Differentiation and development of skeletal muscle are complex processes that are regulated at different levels of gene expression. Two groups of proteins, myogenic transcription factors and myocyte enhancer factors 2 (MEF2), play a major role in skeletal muscle myogenesis. Molecular mechanisms, which regulate protein levels of MEF2 and myogenic factors during skeletal muscle myogenesis, are not well understood. In this application, we will examine the hypothesis that a family of CUG RNA-binding proteins controls muscle differentiation through the regulation of MEF2 and MyoD on post-transcription levels. One of the members of this family, CUGBP1, is highly expressed in skeletal muscle and is implicated in the delay of skeletal muscle differentiation in patients with a neuro-muscular disease, Myotonic Dystrophy. Analysis of CUGBP1 expression during differentiation course revealed a dramatic induction of CUGBP1 mRNA and protein levels in differentiating myoblasts, suggesting a key role of CUGBP1 in differentiation of skeletal muscle. The role of CUGBP1 in myogenesis is also confirmed by the analysis of mouse model overexpressing CUGBP1. The unscheduled expression of CUGBP1 in skeletal muscle in vivo leads to an abnormal expression of myogenic regulators (p21, myogenin, MEF2) that is accompanied by significant loss of mouse weight and by underdevelopment. Searching for molecular pathways of CUGBPl-dependent regulation of myogenesis, we found that CUGBP1 binds to MEF2A mRNA and that this binding induces MEF2A translation in cell culture models. Since CUGBP1 levels are dramatically elevated in differentiated myoblasts and because CUGBP1 induces MEF2 expression, we propose that CUGBP 1 regulates skeletal muscle differentiation through translational control of MEF2 proteins and myogenic factors. This application proposes: 1) to study molecular mechanisms responsible for induction of CUGBP1 expression during differentiation and identify regulatory factors controlling CUGBP1 expression during differentiation (Specific Aim 1); and 2) to determine molecular pathway by which elevated levels of CUGBP1 alter expression of myogenic regulators in skeletal muscle (Specific Aim 2). Non-muscle cells, mouse fibroblasts, will be converted into muscle cells by MyoD and MEF2A. The role of CUGBP1 in the MyoD and MEF2A dependent pathways in myogenesis will be investigated.
