MicroRNAs are small noncoding RNAs that post-transcriptionally reduce protein output from most human mRNAs by mechanisms that are still obscure. This thesis provides insights into three aspects of microRNA biogenesis and function described below. MicroRNA precursors are excised from primary transcripts by the Microprocessor complex containing Drosha and DGCR8. Although most microRNAs are located in introns of protein-coding and noncoding genes, the mechanisms coordinating microprocessing and splicing are unclear. MiR-211 is a microRNA expressed from intron 6 of melastatin, a suspected melanoma tumor suppressor. We demonstrate that miR-211, and not melastatin, is responsible for the tumor suppressive function of this locus, that Drosha-mediated processing of the miR-211 precursor promotes splicing of melastatin exon 6-exon 7 junctions, and that perturbing 5' splice site recognition by the U1 snRNP reduces Drosha recruitment to intron 6 specifically and intronic microRNA levels globally. Thus we identify a novel physical and functional coupling between microprocessing and splicing. Typically, Agos stabilize mature microRNAs and as a complex stoichiometrically bind to complementary mRNAs. We demonstrate an alternative order of events in which Agos bind and repress pre-formed imperfect microRNA-mRNA duplexes in processing bodies of live cells, and cleave pre-formed perfect microRNA-mRNA duplexes in vitro. Our data support a novel catalytic model whereby Agos first deposit microRNAs onto mRNAs and dissociate, thus priming multiple microRNA-mRNA duplexes for concurrent repression by a single Ago. Despite key roles in development and pathogenesis, effectors and regulators of microRNA-mediated repression are still poorly characterized. An RNAi screen revealed that depletion of ribosomal proteins of either small or large ribosomal subunit dissociates microRNA-containing complexes from mRNAs repressed at translation initiation, increasing their polysome association, translation, and stability relative to untargeted mRNAs. Thus ribosomal proteins globally regulate microRNA function. Another RNAi screen revealed that Akt3 phosphorylates Ago2, which negatively regulates cleavage and positively regulates translational repression of microRNA-targeted mRNAs. Thus Ago2 phosphorylation is a molecular switch between its mRNA cleavage and translational repression activities. The following pages will place these novel insights into biological and disease-relevant context, will describe what was known prior to these studies, and will provide perspectives for future studies.

Cardiovascular diseases (CVDs) are the most widely spread diseases of modern civilization. Mechanisms involved in the protection of myocardial tissue are for that very reason in the focus of cardiovascular research. The adaptation to chronic hypoxia has been studied for many years in the context of its positive effects on heart function and its increased tolerance to ischemia-reperfusion (I/R) injury. This Master thesis describes the role of Akt kinase in the mechanisms leading to myocardial protection against I/R injury using the model of adaptation to chronic normobaric hypoxia (CNH). The hearts from male Wistar rats, that were kept in normoxic or hypoxic conditions (O2 0.1) for the period of 3 weeks, were retrogradely perfused by oxygenated Krebs-Henseleit solution and then subjected to 10 min of ischemia and 10 min of reperfusion. Samples prepared from left ventricles (LV) of experimental hearts were later used for protein analyses. The adaptation to CNH leads to increased phosphorylation of Akt kinase on Ser473, but it did not affect the phosphorylation on Thr308 nor the total protein level of Akt. A significant increase in Bcl-2/Bax ratio was also observed in hearts adapted to CNH. This Master thesis further elucidates, how Akt signaling pathway and its activation are affected by short...

Les cassures double-brin (DSB) sont des dommages dangereux de l’ADN et représentent un facteur de risque pour la stabilité du génome. Le maintien de l'intégrité du génome est essentiel pour les cellules souches adultes, qui sont responsables de la régénération des tissus endommagés et de l'homéostasie tissulaire tout au long de la vie. La régénération musculaire chez l'adulte repose sur les cellules souches musculaires (cellules satellites, SCs) qui possèdent une remarquable capacité de réparation des DSB, mais dont le mécanisme sous-jacent reste inconnu. Ce projet de thèse consistait à étudier comment la différenciation musculaire est affectée lorsque la réparation des DSB est altérée, et quels sont le(s) mécanisme(s) et les conséquences de ce défaut de réparation sur la régénération musculaire. Au cours de cette étude, il est apparu de façon originale que les facteurs de réparation des DSB peuvent affecter la myogenèse, indépendamment de leur fonction dans la réparation de l'ADN. La présente étude a porté sur le rôle de la protéine kinase dépendante de l'ADN (DNA-PK), un facteur crucial pour la réparation non-homologue des DSBs (NHEJ), au cours de la différenciation musculaire chez la souris. L’étude a ciblé l'activation des SCs et la régénération musculaire in vitro et in vivo et a également abordé la régulation de cette kinase. Le rôle "canonique" de la DNA-PK, et donc du NHEJ, dans les SCs a également été étudié en présence de lésions de l'ADN radio-induites. Le rôle d’ATM, une kinase qui orchestre les réponses cellulaires aux DSB, a également été abordé dans le contexte de la régénération musculaire. Ces résultats confirment la notion émergente du rôle multifonctionnel des protéines de réparation de l’ADN dans d’autres processus physiologiques que la réparation elle-même, ce qui m’a également permis de réaliser une étude bibliographique. Ce travail i) identifie de nouveaux régulateurs de la myogenèse et ii) contribue à la compréhension de la résistance des cellules souches musculaires au stress génotoxique. Ces résultats pourraient avoir des implications dans l'amélioration des thérapies cellulaires de la dysfonction musculaire en agissant sur les régulateurs nouvellement découverts. / DNA double-strand breaks (DSBs) are dangerous DNA damages and a risk factor for genome stability. The maintenance of genome integrity is crucial for adult stem cells that are responsible for regeneration of damaged tissues and tissue homeostasis throughout life. Muscle regeneration in the adult relies on muscle stem cells (satellite cells, SCs) that have a remarkable DSB repair activity, but the underlying mechanism is not known. The aims of the present PhD project were to investigate how muscle differentiation is affected when DSB repair is impaired, and which are the mechanism(s) and the consequences on muscle regeneration. During this study, a novel possibility has arisen, namely that DSB repair factors affects myogenesis independently of their DNA repair activity, suggesting a novel function, not previously anticipated, of these factors. The present study has addressed the role of DNA-dependent protein kinase (DNA-PK), a crucial factor in non-homologous end-joining (NHEJ) repair of DSBs, in muscle differentiation in the mouse. Studies have targeted SC activation and muscle regeneration in vitro and in vivo and also addressed the regulation of this kinase. In parallel the more “canonical” role of DNA-PK, and thereby of NHEJ, has been investigated in SCs via radiation-induced DNA damage. The role of ATM, a kinase that orchestrates cellular responses to DSBs in muscle regeneration has also been addressed. These results support the emerging notion of multifunctional repair proteins in a variety of physiological processes beyond the repair process itself, on which I have conducted a bibliographical study. This work i) identifies novel regulators of myogenesis, and ii) helps understanding the resistance of muscle stem cells to genotoxic stress. It has potential implications for improving cellular therapies for muscle dysfunction by acting on the newly discovered regulators.