Résumé :
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Communicatiion n° 233 Alteration of correct splicing by disruption of an exonic splicing enhancer is a frequent mechanism by which point mutations cause genetic diseases. Duchenne muscular dystrophy (DMD) is primarily caused by frame-disrupting mutations in the dystrophin gene which abort protein synthesis. We report a patient carrying a nonsense mutation [L1417X in exon 31] which results in the skipping on the exon in the part of the mature transcripts, hence eliminating the mutation. Skipping of exon 31 maintains an open reading frame and leads to the production of a rescued protein. This patient shows a milder phenotype than the expected severe Duchenne phenotype. By performing in vitro splicing assays using Sp1 (E1A) heterologous system, we determined that 5' end of the wild-type exon 31 (nucleotides 1-34) possesses an exonic splicing enhancer (ESE) activity and that the SR protein 9G8 might be involved in an ESE-dependent activation of splicing. We hypothesized that the L1417X interferes with the splicing of exon 31 by altering an ESE element, creating an ESS element, or both. In addition, by UV cross-linking assays with C2C12 and Hela nuclear extract, a 35-40kDa protein was detected with the wild type RNA probes but the intensity of this band was significantly increased in the presence on the mutation. Moreover, the 35-40kDa protein was also detected using a nuclear fraction lacking SR proteins, raising the possibility that this protein, by interacting preferentially with the mutated sequence, acts as a splicing repressor. A better knowledge of the function and the importance of exonic regulatory splicing sequences could contribute to define the pathway for the development of effective therapies based on exon skipping.
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