Combinatorial control of a neuron-specific exon

The mouse c-src gene contains a short neuron-specific exon, N1, N1 exon spl icing is partly controlled by an intronic splicing enhancer sequence that a ctivates splicing of a heterologous reporter exon in both neural and nonneu ral cells. Here we attempt to dissect all of the regulatory elements contro lling the N1 exon and examine how these multiple elements work in combinati on. We show that the 3' splice site sequence upstream of exon N1 represses the activation of splicing by the downstream intronic enhancer, This repres sion is stronger in nonneural cells and these two regulatory sequences comb ine to make a reporter exon highly cell-type specific. Substitution of the 3' splice site of this test exon with sites from other exons indicates that activation by the enhancer is very dependent on the nature of the upstream 3' splice site, In addition, we identify a previously uncharacterized puri ne-rich sequence within exon N1 that cooperates with the downstream introni c enhancer to increase exon inclusion, Finally, different regulatory elemen ts were tested in multiple cell lines of both neuronal and nonneuronal orig in. The individual splicing regulatory sequences from the src gene vary wid ely in their activity between different cell lines. These results demonstra te how a simple cassette exon is controlled by a variety of regulatory elem ents that only in combination will produce the correct tissue specificity o f splicing.