Staufen1 is a double-stranded RNA-binding protein known to be involved in the transport, localization, decay and increased translation of some mRNAs. The goal of the present study is to determine the role of Staufen1 during myogenic differentiation by characterizing the effects of Staufen1 over-expression in C2C12 cells. Immunofluorescence experiments revealed that Staufen1 over-expression causes a decrease in the fusion and differentiation indices and leads to the formation of myotubes with significantly fewer nuclei. We show, by western blot and qRT-PCR, that the protein expression of MyoD, myogenin and MyHC and the mRNA expression of MyoD, myogenin, Mef2A, Mef2C and p35 are significantly decreased during differentiation when Staufen1 is over-expressed. We then found that c-myc protein expression was increased during proliferation but that its mRNA expression remained unchanged. In this study we propose that Staufen1 negatively regulates skeletal muscle differentiation through the posttranscriptional regulation of c-myc, Mef2A, Mef2C and p35 transcripts.

Staufen1 is a double-stranded RNA-binding protein known to be involved in the transport, localization, decay and increased translation of some mRNAs. The goal of the present study is to determine the role of Staufen1 during myogenic differentiation by characterizing the effects of Staufen1 over-expression in C2C12 cells. Immunofluorescence experiments revealed that Staufen1 over-expression causes a decrease in the fusion and differentiation indices and leads to the formation of myotubes with significantly fewer nuclei. We show, by western blot and qRT-PCR, that the protein expression of MyoD, myogenin and MyHC and the mRNA expression of MyoD, myogenin, Mef2A, Mef2C and p35 are significantly decreased during differentiation when Staufen1 is over-expressed. We then found that c-myc protein expression was increased during proliferation but that its mRNA expression remained unchanged. In this study we propose that Staufen1 negatively regulates skeletal muscle differentiation through the posttranscriptional regulation of c-myc, Mef2A, Mef2C and p35 transcripts.

Staufen1 is a double-stranded RNA-binding protein known to be involved in the transport, localization, decay and increased translation of some mRNAs. The goal of the present study is to determine the role of Staufen1 during myogenic differentiation by characterizing the effects of Staufen1 over-expression in C2C12 cells. Immunofluorescence experiments revealed that Staufen1 over-expression causes a decrease in the fusion and differentiation indices and leads to the formation of myotubes with significantly fewer nuclei. We show, by western blot and qRT-PCR, that the protein expression of MyoD, myogenin and MyHC and the mRNA expression of MyoD, myogenin, Mef2A, Mef2C and p35 are significantly decreased during differentiation when Staufen1 is over-expressed. We then found that c-myc protein expression was increased during proliferation but that its mRNA expression remained unchanged. In this study we propose that Staufen1 negatively regulates skeletal muscle differentiation through the posttranscriptional regulation of c-myc, Mef2A, Mef2C and p35 transcripts.

Staufen1 is a double-stranded RNA-binding protein known to be involved in the transport, localization, decay and increased translation of some mRNAs. The goal of the present study is to determine the role of Staufen1 during myogenic differentiation by characterizing the effects of Staufen1 over-expression in C2C12 cells. Immunofluorescence experiments revealed that Staufen1 over-expression causes a decrease in the fusion and differentiation indices and leads to the formation of myotubes with significantly fewer nuclei. We show, by western blot and qRT-PCR, that the protein expression of MyoD, myogenin and MyHC and the mRNA expression of MyoD, myogenin, Mef2A, Mef2C and p35 are significantly decreased during differentiation when Staufen1 is over-expressed. We then found that c-myc protein expression was increased during proliferation but that its mRNA expression remained unchanged. In this study we propose that Staufen1 negatively regulates skeletal muscle differentiation through the posttranscriptional regulation of c-myc, Mef2A, Mef2C and p35 transcripts.

Over the past decade several converging lines of evidence have highlighted the importance of post-transcriptional events in skeletal muscle. This level of regulation is controlled by multi-functional RNA-binding proteins and trans-acting factors. In fact, several RNA-binding proteins are implicated in neuromuscular disorders including myotonic dystrophy type I, spinal muscular atrophy and amyotrophic lateral sclerosis. Therefore, it is necessary to examine the impact of RNA-binding proteins during skeletal muscle development and plasticity in order to understand the consequences linked to their misregulation in disease. Here, we focused on the RNA-binding protein Staufen1, which assumes multiple roles in both skeletal muscle and neurons. We previously demonstrated that Staufen1 is regulated during myogenic differentiation and that its expression is increased in denervated and in myotonic dystrophy type I skeletal muscles. The increased expression of Staufen1 initially appeared beneficial for DM1 since further elevating Staufen1 levels rescued key hallmarks of the disease. However, based on the multi-functional nature of Staufen1, we hypothesized that Staufen1 acts as a disease modifier in DM1. To test this, we investigated the roles of Staufen1 in skeletal muscle biology and their implications for disease. Our data demonstrated that Staufen1 is required during the early stages of muscle development, however its expression must remain low in postnatal skeletal muscle. Interestingly, the overexpression of Staufen1 impaired myogenesis through the regulation of c-myc translation. Since the function of c-myc in oncogenesis is well described, we investigated the role of Staufen1 in cancer biology. In particular, we determined novel functions of Staufen1 in rhabdomyosarcoma tumorigenesis, thus providing the first direct evidence for Staufen1’s involvement in cancer. Moreover, based on Staufen1’s role in myogenic differentiation and in myotonic dystrophy type I, we generated muscle-specific transgenic mice to examine the impact of sustained Staufen1 expression in postnatal skeletal muscle. Staufen1 transgenic mice developed a myopathy characterized by histological and functional abnormalities via atrogene induction and the regulation of PTEN mRNAs. In parallel, we further investigated Staufen1-regulated alternative splicing and our data demonstrated that Staufen1 regulates multiple alternative splicing events in normal and myotonic dystrophy type I skeletal muscles, both beneficial and detrimental for the pathology. Collectively, these findings uncover several novel functions of Staufen1 in skeletal muscle biology and highlight Staufen1’s role as a disease modifier in DM1.

La protéine de liaison à l’ARN double-brin Staufen1 (STAU1) est exprimée dans les cellules de mammifères de manière ubiquitaire. STAU1 est impliqué dans la régulation post-transcriptionnelle de l’expression génique grâce à sa capacité de lier les ARN et moduler leur épissage, leur transport et localisation, leur traduction ainsi que leur dégradation. Des études récentes de notre laboratoire indiquent que l’expression de STAU1 est régulée durant le cycle cellulaire, ayant une abondance maximale au début de la mitose. En prométaphase, STAU1 est lié à des ARNm codant pour des facteurs impliqués dans la régulation de la prolifération, la croissance et la différenciation cellulaires. De plus, des analyses protéomiques menées sur des cellules humaines ont permis d’identifier STAU1 comme un composant de l’appareil mitotique. Cependant, l’importance de cette association n’a pas été investiguée. Par ailleurs, il a été montré qu’une défaillance dans l’expression ou les fonctions de STAU1 pourrait contribuer au développement et l’accélération de plusieurs maladies débilitantes, dont le cancer. Dans cette thèse, nous avons montré la localisation de STAU155 sur le fuseau mitotique dans les cellules de cancer colorectal HCT116 et les cellules non transformées hTERT-RPE1. Nous avons également caractérisé le déterminant moléculaire impliqué dans l’interaction entre STAU155 et les microtubules mitotiques, soit la séquence située dans les 88 premiers acides aminés N-terminaux de RBD2, un domaine qui n’est pas requis pour l’activité de liaison à l’ARN de STAU1. Nous avons ainsi montré que la fraction de STAU1 enrichie sur le fuseau colocalise avec des ribosomes dans des sites actifs de traduction. De plus, notre analyse transcriptomique du fuseau mitotique montre que 1054 transcrits (ARNm, pré-ARNr, lncRNA et snoRNA) sont enrichis sur l’appareil mitotique. De façon intéressante, le knockout de STAU1 entraine la délocalisation des pré-ARNr et de 154 ARNm codants pour des protéines impliquées dans l’organisation du cytosquelette d'actine et la croissance 4 cellulaire. Bien que STAU1 n’est pas essentiel pour la survie et la prolifération des cellules cancéreuses HCT116, nos résultats mettent clairement en évidence l’implication de STAU1 dans la régulation des ARN spécifiques en mitose et suggèrent un nouveau rôle de cette protéine dans la progression mitotique et la cytokinèse par la régulation de la maintenance des pré-ARNr, la ribogenèse et/ou la reconstitution de l’enveloppe nucléaire. / Staufen1 (STAU1) is a double-stranded RNA-binding protein that is ubiquitously expressed in mammals and known for its involvement in the post-transcriptional regulation of gene expression such as splicing, transport and localization, translation, and decay. It has been demonstrated that STAU1 protein expression level is modulated through the cell cycle with peak abundance by the onset of the mitotic phase after which it is degraded. Genome-wide analysis revealed that in prometaphase, STAU1 bound with mRNAs code for factors implicated in cell differentiation, cell growth as well as for cell proliferation. Interestingly, previous large-scale proteomic studies identified STAU1 as a component of the human mitotic spindle apparatus. Altering STAU1 expression patterns or functions may lead to several debilitating human diseases including cancer. In this thesis, we further elucidated the localization of STAU1 at the mitotic spindle of the colorectal cancer HCT116 and the non-transformed hTERT-RPE1 cells. Next, we characterized the molecular determinant required for STAU1/spindle association within the first 88 N-terminal amino acids, a domain that is not required for the RNA binding activity. RNA-Seq analysis of purified mitotic spindles reveals that 1054 mRNAs as well as the precursor ribosomal RNA, lncRNAs and snoRNAs are enriched on spindles compared to cell extracts. Spindle-associated STAU1 partly co-localizes with ribosomes and active sites of translation. Interestingly, the knockout of STAU1 delocalizes pre-rRNA and 154 mRNAs coding for proteins involved in actin cytoskeleton organization and cell growth. Our results highlighting a role for STAU1 in mRNA trafficking to the spindle. These data demonstrate that STAU1 controls the localization of sub-populations of RNA during cell division and suggests a novel role of STAU1 protein in mitotic progression and cytokinesis by regulating pre-rRNA maintenance, ribogenesis and/or nucleoli reassembly.

Hematopoietic stem cells (HSCs) are critical to the development of the hematopoietic system during ontogeny and maintaining hematopoiesis under steady-state. Several genes implicated in asymmetric cell division (ACD) have been found to influence HSC self-renewal in normal hematopoiesis and various leukemias. From a separate survey of genes associated with ACD, I now present the results from dedicated functional studies on two genes – Arhgef2 and Staufen1 – in HSCs and identify their potential contributions to benign hematopoietic disorders. Specifically, I present evidence that demonstrates a conserved role of Arhgef2 in orienting HSC division, the loss of which leads to HSC exhaustion that may underlie and contribute to the pathogenesis of Shwachman-Diamond syndrome. I also identify Staufen1 as a critical RNA-binding protein (RBP) in HSC function, downregulation of which elicits expression signatures consistent with clinical anemias reminiscent of aplastic anemia and/or paroxysmal nocturnal hemoglobinuria. I end by reviewing how RBPs function in HSCs and discuss future research directions that could further elucidate how bone marrow failure syndromes arise at the stem cell level. / Thesis / Doctor of Philosophy (PhD)

Staufen1 (STAU1) est une protéine de liaison à l’ARN essentielle dans les cellules non-transformées. Dans les cellules cancéreuses, le niveau d’expression de la protéine est critique et étroitement lié à des évènements d’apoptose et des altérations dans la prolifération cellulaire. Le dsRBD2 de STAU1 lie des facteurs protéiques qui sont fondamentaux pour les fonctions de la protéine, telles que la liaison aux microtubules qui garantit sa localisation au fuseau mitotique et l’interaction avec les coactivateurs de l’E3 ubiquitine-ligase APC/C, ce qui garantit la dégradation partielle de STAU1 en mitose. Nous avons cartographié un nouveau motif F39PxPxxLxxxxL50 (motif FPL) dans le dsRBD2 de STAU1. Ce motif est fondamental pour l’interaction de la protéine avec les co-activateurs de l’APC/C, CDC20 et CDH1, et sa dégradation subséquente. Nous avons ensuite identifié un total de 15 protéines impliquées dans le processus inflammatoire qui partagent cette séquence avec STAU1. Nous avons prouvé, par des essais de co-transfection et de dégradation, que MAP4K1, l’une des protéines qui partagent ce motif, est aussi dégradé via ce motif FPL. Cependant, le motif de MAP4K1 n’est pas la cible de l’APC/C. Des techniques de biotinylation des protéines à proximité de STAU1 nous ont permis d’identifier TRIM25, une E3 ubiquitine ligase impliquée dans la régulation immunitaire et l’inflammation, comme protéine impliquée dans la dégradation de STAU1 et de MAP4K1 via le motif FPL. Ceci suggère des rôles de STAU1 dans la régulation du processus inflammatoire, ce qui est consistent avec des études récentes qui associent STAU1 à ce processus. Nous considérons que le motif FPL pourrait être à la base de la coordination de la régulation des protéines impliquées dans l’inflammation et la régulation de la réponse immune. Nos études sur l’effet anti-prolifératif de STAU1 lorsque surexprimé dans les cellules transformées ont identifié le domaine dsRBD2 de STAU1 comme responsable de ce phénotype. Des mutants qui miment les différents états de phosphorylation de la serine 20, située dans le domaine dsRBD2, sont à la base des changements dans la régulation de la traduction et la dégradation des ARNm liés à STAU1. Ces changements dans la régulation des ARNm par STAU1 sont associés aux altérations dans la prolifération des cellules transformées observées lors de la surexpression de STAU1. Nous avons aussi découvert que, après la transfection de STAU1, la cellule déclenche rapidement des évènements d’apoptose, et que ces évènements sont aussi dépendants de l’état de phosphorylation de la sérine 20 dans dsRBD2 de STAU1. Ces résultats suggèrent que STAU1 est un senseur qui contrôle la balance entre la survie et la prolifération cellulaire et que l’état de phosphorylation de son dsRBD2 est à la base de ce contrôle. Nos résultats indiquent que le dsRBD2 de STAU1 est le domaine de régulation du niveau d’expression protéique et un modulateurs des rôles de la protéine comme facteur post-transcriptionnel. Nous pensons que cibler la régulation de STAU1 et ses fonctions situées dans son domaine dsRBD2, serait important dans l’étude des maladies qui impliquent des événements d’apoptose, d’inflammation et de prolifération cellulaire telles que le cancer. / Staufen1 (STAU1) is an RNA-binding protein essential in untransformed cells. In cancer cells, the level of expression of the STAU1 protein is critical and it has been closely linked to events of apoptosis and to cell proliferation impairments. STAU1's dsRBD2 binds protein factors that are fundamental for the protein's functions, such as microtubules components that ensure the protein localization to the mitotic spindle and its interaction with E3 ubiquitin-ligase APC/C coactivators, which guarantees the partial degradation of STAU1 during mitosis. By mapping a novel F39PxPxxLxxxxL50 motif (FPL motif) in the dsRBD2 of STAU1, responsible of the interaction with the co-activators of APC/C, CDC20 and CDH1, and its subsequent degradation, we were able to identify a total of 15 proteins mostly involved in the inflammatory process that share this sequence with STAU1. We proved, by co-transfection and degradation assays that, MAP4K1, one of the proteins that shares this motif, is also degraded via this FPL motif. However, we demonstrated that this motif on MAP4K1 is not the target of APC/C. Biotinylation techniques of proteins near STAU1 allowed us to identify TRIM25, an E3 ubiquitin ligase involved in immune regulation and inflammation, as a protein involved in the degradation of STAU1 and MAP4K1 via the FPL motif. This suggests roles of STAU1 in the regulation of the inflammatory events, which is consistent with recent studies that associate STAU1 with this process. We consider that the FPL motif could be at the basis of the coordination of the regulation of proteins involved in inflammation and the regulation of the immune response. Our studies on the anti-proliferative effect of STAU1 when overexpressed in transformed cells identified the domain dsRBD2 of STAU1 as responsible for this phenotype. Mutants 8 that mimic different phosphorylation states of serine 20, located in dsRBD2, underlie changes in the regulation of translation and degradation of STAU1-linked mRNAs. These STAU1-dependent changes in mRNA regulation are associated with the proliferation impairment of transformed cells that is observed upon overexpression of STAU1. We also discovered that, after STAU1 transfection, the cell rapidly triggers apoptotic events, and that these events are also dependent on the phosphorylation state of serine 20 in dsRBD2 of STAU1. These results suggest that STAU1 is a sensor that controls the balance between cell survival and cell proliferation and that the state of phosphorylation of its dsRBD2 is the basis of this control. Our results indicate that the dsRBD2 of STAU1 is the regulatory domain of the level of protein expression and a modulator of the protein roles as a post-transcriptional factor. We believe that targeting the regulation of STAU1 and its functions located in its dsRBD2 domain, would be important in the study of diseases that involve apoptosis, inflammation and cell proliferation events such as cancer.