The Severe Acute Respiratory Syndrome (SARS) virus encodes several unusual RNA processing enzymes, including Nsp15, an endoribonuclease that preferentially cleaves 3? of uridylates through a Ribonuclease A-like mechanism. Crystal structures of Nsp15 confirmed that the Nsp15 active site is structurally similar to that of Ribonuclease A. These similarities and our molecular docking analysis lead us to hypothesize that previously characterized Ribonuclease A inhibitors will also inhibit the SARS-CoV Nsp15. Benzopurpurin B, C-467929, C-473872, N-36711, N-65828, N-103018 and Congo red were tested for effects on Nsp15 endoribonuclease activity. A real-time fluorescence assay revealed that the IC50 values for inhibiting Nsp15 were between 0.2 ?M and 40 ?M. Benzopurpurin B, C-473872, and Congo red are competitive inhibitors, according to kinetic studies and were demonstrated to bind SARS-CoV Nsp15 by a differential scanning fluorimetry assay. Benzopurpurin B also inhibited the Nsp15 orthologs from two other coronaviruses: mouse hepatitis virus (MHV) and infectious bronchitis virus. The three compounds reduced infectivity of MHV in L2 cells by 8 to 26 fold. The more effective drugs also caused a decrease in MHV RNA accumulation.

Dicer endoribonuclease catalyzes the maturation of microRNAs (miRNAs) from double stranded precursors. Studies conditionally inactivating Dicer in the mouse embryonic forebrain continue to shed light on the spectrum of biological processes subject to miRNA regulation. This study looked at defects of brain development following a widespread ablation of Dicer in the early forebrain. The neuroepithelial stem cells failed to specify the radial glia appropriately around the time when the first postmitotic neurons begin to be generated in the neuroepithelium. Ablation of Dicer in only a subset of radial glia was not accompanied by the early apoptosis observed in all other models of Dicer ablation in the cortex. This allowed the study of the role of miRNAs in regulating cell numbers in the cortex. The study revealed that generation of cortical cells is increased during postnatal development. Finally, the study identified a miRNA which is able to negatively regulate the development of neuronal precursor cells of the developing cortex by targeting Tbox transcription factor 2. Together the results presented in this Thesis contribute to the understanding of the roles of endogenous RNA interference in the development of the brain.

Protein synthesis is a fundamental and ultimate process in living cells. Cells possess sophisticated machineries and continuously carry out complex processes. Monitoring protein synthesis in living cells not only inform us about the mechanism of translation but also deepen our insights about all aspects of life. Understanding the structure and mechanism of the ribosome and its associated factors helped us enlarge our knowledge on protein synthesis. Recently, with the dramatic advances of high-throughput sequencing and bioinformatics, a new technique called ribosome profiling emerged. By retrieving mRNA fragments protected by translating ribosomes, ribosome profiling reveals global ribosome occupancy along mRNAs in living cells, which can inform us with the identity and quantity of proteins being made. Easily adapted to other organisms, ribosome profiling technique is expanding its application in revealing various cellular activities as well as the knowledge on protein synthesis. Here, we report the mechanism of translating mRNA cleavage by endoribonuclease RelE in vivo. RelE is an endoribonuclease that is induced during nutrient deficiency stress and specifically cleaves translating mRNAs upon binding to the ribosomal A site. Overexpression of RelE in living cells causes growth arrest by inhibiting global translation. We monitored RelE activity in vivo upon overexpression using ribosome profiling. The data show that RelE actively cuts translating mRNAs whenever the ribosomal A site is accessible, resulting in truncated mRNAs. RelE causes the ribosome complexes to accumulate near the 5' end of genes as the process of ribosome rescue, translation, and cleavage by RelE repeats. RelE cleavage specific sub-codon level ribosome profiling data also represent reading frame in Escherichia coli and sequence specificity of RelE cleavage in vivo. We report another ribosome profiling study on a methyltransferase TrmD in E. coli. TrmD is known to methylate G37 (the residue at 3' side of anticodon) of some tRNAs and be responsible for codon-anticodon interaction. We constructed a TrmD depletion E. coli strain, whose deletion results in lethality of cells. Resulting depletion of m1G37 in the strain leads to growth arrest. Lack of m1G37 of some tRNAs whose codons start with C showed frequent frameshift when translating the gene message in vitro. By using ribosome profiling, we successfully observed significant difference on translation process when codons interact with anticodons of tRNAs lacking m1G37. The data reveal slow translation rate or pauses on the tRNAs when missing the appropriate methylation, which corresponds to the previous biochemical data in vitro.

Upon termination of translation, the fate of ribosomes is determined largely by the rate at which cells are growing. During periods of exponential growth, ribosomes are rapidly recycled, translation is re-initiated, and the ribosomes are extremely stable. However, when nutrient sources become limiting, and ribosomes are not actively translating, they may become substrates for degradation. While this phenomenon is well known, details of how the process is initiated and what are the signals for degradation have, until now, remained elusive. Here, I present in vitro and in vivo data showing that free ribosome subunits are the targets of degradative enzymes, whereas 70S particles that remain associated are protected from such degradation. Conditions that increase the formation of subunits both in vitro and in vivo lead to enhanced degradation. Thus, the simple presence of free 50S and 30S subunits is sufficient to serve as the mechanism that initiates ribosome degradation. In order to identify RNases involved in ribosome degradation, both in vitro and in vivo assays were developed. Together, they have provided evidence for a multi-step degradation process involving both endo- and exoribonucleases. Examination of extracts from strains deficient in known RNases revealed that the endoribonucleases, RNase E and RNase G, may be involved in the initial cleavages. The resulting fragments, some of which are small enough oligoribonucleotides that they remain part of the acid-soluble fraction are degraded to mononucleotides primarily by the 3'-5' exoribonucleases, RNase R and polynucleotide phosphorylase.

No description available.

The non-structural protein 15 (NSP15) locus in Lineage A β-coronaviruses has two important functions during replication. The encoded endoribonuclease is conserved among coronaviruses. The function of the nsp15 protein is still not fully understood, but recent evidence suggests it may be involved in both replication and inhibiting viral sensing of double stranded RNA. In Lineage A β-coronaviruses, the RNA locus contains an inserted packaging signal (P/S). The P/S is essential for selectively packaging viral genomic RNA. While the P/S is required for selective packaging, it is not required for nsp15 protein function or viral replication. Utilizing this region, I studied the interactions of nsp15 protein during infection. Additionally, I studied the effect of selective packaging on virulence. Coronaviruses encode 16 nonstructural proteins in two open reading frames. These proteins are responsible for forming the replication/ transcription complex (RTC) and creating an environment conducive to viral replication. The RTC is an intricate complex of viral and host proteins with a largely unknown composition. While almost all nsps studied to date localize to sites of replication, the interactions between these proteins are not fully understood. In Chapter II, I describe studies of the interactions and localization of Nsp15 by creating an in situ hemagglutinin epitope tag. I found that mouse hepatitis virus nsp15 could tolerate an in situ tag when placed into the P/S (MHVNsp15-HA). MHVNsp15-HA had wild-type like replication in vitro. Nsp15 was localized to sites of replication throughout infection, with no localization to sites of assembly. Finally, nsp15 interacted with the RNA dependent RNA polymerase and putative primase during infection. These data demonstrate that nsp15 is a member of the RTC. During coronavirus replication two species of viral RNA are present, genomic RNA (gRNA) and sub-genomic RNA (sgRNA). These RNAs are co-terminal on both their 5’ and 3’ ends, containing the leader sequence and 3’ UTR/ polyA respectively. Even with these similarities, coronaviruses are adept at selectively packaging gRNA over sgRNA. This selective packaging is determined by the P/S, a 95 base pair stem-loop structure in the nsp15 locus. This RNA motif is sufficient for packaging of nonviral RNAs and has been shown to interact with the M protein from MHV. Moreover, when this RNA motif is deleted from MHV, (MHVPS-) selective packaging is lost during infection as sgRNAs become a large percentage of packaged viral RNA. In chapter IV I determined the effect of selective packaging on pathogenicity in vivo. Immunocompetent mice infected with MHVPS- had significantly better outcomes compared to MHV wild-type (MHVWT) infected mice. Peak viral loads were decreased in MHVPS- compared to MHVWT. Strikingly I found MHVPS- infected bone marrow derived macrophages had significant increases in type-I interferons (IFNs) and pathogenesis of MHVPS- was restored in mice deficient in IFN signaling. These data indicate that the P/S of MHV is an uncharacterized MHV virulence factor, which acts by preventing an increased IFN response during infection. In MHV, the nsp15 locus is translated into a functional protein and contains functional cis acting RNA elements both of which play a role in MHV replication. This work provides understanding of nsp15 localization and interactions which educate our understanding of the function of this conserved endoribonuclease. Additionally, this work demonstrates a unique function for the P/S not previously described. This work informs future studies of nsp15 protein function and the function of selective packaging during coronavirus infection.

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.

No description available.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and associated (Cas) proteins form the basis of a prokaryotic adaptive immune system. Acquired sections of viral DNA are stored within the host genome as ‘spacers' flanked by ‘repeat' sequences. The CRISPR arrays are transcribed and processed to release mature CRISPR RNAs (crRNAs) – containing a single, intact spacer sequence – that are used by effector complexes to base-pair with matching hostile genetic elements and silence future infections. crRNA-biogenesis is thus an essential step within the defence pathway. Within Type I and III systems, the primary processing of the CRISPR transcript at repeat sites is performed almost exclusively by the CRISPR-specific riboendonuclease, Cas6. This thesis seeks to probe the catalytic mechanism of a Cas6 enzyme from the crenarchaeon Sulfolobus solfataricus (sso). Despite analogous generation of crRNA, ssoCas6 paralogues differ from previously characterised Cas6 examples in their lack of a canonical active site histidine residue. The work here builds on recent crystallographic evidence that the ssoCas6-1 paralogue unexpectedly adopts a dimeric conformation (PDB 3ZFV, 4ILR), to show that not only is the ssoCas6-1 dimer stable in solution but that this atypical arrangement is important to the activity of this particular enzyme. Furthermore, the ssoCas6-1 paralogue is shown to be the first in this family of endonucleases to employ multiple-turnover kinetics. The widespread diversity in Cas6 catalytic mechanisms reflects the plastic nature of the Cas6 active site and rapid co-evolution with substrate repeat sequences. The CRISPR/Cas environment within S. solfataricus is highly complex, containing three co-existing system types (Type I-A, III-A, III-B), five Cas6 paralogues and two families of CRISPR loci (AB and CD) that differ by repeat sequence. By probing the activity of an additional ssoCas6 paralogue (ssoCas6-3), which reveals different substrate specificities to those of ssoCas6-1, evidence emerges for functional coupling between ssoCas6 paralogues and downstream effector complexes, sufficient to regulate crRNA uptake and possibly even complex assembly.

L'insulinorésistance, caractérisée par l'incapacité des organes impliqués dans le métabolisme énergétique (tissu adipeux, muscles squelettiques et foie) à répondre à l'insuline, tient une place centrale dans la physiopathologie des complications métaboliques de l'obésité. L'apparition d'une insulinorésistance chez un sujet obèse est plurifactorielle et les mécanismes moléculaires impliqués ne sont à ce jour pas complètement élucidés.L'expansion majoritairement hyperplasique du tissu adipeux conduit à une hypoxie et un stress des adipocytes, induisant un relargage accru de cytokines inflammatoires et d'acides gras libres (AGL). Les AGL se fixent eux-mêmes sur les récepteurs toll-like (TLRs) de l'immunité innée, dont l'activation aboutit également à la sécrétion de cytokines inflammatoires. Ces AGL et cytokines, véhiculés par la circulation systémique, contribuent, avec la coopération des macrophages infiltrant le tissu adipeux, au développement d'une inflammation chronique de bas grade. Ainsi, les perturbations de l'homéostasie énergétique, associées à une activation du système immunitaire sont à l'origine d'une atteinte globale de la sensibilité à l'insuline de l'organisme, particulièrement délétère au métabolisme musculaire.Cette étude porte sur le rôle d'un effecteur de l'immunité innée, l'endoribonucléase latente (RNase L), dont l'expression est régulée par les interférons de type I et l'activation, par un oligoadénylate, le 2-5A. La RNase L clive les ARNs cellulaires conduisant à l'inhibition spécifique de l'expression de certains gènes. Nous montrons par ce travail l'implication de la RNase L dans le contrôle de la différenciation cellulaire et la pathogenèse de l'insulinorésistance associée à l'obésité, via la régulation des voies de l'inflammation au niveau des tissus adipeux et musculaire. / Insulin resistance, which is characterized by the incapacity of organs involved in the energetic metabolism (adipose tissue, skeletal muscles and liver) to respond to insulin, has a central place in the pathophysiology of the metabolic complications associated to obesity. The onset of insulin resistance in obese subjects is multifactorial and the molecular mechanisms involved have not yet been completely elucidated.The mainly hyperplasic expansion of white adipose tissue leads to hypoxia and stress in adipocytes, inducing an increased release of inflammatory cytokines and free fatty acids (FFA). FFA bind and activate the toll-like receptors (TLR) of the innate immunity system, leading to the secretion of inflammatory cytokines. These FFA and cytokines, taken by the systemic circulation, contribute, with the cooperation of macrophages infiltrating the adipose tissue, to the development of a chronic low-grade inflammation. Thus, the disturbances of the energetic homeostasis, associated with an activation of the immune system cause a global impairment of insulin sensitivity of the body, with particularly deleterious effects on muscular metabolism.This study focuses on the role of an effector of innate immunity, the latent endoribonuclease (RNase L). RNase L expression is regulated by type I interferons and is activated by the 2-5A oligoadenylate. RNase L splits cellular RNA, thus leading to the specific inhibition of the expression of certain genes. In this study, we demonstrate the implication of RNase L in the control of cell differentiation and the pathogenesis of obesity-associated insulin resistance, via the regulation of inflammatory pathways in the adipose and muscular tissues.