A Missense Mutation (Q279R) in the Fumarylacetoacetate Hydrolase Gene, Responsible for Hereditary Tyrosinemia, Acts as a Splicing Mutation

Background: Tyrosinemia type I, the most severe disease of the tyrosine catabolic pathway is caused by a deficiency in fumarylacetoacetate hydrolase (FAH). A patient showing few of the symptoms associated with the disease, was found to be a compound heterozygote for a splice mutation, IVS6-1g->t, and a putative missense mutation, Q279R. Analysis of FAH expression in liver sections obtained after resection for hepatocellular carcinoma revealed a mosaic pattern of expression. No FAH was found in tumor regions while a healthy region contained enzymeexpressing nodules. Results: Analysis of DNA from a FAH expressing region showed that the expression of the protein was due to correction of the Q279R mutation. RT-PCR was used to assess if Q279R RNA was produced in the liver cells and in fibroblasts from the patient. Normal mRNA was found in the liver region where the mutation had reverted while splicing intermediates were found in nonexpressing regions suggesting that the Q279R mutation acted as a splicing mutation in vivo. Sequence of transcripts showed skipping of exon 8 alone or together with exon 9. Using minigenes in transfection assays, the Q279R mutation was shown to induce skipping of exon 9 when placed in a constitutive splicing environment. Conclusion: These data suggest that the putative missense mutation Q279R in the FAH gene acts as a splicing mutation in vivo. Moreover FAH expression can be partially restored in certain liver cells as a result of a reversion of the Q279R mutation and expansion of the corrected cells

Reinforcement of a minor alternative splicing event in MYO7A due to a missense mutation results in a mild form of retinopathy and deafness

International audiencePURPOSE: Recessive mutations of the myosin VIIA (MYO7A) gene are reported to be responsible for both a deaf-blindness syndrome (Usher type 1B [USH1B] and atypical Usher syndrome) and nonsyndromic hearing loss (HL; Deafness, Neurosensory, Autosomal Recessive 2 [DFNB2]). The existence of DFNB2 is controversial, and often there is no relationship between the type and location of the MYO7A mutations corresponding to the USH1B and DFNB2 phenotype. We investigated the molecular determinant of a mild form of retinopathy in association with a subtle splicing modulation of MYO7A mRNA. METHODS: Affected members underwent detailed audiologic and ocular characterization. DNA samples from family members were genotyped with polymorphic microsatellite markers. Sequencing of MYO7A was performed. Endogenous lymphoid RNA analysis and a splicing minigene assay were used to study the effect of the c.1935G>A mutation. RESULTS: Funduscopy showed mild retinitis pigmentosa in adults with HL. Microsatellite analysis showed linkage to markers in the region on chromosome 11q13.5. Sequencing of MYO7A revealed a mutation in the last nucleotide of exon 16 (c.1935G>A), which corresponds to a substitution of a methionine to an isoleucine residue at amino acid 645 of the myosin VIIA. However, structural prediction of the molecular model of myosin VIIA shows that this amino acid replacement induces only minor structural changes in the immediate environment of the mutation and thus does not alter the overall native structure. We found that, although predominantly included in mature mRNA, exon 16 is in fact alternatively spliced in control cells and that the mutation at the very last position is associated with a switch toward a predominant exclusion of that exon. This observation was further supported using a splicing minigene transfection assay; the c.1935G>A mutation was found to trigger a partial impairment of the adjacent donor splice site, suggesting that the unique change at the last position of the exon is responsible for the enhanced exon exclusion in this family. CONCLUSIONS: This study shows how an exonic mutation that weakens the 5' splice site enhances a minor alternative splicing without abolishing a complete exclusion of the exon and therefore causes a less severe retinitis pigmentosa than the USH1B-associated alleles. It would be interesting to examine a possible correlation between intrafamilial phenotypic variability and the subtle variation in exon 16 inclusion, probably related to genetic background specificities

Protein 4.1R expression in normal and dystrophic skeletal muscle

4.1R pre-mRNA alternative splicing results in multiple mRNA and protein isoforms that are expressed in virtually all tissues. More specifically, isoforms containing the alternative exon 17a, are exclusively expressed in muscle tissues. In this report, we show that these isoforms are preferentially present in the myoplasm of fast myofibres. 4.1R epitopes are also found at the sarcolemma of both slow and fast myofibres in normal muscle. Interestingly, they are absent from dystrophin-deficient sarcolemma of DMD muscle, and colocalize with partially expressed dystrophin in BMD muscle. We also show that alternative splicing of exons 16 and 17a is regulated during muscle differentiation in an asynchronous fashion, with an early inclusion of exon 16 in forming myotubes, and a late inclusion of exon 17a. Consistently, Western blot analysis led to characterize mainly an approximately 96/98-kDa doublet bearing exons 16-17a-encoding peptide, exclusively occurring in the differentiated muscle

Implication des facteurs d'épissage agissant en trans dans la régulation de l'épissage alternatif de l'exon 16 du pré-messager 4.1R80 au cours de la différenciation érythroïde

L'inclusion de l'exon 16 permet la production d une protéine 4.1R fonctionnelle, essentielle à l intégrité des membranes cytoplasmiques dans les érythroblastes mature. Dans notre étude, nous avons identifié KSRP comme protéine majoritairement fixée sur l ESS de l exon 16. Sa fixation requiert hnRNP A1 in vitro mais n influe pas sur l épissage de l exon 16 de manière directe et indirecte. De plus, nous avons observé que dans les protéines hnRNP A/B uniquement hnRNP A2 régule in vivo l inclusion de l exon 16 dans le système érythroïde. Ces données ont permis de créer un modèle de régulation de l épissage de l exon 16 permettant de mieux apréhender les mécanismes d épissage essentiels contribuant à la formation des cellules érythroïdes différenciées. Ce modèle montre que les protéines hnRNP A1, hnRNP A2 et KSRP font partie du complexe ribonucléoprotéique fixé sur l ESS in vitro mais que seul hnRNP A2 inhibe l inclusion de l exon 16 in vivoThe inclusion of exon 16 in the mature protein 4.1R messenger RNA (mRNA) is a critical event in red blood cell membrane biogenesis. It occurs during late erythroid development and results in inclusion of the 10kDa domain needed for stabilization of the spectrin/actin lattice. We here identified KSRP as a predominant splicing factor that binds to ESS16, a splicing silencer within exon 16. Its binding requires hnRNP A1 protein in vitro but does not affect exon 16 splicing in intact cells. In addition, we observed that the regulation of the exon 16 inclusion in the erythroid system is not affected by hnRNP A1 in vivo, but rather is modulated by hnRNP A2 in an isoform specific mannerLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Rôle de Spi-1 / PU.1 dans la régulation de l'épissage alternatif des pré-messagers 4.1R au cours de la différenciation érythroïde

L'inclusion de l'exon 16 dans le messager mature 4.1R est un évènement spécifique de la phase terminale de la différenciation érythroïde. Mon travail de thèse s est focalisé sur la recherche des éléments impliqués dans la régulation de l'epissage érythroïde et en particulier sur le rôle du facteur de transcription Spi-1/PU.1 codé par le proto-oncogène Spi-1/PU.1. La surexpression des oncoprotéines PU.1 ou Fli-1 dans les cellules MEL SFFV inhibe la production d'hémoglobine, mais seule la surexpression de PU.1 inhibe l'inclusion de l'exon 16 dans l'ARNm 4.1R. J ai prouvé que l extinction partielle inductible de Spi-1/PU.1 par RNA interférence est associée à une augmentation de l inclusion de l exon 16 dans l ARNm 4.1R ainsi qu une activation de HERF-1 en absence d un inducteur chimique. HERF-1 (Hematopoietic Ring Finger-1) est un facteur indispensable à la production d hémoglobine et à la viabilité des cellules enfin de différenciation érythroïde. J ai également montré que le facteur HERF-1 est nécessaire mais pas suffisant à la reconnaissance de l exon 16 4.1R. Spi-1/PU.1 n inhibe pas l épissage de l exon 16 par fixation sur le promoteur mais il agit par répression de l expression de herf-1Inclusion of exon 16 in the mature 4.1R RNA arises from a stage specific splicing event that occurs during late erythroid development. My project was to investigate the factors implicated and especially the role of transcription factor Spi-1/PU.1 encoded by the pu.1 in the regulation of this erythroid specific splicing event. Sustained expression of the Spi-1/PU.1 and Fli-1 oncoproteins blocks globin gene activation in MEL cells; however, only Spi-1/PU.1 expression inhibits the inclusion of exon 16 in the mature 4.1R mRNA. We demonstrate that Spi-1/PU.1 downregulation induces the activation of HERF-1 (Hematopoietic Ring Finger 1) and inclusion of exon 16 in 4.1R mRNA in the absence of a chemical inducer. HERF-1 is a member of the tripartite motif (TRIM)/ RBCC family needed for globin gene transcription and viability of cells undergoing terminal erythroid differentiation. We demonstrate that HERF-1 is required but is insufficient for exon 16 retention and that Spi-1/PU.1 inhibits exon 16 inclusion in a promoter independent manner during erythroid differentiation and by repression of herf-1 expression.LYON1-BU.Sciences (692662101) / SudocSudocFranceF

Recherche de voies de régulation de l'épissage alternatif du pré-messager 4.1R au cours de la différenciation érythroïde

Au cours de la différenciation érythroïde, la reconnaissance de l'exon 16 est indispensable à la fonction de la protéine 4.1R du globule rouge mature. La rétention de cet exon est un processus d'épissage alternatif dont la régulation est encore totalement inconnue. Mon projet de thèse avait pour but la recherche de voies de régulation de l'inclusion de l'exon 16. Pour cela, j'ai étudié à la fois l'implication éventuelle de voies de signalisation par inhibition de kinases déterminées et de facteurs de transcription impliqués dans la différenciation érythroïde, en général, par surexpression. Ce travail a permis d'impliquer dans la régulation positive la kinase P38 et un facteur érythroïde à la fonction inconnue Herf1 et dans la régulation négative la kinase PI3K et surtout le facteur de transcription Spi-1. Ce dernier capable d'interagir directement avec l'exon 16 et ses introns a été impliqué dans l'évènement d'épissage en lui-mêmeLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Régulation de l'épissage alternatif de l'exon 16 du pré-messager 4.1 R au cours de la différenciation érythroïde

L'épissage alternatif des pré-ARNm est un mécanisme majeur de diversification de l'information génétique et ses dérégulations sont liées à des maladies. L'inclusion de l'exon 16 est essentielle à la protéine 4.1R cytosquelettique pour la stabilité de l'hématie, elle n'a lieu qu'en fin de différenciation érythroïde. Nous avons cherché les séquences, éléments cis, et leurs ligands, facteurs trans, requis pour cet événement. Des stratégies in culturo (transfections de minigènes 4.1 R, surexpression de facteurs d'épissage et étude de l'inclusion de l'exon 16 sur les ARNm 4.1 R endogènes ou exogènes) et in vitro (gels retard, pontage aux UV) ont été appliquées. Nous avons établi un modèle de régulation in cis. Puis j'ai focalisé mon travail sur le répresseur portant 2 motifs UAG, sites putatifs d'hnRNP A1. Nous avons montré que le rôle de hnRNP A1 est minime dans cette répression mais qu'en revanche, le répresseur recrute KSRP, un autre facteur d'épissage, de façon indépendante des motifs UAGLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Des oncogènes à l’interface entre transcription et épissage

Le développement de cellules cancéreuses est depuis longtemps associé à des dérèglements de l’activité transcriptionnelle de nombreux gènes, mais aussi, plus récemment, à l’apparition d’anomalies d’épissage des ARN prémessagers. La contribution de ces anomalies d’épissage au développement de cellules tumorales, ainsi que le pouvoir tumorigène de certaines des isoformes protéiques qui en sont issues sont encore très peu explorés. Toutefois, depuis la découverte récente du couplage des deux mécanismes - transcription et épissage - des efforts de recherche ont permis de démontrer que des facteurs de transcription oncogéniques affectent aussi l’épissage des prémessagers. Ces observations suscitent des interrogations quant aux mécanismes d’action des oncogènes à activité transcriptionnelle et, à plus long terme, quant à la recherche de cibles cellulaires nouvelles à appréhender dans de futurs protocoles thérapeutiques anticancéreux.Oncogene activity ranges from transduction signals to transcription factors. Altered expression of oncogenes, either by chromosomal translocation, proviral insertion or point mutations, can lead to tumor formation. More specifically, data accumulated through the last two decades have shown that disregulation of oncogenic transcription factors can interfere with regulatory cascades that control the growth, differentiation, and survival of normal cells. There is also evidence that alterations of oncogene activity are associated with pre-mRNA splicing defects. The insights gained from the pivotal role of RNA polymerase II in coupling transcription and splicing have instigated a new line of research regarding the possible role of oncogenic transcription factors in pre-mRNA splicing regulation. This review focuses on recent advances addressing this question. Understanding the impact of alterations in the expression and/or function of oncogenes have important prognostic implications that can guide the design of new therapeutic drugs to promote differentiation and/or apoptosis over cell proliferation

Proteasome-mediated proteolysis of SRSF5 splicing factor intriguingly co-occurs with SRSF5 mRNA upregulation during late erythroid differentiation.

SR proteins exhibit diverse functions ranging from their role in constitutive and alternative splicing, to virtually all aspects of mRNA metabolism. These findings have attracted growing interest in deciphering the regulatory mechanisms that control the tissue-specific expression of these SR proteins. In this study, we show that SRSF5 protein decreases drastically during erythroid cell differentiation, contrasting with a concomitant upregulation of SRSF5 mRNA level. Proteasome chemical inhibition provided strong evidence that endogenous SRSF5 protein, as well as protein deriving from stably transfected SRSF5 cDNA, are both targeted to proteolysis as the cells undergo terminal differentiation. Consistently, functional experiments show that overexpression of SRSF5 enhances a specific endogenous pre-mRNA splicing event in proliferating cells, but not in differentiating cells, due to proteasome-mediated targeting of both endogenous and transfection-derived SRSF5. Further investigation of the relationship between SRSF5 structure and its post-translation regulation and function, suggested that the RNA recognition motifs of SRSF5 are sufficient to activate pre-mRNA splicing, whereas proteasome-mediated proteolysis of SRSF5 requires the presence of the C-terminal RS domain of the protein. Phosphorylation of SR proteins is a key post-translation regulation that promotes their activity and subcellular availability. We here show that inhibition of the CDC2-like kinase (CLK) family and mutation of the AKT phosphorylation site Ser86 on SRSF5, have no effect on SRSF5 stability. We reasoned that at least AKT and CLK signaling pathways are not involved in proteasome-induced turnover of SRSF5 during late erythroid development

SRSF5 expression in primary ex-vivo erythroid precursors.

<p>A. Flow cytometric analysis of proliferating (black line) and differentiating (grey line) cells. In each graph, the intensity of scatter (FSC) or fluorescence (kit (CD117), CD71, Ter119) is plotted on the Y-axis. The number of events (number of cells) is plotted on the X-axis; it is expressed as 10³-fold (K) in FSC histogram. Proliferating erythroblasts are large cells (high FSC). They express moderate to high levels of Kit and CD71, and low levels of Ter119 on their cell surface. After 2 days of maturation, these cells display a cell size decrease (low FSC), and cell surface phenotype changes, including Kit decrease and higher levels of CD71 and Ter119.B. Immunoblot analysis of SRSF5 expression in fetal liver-derived erythroblasts. Proteins were collected from proliferating cultured erythroblasts (Prolif.) and 2 days after ex-vivo differentiation (Diff.). Western blot analysis was performed using anti-SRSF5 antibody "pAb-SRSF5". Note that SRSF5 virtually vanishes as the cells differentiate.</p