The myotonic dystrophies, DM1 and DM2, result from the expansion of CTG and CCTG repeats located in the untranslated regions of two genes, DMPK and ZNF9. Importantly, both DM1 and DM2 are characterized by muscle disease. This congruence in genetics and pathology supports a dominant RNA model in which DM muscle disease results from the expression of mutant mRNAs encoding expanded CUG/CCUG repeats. Consistent with this hypothesis we and others have demonstrated that expression of mRNAs encoding large CUG tracts results in DM1 skeletal muscle pathology. Significantly, in both DM1 and DM2 the expanded CUG/CUGG repeats complex with two novel RNA binding proteins, known as the muscleblind (MBNL) and the muscleblind-like (MBLL) proteins, to form abnormal RNA-protein complexes. Importantly, loss of Mbnl function has been shown to result in myotonia and central nuclei. However all features of DM1 skeletal muscle disease are not observed in Mbnl knockout mice. Taken together these data support the hypothesis that DM1 pathology results from the ability of the CUG repeats to bind and functionally inactivate the RNA- binding proteins, MBNL and MBLL. Growing evidence demonstrates that abnormal function of a subset of physiologically important mRNAs plays a pivotal role in DM1 pathogenesis. However the identity of such RNAs, the mechanism whereby RNA discrimination occurs and the nature of the RNA defects that manifest are currently unclear. We therefore hypothesize that expression of CUG repeats causes the sequestration and loss of function of MBNL and MBLL, which in turn is predicted to result in abnormal function of a set of mRNAs that are normally processed by MBNL and MBLL. To test this hypothesis In Aim1 mutually reinforcing technologies will be used to identify Mbnl/Mbll mRNA targets in vivo. In complimentary experiments both the RNA sequence and the muscleblind structural domains required for high affinity binding will be determined.
In Aim 2 we will test the hypothesis that expression of CUG repeats compromises the function of Mbnl/Mbll target mRNAs. Specifically, we will study the fate of high affinity Mbnl/Mbll target mRNAs in skeletal muscle expressing expanded CUG repeats and define the nature of the RNA defects that manifest.
In Aim 3 we will test the hypothesis that the dual loss of Mbnl and Mbll plays a causal role in the development of RNA defects in vitro and skeletal muscle disease in vivo. ? ? ? ?
