Ribonuclear inclusions and MBNL1 nuclear sequestration do not affect myoblast differentiation but alter gene splicing...

Myotonic dystrophy type 2 (DM2) is an autosomal dominant multisystemic disorder caused by a CCTG expansion in intron 1 of the zinc finger protein 9 gene on chromosome 3. Mutant transcripts are retained in muscle nuclei producing ribonuclear inclusions, which can bind specific RNA-binding proteins leading to a reduction in their activity. The nuclear sequestration of muscleblind-like proteins appears to be involved in splicing defects of genes directly related to the myotonic dystrophy phenotypes. Experimental evidence suggests that ribonuclear inclusions and muscleblind-like protein 1 (MBNL1) sequestration are strongly involved in DM2 pathogenesis. By using fluorescence in situ hybridization in combination with MBNL1-immunofluorescence, we have observed the presence of ribonuclear inclusions and MBNL1 nuclear sequestration at different time points of in vitro myoblast differentiation in each DM2 patient examined. Immunofluorescence and Western blot analysis of several markers of skeletal muscle differentiation reveal that the degree of differentiation of DM2 myoblasts is comparable to that observed in controls. Nevertheless the splicing pattern of the insulin receptor and MBNL1 transcripts, directly related to the DM2 phenotype, appears to be altered in in vitro differentiated DM2 myotubes. Our data seem indicate that the presence of ribonuclear inclusions and MBNL1 nuclear foci are involved in alteration of alternative splicing but do not impair DM2 myogenic differentiation.