Myotonic dystrophy (DM) is a neuro-muscular disease with a complex inheritance and an extremely complex molecular pathophysiology. We have suggested that unstable CTG repeats responsible for DM cause the disease at the RNA level via recruitment of specific RNA-binding proteins. We identified a CUG RNA-binding protein, CUGBP 1, and showed that this protein is recruited by CUG repeats into heavy RNA-protein complexes in cardiac tissue from DM patients. In order to investigate the role of CUGBP 1 in skeletal muscle differentiation, we generated primary skeletal muscle lines from DM patients and showed that a significant portion of DM cells fail to exit cell cycle during differentiation and that DM cells are able to proliferate. The failure of DM cells to differentiate is accompanying with a failure to accumulate CUGBP1 in cytoplasm. Our data show that CUGBP1 regulates translation of several mRNAs, including mRNA coding for an inhibitor of cell cycle, p21. p21 plays a key role in the differentiation of skeletal muscle. The major hypothesis of this application is that, under normal conditions, 1) CUGBP1 regulates cell cycle withdrawal in skeletal muscle via induction of p21 translation. 2) In DM skeletal muscle cells, expansion of CUG repeats within the mutant DMPK mRNA recruits CUGBP1 leading to the trapping CUGBP1 in nuclei and to inhibition of its cytoplasmic function: induction of p21. 3) The reduction of p21 in DM muscle cells results in a delay in exit from the cell cycle during muscle differentiation.
Specific Aim I will define molecular mechanisms by which CUGBP1 is trapped in nuclei of DM differentiated cells.
Specific Aim II examines the role of cytoplasmic CUGBP1 in p21-dependent regulation of skeletal muscle differentiation. Myoblast cell cycle progression and exit from the cell cycle will be examined in DM patients and in cell cultures with reduced levels of CUGBP 1.
Specific Aim III will examine whether an increase of CUGBP 1 in nuclei of DM cells affects skeletal muscle differentiation. Myoblast cell cycle withdrawal and efficiency of myoblast fusion will be examined in cells derived from transgenic mice overexpressing CUGBP1. p21-dependent and p21-independent pathways will be examined. We will test whether other CUG repeats binding proteins are also affected in DM skeletal muscle.
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