Trans-splicing requires that 5′ and 3′
splice sites be independently recognized. Here, we have
used mutational analyses and a sensitive nuclease protection
assay to determine the mechanism of trans-3′
splice site recognition in vitro. Efficient recognition
of the 3′ splice site is dependent upon both the
sequence of the 3′ splice site itself and enhancer
elements located in the 3′ exon. We show that the
presence of three distinct classes of enhancers results
in increased binding of U2 snRNP to the branchpoint region.
Several lines of evidence strongly suggest that the increased
binding of U2 snRNP is mediated by U2AF. These results
expand the roles of enhancers in constitutive splicing
and provide direct support for the recruitment model of
enhancer function.