Protein binding by the 3' untranslated region of alpha-striated tropomyosin

Tropomyosin is a component protein of the thin filament system in striated muscle, regulating the interaction between actin and myosin. The 3' untranslated region of the alpha-striated tropomyosin gene (TM UTR) induces muscle differentiation when expressed in primary fibroblasts, but the mechanism has not been defined. We hypothesize that fibroblasts utilize resident proteins to effect this response, perhaps by TM UTR binding to protein(s). In order to facilitate identification of protein(s) involved in mediating this differentiation response, we investigated the potential for this sequence to bind to cellular protein utilizing electrophoretic mobility gel shifting analysis (EMSA) with and without UV cross-linking. Under very specific conditions (including pH, KCl, and Mg concentration and extent of phosphorylation of protein), the TM UTR is able to bind protein in cells that differentiate upon TM UTR expression. Protein binding is significantly more extensive in cytoplasmic than nuclear protein preparations. Secondary structure of the RNA probe facilitates protein binding. The molecular masses of bound proteins are approximately 42 and 115 kDa under basal conditions. EMSA analysis of extract from cultured skeletal muscle confirms that protein binding by the TM UTR occurs in this cell type, and is more extensive in less differentiated cells. The demonstration of highly regulated protein binding by the TM UTR raises the possibility that this sequence may cause differentiation by binding to endogenous proteins, and further that this sequence may play a role in normal differentiation. Identification of proteins bound by the TM UTR will be necessary to completely define the mechanism by which it causes differentiation.