NATURAL BASE-PAIRING INTERACTIONS BETWEEN 5'-SPLICE-SITE AND BRANCH SITE SEQUENCES AFFECT MAMMALIAN 5'-SPLICE-SITE SELECTION

In the murine gene encoding the neuronal cell adhesion molecule (NCAM) , the integrity of the 5' splice site of exon 18 (E18) is essential fo r regulation of alternative splicing. To further study the contributio n of 5' splice site sequences, we used a simple NCAM pre-mRNA containi ng a portion of E18 fused to E19 and separated by a shortened intron. This RNA is spliced in vitro to produce five sets of lariat intermedia tes and products, the most abundant set displaying aberrant migration in acrylamide/urea gels. Base pairing interactions between positions 5 and +8 of the intron and positions -3 and -6 from the branch point w ere responsible for the faster migration of this set of lariat molecul es. To test whether the duplex structure forms earlier and contributes to 5' splice site selection, we used NCAM substrates carrying the 5' splice sites of E17 and E18 in competition for the 3' splice site of E 19. Mutations upstream of the major branch site improve E18/E19 splici ng in NIH3T3 extracts, whereas compensatory mutations at positions +7 and +8 neutralize the effect of branch site mutations and curtail E18/ E19 splicing. Our data suggest that duplex formation occurs early and interferes with the assembly of complexes initiated on the 5' splice s ite of NCAM E18. This novel type of intron interaction may exist in th e introns of other mammalian pre-mRNAs.