Régulation de l'épissage alternatif par des facteurs d'épissage de la famille des protéines SR dans des situations physiologiques ou pathologiques

Les protéines SR jouent un rôle crucial dans les processus de maturation des précurseurs d'ARNm, et plus particulièrement dans la régulation de l'épissage alternatif. Ce mécanisme permet la synthèse de multiples ARNm à partir d'un unique précurseur, et constitue un moteur essentiel de la diversité protéique chez les eucaryotes supérieurs. L'activité des protéines SR est régulée par la phosphorylation de leur domaine RS, riche en résidus arginines et sérines, par plusieurs familles de protéines kinases et dont l'ADN topoisomérase I (Topo I). En utilisant des cellules résistantes à un inhibiteur de la Topo I, nous avons observé que la résistance à la camptothécine est étroitement liée à l'extinction de l'expression de cette enzyme. L'absence de Topo I est corrélée à une hypophosphorylation des protéines SR et à des défauts d'épissage alternatif, ce qui suggère que l'activité kinase de la Topo I n'est pas redondante avec celle d'autres protéines et est nécessaire pour les processus d'épissage alternatif in vivo. Les protéines SR jouent un rôle essentiel au cours du développement, mais paradoxalement, leur fonction dans ce processus reste élusive. Chez la drosophile, la surexpression de diverses protéines SR, dans les disques imaginaux d'œil et dans le cerveau de larves, induit des défauts de développement de ces tissus. Nous avons identifié les ARNm associés à dASF/SF2 et à B52, et nous avons montré que la surexpression de ces deux protéines entraîne des altérations du profil d'épissage de certains d'entre eux impliqués, en particulier, dans le développement de l'œil et du cerveau. L'analyse du développement de ces deux tissus, au stade larvaire, a révélé que la surexpression de dASF/SF2 perturbe la différentiation de cellules de l'ommatidie, et que l'expression de B52-GFP interfère également avec le développement du cerveau. Les protéines SR régulent l'épissage des précurseurs d'ARNm en interagissant avec des séquences activatrices introniques ou exoniques, dont la fonction peut être altérée par des mutations, dans le cas de maladies génétiques. Nous avons étudié une mutation qui localisée dans l'intron 7 du gène E1a du complexe PDH, chez un patient atteint du syndrome de Leigh. Cette mutation créée une séquence spécifiquement reconnue par la protéine SC35 qui est responsable d'un épissage aberrant de l'ARNm E1aPDH à l'origine de la maladie. Nous avons montré que la réduction de l'expression de SC35 par ARN interférence permet de restaurer un épissage normal du gène E1aPDH dans des fibroblastes primaires de patients. Enfin, nous avons identifié des composés chimiques qui inhibent spécifiquement des évènements d'épissage alternatif dépendants d'une ou plusieurs protéines SR, in vitro et in vivo. De telles molécules pourraient ouvrir la voie à de nouvelles approches thérapeutiques pour traiter des pathologies humaines résultant de mutations génétiques qui affectent les processus d'épissage.SR proteins play a crucial role in messenger RNA maturation processes, and more precisely in the regulation of alternative splicing. This mechanism allows the synthesis of multiple mRNAs from a single precursor and is a major source of proteomic diversity. The activities of SR proteins are regulated by phosphorylation of the RS domain, rich in arginine and serine residues, by several protein kinases, including the DNA topoisomerase I (Topo I). Using cells resistant to a Topo I inhibitor, we have shown that resistance to camptothecin is correlated to the downregulation of the expression of this enzyme. Topo I depletion induces the hypophosphorylation of SR proteins as well as alternative splicing alterations, suggesting thereby that the kinase activity of Topo I is not redundant and is necessary for alternative splicing processes in vivo. As key splicing regulators, SR proteins also play an essential role during development, even if this function remains elusive. In drosophila, the overexpression of several SR proteins, in larval eye imaginal discs and brain, is responsible for developmental alterations of both tissues. We have identified candidate target mRNAs associated to dASF/SF2 and B52, and we have shown that overexpression of both SR proteins induces splicing patterns alterations for some of these mRNAs that are involved in eye and brain development. The analysis of both tissues, at the larval stage, revealed that overexpression of dASF/SF2 impairs cell differentiation within the ommatidia, when the overexpression of B52 also alters brain development. SR proteins regulate the splicing of mRNA precursors through binding of intronic or exonic enhancer sequences, which can be altered by mutations in genetic diseases. We have investigated the consequences of a mutation, located in the intron 7 of the PDH complex E1a subunit encoding gene, in a case of Leigh syndrome. This mutation creates an enhancer sequence specific for SC35, and is responsible for an aberrant splicing pattern of the E1aPDH mRNA causing the disease. Using RNA interference, we were able to restore the normal splicing pattern of the E1aPDH gene by reducing the expression level of SC35. At last, we have characterized small chemical molecules that specifically inhibit, both in vitro and in vivo, alternative splicing events regulated by one or more SR protein. Such molecules open exciting perspectives concerning therapeutical approaches to treat human diseases resulting from genetic mutations that impair splicing processes.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

The SR Family Proteins B52 and dASF/SF2 Modulate Development of the Drosophila Visual System by Regulating Specific RNA Targets▿ †

Deciphering the role of alternative splicing in developmental processes relies on the identification of key genes whose expression is controlled by splicing regulators throughout the growth of a whole organism. Modulating the expression levels of five SR proteins in the developing eye of Drosophila melanogaster revealed that these splicing factors induce various phenotypic alterations in eye organogenesis and also affect viability. Although the SR proteins dASF/SF2 and B52 caused defects in ommatidia structure, only B52 impaired normal axonal projections of photoreceptors and neurogenesis in visual ganglia. Microarray analyses revealed that many transcripts involved in brain organogenesis have altered splicing profiles upon both loss and gain of B52 function. Conversely, a large proportion of transcripts regulated by dASF/SF2 are involved in eye development. These differential and specific effects of SR proteins indicate that they function to confer accuracy to developmental gene expression programs by facilitating the cell lineage decisions that underline the generation of tissue identities

Post-transcriptional regulations of cancer stem cell homeostasis

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Ribosome and Translational Control in Stem Cells

International audienceEmbryonic stem cells (ESCs) and adult stem cells (ASCs) possess the remarkable capacity to self-renew while remaining poised to differentiate into multiple progenies in the context of a rapidly developing embryo or in steady-state tissues, respectively. This ability is controlled by complex genetic programs, which are dynamically orchestrated at different steps of gene expression, including chromatin remodeling, mRNA transcription, processing, and stability. In addition to maintaining stem cell homeostasis, these molecular processes need to be rapidly rewired to coordinate complex physiological modifications required to redirect cell fate in response to environmental clues, such as differentiation signals or tissue injuries. Although chromatin remodeling and mRNA expression have been extensively studied in stem cells, accumulating evidence suggests that stem cell transcriptomes and proteomes are poorly correlated and that stem cell properties require finely tuned protein synthesis. In addition, many studies have shown that the biogenesis of the translation machinery, the ribosome, is decisive for sustaining ESC and ASC properties. Therefore, these observations emphasize the importance of translational control in stem cell homeostasis and fate decisions. In this review, we will provide the most recent literature describing how ribosome biogenesis and translational control regulate stem cell functions and are crucial for accommodating proteome remodeling in response to changes in stem cell fate

SRP20 and ESC

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Inheritance of Polycomb-dependent chromosomal interactions in Drosophila

Maintenance of cell identity is a complex task that involves multiple layers of regulation, acting at all levels of chromatin packaging, from nucleosomes to folding of chromosomal domains in the cell nucleus. Polycomb-group (PcG) and trithorax-group (trxG) proteins maintain memory of chromatin states through binding at cis-regulatory elements named PcG response elements or cellular memory modules. Fab-7 is a well-defined cellular memory module involved in regulation of the homeotic gene Abdominal-B (Abd-B). In addition to its action in cis, we show here by three-dimensional FISH that the Fab-7 element leads to association of transgenes with each other or with the endogenous Fab-7, even when inserted in different chromosomes. These long-distance interactions enhance PcG-mediated silencing. They depend on PcG proteins, on DNA sequence homology, and on developmental progression. Once long-distance pairing is abolished by removal of the endogenous Fab-7, the derepressed chromatin state induced at the transgene locus can be transmitted through meiosis into a large fraction of the progeny, even after reintroduction of the endogenous Fab-7. Strikingly, meiotic inheritance of the derepressed state involves loss of pairing between endogenous and transgenic Fab-7. This suggests that transmission of nuclear architecture through cell division might contribute to inheritance of chromatin states in eukaryotes

Profiling Anti-Apoptotic BCL-xL Protein Expression in Glioblastoma Tumorspheres

International audienceGlioblastoma (GBM) is one of the cancers with the worst prognosis, despite huge efforts to understand its unusual heterogeneity and aggressiveness. This is mainly due to glioblastoma stem cells (GSCs), which are also responsible for the frequent tumor recurrence following surgery, chemotherapy or radiotherapy. In this study, we investigate the expression pattern of the anti-apoptotic BCL-xL protein in several GBM cell lines and the role it might play in GSC-enriched tumorspheres. We report that several GBM cell lines have an increased BCL-xL expression in tumorspheres compared to differentiated cells. Moreover, by artificially modulating BCL-xL expression, we unravel a correlation between BCL-xL and tumorsphere size. In addition, BCL-xL upregulation appears to sensitize GBM tumorspheres to newly developed BH3 mimetics, opening promising therapeutic perspectives for treating GBM patients

2′O-Ribose Methylation of Ribosomal RNAs: Natural Diversity in Living Organisms, Biological Processes, and Diseases

International audienceRecent findings suggest that ribosomes, the translational machineries, can display a distinct composition depending on physio-pathological contexts. Thanks to outstanding technological breakthroughs, many studies have reported that variations of rRNA modifications, and more particularly the most abundant rRNA chemical modification, the rRNA 2′O-ribose methylation (2′Ome), intrinsically occur in many organisms. In the last 5 years, accumulating reports have illustrated that rRNA 2′Ome varies in human cell lines but also in living organisms (yeast, plant, zebrafish, mouse, human) during development and diseases. These rRNA 2′Ome variations occur either within a single cell line, organ, or patient’s sample (i.e., intra-variability) or between at least two biological conditions (i.e., inter-variability). Thus, the ribosomes can tolerate the absence of 2′Ome at some specific positions. These observations question whether variations in rRNA 2′Ome could provide ribosomes with particular translational regulatory activities and functional specializations. Here, we compile recent studies supporting the heterogeneity of ribosome composition at rRNA 2′Ome level and provide an overview of the natural diversity in rRNA 2′Ome that has been reported up to now throughout the kingdom of life. Moreover, we discuss the little evidence that suggests that variations of rRNA 2′Ome can effectively impact the ribosome activity and contribute to the etiology of some human diseases