Multisite RNA binding and release of polypyrimidine tract binding protein during the regulation of c-src neural-specific splicing

We studied the role of polypyrimidine tract binding protein in repressing s plicing of the c-src neuronspecific N1 exon. Immunodepletion/add-back exper iments demonstrate that PTB is essential for splicing repression in HeLa ex tract. When splicing is repressed, PTB cross-links to intronic CUCUCU eleme nts flanking the N1 exon. Mutation of the downstream CLI elements causes di ssociation of PTB from the intact upstream CU elements and allows splicing. Thus, PTB molecules bound to multiple elements cooperate to repress splici ng. Interestingly, in neuronal WERI-1 cell extract where N1 is spliced, PTB also binds to the upstream CU elements but is dissociated in the presence of ATP. We conclude that splicing repression by PTB is modulated in differe nt cells by a combination of cooperative binding and ATP-dependent dissocia tion.