Abstract
In myotonic dystrophy 1 (DM1), aggregation of the mutant DMPK RNA into RNA-protein complexes containing MBNL1 and MBNL2 has been linked to aberrant splicing of the insulin receptor (IR) RNA. In a parallel line of investigation, elevated levels of CUG-binding protein (CUG-BP) have been shown to result in altered IR splicing in DM1. The relative importance of MBNL1, MBNL2, and CUG-BP in DM1 pathogenesis is, however, unclear. Here we have demonstrated that either small interfering RNA-mediated down-regulation of MBNL1 and MBNL2 or the overexpression of CUG-BP in normal myoblasts results in abnormal IR splicing. Our results suggest that CUG-BP regulates the equilibrium of splice site selection by antagonizing the facilitatory activity of MBNL1 and MBNL2 on IR exon 11 splicing in a dose-dependent manner. We have shown that CUG-BP levels are elevated in DM1 cells by mechanisms that are independent of MBNL1 and MBNL2 loss. Importantly, rescue experiments in DM1 myoblasts demonstrated that loss of MBNL1 function is the key event, whereas the overexpression of CUG-BP plays a secondary role in the aberrant alternative splicing of IR RNA in DM1. Small interfering RNA-mediated down-regulation of MBNL1, MBNL2, and CUG-BP in DM1 myoblasts demonstrated that MBNL1 plays a critical role in the maintenance of DM1 focus integrity. Thus, these experiments demonstrate that sequestration of MBNL1 by the expanded CUG repeats is the primary determinant of both DM1 focus formation and the abnormal splicing of the IR RNA in DM1 myoblasts. The data therefore support MBNL1-mediated therapy for DM1.
Highlights
In myotonic dystrophy 1 (DMI), aggregation of the mutant DMPK RNA into RNA-protein complexes containing MBNLI and MBNL2 has been linked to aberrant splicing of the insulin receptor (IR) RNA
Elevated levels of CUG-binding protein (CUG-BP) act to negatively regulate the inclusion of IR exon 11, whereas a similar increase in the expression of MBNL1 and MBNL2 does not alter the equilibrium of IR exon 11 splice site selection in human myoblasts
In this study we have shown that siRNA-mediated downregulation of MBNL1 and MBNL2 and the overexpression of CUG-BP in normal myoblasts resulted in abnormal IR splicing
Summary
To be sufficient to result both in the for mation of intranuclear foci and t he development of myotonia and other histopathological ch anges th at ar e characteristic of DM1 [7, 8]. Aberrant splicing and function of the chloride cha nnel RNA has been shown to be the mech anistic basis for the myotonia observed both in mice expressing expanded CUG repeat tracts and in DM1 patients [9, 10] Taken together these experiments support a dominant RNA mechanism for DMl. the data suggest th at binding and sequestration of physiologically important RNA-binding proteins by the expanded CUG repeat tracts in DM1 cells r esults in the abnorm al function of RNAs that are normally processed by the sequestered proteins. Functional inactivation of Mbnll in mice results in skeletal muscle myotonia and abnormal chloride ch annel RNA splicing [13] These data provide strong su pport for t he dominant RNA model and demonstrate that sequestration and functional inactivation of MBNL1 by the expanded CUG repeat tracts may play an important role in the development of key features ofDM1 pathology.
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