Résumé :
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Myotonic Dystrophy (DM) is the most frequent muscular dystrophy in adult. Myotonic Dystrophy type 1 (DM1) is caused by an expansion of CTG repeats located within the 3'-untranslated region of the DMPK gene, while DM2 is caused by an expansion of CCTG repeats in the intron 1 of the ZNF9 gene. Mutant DMPK or ZNF9 mRNAs, which contain expanded CUG or CCUG repeats, respectively, are retained in nuclear aggregates that sequester the splicing factor MBNL1. Sequestration of MBNL1 leads to splicing mis-regulation and plays an important role in DM1/DM2 pathology. While the basic pathogenesis of DM is now well understood, a paradox remains unexplained: despite a higher number of CCUG repeats, DM2 is less severe than DM1.Fox1 (A2BP1) and Fox2 (RBM9) proteins are splicing regulators. We identified that Fox proteins were specifically recruited within CCUG aggregates in muscle cells and biopsies of DM2 patients. In contrast, Fox1 and Fox2 did not co-localize with CUG aggregates in muscle cells and biopsies of DM1 patients. We confirmed by UV-crosslinking experiment that Fox proteins interacted in vitro with expanded CCUG repeat but not with CUG repeats. Next, analysis of alternative splicing events regulated by Fox but not by MBNL1 revealed that Fox1 and Fox2 were not sequestered within CCUG aggregates. Fluorescence recovery after photobleaching (FRAP) analysis confirmed that Fox proteins were not immobilized within CCUG aggregates. Finally, over-expression of Flag-tagged Fox1 liberated MBNL1 from CCUG aggregates, but not from CUG aggregates. In vitro competition assays confirmed that Fox competed binding of MBNL1 to CCUG repeats. In conclusion, these results suggest that Fox proteins are present within CCUG aggregates but are not sequestered. Importantly, Fox1 and Fox2 chase MBNL1 out of CCUG aggregates. We propose that the competition between Fox and MBNL1 proteins to bind to CCUG repeats may explain the DM paradox and the absence of correlation between the number of CCUG repeat and the severity of DM2 disease.
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