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No description available.

Ribonucleoprotein (RNP) granules are membraneless compartments that form inside the cyto- or nucleoplasm by the process of phase separation. RNP granules play essential roles in cell organization and physiology and their misregulation can have detrimental effects including protein misfolding and aggregation. Even though our knowledge about RNP granules has increased greatly in the past years there are still many open questions about the mechanisms involved in their formation, dissolution, and properties. There are indications that RNA helicases could play a crucial role in regulating the properties of RNP granules. Many RNA helicases can be found in different types of RNP granules. Furthermore, they have the ability to bind and remodel secondary structures of RNA, one of the main components of RNP granules. This gives them the potential ability to influence RNP granule formation, dissolution, and properties. In my PhD thesis, I analysed the human DEAD-box helicase DDX3X, which is known to be present in stress-inducible RNP granules. For this, I established a purification method for DDX3X-WT as well as two cancer related-variants of DDX3X. By biochemical analysis, I could demonstrate that the cancer-related variants are partially or fully defective in their enzymatic activity. I could show that DDX3X forms condensates at physiologically relevant conditions in vitro and that the phase separation propensity is independent on the enzymatic activity. However, analysis of the material properties of condensates formed by DDX3X-WT and the two cancer-related variants in different conditions revealed significant differences. This indicates that the enzymatic activity of DDX3X is relevant for the material properties of these condensates. Furthermore, I set up an in vitro system to mimic stress-inducible RNP granules, using G3BP1 and RNA. This assay revealed that condensates formed by G3BP1 in the presence of heat-aggregated RNA exhibit solid-like features. I could show that DDX3X-WT, but not the cancer-related variants, collaborates with G3BP1 and promotes a solid to liquid phase transition of these solid-like condensates. Taken together, this suggests that the RNA helicase DDX3X can regulate the material properties of RNP granules using its enzymatic activity. This reveals a potential mechanism how DEAD-box helicases could regulate RNP granules formation, dissolution and their material properties.

The 3' terminal stem loop of the WNV minus-strand [WNV3'(-) SL] RNA was previously shown to bind the cell protein, T-cell intracellular antigen-1 (TIA-1), and the related protein, TIAR. These two proteins are known to bind AU-rich sequences in the 3' UTRs of some cellular mRNAs. AU stretches are located in three single-stranded loops (L1, L2, and L3) of the WNV3'(-) SL RNA. The RNA binding activity of both proteins was reduced when L1 or L2, but not L3, AU sequences were deleted or substituted with Cs. Deletion or substitution with Cs of the entire AU-rich sequence in either L1 or L2 in a WNV infectious clone was lethal for the virus while mutation of some of these nt decreased the efficiency of virus replication. Mutant viral RNAs with small plaque or lethal phenotypes had similar translational efficiencies to wildtype RNA, but showed decreased levels of plus-strand RNA synthesis. These results correlated well with the efficiency of TIA-1 and/or TIAR binding in in vitro assays. In normal cells, TIA-1 and TIAR are evenly distributed in the cytoplasm and nucleus. Between 6 and 24 hr after WNV infection, TIAR concentrated in the perinuclear region and TIA-1 localization to this region began by 24 hr. Similar observations were made in DV2 infected cells but at later times after infection. In infected cells, both proteins colocalized with dsRNA, a marker for viral replication complexes, and with viral non-structural proteins. Anti-TIAR or anti-TIA-1 antibody coimmunoprecipitated viral NS3 and possibly other viral nonstructural proteins. In response to different types stress, TIA-1 and TIAR recruit cell mRNA poly(A)+ into cytoplasmic stress granules (SG) leading to general translational arrest in these cells. SG were not induced by flavivirus infection and cells became increasingly resistant to arsenite induction of SG with time after infection. Processing Body (PB) assembly was also decreased beginning at 24 hr. These data suggest that the sequestration of first TIAR and then TIA-1 via their interaction with viral components in flavivirus infected cells inhibits SG formation and prevents the shutoff of host translation.

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La dégradation des ARN est un mécanisme essentiel à la régulation de l’expression des génomes. L’importance de l’uridylation dans les mécanismes de dégradation des ARN commence juste à être appréciée. Cette thèse présente l’étudede l’UTP :RNA Uridylyltransferase 1 (URT1) et de son rôle dans la dégradation des ARN chez Arabidopsis thaliana. L’étude des propriétés catalytiques de URT1 montre que cette uridylyltransférase est intrinsèquement spécifique des UTP et distributive pour les premières uridines ajoutées. URT1 est responsable in vivo de l’uridylation des ARNm après une étape de déadénylation, protégeant leur extrémité 3’ et polarisant la dégradation de 5’ en 3’. URT1 est localisée dans le cytosol au niveau des granules de stress et des processing bodies. Le mécanisme d’adressage de URT1 dans les processing bodies implique une partie de la région N terminale prédite comme intrinsèquement désorganisée, alors que le domainenucléotidyltransférase C terminal semble suffisant pour permettre l’adressage de URT1 au niveau des processing bodies et granules de stress en réponse à un stress thermique. Ces travaux de thèse ont permis de mieux comprendre les mécanismes et les rôles de l’uridylation dans la dégradation des ARNm chez Arabidopsis. Ils ouvrent des perspectives dans l’étude d’autres fonctions de l’uridylation comme l’inhibition de la traduction. / RNA degradation is an essential mechanism for the regulation of genome expression. The importance of uridylation for RNA degradation is just emerging. This thesis presents the study of URT1 (UTP :RNA Uridylyltransferase 1) and its role in RNA degradation in Arabidopsis thaliana. URT1 is an uridylyltransferase intrinsically and strictly specific for UTP and is distributive for the first nucleotides added. URT1 uridylates mRNA in vivo after a deadenylation step. This uridylation protects mRNA’s3’ end from further attacks and polarise degradation in the 5’ to 3’ direction. This protection of 3’ ends by uridylation and its conferred polarity of 5’ to 3’ degradation are also detected in polysomes. Uridylation is therefore likely important in case of cotranslational degradation of mRNAs. A region in URT1’s N terminal region predicted to be intrinsically disorganised is required for addressing URT1 to processing bodies. However, following heat shock, the nucleotidyltransferase domain present in the C terminal region of URT1 is sufficient to address URT1 to both processing bodies and stress granules, This work contributes to a better understanding of the mechanisms and roles of uridylation in RNA degradation in Arabidopsis thaliana. These results also open perspectives for studying other functions of uridylation such as translation inhibition.

Eukaryotic initiation factor 4E (eIF4E) is a key part of initiation and regulation of translation in human cells. Three members of human eIF4E proteins have been characterized: eIF4E1, eIF4E2 and eIF4E3. Cellular stress causes translation initiation inhibition followed by disassembly of the polysomes, those processes are accompanied by the assembly of cytoplasmic RNA granules, called stress granules (SG). Stress granules are dynamic structures whose composition may vary depending on the cell type and the stress stimulus. In this study, human cells were subjected to the following stress conditions: high temperature (HS), sodium arsenite (AS) or hypoxia. Using fluorescence microscopy, pairs of human translational initiation factors from the 4E protein family were visualized and their localization to SG was assessed with one GFP- 4E incorporated in the stable cell line and the other one detected endogenously. Here we show eIF4E1 being a part of all the SGs, both in HS and AS conditions. Next, the eIF4E1 and eIF4E3 proteins together form more SGs than proteins eIF4E1, respectively eIF4E3, with eIF4E2. And last, that the presence of the particular 4E protein has no effect on the composition of SGs. Furthermore, selected groups of proteins were assessed for their potential to localize to the SGs under HS...

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Under environmental stress, such as glucose deprivation, cells form stress granules - the accumulation of cytoplasmic aggregates of repressed translational initiation complexes, proteins, and stalled mRNAs. Recent research implicates stress granules in various diseases, such as neurodegenerative disease, but the exact regulators responsible for the assembly and disassembly of stress granules are unknown. Studies detect post-translational modifications on core stress granule proteins. One modification is lysine acetylation, in which a substrate is regulated by a lysine acetyltransferase (KAT) and lysine deacetylase (KDAC). My project deciphers the impact of lysine acetylation on an essential protein found in stress granules, poly(A) binding protein (Pab1) in Saccharomyces cerevisiae. In this work, I demonstrated that acetylation mimic of Pab1-K131 reduces stress granule formation upon glucose deprivation, and other stressors such as ethanol, raffinose, and vanillin. A potential KDAC that might be facilitating this role is Rpd3. Further, electromobility shift assay studies suggest that acetylation mimic of Pab1-K131 negatively impacts poly(A) RNA binding. This work will be useful when exploring therapeutic options when combating diseases linked to stress granules.