Nouveaux rôles du complexe CCR4-NOT dans le contrôle de l'expression des gènes eucaryotes

The multi-subunit CCR4-NOT complex has been implicated in all aspects of the mRNA life cycle, from synthesis of mRNAs in the nucleus to their degradation in the cytoplasm. The CAF1 protein is a catalytic subunit which plays a central role inside the complex. Human CAF1 is a deadenylase, modulates arginine methylation, and is a transcriptional cofactor of several nuclear receptors. The main objective of the thesis was to elucidate the molecular mechanism of hCAF1- mediated gene expression. We reported that hCAF1 is an important negative regulator of the interferon pathway and that hCAF1 is associated in the cytoplasm of resting cells with STAT1, a crucial transcription factor of this pathway. We found that hCAF1 participates in the extinction of the IFN signal via its deadenylase activity, by speeding up the degradation of some STAT1-induced mRNAs. Our findings are important because abnormal activations of this pathway are frequently associated with cancer and auto-immune diseases. In parallel, we characterized a novel isoform called hCAF1v2 produced by alternative splicing of the Caf1 gene. We reported that hCAF1v2 displays divergent functions compared with hCAF1. In fact hCAF1v2 does not have a deadenylase activity and is preferentially associated with PRMT1 to modulate arginine methylation. Altogether, our findings identify a new signalling pathway which is regulated by hCAF1, and reveal novel mechanisms utilized by the CCR4-NOT complex to control gene expressionDe la synthèse des ARNm jusqu'à leur dégradation, le complexe CCR4-NOT est un régulateur essentiel de l'expression des gènes eucaryotes. CAF1 est une sous-unité catalytique qui joue un rôle central dans la fonction de ce complexe. La protéine humaine hCAF1 possède une activité déadénylase, régule la méthylation des arginines dépendante de PRMT1 et est un régulateur transcriptionnel des récepteurs nucléaires. Bien que l'ensemble des travaux publiés sur hCAF1 lui confère une place importante dans la régulation de l'expression des gènes, son mécanisme d'action et surtout les voies de signalisation qu'elle régule restent encore mal compris dans les cellules humaines. Lors de ce travail de thèse, nous avons mis en évidence une nouvelle fonction de la protéine hCAF1 comme régulateur de la voie des interférons via le contrôle du facteur de transcription STAT1 et la dégradation de ses ARNm cibles. L'identification de hCAF1 comme régulateur de l'activité de STAT1 et de la réponse aux interférons est très importante car des activations anormales de ces voies sont associées à de nombreuses pathologies telles que le cancer ou des maladies immunitaires. En parallèle, nous avons caractérisé un nouvel isoforme nommé hCAF1v2 produit par le gène humain Caf1 suite à un évènement d'épissage alternatif. Nos résultats indiquent que hCAF1v2 présente une divergence fonctionnelle vis-à-vis de hCAF1 puisqu'elle ne possède pas d'activité déadénylase intrinsèque et s'avère requise pour la régulation de la méthylation des arginines via son interaction avec l'enzyme PRMT1. L'ensemble des résultats obtenus identifient une nouvelle voie de signalisation régulée par la protéine hCAF1 dans les cellules humaines et permettent de mieux comprendre l'implication du complexe CCR4-NOT dans les mécanismes de régulation de l'expression des gène

Cap-binding protein 4EHP effects translation silencing by microRNAs

Significance miRNAs are important components of gene regulatory networks and affect all aspects of cell biology by controlling the stability and translation efficiency of their target mRNAs. Here, we identified the mRNA cap-binding eIF4E-related protein 4EHP as an effector of miRNA-mediated translation repression. Through screening for protein interactions in cells via the BioID method, we identified 4EHP as a component of the CCR4–NOT/DDX6/4E-T axis. Direct interaction between 4E-T and 4EHP increases the latter’s cap-binding affinity, suggesting that this interaction potentiates its competition with the eIF4F complex for binding to the mRNA 5′ cap. Our findings suggest that 4EHP facilitates the formation of a closed-loop structure between the 3′ UTR of the mRNA and its 5′ cap, which causes repression of mRNA translation.</jats:p

Novel roles of CCR4-NOT complex in the control of eukaryotic gene expression

De la synthèse des ARNm jusqu'à leur dégradation, le complexe CCR4-NOT est un régulateur essentiel de l'expression des gènes eucaryotes. CAF1 est une sous-unité catalytique qui joue un rôle central dans la fonction de ce complexe. La protéine humaine hCAF1 possède une activité déadénylase, régule la méthylation des arginines dépendante de PRMT1 et est un régulateur transcriptionnel des récepteurs nucléaires. Bien que l'ensemble des travaux publiés sur hCAF1 lui confère une place importante dans la régulation de l'expression des gènes, son mécanisme d'action et surtout les voies de signalisation qu'elle régule restent encore mal compris dans les cellules humaines. Lors de ce travail de thèse, nous avons mis en évidence une nouvelle fonction de la protéine hCAF1 comme régulateur de la voie des interférons via le contrôle du facteur de transcription STAT1 et la dégradation de ses ARNm cibles. L'identification de hCAF1 comme régulateur de l'activité de STAT1 et de la réponse aux interférons est très importante car des activations anormales de ces voies sont associées à de nombreuses pathologies telles que le cancer ou des maladies immunitaires. En parallèle, nous avons caractérisé un nouvel isoforme nommé hCAF1v2 produit par le gène humain Caf1 suite à un évènement d'épissage alternatif. Nos résultats indiquent que hCAF1v2 présente une divergence fonctionnelle vis-à-vis de hCAF1 puisqu'elle ne possède pas d'activité déadénylase intrinsèque et s'avère requise pour la régulation de la méthylation des arginines via son interaction avec l'enzyme PRMT1. L'ensemble des résultats obtenus identifient une nouvelle voie de signalisation régulée par la protéine hCAF1 dans les cellules humaines et permettent de mieux comprendre l'implication du complexe CCR4-NOT dans les mécanismes de régulation de l'expression des gènesThe multi-subunit CCR4-NOT complex has been implicated in all aspects of the mRNA life cycle, from synthesis of mRNAs in the nucleus to their degradation in the cytoplasm. The CAF1 protein is a catalytic subunit which plays a central role inside the complex. Human CAF1 is a deadenylase, modulates arginine methylation, and is a transcriptional cofactor of several nuclear receptors. The main objective of the thesis was to elucidate the molecular mechanism of hCAF1- mediated gene expression. We reported that hCAF1 is an important negative regulator of the interferon pathway and that hCAF1 is associated in the cytoplasm of resting cells with STAT1, a crucial transcription factor of this pathway. We found that hCAF1 participates in the extinction of the IFN signal via its deadenylase activity, by speeding up the degradation of some STAT1-induced mRNAs. Our findings are important because abnormal activations of this pathway are frequently associated with cancer and auto-immune diseases. In parallel, we characterized a novel isoform called hCAF1v2 produced by alternative splicing of the Caf1 gene. We reported that hCAF1v2 displays divergent functions compared with hCAF1. In fact hCAF1v2 does not have a deadenylase activity and is preferentially associated with PRMT1 to modulate arginine methylation. Altogether, our findings identify a new signalling pathway which is regulated by hCAF1, and reveal novel mechanisms utilized by the CCR4-NOT complex to control gene expressio

m6A mRNA Destiny: Chained to the rhYTHm by the YTH-Containing Proteins

The control of gene expression is a multi-layered process occurring at the level of DNA, RNA, and proteins. With the emergence of highly sensitive techniques, new aspects of RNA regulation have been uncovered leading to the emerging field of epitranscriptomics dealing with RNA modifications. Among those post-transcriptional modifications, N6-methyladenosine (m6A) is the most prevalent in messenger RNAs (mRNAs). This mark can either prevent or stimulate the formation of RNA-protein complexes, thereby influencing mRNA-related mechanisms and cellular processes. This review focuses on proteins containing a YTH domain (for YT521-B Homology), a small building block, that selectively detects the m6A nucleotide embedded within a consensus motif. Thereby, it contributes to the recruitment of various effectors involved in the control of mRNA fates through adjacent regions present in the different YTH-containing proteins

The SARS-CoV-2 protein NSP2 impairs the microRNA-induced silencing capacity of human cells

Abstract The coronavirus SARS-CoV-2 is the cause of the ongoing pandemic of COVID-19. Given the absence of effective treatments against SARS-CoV-2, there is an urgent need for a molecular understanding of how the virus influences the machineries of the host cell. The SARS-CoV-2 generates 16 Non-Structural Proteins (NSPs) through proteolytic cleavage of a large precursor protein. In the present study, we focused our attention on the SARS-CoV-2 protein NSP2, whose role in the viral pathogenicity is poorly understood. Recent proteomic studies shed light on the capacity of NSP2 to bind the 4EHP-GIGYF2 complex, a key factor involved in microRNA-mediated silencing of gene expression in human cells. In order to gain a better understanding of the function of NSP2, we attempted to identify the molecular basis of its interaction with 4EHP-GIGYF2. Our data demonstrate that NSP2 physically associates with the endogenous 4EHP-GIGYF2 complex in the cytoplasm. Using co-immunoprecipitation and in vitro interaction assays, we identified both 4EHP and a central segment in GIGYF2 as binding sites for NSP2. We also provide functional evidence that NSP2 impairs the function of GIGYF2 in mediating mRNA silencing using reporter-based assays, thus leading to a reduced activity of microRNAs. Altogether, these data reveal the profound impact of NSP2 on the post-transcriptional silencing of gene expression in human cells, pointing out 4EHP-GIGYF2 targeting as a possible strategy of SARS-CoV-2 to take over the silencing machinery and to suppress host defenses

The SARS-CoV-2 protein NSP2 impairs the silencing capacity of the human 4EHP-GIGYF2 complex

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Beta-nerve growth factor participates in an auto/paracine pathway of regulation of the meiotic differentiation of rat spermatocytes

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Alternative splicing of CNOT7 diversifies CCR4–NOT functions

International audienceThe CCR4-associated factor CAF1, also called CNOT7, is a catalytic subunit of the CCR4–NOT complex , which has been implicated in all aspects of the mRNA life cycle, from mRNA synthesis in the nucleus to degradation in the cytoplasm. In human cells, alternative splicing of the CNOT7 gene yields a second CNOT7 transcript leading to the formation of a shorter protein, CNOT7 variant 2 (CNOT7v2). Biochemical characterization indicates that CNOT7v2 interacts with CCR4–NOT subunits, although it does not bind to BTG proteins. We report that CNOT7v2 displays a distinct expression profile in human tissues , as well as a nuclear sub-cellular localization compared to CNOT7v1. Despite a conserved DEDD nuclease domain, CNOT7v2 is unable to degrade a poly(A) tail in vitro and preferentially associates with the protein arginine methyltransferase PRMT1 to regulate its activity. Using both in vitro and in cellulo systems, we have also demonstrated that CNOT7v2 regulates the inclusion of CD44 variable exons. Altogether , our findings suggest a preferential involvement of CNOT7v2 in nuclear processes, such as argi-nine methylation and alternative splicing, rather than mRNA turnover. These observations illustrate how the integration of a splicing variant inside CCR4–NOT can diversify its cell-and tissue-specific functions