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La biogenèse du ribosome est un processus complexe et dynamique qui nécessite de nombreuses étapes de maturation et de modification des ARNr ainsi que l’assemblage et le transport des RNPs précurseurs. Un ribosome mature contient une centaine de pièces, ARN et protéines confondus, mais son assemblage requiert l’intervention de plus de 400 facteurs de synthèse. De part le coût énergétique important de ce processus, plusieurs voies de régulation interviennent pour contrôler la biogenèse des ribosomes en fonction des conditions nutritives. L’une des voies les plus connue est la voie TOR (Target of rapamycin). Cette voie de régulation agît principalement au niveau de la transcription des différents intervenants de la biogenèse : les ARNr, les protéines ribosomiques mais aussi les facteurs de synthèse. Ces facteurs, ayant une action transitoire dans la maturation des ribosomes, sont, par économie, recyclés pour la synthèse de nouveaux ribosomes. Nous nous sommes donc intéressés au devenir de ces facteurs, plus particulièrement de ceux intervenants dans la biogenèse de la petite sous unité, lorsque les conditions environnementales sont inadaptées à la croissance cellulaire. Ainsi, nous avons pu montré, pour quatre facteurs particuliers : Dim2, Rrp12, Hrr25 et Fap7, que leur localisation est dépendante de la synthèse ribosomique. Ainsi, lors de carence en sources nutritives, l’inhibition de la synthèse et de l’activité ribosomique entraîne un confinement de ces facteurs ribosomiques dans le nucléole ou dans des corps cytoplasmiques. En outre, la localisation particulière des facteurs ribosomiques Hrr25 et Fap7 dans les P-bodies en phase de croissance saturée laisse penser que ces corps cytoplasmiques sont le lieu de dégradation des pré-ribosomes lorsque les carences nutritives perdurent.

Swine influenza viruses (SIV) are a common and an important cause of respiratory disease in pigs. Pigs can serve as mixing vessels for the evolution of reassortment viruses containing both avian and human signatures, which have the potential to cause pandemics. NS1 protein of influenza A viruses is a major antagonist of host defence and it regulates multiple functions during infection by interacting with a variety of host proteins. Therefore, it is important to study swine viruses and NS1-interacting host factors in order to understand the mechanisms by which NS1 regulates virus replication and exerts its host defense functions. Influenza A viruses enter the host through the respiratory tract and infect epithelial cells in the respiratory tract, which form the primary sites of virus replication in the host. Thus, studying SIV infection in primary swine respiratory epithelial cells (SRECs) would resemble conditions similar to natural infection. The objectives of this study were to identify NS1-interacting host factors in the virus-infected SRECs and to understand the physiological role of at least one of the factors in influenza virus infection. The approaches to meet this objective were to generate a recombinant SIV carrying a Strep-tag in the NS1 protein, infect SRECs with the Strep-tag virus, purify NS1-interacting host protein complex from the infected cells by pull-down using strep-tactin resin and then study the physiological role of one of the NS1-interacting partners during influenza infection. Using a reverse-genetics strategy, a recombinant virus carrying the Strep-tag NS1 was successfully rescued and the SRECs were infected with this recombinant virus. The Strep-tag in the NS1 protein facilitated the isolation of an intact NS1-interacting protein complex and the proteins present in the complex were identified by liquid chromatography-tandem mass spectrometry. The identified proteins were grouped to enrich for different functions using bioinformatics. This gave an insight into the different functions that NS1 may regulate during infection and the potential host partners involved in these functions. Among the host proteins identified as potential interaction partners, RNA helicases were particularly of interest to study. Influenza being an RNA virus, RNA helicases could have important functions in the virus life cycle. Among the identified RNA helicases, DDX3 has been shown to regulate IFNβ induction and affect the life cycle of a number of viruses. However, its function in influenza A virus life cycle has not been studied. Hence, this study explored whether DDX3 has any role in the influenza A virus life cycle. Immunoprecipitation studies revealed viral proteins NP and NS1 as direct interaction partners with DDX3. DDX3 is a known component of stress granules (SGs) and influenza A virus lacking the NS1 gene is reported to induce SG formation. Therefore, the role of DDX3 in SG formation, induced by PR8 influenza A virus lacking NS1 (PR8 del NS1) was explored. The results from this study showed that DDX3 co-localized with NP in SGs indicating that DDX3 may interact with NP in the SGs. NS1 protein was found to inhibit virus-induced SGs and DDX3 downregulation impaired virus-induced SG formation. The contribution of the different domains of DDX3 to viral protein interaction and virus-induced SG formation was also studied. While DDX3 helicase domain did not interact with NS1 and NP, it was essential for DDX3 localization in virus induced SGs. Moreover, DDX3 downregulation resulted in the increased replication of PR8 del NS1virus, accompanied by an impairment of SG induction in infected cells. Since DDX3 is reported to regulate IFNβ induction, the role of DDX3 in influenza A virus induced IFNβ induction was also examined. Using small molecule inhibitors and siRNA-mediated gene knockdown, the RIG-I pathway was identified as the major contributor of influenza induced IFNβ induction in newborn porcine tracheal epithelial (NPTr) cells. DDX3 downregulation and overexpression also showed that DDX3 has an inhibitory effect on IFNβ expression induced by both influenza infection and low molecular weight (LMW) poly I:C treatment, which is also a RIG-I ligand. RNA competition assay to identify the mechanism of DDX3-mediated inhibition, showed that RIG-I binding affinity to its ligands LMW poly I:C and influenza viral RNA (vRNA) is much higher than that of DDX3. Furthermore, DDX3 downregulation enhanced titers of the PR8 del NS1 virus, while it did not affect the titers of the wild-type strains of PR8 and SIV/SK viruses. Overall, the results show that DDX3 has an antiviral role and the SG regulatory function of DDX3 has a profound effect on virus replication than the IFNβ regulatory function.

eIF5A seems to be involved in both, translation initiation and elongation. It was also reported to affect assembly of P-bodies. Given similarities of P-bodies with stress granules (SGs) we decided to test the role of eIF5A in dynamics of heat-induced SGs and its implications for the cell recovery. For the evaluation of eIF5A function in SGs formation was used the temperature- sensitive (ts) mutant eIF5A-3 (C39Y/G118D) cultivated under permissive temperature 25řC and Rpg1-GFP fusion protein as a marker of SGs. The cells were exposed to robust heat shock at 46řC for 10 minutes. The ability of the mutant cells to recover was tested by propidium iodine staining and colony forming units plating. We found that the eIF5A-3 mutant forms heat-induced SGs more loosely aggregated, indicating that the fully functional eIF5A is necessary for SGs assembly. However, it does not seem to affect the rate of SGs dissolution. Survival tests indicate that eIF5A-3 mutant cells are susceptible to dying in a similar way as WT cells; nevertheless, their ability to resume proliferation is significantly better. We also observed a loss of the ts phenotype of the eIF5A-3 mutant. This loss cannot be explained by reversion of mutated eIF5A sequence into normal. Probable cause lies in the adaptive evolution. Our results indicate role of...

The cells are capable of very quick and specific reactions on stress conditions. Influence of translation, specifically initiation of translation by inhibition factors, is one of the main regulatory process. Two of eIF4E-binding proteins (4E-BP), Eap1p and Caf20p, are known as cap-dependent translation repressors in yeast Saccharomyces cerevisiae. We used in vivo fluorescent microscopy analysis to show different reaction of Caf20p and Eap1p to heat stress. Protein Caf20p does not react on heat shock and stays difused in cytoplasm. Contrary to Caf20p reaction, protein Eap1p accumulates in cytoplasm close to stress granules (SGs). This work shows that Eap1p is involved in stress granules assembly. In the absence of Eap1p, yeast cells react to the heat stress with small and less focused SGs. Dele- tion of CAF20 does not affect SG assembly. This points to specific function of SG in distribution of factors connected with stress reaction. Polysomal analysis shows that deletion of one of initiation translation repressors does not affect heat induced global repression of translation. In permisive condition deletion of EAP1 may cause defect in addition of 60S ribosomal subunits. Absence of protein Eap1p causes morphological defect. That point to a different reactions of Eap1p and Caf20p on heat stress and possible...

Human Caprin-1 and Caprin-2 are prototypic members of the caprin (cytoplasmic activation/proliferation-associated protein) protein family. Vertebrate caprin proteins contain two highly conserved homologous regions (HR1 and HR2) and C-terminal RGG motifs. Drosophila caprin (dCaprin) shares HR1 and RGG motifs but lacks HR2. Caprin-1 and Caprin-2 have important and non-redundant functions. The detailed molecular mechanisms of their actions remain largely unknown.

RNA binding proteins (RBPs) have been found to be frequently involved in neurodegenerative diseases (Ash 2014). Mutations in RBPs cause amyotrophic lateral sclerosis (ALS), spinocerebellar ataxia, frontotemporal dementia (FTD) and myopathies (Ash 2014), and recent studies suggest that aggregation of RBPs are a pathological feature frequently encountered in tauopathies (Vanderweyde 2016). Emerging studies on neurodegenerative diseases are now showing an increasingly important role for tau in regulating the biology of RBPs. In this study, we examine findings that show that reducing the RBP T-cell intracellular antigen 1 (TIA1) in vivo not only protects against neurodegeneration, but also prolongs the survival rate in transgenic P301S tau mice. Furthermore, the reduction of TIA1 decreases the number and size of granules co-localizing with stress granule markers, and inhibits the accumulation of tau oligomers, although at the expense of an increased number of neurofibrillary tangles. However, despite the observed increase in neurofibrillary tangles, this TIA1 reduction still manages to increase neuronal survival, rescue behavioral deficits and prolong lifespan. The in vivo data presented in this study suggests an important role for TIA1 in mediating toxicity and provides evidence that RBPs orchestrate a pathway to tau aggregation and the resulting neurodegeneration.

Sébastien Legros (2010). La protéine Tax du virus HTLV-1 inhibe la formation des granules de stress en interagissant avec lhistone désacétylase 6 (HDAC6) (thèse de doctorat, en anglais). Université de Liège - Gembloux Agro-Bio Tech, 153 p., 2 tabl., 24 fig. Résumé Le virus T-lymphotrope humain qui infecte 20 millions de personnes dans le monde, est responsable de deux pathologies : une leucémie fatale, appelée leucémie des cellules T de ladulte (ATL) et une maladie neurodégénérative, la paraparésie tropicale spastique (TSP). Loncoprotéine virale Tax-1 constitue une cible thérapeutique privilégiée car elle joue un rôle crucial dans les pathologies induites par HTLV-1. En réponse à un stress comme une infection virale, un stress oxydatif ou une exposition aux UV, la cellule bloque la traduction des ARNm et les séquestre dans des structures cytoplasmiques spécifiques appelées granules de stress (GS). Ces granules sont caractérisés par la présence de protéines spécifiques telles que G3BP et Tiar. Récemment, lhistone désacétylase HDAC6 a été identifiée comme un composant critique de ces GS. Dans ce travail, nous démontrons qu'en présence d'un stress cellulaire, Tax-1 relocalise du noyau vers le cytoplasme. Dans le cytoplasme, Tax-1 colocalise avec G3BP et Tiar dans les GS de certaines cellules. De plus, la protéine Tax-1 exprimée dans le cytoplasme, empêche la formation des GS en interagissant avec HDAC6. Finalement, nous avons montré que les lymphocytes T infectés par HTLV-1 contiennent moins de GS et que ceux-ci sont de taille réduite par rapport aux cellules contrôles Jurkat. Nos résultats révèlent une nouvelle stratégie développée par HTLV-1 et nous postulons que cette nouvelle fonction pourrait avoir un rôle important dans la transformation et loncogenèse induite par le virus HTLV-1. Sébastien Legros (2010). HTLV-1 Tax inhibits stress granules formation by interacting with histone deacetylase 6 (HDAC6) (thèse de doctorat, in English). University of Liège - Gembloux Agro-Bio Tech, 153 p., 2 tabl., 24 fig. Summary Human T cell leukaemia virus type-1 (HTLV-1) which infects 20 million people worldwide is the causative agent of two major diseases: a rapidly fatal leukaemia designated adult T-cell leukaemia (ATL) and a neurological degenerative disease known as tropical spastic paraparesis (TSP). The viral transcriptional activator and oncoprotein Tax-1 has been the major focus of scientific investigation because of its numerous and crucial roles in the pathogenesis of HTLV-1-induced diseases. In response to stress such as viral infection, oxidative stress or UV exposure, the cell blocks mRNA translation and sequesters mRNAs in specific cytoplasmic structures called stress granules (SGs). Stress granules are characterized by the presence of specific proteins such as G3BP and Tiar. Recently, the histone deacetylase HDAC6 was identified as a critical component of SGs. In this report, we demonstrated that in response to cellular stress, Tax-1 relocalizes in the cytoplasm, where it can be found colocalizing with G3BP and Tiar in SGs. Moreover, cytoplasmic Tax-1 prevents SGs formation by interacting with HDAC6. Finally, we have shown that HTLV-1 infected cells exhibit less and smaller SGs than the control Jurkat cells. Our findings thus unravel a new strategy developed by HTLV-1 and we postulate that this new function of Tax might have important role in HTLV-I-induced transformation and oncogenesis. Copyright : Aux termes de la loi belge du 30 juin 1994, sur le droit d'auteur et les droits voisins, seul l'auteur a le droit de reproduire partiellement ou complètement cet ouvrage de quelque façon et forme que ce soit ou d'en autoriser la reproduction partielle ou complète de quelque manière et sous quelque forme que ce soit. Toute photocopie ou reproduction sous autre forme est donc faite en violation de la dite loi et de des modifications ultérieures.

Cellular T-cell intracellular antigen-1 related protein (TIAR) binds to the 3' terminal stem-loop of the West Nile virus minus-strand RNA [WNV 3'(-) SL RNA]. TIAR binding sites were previously mapped on loop 1 (L1) and loop 2 (L2) of the 3' (-) SL RNA and mutations of these sites in a WNV infectious clone inhibited virus replication. In the present study, data from in vitro binding assays suggested that multiple TIAR proteins bind to each WNV 3′ (-) SL RNA in a positively cooperative manner. The tertiary structure of WNV 3′ (-) SL RNA was predicted and it was suggested that L2 forms an exposed loop while L1 forms an embedded loop. We propose that TIAR binds first to L2 and that this interaction facilitates the binding of a second TIAR molecule to L1. Data from in vitro assays also showed that TIAR binds specifically to the WNV 3' (-) SL RNA but not to the complementary WNV 5' (+) SL RNA and that the C-terminal prion domain of TIAR contributes to RNA binding specificity. Immunoprecipitation experiments indicated that TIAR interacts with the WNV 3' (-) SL RNA in cells. Colocalization of TIAR and viral dsRNA in the perinuclear region of WNV-infected cells was visualized using a proximity ligation assay. In WNV-infected, TIAR-overexpressing cells, increased extracellular virus yields, intracellular viral protein and RNA levels, and an increased ratio of viral plus-strand RNA to minus-strand RNA were observed. These data suggest that TIAR enhances WNV plus-strand RNA synthesis from the minus-strand template. WNV infections induce small TIAR foci formation in primate cells but not rodent cells. The TIAR foci are located in the perinuclear region and differ in size and location from arsenite-induced stress granules (SGs). However, the small TIAR foci contain many SG components, such as G3BP, PABP, and eIF3A, but not HuR. Arsenite-induced SG formation is still inhibited by WNV infection in these cells. eIF2a phosphorylation was observed in some infected cells that contained WNV-induced TIAR foci but viral NS3 protein accumulation was not inhibited. The data suggest that WNV-induced TIAR foci in primate cells are not canonical SGs.

A recurring theme in biology is the aggregation of mRNA-protein complexes (mRNPs) into higher order assemblies. Often these complexes play important roles in the regulation of gene expression, but the function of the conserved cytoplasmic mRNP assemblies - P bodies and stress granules, is not known. It is believed that the misregulation of granule assembly is related to disorders like Amyotrophic Lateral Sclerosis and Frontotemporal Lobe Degeneration. Determining the complete composition of these granules may hold the key to understanding the function and mechanism of assembly of these granules. This work describes multiple approaches taken to identify new protein and mRNA components of P bodies and stress granules. New members of the P body and stress granule proteome reveal a role for these granules in diverse cellular processes including signal transduction, transcription and metabolism. Additionally, a new stress granule resident complex - the CCT complex, was also identified as a novel regulator of granule disassembly. This work also describes the first purification scheme for stress granules and presents a new system for in vitro study of stress granules. Together, the findings shed new light on the composition, function, structure and regulation of P bodies and stress granules in yeast.