3 ' splice site recognition in nematode trans-splicing involves enhancer-dependent recruitment of U2 snRNP

Trans-splicing requires that 5' and 3' splice sites be independently recogn ized. Here, we have used mutational analyses and a sensitive nuclease prote ction assay to determine the mechanism of trans-3' splice site recognition in vitro. Efficient recognition of the 3' splice site is dependent upon bot h the sequence of the 3' splice site itself and enhancer elements located i n the 3' exon. We show that the presence of three distinct classes of enhan cers results in increased binding of U2 snRNP to the branchpoint region. Se veral lines of evidence strongly suggest that the increased binding of U2 s nRNP is mediated by USAF. These results expand the roles of enhancers in co nstitutive splicing and provide direct support for the recruitment model of enhancer function.