A COMPLEX INTRONIC SPLICING ENHANCER FROM THE C-SRC PRE-MESSENGER-RNAACTIVATES INCLUSION OF A HETEROLOGOUS EXON

The mouse c-src gene contains a short neuron-specific exon, N1. To cha racterize the sequences that regulate N1 splicing, we used a heterolog ous gene, derived from the human beta-globin gene, containing a short internal exon that is usually skipped by the splicing machinery. Vario us fragments from the src gene were inserted into the globin substrate to measure their effects on the splicing of the test exon, These clon es were transiently expressed in neuronal and nonneuronal cell lines, and the level of exon inclusion was measured by primer extension. Seve ral sequences from the N1 exon region induced the splicing of the hete rologous exon, The most powerful effect was seen with a sequence from the intron downstream of the N1 exon. This sequence acted as a strong splicing enhancer, activating splicing of the test exon when placed in the intron downstream. The enhancer was strongest in neuronal LA-N-5 cells but also activated splicing in nonneuronal HEK293 cells. Deletio n and linker scanning mutagenesis indicate that the enhancer is made n p of multiple smaller elements that must act in combination, One of th ese elements was identified as the sequence UGCAUG, Three copies of th is element can strongly activate splicing of the test exon in LA-N-5 n euroblastoma cells. These component elements of the src splicing enhan cer are also apparently involved in the spicing of other short cassett e exons.