RNA interference (RNAi) is a pathway, which employs Dicer to process long double stranded RNAs (dsRNA) from endogenous or exogenous sources into short interfering RNAs (siRNA). siRNAs are loaded onto Argonaute proteins to mediate sequence-specific post-transcriptional RNA targeting resulting in regulation of protein-coding genes and retrotransposons or antiviral immune response. Another small RNA pathway - PIWI-associated RNA (piRNA) pathway is suppressing retrotransposons in the germline. In mice, canonical RNAi pathway activity is negligible in somatic cells where a full-length Dicer produces gene-regulatory microRNAs (miRNA) but RNAi is highly active in oocytes, which express a truncated oocyte-specific Dicer isoform (DicerO ). DicerO lacks an N-terminal DExD helicase domain and has higher cleavage activity of long dsRNAs. Deletion of oocyte specific DicerO promoter leads to transcriptome aberrations, which include upregulation of putative RNAi targets and MT retrotransposons and, consequently, to meiotic spindle defects and female sterility. In contrast, the piRNA pathway is non-essential in mouse oocytes, potentially because of overlapping functions of RNAi. The PhD thesis aims to understand biological significance of mammalian endogenous RNAi and to explore consequences of re-activated RNAi...

Je me suis intéressé au système de défense anti-viral majeur de Drosophila melanogaster qui est la voie du RNA silencing (siRNA). A ce jour, le seul senseur d’acide nucléique viral et activateur de la voie siRNA est Dicer-2. Ainsi, le travail que j’ai effectué́ a permis d’apporter de nouvelles informations concernant la détection des ARNs viraux par Dicer-2. L’utilisation de méthodes de séquençage à haut débit (HTS) des petits ARNs dans des cellules S2 infectées par le Drosophila C Virus (DCV) à des temps précoces m’a permis de proposer un point d’entrée précis et interne de Dicer-2 sur l’ARN double brin de ce dicistrovirus. La validation de ce point faible dans la défense du virus a été effectuée en réalisant un HTS des petits ARNs dans des mouches de différents génotypes infectées avec DCV. J’ai ensuite caractérisé plus en profondeur cette région du génome virale en déterminant tout d’abord sa structure 2D puis sa sensibilité à des clivages médiés par des extraits embryonnaires de mouches. Finalement, l’utilisation de différents variants de Dicer-2 présentant des mutations du domaine DRA m’a permis de proposer un nouveau mécanisme de fonctionnement de cette protéine. / My Ph.D revolved around the study of the major antiviral defense system of Drosophila melanogaster: the siRNA pathway. To date, the only viral nucleic acid sensor and siRNA pathway activator in drosophila is Dicer-2. Thus, the work I have done has provided new information regarding the detection of viral RNAs by Dicer-2. The use of high throughput sequencing (HTS) methods of small RNAs in S2 cells infected with Drosophila C Virus (DCV) at early time points has allowed me to propose a precise and internal entry point for Dicer-2 on the double-stranded RNA of this dicistrovirus. The validation of this weak point in the defence of the virus was carried out by performing an HTS of small RNAs in flies of different genotypes infected with DCV. I then characterized this region of the viral genome in more depth by first determining its 2D structure and then its sensitivity to cleavages mediated by embryonic fly extracts. Finally, the use of different variants of Dicer-2 with mutations in the DRA domain allowed me to propose a new mechanism of action for this protein.

No description available.

La famille des microARN (miARN) let-7 comprend treize membres qui jouent des rôles critiques dans de nombreux processus biologiques, notamment la différenciation et le développement cellulaires. Plus spécifiquement, ils fonctionnent comme des suppresseurs de tumeurs en ciblant plusieurs oncogènes. La dérégulation des niveaux de miARN let-7 a été associée à diverses maladies humaines, y compris des cancers et des troubles neurodégénératifs. Il est bien établi que Drosha et Dicer, appartenant à la famille des RNases III, sont deux enzymes clés de la voie de maturation des miARN, et qu'un traitement défectueux par ces endoribonucléases pourrait affecter l'expression des gènes. Au cours des dix dernières années, plusieurs recherches ont permis d'identifier les caractéristiques structurales de l'ARN et les protéines qui régulent la voie de maturation des miARN. Cependant, les détails moléculaires menant à la régulation des niveaux d’expression des miARN nécessitent des investigations supplémentaires. L'objectif principal de cette thèse est d'étudier l'activité de clivage in vitro des endoribonucléases Drosha et Dicer envers leurs substrats let-7, en se concentrant sur la façon dont diverses caractéristiques de séquence et de structure affectent leurs activités. Tout d'abord, un criblage structural de type SHAPE suivi d'investigations thermodynamiques et cinétiques détaillées pour les treize pré-miARN de la famille let-7 ont été réalisés avec une enzyme Dicer purifiée in vitro. Cette étude a révélé que malgré les différences structurales des membres de la famille let-7, Dicer ne discrimine pas entre ses substrats, y compris les pré-miARN avec une extension de 1-nt et 2-nt à leur extrémité 3'. L'ensemble de ces travaux met en évidence la remarquable promiscuité de Dicer vis-à-vis divers pré-miARN de la famille let-7. Deuxièmement, le mécanisme enzymatique du clivage du pré-let-7a-1 a été examiné. Les résultats de la cinétique de l'état stable, de l'état pré-stable et de l'impulsion-chase sont conformes à l'opinion dominante, soutenue par de récentes structures de cryo-EM, selon laquelle le ou les changements de conformation d'un complexe enzyme-substrat dans une conformation catalytiquement productive sont importants pour l'activité de clivage. Troisièmement, nous avons étudié la séquence et les déterminants structuraux du clivage du pri-let-7 par le complexe microprocesseur (MP) composé de Drosha et de son partenaire obligatoire DGCR8. Sur la base d'études de clivage de plusieurs substrats pri-let-7 avec un complexe MP reconstitué in vitro, il a été constaté que le clivage du pri-let-7g donne des produits multiples. En utilisant des variantes de pri-let-7g, il a été révélé qu'un élément structural conservé de pri-let-7g favorise un clivage improductif, peut-être en raison du clivage de son substrat par la MP dans l'orientation inverse. Cette étude fournit un cadre pour des investigations futures dans l'étude du clivage de pri-let-7g par Drosha et éventuellement l'identification de nouveaux mécanismes de régulation. Dans l'ensemble, nos résultats donnent un aperçu de la façon dont les caractéristiques structurales des pri-miARN et des pré-miARN de la famille let-7 modulent le traitement par Drosha et Dicer et ouvrent la voie à de futures études visant à examiner le rôle des facteurs protéiques dans la régulation de la maturation des miARN let-7. / The let-7 family of microRNAs (miRNAs) comprises of thirteen members that play critical roles in many biological processes, including cell differentiation and development. More specifically, they function as tumor suppressors by targeting several oncogenes. Deregulation in let-7 miRNA levels has been associated with various human diseases, including cancers and neurodegenerative disorders. It is well established that Drosha and Dicer are the two key enzymes of the miRNA maturation pathway, and that faulty processing by these endoribonucleases could affect gene silencing. Thus, it is crucial to better understand how Drosha and Dicer respectively process the primary miRNAs (pri-miRNAs) and precursor miRNAs (pre-miRNAs) to yield mature miRNAs, and how these enzymes are regulated. In the last decade of miRNA research, several investigations have identified RNA structural features and RNA-binding proteins that regulate the miRNA maturation pathway, adding another layer of regulation in this pathway. However, the molecular detail of this regulation requires further investigations. The main goal of this thesis is to investigate the in vitro processing activity of Drosha and Dicer toward their let-7 substrates, focusing on how diverse sequence and structural features affect their activities. First, SHAPE structural probing followed by detailed thermodynamic and kinetic investigations for all thirteen pre-miRNAs of the let-7 family were performed with in vitro purified Dicer. Surprisingly, this study revealed that despite structural differences in the pre-let-7 members, Dicer does not discriminate between these substrates, including pre-miRNAs with a 1 nt and a 2-nt overhang at their 3'-end. Additional binding and cleavage investigations of pre let-7 substrates carrying 3'-end modifications (mono- and oligo-uridylation, mono- and oligo-adenylation) were performed to clarify how these modifications affect Dicer binding and cleavage activities. Together, this work highlights the remarkable substrate promiscuity of Dicer toward diverse pre-miRNAs of the let-7 family. Second, the enzymatic mechanism of pre-let-7 cleavage by Dicer was examined using pre-let-7a-1 as a model substrate. The results from the steady-state, pre-steady state and pulse-chase kinetics are consistent with the prevailing view, supported by recent cryo-EM structures, that the conformational change(s) of an enzyme-substrate complex into a catalytically productive conformation are important for cleavage activity. Third, the sequence and structural determinants of pri-let-7 processing by the Microprocessor (MP) complex composed of Drosha and its obligatory partner DGCR8 were investigated. Based on cleavage studies of several pri-let-7 substrates with an in vitro reconstituted MP complex, it was found that cleavage of pri-let-7g yields multiple products. Using pri-let-7g variants, it was revealed that a conserved structural element of pri-let-7g promotes unproductive cleavage, possibly as a result of the MP cleaving its substrate in the reverse orientation. This study provides the framework for future investigations in studying pri-let-7g processing by Drosha and possibly identifying novel mechanisms of regulation. Overall, our findings provide insights on how the structural features of pri-miRNAs and pre-miRNAs of the let-7 family modulate processing by Drosha and Dicer and pave the way for future studies aimed at examining the role of protein factors in regulating the maturation of let-7 miRNAs.

Since their discovery in 1953, adenoviruses have significantly contributed to the understanding of virus-host cell interactions, including mechanistic details of cellular processes such as cell cycle control and alternative RNA splicing. Among the first characterized adenoviral genes were the virus-associated RNAs (VA RNAI/II), which are produced in massive amount during a lytic infection. The VA RNAs perform multiple functions and are required for a successful adenovirus life cycle. More recently, it was shown that the VA RNAs are processed into small viral miRNAs, so-called mivaRNAs, which interfere with the function of the cellular RNAi/miRNA machinery. In papers I and II, we focused on a structural and functional characterization of the mivaRNAs using two approaches. Firstly, we created a model system where the predicted miRNA-like function of mivaRNAI could be investigated, without interfering with other VA RNA functions. This was accomplished by construction of recombinant adenoviruses, in which the seed sequence of mivaRNAI was altered. The results showed that in cell culture experiments the mivaRNAI seed sequence mutants grew as the wild type virus, suggesting that the mivaRNAs are not required during the lytic phase of an adenovirus infection. Secondly, we showed that the VA RNAs from different human adenoviruses (Ad4, Ad5, Ad11 and Ad37) undergo the same type of Dicer-dependent processing into mivaRNAs, which subsequently are loaded onto the RNA induced silencing complex (RISC), albeit with different efficiencies. In paper III, we demonstrated that the promoter proximal region of the adenovirus major late promoter (MLP) produces a novel non-canonical class of small RNAs, which we termed the MLP-TSS-sRNAs. Surprisingly the MLP-TSS-sRNA maintains the m7G-cap structure while bound to Ago2 containing RISC. These complexes are functional suppressing expression of target mRNAs with complementary binding site. Most importantly, the MLP-TSS-sRNA limits the efficiency of viral DNA replication probably through a targeting of the E2B mRNAs, which are transcribed in the antisense orientation. In conclusion, the MLP-TSS-sRNA represents the first viral small RNA, which has been shown to have a function as a regulator of an adenovirus infection.

No description available.

Melanoma differentiation associated gene 7/Interleukin-24 (MDA-7/IL-24) is a secreted cytokine which acts as a tumor suppressor. It is capable of selectively killing cancer cells, regardless of anatomic origin, while sparing normal cells. miRNAs are master regulators of gene expression that can play two roles in cancer: tumor-suppression and oncogenesis. We identified a number of miRNAs that are regulated by MDA-7/IL-24 using a PCR plate array containing probes for miRNAs known to play a role in prostate cancer. We independently validated the array with qRT-PCR to identify three miRNAs which are downregulated by MDA-7/IL-24 treatment in DU145, PC3, and PC3ML prostate cancer lines. These miRNAs were miR-125a, miR-145, and miR-23b. Their gene targets were identified using TargetScan and confirmed to be regulated in our prostate cancer model. NLRC5, KLF4, and KLF15, respectively, were upregulated after treatment with MDA-7/IL-24. We focused on NLRC5 as a novel target of MDA-7/IL-24, which plays a role in immune evasion by cancer cells. NLRC5 is upregulated following inhibition of miR-125a. It is not downregulated by overexpression of miR-125a which suggests that more than one miRNA may be acting to regulate its expression. Finally, we determined that miR-125a is downregulated by MDA-7 through DICER, an important processing enzyme for miRNA biogenesis.

Cellular messenger RNAs (mRNAs) exist almost exclusively in the context of ribonucleoprotein complexes (RNPs), which are largely responsible for the coordinated regulation of mRNA fate, and in particular, the post-transcriptional regulation of mRNA stability and translation. RNA- binding proteins, antisense RNAs, and microRNAs represent three major classes of post- transcriptional regulatory factors that interact with target mRNAs. Significantly, these interactions are dynamically regulated under both basal and stress conditions, such as hypoxia. Given the prominent contributions of post-transcriptional regulation to overall gene expression, a more comprehensive understanding of the underlying mechanisms is required. In this thesis, we present exciting new evidence for the functional importance of post- transcriptional gene regulation, especially in the vascular endothelium. Firstly, we show that the formation of hnRNP E1-containing RNPs contributes significantly to the remarkable basal stability of endothelial nitric oxide synthase (eNOS) mRNAs in endothelial cells by protecting them from inhibitory post-transcriptional forces. However, hypoxia impairs such RNP formation through hnRNP E1 serine phosphorylation and nuclear localization. Together, these mechanisms contribute significantly to decreased eNOS expression and activity in chronic hypoxia. ii Secondly, we reveal an important functional relationship between the microRNA pathway and the HIF-mediated cellular hypoxic response. Specifically, the down-regulation of Dicer and an important number of Dicer-dependent microRNAs in chronic hypoxia represents an important adaptive mechanism that serves to maintain the cellular hypoxic response through HIF-α- and microRNA-dependent mechanisms, with significant implications for the development of RNAi- based therapies. Finally, we provide evidence that the up-regulation of specific microRNAs in acute hypoxia is a potentially important mechanism that serves to suppress global translation initiation in order to conserve energy and ensure cellular survival. Collectively, the findings presented in this thesis provide important new mechanistic insight into the post-transcriptional regulation of eNOS, as well as the functional integration of the microRNA and the cellular hypoxic response pathways.

Cellular messenger RNAs (mRNAs) exist almost exclusively in the context of ribonucleoprotein complexes (RNPs), which are largely responsible for the coordinated regulation of mRNA fate, and in particular, the post-transcriptional regulation of mRNA stability and translation. RNA- binding proteins, antisense RNAs, and microRNAs represent three major classes of post- transcriptional regulatory factors that interact with target mRNAs. Significantly, these interactions are dynamically regulated under both basal and stress conditions, such as hypoxia. Given the prominent contributions of post-transcriptional regulation to overall gene expression, a more comprehensive understanding of the underlying mechanisms is required. In this thesis, we present exciting new evidence for the functional importance of post- transcriptional gene regulation, especially in the vascular endothelium. Firstly, we show that the formation of hnRNP E1-containing RNPs contributes significantly to the remarkable basal stability of endothelial nitric oxide synthase (eNOS) mRNAs in endothelial cells by protecting them from inhibitory post-transcriptional forces. However, hypoxia impairs such RNP formation through hnRNP E1 serine phosphorylation and nuclear localization. Together, these mechanisms contribute significantly to decreased eNOS expression and activity in chronic hypoxia. ii Secondly, we reveal an important functional relationship between the microRNA pathway and the HIF-mediated cellular hypoxic response. Specifically, the down-regulation of Dicer and an important number of Dicer-dependent microRNAs in chronic hypoxia represents an important adaptive mechanism that serves to maintain the cellular hypoxic response through HIF-α- and microRNA-dependent mechanisms, with significant implications for the development of RNAi- based therapies. Finally, we provide evidence that the up-regulation of specific microRNAs in acute hypoxia is a potentially important mechanism that serves to suppress global translation initiation in order to conserve energy and ensure cellular survival. Collectively, the findings presented in this thesis provide important new mechanistic insight into the post-transcriptional regulation of eNOS, as well as the functional integration of the microRNA and the cellular hypoxic response pathways.

Les microARNs sont une classe de petits ARNs non codants qui régulent l'expression des gènes via un mecanisme d'interference par ARN. Les microARNs humains sont produits par une série de réactions enzymatiques. En particulier, dans le cytoplasme le precurseur de miRNA (pre-miRNA) est reconnu et clivé par un complexe contenant l'enzyme RNAse III Dicer et plusieurs cofacteurs protéiques. La proteine TRBP (HIV TAR RNA binding protein) est l'un de ces cofacteurs et augmente la stabilité du complexe, influe sur la cinétique, la position du clivage et a role potentiel dans la reconnaissance du substrat et dans le transfet du produit vers le complexe RISC (RNA-induced silencing complex) effecteur de l'interference par ARN. TRBP est composé de 3 domaines de liaison aux ARN doubles brin (dsRBDs). La région d'interaction de TRBP avec les ARNs est composé des deux premiers dsRBDs liés par une région interdomaine non charactérizée. La présente étude rapporte la caractérisation biophysique in vitro de la région d'interaction avec les ARNs de TRBP dans l'état apo de TRBP ou dans l'état lié avec les deux precurseurs cytoplasmique successifs du microARN oncogène miR-155 comprenant la tige boucle pre-miR-155 et le duplex miR-155/miR-155* résultat du clivage de pre-miR-155 par Dicer. L'étude montre que la région d'intéraction de TRBP avec les ARNs est monomerique, est composée de deux dsRBDs independants en solution et que la région interdomaine de 60 résidus est flexible. Le premier dsRBD, non caractérisé précédement en solution est le siège d'un equilibre plié/déplié integral dans une grande gamme de conditions physico-chimiques. Les deux premiers dsRBDs de TRBP peuvent interagir avec un même precurseur de microARN et deux régions d'interaction de TRBP avec les ARNs peuvent interagir avec un même precuseur. La région d'interaction de TRBP avec les ARNs interagit avec pre-miR-155 et le duplex miR-155/miR-155* avec des affinités très similaires. Dans le complexe avec une région d'interaction de TRBP avec les ARN liée à pre-miR-155 ou au duplexe miR-155/miR-155*, aucune indice de contact entre les deux dsRBDs n'a été detecté et la protéine interagit avec les deux precurseurs par la même surface d'interaction. Les informations récoltées suggèrent que TRBP peut jouer un rôle avant et après le clivage des pre-miARN par Dicer, notamment dans le complexe de chargement de RISC.