In the cell, the genetic information encoded in the DNA is transcribed to RNA. All RNAs that are transcribed in the cell are initially produced as precursor RNAs, which have to undergo various steps of processing to obtain their mature form. The maturation and processing for all RNA classes requires the activity of multiple RNA binding proteins (RBPs). An important family of RBPs that is involved in RNA maturation and processing is the SR-protein family. SR proteins are important for the regulation of a multitude of processes that include: splicing, transcription, export, RNA stabilization, translation and ncRNA processing. As of yet, there have been no comprehensive studies that describe how SR proteins dynamically regulate the maturation of RNAs. The results presented in this thesis provide new insights into the function and activity of SR proteins during RNA maturation. My experiments greatly expand the knowledge surrounding the action of RNA-binding proteins in vivo and in different cell compartments. To study the action of two different SR proteins in different cell compartments, I developed a new technique that combines cell fractionation and iCLIP, which I named FRACKING. For the first time, this method allowed me to collect information regarding the subcellular location where the RNA-protein interactions are taking place, giving a dynamic picture of the in vivo binding of SR proteins and of RNA binding proteins (RBP) in general. By using FRACKING on two heavily shuttling SR proteins, SRSF3 and SRSF7, I showed that both SR proteins are very dynamic in their binding behavior with RNAs. My research showed that both SRSF3 and SRSF7 strongly associate with RNAs during transcription (co-transcriptionally) and that they often remain bound to these transcripts until they are exported to the cytoplasm. The functions of SRSF3 and SRSF7 are closely related to their binding location on the target RNAs. I identified a subset of highly conserved introns that associated with SR proteins and are retained in their transcripts. These intron-retaining isoforms, contrary to textbook knowledge, are exported to the cytoplasm. I showed, for the first time, that SRSF3 and SRSF7 strongly interact with snoRNAs in the chromatin, and that this snoRNA-SR-protein binding behavior is distinct between SRSF3 and SRSF7. SRSF3 binds to the mature snoRNA sequence, and also to the surrounding intronic sequences, pointing towards a possible activity in guiding snoRNA maturation. Whereas SRSF7 associates to mature snoRNA sequences. Taken together, my study identified a dynamic pool of interactions for two SR proteins, in different cell compartments and discovered new activities for the two SR proteins. Importantly, this study challenges textbook knowledge on splicing and export of mRNAs by identifying a subset of transcripts that can be exported even when they retain introns.

In the cell, the genetic information encoded in the DNA is transcribed to RNA. All RNAs that are transcribed in the cell are initially produced as precursor RNAs, which have to undergo various steps of processing to obtain their mature form. The maturation and processing for all RNA classes requires the activity of multiple RNA binding proteins (RBPs). An important family of RBPs that is involved in RNA maturation and processing is the SR-protein family. SR proteins are important for the regulation of a multitude of processes that include: splicing, transcription, export, RNA stabilization, translation and ncRNA processing. As of yet, there have been no comprehensive studies that describe how SR proteins dynamically regulate the maturation of RNAs. The results presented in this thesis provide new insights into the function and activity of SR proteins during RNA maturation. My experiments greatly expand the knowledge surrounding the action of RNA-binding proteins in vivo and in different cell compartments. To study the action of two different SR proteins in different cell compartments, I developed a new technique that combines cell fractionation and iCLIP, which I named FRACKING. For the first time, this method allowed me to collect information regarding the subcellular location where the RNA-protein interactions are taking place, giving a dynamic picture of the in vivo binding of SR proteins and of RNA binding proteins (RBP) in general. By using FRACKING on two heavily shuttling SR proteins, SRSF3 and SRSF7, I showed that both SR proteins are very dynamic in their binding behavior with RNAs. My research showed that both SRSF3 and SRSF7 strongly associate with RNAs during transcription (co-transcriptionally) and that they often remain bound to these transcripts until they are exported to the cytoplasm. The functions of SRSF3 and SRSF7 are closely related to their binding location on the target RNAs. I identified a subset of highly conserved introns that associated with SR proteins and are retained in their transcripts. These intron-retaining isoforms, contrary to textbook knowledge, are exported to the cytoplasm. I showed, for the first time, that SRSF3 and SRSF7 strongly interact with snoRNAs in the chromatin, and that this snoRNA-SR-protein binding behavior is distinct between SRSF3 and SRSF7. SRSF3 binds to the mature snoRNA sequence, and also to the surrounding intronic sequences, pointing towards a possible activity in guiding snoRNA maturation. Whereas SRSF7 associates to mature snoRNA sequences. Taken together, my study identified a dynamic pool of interactions for two SR proteins, in different cell compartments and discovered new activities for the two SR proteins. Importantly, this study challenges textbook knowledge on splicing and export of mRNAs by identifying a subset of transcripts that can be exported even when they retain introns.

TDP-43 est une protéine de liaison aux acides nucléiques qui joue un rôle essentiel dans le métabolisme de l'ARN. À l'état physiologique, un contrôle strict des niveaux d’expression de cette protéine est critique pour la fonction et la survie cellulaire. Une boucle d'autorégulation négative est à la base de ce contrôle du taux intracellulaire de TDP-43. Laquelle a été identifiée comme le constituant principal des inclusions observées chez une majorité des patients atteints de Sclérose Latérale Amyotrophique (SLA) ou de Dégénérescence Lobaire Fronto-Temporale (DLFT). A ce jour, plus de 50 mutations faux-sensdu gène TARDBP/TDP-43 ont été décrites chez des patients DLFT/SLA, démontrant le rôle clé de TDP-43 dans ces pathologies neurodégénératives. Notons cependant que les conséquences fonctionnelles de ces mutations ne sont pas complètement déterminées. Plusieurs études suggèrent qu’une élévation des niveaux d’accumulation de TDP-43 pourraitparticiper aux mécanismes physiopathologiques. La modulation du cycle de production de TDP-43 pourrait donc constituer une nouvelle stratégie thérapeutique. Ce travail de recherche avait donc pour principal objectif d’identifier des modulateurs génétiques de la production de TDP-43 en utilisant un nouveau modèle de drosophile transgénique mimant les principales étapes d’autorégulation de TDP-43. Nous avons ainsi pu montrer que la modulation des niveaux d’expression de la protéine TCERG1 et de plusieurs facteurs d'épissage, parmi lesquels SRSF1, SRSF3 et SF3B1, influe sur les niveaux de production deTDP-43. Nous avons également montré que la présence des mutations DLFT/SLA n’altère pas la capacité de la protéine à s’autoréguler. / TDP-43 is a DNA/RNA binding protein that plays an important role in RNA metabolism. In the physiological state, strict control of its expression levels is critical for cell function and survival. TDP-43 expression is tightly regulated through an autoregulatory negative feedback loop. This protein has been identified as the principal component of the inclusions observed in a majority of patients with Amyotrophic Lateral Sclerosis (ALS) or FrontoTemporal Lobar Degeneration (FTLD). To date, more than 50 missense mutations of the TARDBP / TDP-43 gene have been described in FTLD / ALS patients, demonstrating the key role of TDP-43 in these neurodegenerative pathologies. However, the functional consequences of TDP-43 mutations are not completely determined. Several studies suggest that high accumulation levels of TDP-43 may participate in pathophysiological mechanisms. The modulation of the production cycle of TDP-43 may therefore provide a new therapeutic strategy. The main goal of this research project was to identify genetic modulators of TDP-43 production by using a novel transgenic Drosophila model mimicking main steps of TDP-43 the autoregulatory mechanism. We identified several splicing factors, including SF2, Rbp1 and Sf3b1, as genetic modulators of TDP-43 production. We have also shown that modulation of TCERG1 expression levels affect TDP-43 production levels in flies. Finally, we found that FTLD/ALSlinked TDP-43 mutations do not alter TDP-43’s ability to self-regulate its expression and consequently of the homeostasis of TDP-43 protein levels.