Chez la levure fissipare S. pombe, certains gènes méiotiques sont exprimés de façon constitutive pendant la croissance végétative. Cependant, pour empêcher le déclenchement prématuré de la méiose, la cellule a mis en place un système de dégradation sélective des ARN messagers correspondant. La protéine de liaison à l’ARN Mmi1, de la famille YTH, reconnaît des répétitions de motifs spécifiques (UNAAAC) au sein des transcrits et dirige ces derniers vers la dégradation par l’exosome nucléaire. Lors de l’entrée en méiose, Mmi1 est séquestré par un complexe ribonucléoprotéique comprenant la protéine de méiose Mei2 et l’ARN noncodant meiRNA, ce qui permet aux ARNm méiotiques d’être exportés et traduits. Au cours de ma thèse, je me suis intéressé au rôle de Mmi1 dans la dégradation des transcrits méiotiques pendant la croissance végétative. En accord avec des études récentes, nos travaux montrent que Mmi1 interagit étroitement avec le complexe Ccr4-Not de déadenylation des ARNm. Cette interaction est fonctionnelle car Ccr4-Not est requis pour la dégradation des ARNs méiotiques. De façon surprenante, cependant, l’activité de déadénylation n’est pas requise. Nos analyses génétiques et biochimiques suggèrent que la sous-unité E3 ubiquitin ligase Mot2 de Ccr4-Not ubiquitine un pool de l’inhibiteur de Mmi1, la protéine Mei2, pour faciliter sa dégradation par le protéasome. Cette voie de régulation permet de maintenir la fonction de Mmi1 et donc la répression des ARNm méiotiques dans les cellules mitotiques. Ainsi, Mmi1 a une double fonction: cibler les ARNm méiotiques vers la dégradation par l’exosome nucléaire, et recruter Ccr4-Not pour ubiquitiner et dégrader son propre inhibiteur Mei2. Ces résultats mettent également en avant un nouveau rôle pour la sous-unité E3 ligase du complexe Ccr4-Not dans le contrôle de la différenciation sexuelle. Des expériences supplémentaires indiquent que le domaine YTH de liaison à l’ARN de Mmi1, mais pas l’ARN noncodant meiRNA, est requis pour la dégradation de Mei2. De façon importante, nos données révèlent aussi que le domaine YTH de Mmi1 a un rôle clé dans l’interaction avec Mei2. Ceci suggère fortement que le domaine YTH agit comme un module bifonctionnel, permettant la liaison non seulement aux ARNs méiotiques mais aussi aux protéines comme Mei2. Nous discutons ces résultats dans le contexte de la littérature actuelle et proposons un nouveau modèle du contrôle de la différenciation sexuelle par le système Mmi1-Mei2. / In the fission yeast S. pombe, several meiotic mRNAs are constitutively expressed during the mitotic cell cycle. In order to avoid untimely entry into meiosis, cells have adopted a degradation system that selectively eliminates the corresponding mRNAs. The YTH family RNA-binding protein Mmi1 recognizes specific sequence motifs within these transcripts (UNAAAC) and allows their targeting to the nuclear exosome for degradation. Upon entry into meiosis, Mmi1 is sequestered in a ribonucleoprotein complex, composed by the meiotic protein Mei2 and the non-coding RNA meiRNA, allowing meiotic mRNAs to be exported and translated. During my PhD studies, I focused my interest on the role of Mmi1 in the degradation of meiotic transcripts during vegetative growth. In accord with recent studies, our results show that Mmi1 stably interacts with the mRNA deadenylation complex Ccr4-Not. This interaction has a functional relevance since Ccr4-Not is involved in the degradation of meiotic mRNAs. Surprisingly, however, the deadenylation activity is not required. Our genetic and biochemical analyses indicate that the E3 ubiquitin ligase Mot2, subunit of the Ccr4-Not complex, ubiquitinate a pool of the inhibitor of Mmi1, the Mei2 protein, to favor its degradation by the proteasome. This regulatory mechanism ensures the maintenance of Mmi1 in a functional state, leading to the persistent repression of meiotic mRNAs in mitotic cells. Thus, Mmi1 has a dual role: in nuclear mRNA surveillance, by targeting meiotic transcripts for degradation by the exosome, and in protein degradation, by recruiting Ccr4-Not to its own inhibitor Mei2. These results have also revealed a novel role for the ubiquitin ligase activity of the Ccr4-Not subunit Mot2 in the control of sexual differentiation in fission yeast. Our supplemental results indicate that the YTH RNA-binding domain of Mmi1, but not the non-coding RNA meiRNA, is required for the degradation of Mei2. Remarkably, our results also revealed that the YTH domain of Mmi1 has a key role in the interaction with Mei2. This strongly suggests that the YTH domain acts as a bifunctional module, allowing the binding not only to meiotic RNAs but also to proteins, such as Mei2. We discuss these results within the context of the current literature and we propose a novel model for the control of sexual differentiation by the Mmi1-Mei2 system.

The study of RNA-binding proteins has recently increased in importance due to discoveries of their larger role in cellular processes. One study currently conducted at Umeå University involves constructing a model that will be able to improve our knowledge about T-cells by explaining how these cells work in different diseases. But before this model can become a reality, Umeå Univerity needs to investigate the relation between RNA and RNA-binding proteins and find proteins of which highly contribute to the activity of the RNA-binding proteins. To do so, they have decided to use four penalized regression Machine Learning models to analyse protein sequences from CD4 cells. These models consist of a ridge penalized model, an elastic net model, a neural network model, and a Bayesian model. The results show that the models have a number of RNA-binding protein sequences in common which they list as highly decisive in their predictions.

Many intricate and highly conserved mechanisms have evolved to safeguard organisms against errors in gene expression. The nonsense-mediated mRNA decay pathway (NMD) exemplifies one such mechanism, specifically by eliminating mRNAs containing premature translation termination codons within their protein coding regions, thereby limiting the synthesis of potentially deleterious truncated polypeptides. Studies in Saccharomyces Cerevisiae have found that the activity of at least three trans-acting factors, known as UPF1, UPF2/NMD2, and UPF3is necessary for the proper function of the NMD pathway. Further research conducted in yeast indicates that the degradation of substrates of the NMD pathway is dependent on their translation, and that the sub-cellular site of their degradation in the cytoplasm. Although most evidence in yeast suggests that substrates of the NMD pathway are degraded in the cytoplasm while in association with the translation apparatus, some mammalian studies have found several mRNAs whose decay appears to occur within the nucleus or before their transport to the cytoplasm has been completed. In addition, study of the mammalian TPI mRNA found that this transcript was unavailable as a substrate for the NMD pathway once it had been successfully exported to the cytoplasm, further supporting the notion that the degradation of mammalian substrates of the NMD pathway occurs in association with the nucleus, or during export from the nucleus to the cytoplasm. To determine if yeast cytoplasmic nonsense-containing mRNA can become immune to the NMD pathway we examined the decay kinetics of two NMDS substrate mRNAs in response to repressing or activating the NMD pathway. Both the ade2-1 and pgk1-UAG-2nonsense-containing mRNAs were stabilized by repressing this pathway, while activation of NMD resulted in the rapid and immediate degradation of each transcripts. These findings demonstrate that nonsense-containing mRNAs residing in the nucleus are potentially susceptible to NMD at each round of translation. The remainder of this thesis utilizes protein overexpression studies to gain understanding into the function of factors related to the processes of nonsense-mediated mRNA decay and translation in Saccharomyces cerevisiae. Overexpression of a C-terminal truncated form of Nmd3p was found to be dominant-negative for cell viability, translation and the normal course of rRNA biogenesis. Overexpression studies conducted with mutant forms of the nonsense-mediated mRNA decay protein Upf1p, found that overexpression of mutants in the ATP binding and ATP hydrolysis region ofUpflp were dominant-negative for growth in an otherwise wild-type yeast strain. Furthermore, overexpression of the ATP hydrolysis mutant of Upf1p (DE572AA), resulted in the partial inhibition of NMD and a general perturbation of the translation apparatus. These results support previous studies suggesting a general role for Upf1p function in translation.

L’aldostérone et le Récepteur Minéralocorticoïde (MR) participent au contrôle de la balance hydrosodée et de la pression artérielle. Les altérations de l’expression du MR ou de la signalisation minéralocorticoïde sont associées à de nombreuses pathologies chez l’Homme. Dans ce travail, nous avons démontré, le rôle majeur de protéines de liaison à l’ARN, Tis11b et HuR, dans le contrôle post-transcriptionnel de l’expression du MR en réponse aux variations de tonicité extracellulaire dans un modèle de cellules principales rénales et chez la souris. L’hypertonicité (500 mOsmol/L) induit l’expression de la protéine Tis11b, qui lie la région 3’-non traduite du transcrit MR afin d’accélérer sa dégradation, diminuant ainsi l’expression rénale de la protéine MR et de la signalisation minéralocorticoïde. A l’opposé, l’hypotonicité (150 mOsmol/L) stimule la translocation nucléo-cytoplasmique de HuR, qui stabilise le transcrit MR, augmentant ainsi l’expression du MR et la sensibilité rénale à l’aldostérone. De plus, HuR est responsable de l’édition d’un nouveau variant d’épissage du MR, le variant MR Δ6, obtenu par l’exclusion de l’exon 6.Ce variant d’épissage exerce un effet dominant négatif sur la signalisation minéralocorticoïde. Enfin, l’identification de microARN modulés par l’hypertonicité suggère leur rôle potentiel dans le contrôle de la signalisation minéralocorticoïde rénale. La caractérisation de ces mécanismes inédits modulant l’action du MR améliore notre compréhension de la physiopathologie de la signalisation minéralocorticoïde, et pourrait aboutir, à terme, à de nouvelles stratégies thérapeutiques. / Aldosterone and the Mineralocorticoid Receptor (MR) participate to the control of salt and water balance and the arterial pressure. Alteration of renal MR expression or mineralocorticoid signaling pathway contributes to the development of numerous human disorders. In this work, we have demonstrated the major role played by the RNA-Binding Proteins, Tis11b and HuR, in the control of MR expression in response to variations of extracellular tonicity in a model of principal tubular cells and in vivo. Hypertonicity (500 mOsmol/L) increases the expression ofTis11b, which binds the 3’-untranslated region of MR transcript and accelerates the degradation of MR transcript, leading to the reduction of the mineralocorticoid signaling. Conversely, hypotonicity (150 mOsmol/L) stimulates nuclear-cytoplasmic shuttling of HuR protein, which stabilizes MR transcript increasing its expression and renal sensitivity to aldosterone action. Furthermore, HuR participates to the editing of the novel MR Δ6 splice variant, which lacks exon 6, and exerts a dominant negative effect on mineralocorticoid signaling. Finally, we have provided evidence that hypertonicity modulates expression of microRNA, which may control mineralocorticoid signaling pathway. Characterization of these original mechanisms modulating MR action is pivotal for a better understanding of mineralocorticoid-related pathophysiology, and should ultimately lead to the development of new therapeutic strategies.

The development of an organism and its adult homeostasis rely on regulatory mechanisms that control the underlying gene expression programs. In certain biological contexts, such as germ cell development, gene expression regulation is largely executed at the post-­‐transcriptional level. This relies on RNA-­‐binding proteins (RBPs), whose activity and expression are also heavily controlled. While the RNA-­‐binding potential of RBPs is currently of intense scrutiny, surprisingly little is known to date about the molecular mechanisms that control RNA-­‐binding proteins abundance in the context of germ cell development. This work identifies the molecular mechanisms that shape expression patterns of two evolutionarily conserved RNA-­‐binding proteins, CPB-­‐3 and GLD-­‐ 1, which belong to CPEB and STAR protein family, respectively. By focusing on their regulation in the C. elegans germ line, this work reveals an involvement of the proteasome in reducing levels of CPB-­‐3/CPEB and GLD-­‐1/STAR at the pachytene-­‐to-­‐diplotene transition during meiotic prophase I. Furthermore, it documents that CPB-­‐3 and GLD-­‐1 are targeted to proteasomal degradation by a conserved SCF ubiquitin ligase complex that utilises SEL-­‐10/Fbxw7 as a substrate recognition subunit. Importantly, destabilisation of both RBPs is likely triggered by their phosphorylation, which is regulated by the mitogen-­‐activated protein kinase, MPK-­‐1, and restricted to the meiotic timepoint of pachytene exit. Lastly, this work investigates the potential consequences of target mRNA regulation upon delayed RBP degradation. Altogether, the collected data characterise a molecular pathway of CPEB and STAR protein turnover, and suggest that MPK-­‐1 signaling may couple RBP-­‐mediated regulation of gene expression to progression through meiosis during oogenesis.

Maternally supplied mRNAs encode for necessary developmental regulators that pattern early embryos in many species until zygotic transcription is activated. In Caenorhabditis elegans, post-transcriptional regulatory mechanisms guide early development during embryogenesis. Maternal transcripts remain in a translationally silenced state until fertilization. A suite of RNA-binding proteins (RBP’s) regulate these maternally supplied mRNAs during oogenesis, the oocyte-to-embryo transition, and early embryogenesis. Identifying the target specificity of these RNA-binding proteins will reveal their contribution to patterning of the embryo. We are studying post-transcriptional regulation of maternal mRNAs during oocyte maturation, which is an essential part of meiosis that prepares oocytes for fertilization. Although the physiological events taking place during oocyte maturation have been well studied, the molecular mechanisms that regulate oocyte maturation are not well understood. OMA-1 and OMA-2 are essential CCCH-type tandem zinc finger (TZF) RBP’s that function redundantly during oocyte maturation. This dissertation shows that I defined the RNA-binding specificity of OMA-1, and demonstrated that OMA-1/2 are required to repress the expression of 3ʹUTR reporters in developing oocytes. The recovered sequences from in vitro selection demonstrated that OMA-1 binds UAA and UAU repeats in a cooperative fashion. Interestingly, OMA-1 binds with high affinity to a conserved region of the glp-1 3ʹUTR that is rich in UAA and UAU repeats. Multiple RNA-binding proteins regulate translation of GLP-1 protein, a homolog of Notch receptor. In addition to previously identified RBP’s, we showed that OMA-1 and OMA-2 repress glp-1 reporter expression in C. elegans oocytes. Mapping the OMA-1 dependent regulatory sites in the glp-1 mRNA and characterizing the interplay between OMA-1 and other factors will help reveal how multiple regulatory signals coordinate the transition from oocyte to embryo but the abundance of OMA-1 binding motifs within the glp-1 3ʹUTR makes it infeasible to identify sites with a functional consequence. I therefore first developed a strategy that allowed us to generate transgenic strains efficiently using a library adaptation of MosSCI transgenesis in combination with rapid RNAi screening to identify RBP-mRNA interactions with a functional consequence. This allowed me to identify five novel mRNA targets of OMA-1 with an in vivo regulatory connection. In conclusion, the findings in this dissertation provide new insights into OMA-1 mediated mRNA regulation and provide new tools for C. elegans transgenesis. Development of library MosSCI will advance functional mapping of OMA-1 dependent regulatory sites in the target mRNAs. Extending this strategy to map functional interactions between mRNA targets and RNAbinding proteins in will help reveal how multiple regulatory binding events coordinate complex cellular events such as oocyte to embryo transition and cell-fate specification.

Post-transcriptional regulation of gene expression sits at the core of proteomic complexity; trans-acting factors that regulate RNA localization and translation capacity are thus indispensible. In this thesis, I present studies of the cytoplasmic polyadenylation element binding protein (CPEB), a sequence specific RNA-binding protein important for cell cycle progression and neural synaptic plasticity. I focus on CPEB because the activity of RNA-binding proteins affects the destiny of their mRNA substrates. As presented in Chapter II, CPEB, though mostly cytoplasmic at steady state, shuttles between the nucleus and the cytoplasm. Surprisingly, the RNA recognition motifs are essential for the nuclear localization. CPEB associates with the polyadenylation machinery in both compartments, suggesting it is involved in both nuclear mRNA processing and cytoplasmic translational regulation. Moreover, the nuclear translocalization is critical to relay a tight translation repression on CPE-containing mRNAs. Chapter III focuses on the regulation of CPEB dimerization. CPEB dimerizes through the RNA-binding domains to inhibit its own RNA binding ability in a cell cycle-dependent manner. By dimerizing, CPEB has enhanced binding to protein destruction factors so that robust active degradation occurs in the later cell cycle. The degradation of CPEB is required for translation activation of a subset of mRNAs and cell cycle progression. In addition, dimerization protects cells from being overloaded with excess CPEB. In sum, the localization and dimerization status of CPEB is dynamic and highly regulated; they in turn regulate the activity of CPEB, which results in responsive translation control. These studies provide a strong foundation to decipher CPEB-mediated gene expression.

Post-transcriptional regulation of gene expression is a mechanism that governs developmental and cellular events in metazoans. In early embryogenesis, transcriptionally quiescent cells depend upon maternally supplied factors such as RNA binding proteins and RNA that control key decisions. Morphogen gradients form and in turn pattern the early embryo generating different cell types and spatial order. In the nematode Caenorhabditis elegans, the early embryo relies upon several RNA binding proteins that control mRNA stability, translation efficiency, and/or mRNA localization of cell fate determinants essential for proper development. MEX-5 and MEX-3 are two conserved RNA-binding proteins required to pattern the anterior/posterior axis and early embryo. Mutation of either gene results in a maternal effect lethal phenotype with proliferating posterior muscle into the anterior blastomeres (Muscle EXcess). Several cell-fate determinants are aberrantly expressed in mex-5 and mex-3 embryos. Both proteins are thought to interact with cis-regulatory elements present in 3’-UTRs of target RNAs controlling their metabolism. However, previous studies failed to demonstrate that these proteins regulate maternal transcripts directly. This dissertation presents a thorough assessment of the RNA binding properties of MEX-5 and MEX-3. Quantitative biochemical approaches were used to determine the RNA binding specificity of both proteins. MEX-5 has a relaxed specificity, binding with high affinity to linear RNA containing a tract of six or more uridines within an eight-nucleotide window. This is very different from its mammalian homologs Tristetraprolin (TTP) and ERF-2. I was able to identify two amino acids present within the MEX-5 RNA binding domain that are required for the differential RNA recognition observed between MEX-5 and TTP. MEX-3 on the other hand is a specific RNA binding protein, recognizing a bipartite element with flexible spacing between two four-nucleotide half-sites. I demonstrate that this element is required for MEX-3 dependent regulation in vivo. Previous studies only identify a small number of candidate regulatory targets of MEX-5 and MEX-3. The defined sequence specificity of both proteins is used to predict new putative targets that may be regulated by either protein. Collectively, this study examines the RNA binding properties of MEX-5 and MEX-3 to clarify their role as post-transcriptional regulators in nematode development.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the death of motor neurons, generally leading to paralysis and death within 3-5 years of onset. Over 50 different mutations in the gene encoding FUS/TLS (or FUS) will result in ALS, accounting for ~4% of all inherited cases. FUS is a multifunctional protein with important functions in DNA/RNA processing and stress response. How these mutations affect the structure or function of FUS protein and ultimately cause ALS is not known. The fact that mutations cause the protein to mislocalize from the nucleus to the cytoplasm of cells suggests that ALS pathogenesis may occur through a loss of nuclear function, gain of toxic cytoplasmic function, or both. Several FUS knockout animal models have been utilized for investigating a loss of function hypothesis and show phenotypes such as early lethality, reduced lifespan, and locomotor defects. To uncover cellular pathways affected by loss of FUS function, I have characterized the knockdown of FUS in a motor neuron-like cell line, NSC-34. In NSC-34 cells, the depletion of FUS severely impacts cellular proliferation and potentially causes increased levels of DNA damage. A quantitative proteomics analysis performed on cells undergoing various degrees of FUS knockdown revealed protein expression changes for known RNA targets of FUS, consistent with a loss of FUS function with respect to RNA processing. Proteins that changed in expression as a function of FUS knockdown were associated with vii multiple processes, some of which influence cell proliferation including cell-cycle regulation, cytoskeletal organization, oxidative stress and energy homeostasis. Importantly, cellular proliferation could be rescued by the re-expression of FUS and by treatment with the small-molecule, rolipram, indicative of potential therapeutic approaches. Collectively, the work presented in this dissertation demonstrates the importance of FUS for cell health and homeostasis, is suggestive of a role for FUS in DNA damage repair and identifies additional cellular pathways influenced by FUS depletion. Overall, this work provides mechanistic insight into ALS pathogenesis through loss of FUS/TLS function.

A cell integrates mitogenic signals received at the plasma membrane with intracellular biochemical changes to direct the events of cell division. Oocytes from Xenopus laevis offer a system that allows molecular dissection of pathways controlling cell growth and division in response to extracellular cues. Xenopus oocytes, physiologically arrested in a G2 like state, respond to the hormone progesterone to reinitiate meiosis and mature into a fertilizable egg. Signals received at the oocyte membrane induce translation of dormant maternal mRNAs that not only drive meiotic entry but also maintain the cell cycle arrest in an egg. A major pathway controlling the translation of these mRNAs is cytoplasmic polyadenylation, facilitated by the Cytoplasmic Polyadenylation Element Binding protein (CPEB) through cis-acting elements in their 3'untranslated regions (3'UTRs). Cytoplasmic polyadenylation requires the phosphorylation of serine174 on CPEB by Aurora-A as well as the translation of a hitherto unknown mRNA. The transcript of the RINGO/Spy gene is a putative candidate for this unknown upstream regulator of CPEB function. RINGO/Spy mRNA is translationally repressed in immature oocytes by a ribonucleoprotein (RNP) complex consisting of the repressor Pumilio-2, the putative activator Deleted in Azoospermia-like (DAZl) and embryonic poly A binding protein (ePAB). Progesterone signaling leads to the dissociation of Pumilio-2 from the mRNP and the ensuing RINGO/Spy protein synthesis, in turn, promotes cytoplasmic polyadenylation and oocyte maturation. Pumilio and its associated proteins, such as Drosophila Brain tumor (Brat) and DAZl, in addition to their cytoplasmic roles have ill-defined functions within the nucleus. We detected DAZl within the nucleoli of telomerase-immortalized human retinal pigment epithelial (RPE) cells in interphase and on acrocentric chromosomes during mitosis. DAZl colocalizes with the RNA polymerase I associated Upstream Binding transcription Factor (UBF), most likely through pre-ribosomal RNA and is a likely component of the Nucleolar Organization Region (NOR). Stably knocking down DAZl in RPEs using short hairpin RNAs results in loss of nucleolar segregation, the physiological outcome of which is under investigation. These preliminary findings indicate an additional role for DAZl within the nucleolus, one likely to be independent from cytoplasmic translational control.