Etude de la régulation de la traduction dans les macrophages au cours de la réponse inflammatoire

The dynamic regulation of the protein synthesis process participates in the cell adaptation to a constantly evolving environment. Despite its critical role in gene expression regulation, the understanding of translational control in fundamental biological processes, such as immune responses, is still incomplete. The implementation of new approaches based on deep sequencing can be used to fill the gap in the knowledge of protein synthesis regulation. Notably, monosome vs polysome footprinting is an innovative approach derived from ribosome profiling that allow the characterization of 80S footprints derived either from monosomes or polysomes associated ribosomes. In this work, I identified the key parameters required to obtain a robust picture of ribosomal densities across cellular mRNAs using monosome vs polysome footprinting in murine primary bone-marrow derived macrophages (pBMDM). These immune cells are particularly interesting to study protein synthesis regulation in evolving conditions as they display a high sensitivity towards their environment and have the ability to trigger different gene expression programs depending on external cues. Their high phenotypic plasticity is in fact essential to ensure their protective functions in the organism such as the triggering and the resolution of the inflammatory response. As monosome vs polysome footprinting was initially developed in yeast, the adaptation of this method to study murine immune cells required extensive optimizations. The resulting protocol developed in this work was used to confirm that, contrary to a long lasting belief in the scientific community, murine pBMDM monosomes are actively involved in the translation process. Interestingly, we were able to recapitulate similar observations to what was previously observed in yeast regarding the features of mRNAs preferentially bound to monosomes or polysomes in murine pBMDM. This could suggest that the differential trafficking of ribosomes depending on specific features of the cellular mRNAs is a conserved mechanism of translational control. Importantly, the distribution of ribosomes across the different mRNAs is not random and the proper ribosome allocation pattern could be critical to adapt protein synthesis levels to the cellular needs. Here we developed a robust strategy to study this overlooked transcript-specific mechanism of translational control. Moreover, our optimized protocol can now be used to study the impact of translation through monosomes or polysomes at different stages of the inflammatory response in murine macrophages.La régulation dynamique de la synthèse des protéines en fonction des besoins de la cellule facilite son adaptation face aux fluctuations de l’environnement. Malgré l’importance de la régulation de la traduction au cours du processus d’expression des gènes, l’impact de ce mécanisme sur des processus biologiques fondamentaux, comme la mise en place d’une réponse immunitaire, reste mal compris. Grâce au développement de nouvelles technologies basées sur l’utilisation du séquençage à haut débit, comme le ribosome profiling, il est désormais possible d’étudier en détails la façon dont la synthèse des protéines est contrôlée. Le monosome vs polysome footprinting est une nouvelle méthode qui permet d’étudier la traduction des ARN messagers (ARNm) selon leur association avec un seul ribosome (monosome) ou avec plusieurs ribosomes (polysomes). Au cours de ma thèse, j’ai identifié les paramètres essentiels pour la mise en place d’une expérience de monosome vs polysome footprinting donnant des résultats fiables en utilisant des macrophages primaires dérivés de la moëlle osseuse de souris. Je me suis intéressée à ce type de cellules immunitaires particulier car elles présentent une grande capacité à détecter des modifications dans leur environnement et à modifier leur taux d’expression de protéines en fonction des signaux reçus. Leur grande plasticité est notamment essentielle pour assurer leurs diverses fonctions de protection de l’organisme, comme le déclenchement et la résolution de la réponse inflammatoire. La méthode de monosome vs polysome footprinting ayant été initialement développée chez la levure, son utilisation avec un modèle d’étude différent a nécessité de nombreuses modifications du protocole. Suite à cette phase de développement technologique, j’ai pu confirmer que les monosomes, une population de ribosomes historiquement considérés comme inactifs, sont activement impliqués dans le processus de traduction dans les macrophages primaires de souris. Les données obtenues ont également permis d’identifier des caractéristiques communes entre les ARNm enrichis dans les monosomes chez la levure et dans les macrophages murins. La régulation de la synthèse des protéines via l’association à des monosomes ou à des polysomes pourrait donc être un mécanisme conservé chez les organismes eucaryotes. Enfin, le travail d’optimisation réalisé dans les macrophages primaires murins ouvre la possibilité d’étudier l’effet de la régulation de la traduction sur la mise en place et la résolution de la réponse inflammatoire de façon très détaillée

Le profilage ribosomique

L’explosion du nombre de techniques basées sur le séquençage massif parallèle est actuellement en train de révolutionner l’étude des systèmes biologiques en permettant à l’expérimentateur d’avoir une vision globale des processus se déroulant à l’échelle moléculaire. Parmi ces nouvelles approches, le profilage ribosomique est un outil particulièrement puissant pour l’étude de la traduction à un niveau de détail jamais égalé auparavant. Cette technique permet notamment de cartographier très précisément la position des ribosomes sur l’ensemble des ARN messagers en cours de traduction dans la cellule à un moment donné. Dans le cas d’une infection virale, il est ainsi possible d’étudier les mécanismes souvent très complexes et encore mal compris qui sont mis en place par les virus pour assurer la production des protéines nécessaires à leur multiplication. Cette synthèse a pour but de discuter la manière dont le profilage ribosomique peut nous permettre de mieux comprendre le cycle de réplication virale, mais aussi de montrer les biais liés à la technique à prendre en compte lors de l’analyse des résultats

Perspective: The RNA exosome, cytokine gene regulation and links to autoimmunity

The RNA exosome is a highly conserved exoribonuclease complex that is involved in RNA processing, quality control and turnover regulation. The exosome plays pleiotropic functions by recruiting different cofactors that regulate its target specificity. Recently, the exosome has been implicated in the regulation of immune processes including cytokine production and negative regulation of innate sensing of nucleic acids. Careful regulation of such mechanisms is critical to avoid a breakdown of self-tolerance and the pathogenesis of autoimmune disorders. This perspective briefly introduces the exosome, its its normal function in RNA biology and summarizes regulatory roles of the RNA exosome in immunity. Finally we discuss how dysregulation of exosome function can lead to autoimmune disease

PDZ domain-binding motif of Tax sustains T-cell proliferation in HTLV-1-infected humanized mice.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), an aggressive malignant proliferation of activated CD4+ T lymphocytes. The viral Tax oncoprotein is critically involved in both HTLV-1-replication and T-cell proliferation, a prerequisite to the development of ATLL. In this study, we investigated the in vivo contribution of the Tax PDZ domain-binding motif (PBM) to the lymphoproliferative process. To that aim, we examined T-cell proliferation in humanized mice (hu-mice) carrying a human hemato-lymphoid system infected with either a wild type (WT) or a Tax PBM-deleted (ΔPBM) provirus. We observed that the frequency of CD4+ activated T-cells in the peripheral blood and in the spleen was significantly higher in WT than in ΔPBM hu-mice. Likewise, human T-cells collected from WT hu-mice and cultivated in vitro in presence of interleukin-2 were proliferating at a higher level than those from ΔPBM animals. We next examined the association of Tax with the Scribble PDZ protein, a prominent regulator of T-cell polarity, in human T-cells analyzed either after ex vivo isolation or after in vitro culture. We confirmed the interaction of Tax with Scribble only in T-cells from the WT hu-mice. This association correlated with the presence of both proteins in aggregates at the leading edge of the cells and with the formation of long actin filopods. Finally, data from a comparative genome-wide transcriptomic analysis suggested that the PBM-PDZ association is implicated in the expression of genes regulating proliferation, apoptosis and cytoskeletal organization. Collectively, our findings suggest that the Tax PBM is an auxiliary motif that contributes to the sustained growth of HTLV-1 infected T-cells in vivo and in vitro and is essential to T-cell immortalization

A Long Noncoding RNA lincRNA-EPS Acts as a Transcriptional Brake to Restrain Inflammation

Long intergenic noncoding RNAs (lincRNAs) are important regulators of gene expression. Although lincRNAs are expressed in immune cells, their functions in immunity are largely unexplored. Here, we identify an immunoregulatory lincRNA, lincRNA-EPS, that is precisely regulated in macrophages to control the expression of immune response genes (IRGs). Transcriptome analysis of macrophages from lincRNA-EPS-deficient mice, combined with gain-of-function and rescue experiments, revealed a specific role for this lincRNA in restraining IRG expression. Consistently, lincRNA-EPS-deficient mice manifest enhanced inflammation and lethality following endotoxin challenge in vivo. lincRNA-EPS localizes at regulatory regions of IRGs to control nucleosome positioning and repress transcription. Further, lincRNA-EPS mediates these effects by interacting with heterogeneous nuclear ribonucleoprotein L via a CANACA motif located in its 3\u27 end. Together, these findings identify lincRNA-EPS as a repressor of inflammatory responses, highlighting the importance of lincRNAs in the immune system

A Long Noncoding RNA lincRNA-EPS Acts as a Transcriptional Brake to Restrain Inflammation

International audienceLong intergenic noncoding RNAs (lincRNA) are important regulators of gene expression. Although lincRNAs are expressed in immune cells, their functions in immunity are largely unexplored. Here we identify an immunoregulatory lincRNA, lincRNA-EPS, that is precisely regulated in macrophages to control the expression of immune response genes (IRGs). Transcriptome analysis of macrophages from lincRNA-EPS-deficient mice, combined with gain-of-function and rescue experiments, revealed a specific role for this lincRNA in restraining IRG expression. Consistently, lincRNA-EPS-deficient mice manifest enhanced inflammation and lethality following endotoxin challenge in vivo. lincRNA-EPS associates with chromatin at regulatory regions of IRGs to control nucleosome positioning and repress transcription. Further, lincRNA-EPS mediates these effects by interacting with heterogeneous nuclear ribonucleoprotein L via a CANACA motif located in its 3′ end. Together, these findings identify lincRNA-EPS as a repressor of inflammatory responses highlighting the importance of lincRNAs in the immune system

RSL24D1 sustains steady-state ribosome biogenesis and pluripotency translational programs in embryonic stem cells

Abstract Embryonic stem cell (ESC) fate decisions are regulated by a complex circuitry that coordinates gene expression at multiple levels from chromatin to mRNA processing. Recently, ribosome biogenesis and translation have emerged as key pathways that efficiently control stem cell homeostasis, yet the underlying molecular mechanisms remain largely unknown. Here, we identified RSL24D1 as highly expressed in both mouse and human pluripotent stem cells. RSL24D1 is associated with nuclear pre-ribosomes and is required for the biogenesis of 60S subunits in mouse ESCs. Interestingly, RSL24D1 depletion significantly impairs global translation, particularly of key pluripotency factors and of components from the Polycomb Repressive Complex 2 (PRC2). While having a moderate impact on differentiation, RSL24D1 depletion significantly alters ESC self-renewal and lineage commitment choices. Altogether, these results demonstrate that RSL24D1-dependant ribosome biogenesis is both required to sustain the expression of pluripotent transcriptional programs and to silence PRC2-regulated developmental programs, which concertedly dictate ESC homeostasis

RSL24D1 sustains steady-state ribosome biogenesis and pluripotency translational programs in embryonic stem cells

Abstract Embryonic stem cell (ESC) fate decisions are regulated by a complex circuitry that coordinates gene expression at multiple levels from chromatin to mRNA processing. Recently, ribosome biogenesis and translation have emerged as key pathways that efficiently control stem cell homeostasis, yet the underlying molecular mechanisms remain largely unknown. Here, we identified RSL24D1 as highly expressed in both mouse and human pluripotent stem cells. RSL24D1 is associated with nuclear pre-ribosomes and is required for the biogenesis of 60S subunits in mouse ESCs. Interestingly, RSL24D1 depletion significantly impairs global translation, particularly of key pluripotency factors and of components from the Polycomb Repressive Complex 2 (PRC2). While having a moderate impact on differentiation, RSL24D1 depletion significantly alters ESC self-renewal and lineage commitment choices. Altogether, these results demonstrate that RSL24D1-dependant ribosome biogenesis is both required to sustain the expression of pluripotent transcriptional programs and to silence PRC2-regulated developmental programs, which concertedly dictate ESC homeostasis

RSL24D1 sustains steady-state ribosome biogenesis and pluripotency translational programs in embryonic stem cells

Abstract Embryonic stem cell (ESC) fate decisions are regulated by a complex circuitry that coordinates gene expression at multiple levels from chromatin to mRNA processing. Recently, ribosome biogenesis and translation have emerged as key pathways that efficiently control stem cell homeostasis, yet the underlying molecular mechanisms remain largely unknown. Here, we identified RSL24D1 as highly expressed in both mouse and human pluripotent stem cells. RSL24D1 is associated with nuclear pre-ribosomes and is required for the biogenesis of 60S subunits in mouse ESCs. Interestingly, RSL24D1 depletion significantly impairs global translation, particularly of key pluripotency factors and of components from the Polycomb Repressive Complex 2 (PRC2). While having a moderate impact on differentiation, RSL24D1 depletion significantly alters ESC self-renewal and lineage commitment choices. Altogether, these results demonstrate that RSL24D1-dependant ribosome biogenesis is both required to sustain the expression of pluripotent transcriptional programs and to silence PRC2-regulated developmental programs, which concertedly dictate ESC homeostasis

Genome editing in primary cells and in vivo using viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins

A current challenge in genome editing is delivering Cas9 and sgRNA into target cells. Here the authors engineer a delivery system based on murine leukemia virus-like particles loaded with Cas9-sgRNA ribonucleoproteins to induce efficient genome editing in both cell culture and in vivo in mouse