). Finally, we found that the levels of ER molecular chaperones calnexin, calreticulin, and particularly protein disulfide isomerase are regulated by muscle activity and they correlate with the levels of ColQ-AChE. More importantly, our results suggest that newly synthesized proteins compete for chaperone assistance during the folding process.