In eukaryotes, the regulation of mRNA translation and decay provides a mechanism which can be finely tuned to control gene expression. This ability to control the life cycle of an mRNA begins with the control of mRNA export from the nucleus and extends to the processes which regulate the degradation of the message. In my work, summarized below, I describe how some of the proteins involved in cytoplasmic decay regulate many aspects of the control of mRNA and also describe a novel regulatory mechanism involving the relocation of cytoplasmic mRNA back into the nucleus of the cell.Firstly, I have identified that the protein Pat1, which has been shown to be critical for translational repression and activation of decapping, consists of essentially 3 major domains. By means of a deletional functional analysis, I show that two of these domains are the primary functional domains responsible for all of the currently ascribed function of Pat1. One domain promotes translational repression and P-body assembly, while the second domain promotes mRNA decapping after assembly of the mRNA into a P-body mRNP. Along with the first evidence that Pat1 binds to RNA, we also determine numerous domain-specific interactions with mRNA decapping factors.In eukaryotic cells mRNAs are produced in the nucleus followed by what is thought to be unidirectional export to the cytoplasm. In the cytosol, mRNAs either associate with ribosomes for translation or can be found in cytoplasmic RNP granules, termed P-bodies, when they are translationally repressed. I have now demonstrated that yeast mRNAs can be re-imported into the nucleus. Import of mRNAs into the nucleus is in competition with translation and increased in strains lacking specific components of cytoplasmic processing bodies, which also exhibit nuclear-cytoplasmic shuttling. This indicates that one function of cytoplasmic granules is to limit the import of cytoplasmic mRNAs back into the nucleus. These results demonstrate a novel pathway for mRNA import into the nucleus and suggest distinct pathways of mRNA export of nascent mRNAs and imported mRNAs.

Ce travail montre qu’une protéine non encore décrite dans les phagocytes, la PAT1 « Protein interacting with APP Tail 1 », interagit avec la partie cytosolique de la p22phox (composant du cytochrome b558 membranaire de la NADPH oxydase). Nous avons utilisé différentes approches pour montrer cette interaction : le système double hybride, la technique de GST-pull down, la microscopie confocale et la technique de co-immunoprécipitation. De plus, nous avons montré que la PAT1a recombinante augmente l’activité de la NADPH oxydase, in vitro dans un système acellulaire reconstitué, et dans les cellules intactes (monocytes et cellules COS-phox). Cette nouvelle interaction régule donc l’activation de la NADPH oxydase et la production des FRO. Par ailleurs, la liaison de PAT1 aux microtubules pourrait favoriser l’assemblage du complexe NADPH oxydase pendant son activation. Ceci pourrait conduire à l’identification de nouvelles cibles thérapeutiques qui préviennent la survenue des lésions tissulaires dans les maladies inflammatoires. / Reactive oxygen species (ROS) production by the phagocyte NADPH oxidase plays a crucial role in host defenses. NADPH oxidase is composed of the membrane flavocytochrome b558 components (p22phox and gp91phox/NOX2), and cytosolic components (p40phox, p47phox, p67phox and a small GTPase Rac1 or Rac2). In this work we identified PAT1 by double hybrid system as a potential partner of p22phox. The interaction between p22phox and PAT1a was further confirmed by in vitro GST pull-down assay, confocal microscopy and co-immunoprecipitation. Addition of recombinant PAT1a to the cell free-system enhanced NADPH oxidase activation and it’s over-expression in human monocytes and in COSphox cells increased ROS production in resting and fMLP-stimulated cells.These data clearly identify PAT1 as a novel regulator of NADPH oxidase activation in phagocytes.Inhibition of p22phox/PAT1 interaction could be used as new approach to limit ROS production by phagocytes at inflammatory sites.

Certaines protéines régulatrices dites nucléo-cytoplasmique naviguent entre le noyau et le cytoplasme. En réponse à une perturbation de l'environnement de la cellule, ces protéines relocalisent massivement dans le noyau pour y activer des mécanismes permettant la survie cellulaire. Chez la levure S. pombe, 285 protéines présentent la particularité d'être nucléo-cytoplasmiques. Une étude exhaustive de certaines de ces protéines a été ici entreprise. L'objectif était d'identifier celles présentes chez S. cerevisiae qui sont vitales dans la réponse aux stress endommageant l'ADN. Parmi les protéines candidates, celles i) étant les plus conservées chez l'ensemble des organismes eucaryotes, ii) de fonction inconnue ou peu décrite et iii) dont la répartition nucléo-cytoplasmique change après stress ont été sélectionnées à l'aide d'un crible génétique mis au point chez S. cerevisiae. Douze protéines ont été identifiées comme relocalisant au noyau sous l'effet du stress radiatif ou métaux lourds. Parmi elles, la protéine Pat1 (YCR077C) décrite à ce jour comme un activateur de la dégradation cytoplasmique des ARNs messagers a été choisie et une étude plus approfondie de son activité entreprise. Par une approche TAP-tag couplée à une stratégie de protéomique de type shotgun, le réseau de partenaires protéiques de Pat1 a été établi en absence de stress et en condition de stress UV. La région potentiellement impliquée dans la localisation cellulaire de la protéine Pat1 est sujette à de multiples phosphorylations dont le degré augmente après stress UV. Enfin, les résultats sur les partenaires spécifiques de Pat1 identifiés par protéomique en condition de stress ont été corroborés par une analyse de sa relocalisation chez les différents mutants correspondants. L'ensemble de nos résultats mettent en exergue une nouvelle fonctionnalité pour la protéine Pat1 spécifiquement menée au noyau des cellules qu'il reste désormais à préciser. / Some regulatory proteins called nucleo-cytoplasmic proteins, shuttle between the nucleus and the cytoplasm. Upon environmental stress, these proteins relocate massively to the nucleus where they activate pro-survival mechanisms. In the yeast S. pombe, 285 proteins are nucleo-cytoplasmic. An exhaustive study of some of these proteins was carried out herein. The goal was to identify the ones that are present in S. cerevisiae and vital in the DNA damage response. Among the candidate proteins, the ones i) that are the most conserved in the eukaryotic cells, ii) with unknown function or poorly characterized, and iii) whose nucleo-cytoplasmic repartition changes upon stress were selected by the use of a genetic screen monitored in S. cerevisiae. Twelve proteins were found to accumulate in the nucleus upon irradiating or heavy metal stresses. Pat1 (YCR077C) currently described as a cytosolic mRNA decay activator was chosen and a more complete investigation about its activity was undertaken. By the mean of a TAP-tag approach combined with a shotgun proteomic analysis, the Pat1 interaction network was established without any stress and after UV stress illumination. Pat1 exhibits a domain potentially involved in its relocation that is subjected to multiple phosphorylations whose state enhances after UV stress. Finally, the data about the specific partners of Pat1 identified by proteomic analysis in stress condition were confirmed by the study of Pat1 relocation in the corresponding deleted strains. Altogether, our data suggest a novel function for the Pat1 protein that needs to be further investigated.

Messenger RNA degradation is crucial for the regulation of eukaryotic gene expression. It not only modulates the basal mRNA levels but also functions as a quality control system, thereby controlling the availability of mRNA for protein synthesis. In Saccharomyces cerevisiae, the first and the rate-limiting step in the process of mRNA degradation is the shortening of the poly(A) tail by deadenylation complex. After the poly(A) tail shortens, mRNA can be degraded either through the major 5' to 3' decapping dependent or the 3' to 5' exosome-mediated degradation pathway. In this thesis, we show some of the means by which mRNA decay factors can modulate gene expression. First, Pat1 is a major cytoplasmic mRNA decay factor that can enter the nucleus and nucleo-cytoplasmically shuttle.  Recent evidence suggested several possible nuclear roles for Pat1. We analyzed them and showed that Pat1 might not function in pre-mRNA decay or pre-mRNA splicing, but it is required for normal rRNA processing and transcriptional elongation. We show that the mRNA levels of the genes related to ribosome biogenesis are dysregulated in the strain lacking Pat1, a possible cause of the defective pre-rRNA processing. In conclusion, we theorize that Pat1 might regulate gene expression both at the level of transcription and mRNA decay. Second, Edc3 and Lsm4 are mRNA decapping activators and mRNA decay factors that function in the assembly of RNA granules termed P bodies. Mutations in mRNA degradation factors stabilize mRNA genome-wide or stabilize individual mRNAs. We demonstrated that paradoxically, deletion of Edc3 together with the glutamine/asparagine-rich domain of Lsm4 led to a decrease in mRNA stability. We believe that the decapping activator Edc3 and the glutamine/asparagine-rich domain of Lsm4 functions together, to modify mRNA decay pathway by altering cellular mRNA decay protein abundance or changing the mRNP composition or by regulating P bodies, to enhance mRNA stability. Finally, mRNA decay was recently suggested to occur on translating ribosomes or within P bodies. We showed that mRNA degradation factors associate with large structures in sucrose density gradients and this association is resistant to salt and sensitive to detergent. In flotation assay, mRNA decay factors had buoyancy consistent with membrane association, and this association is independent of stress, translation, P body formation or RNA. We believe that such localization of mRNA degradation to membranes may have important implications in gene expression. In conclusion, this thesis adds to the increasing evidence of the importance of the mRNA degradation factors in the gene expression.

Ce travail montre qu'une protéine non encore décrite dans les phagocytes, la PAT1 " Protein interacting with APP Tail 1 ", interagit avec la partie cytosolique de la p22phox (composant du cytochrome b558 membranaire de la NADPH oxydase). Nous avons utilisé différentes approches pour montrer cette interaction : le système double hybride, la technique de GST-pull down, la microscopie confocale et la technique de co-immunoprécipitation. De plus, nous avons montré que la PAT1a recombinante augmente l'activité de la NADPH oxydase, in vitro dans un système acellulaire reconstitué, et dans les cellules intactes (monocytes et cellules COS-phox). Cette nouvelle interaction régule donc l'activation de la NADPH oxydase et la production des FRO. Par ailleurs, la liaison de PAT1 aux microtubules pourrait favoriser l'assemblage du complexe NADPH oxydase pendant son activation. Ceci pourrait conduire à l'identification de nouvelles cibles thérapeutiques qui préviennent la survenue des lésions tissulaires dans les maladies inflammatoires.