POLYPYRIMIDINE TRACT SEQUENCES DIRECT SELECTION OF ALTERNATIVE BRANCHSITES AND INFLUENCE PROTEIN-BINDING

IVS1, an intron derived from the rat fibronectin gene, is spliced inef ficiently in vitro, involving the use of three alternative branch site s. Mutation of one branch point site, BP3, so as to increase complemen tarity to U2 snRNA resulted in exclusive use of that site and improved splicing efficiency, indicating that the wild type BP3 site is one de terminant of poor IVS1 splicing. Deletions within the polypyrimidine t ract had a variable effect on splicing efficiency and altered the patt ern of branch site usage. Selection of each branch site was influenced negatively by purine substitutions ca. 20 nucleotides downstream. It is proposed that all three IVS1 branch sites are pyrimidine tract-depe ndent. Pyrimidine tract deletions also influenced the crosslinking of PTB (the polypyrimidine tract-binding protein), hnRNP C, and splicing factor U2AF65. All three proteins bound preferentially to distinct reg ions within the polypyrimidine tract and thus are candidates for media ting pyrimidine tract-dependent branch site selection. The findings in dicate the complexity of the IVS1 polypyrimidine tract and suggest a c rucial role for this region in modulating branch site selection and IV S1 splicing.