Polypyrimidine tract-binding protein positively regulates inclusion of an alternative 3 '-terminal exon

Polypyrimidine tract-binding protein (PTB) is an abundant vertebrate hnRNP protein. PTB binding sites have been found within introns both upstream and downstream of alternative exons in a number of genes that are negatively c ontrolled by the binding of PTB. We have previously reported that PTB binds to a pyrimidine tract within an RNA processing enhancer located adjacent t o an alternative 3'-terminal exon within the gene coding for calcitonin and calcitonin gene-related peptide. The enhancer consists of a pyrimidine tra ct and CAG directly abutting on a 5' splice site sequence to form a pseudoe xon. Here we show that the binding of PTB to the enhancer pyrimidine tract is functional in that exon inclusion increases when in vivo levels of PTB i ncrease. This is the first example of positive regulation of exon inclusion by PTB. The binding of PTB was antagonistic to the binding of U2AF to the enhancer-located pyrimidine tract. Altering the enhancer pyrimidine tract t o a consensus sequence for the binding of U2AF eliminated enhancement of ex on inclusion in vivo and exon polyadenylation in vitro. An additional PTB b inding site was identified close to the AAUAAA hexanucleotide sequence of t he exon 4 poly(A) site. These observations suggest a dual role for PTB in f acilitating recognition of exon 4: binding to the enhancer pyrimidine tract to interrupt productive recognition of the enhancer pseudoexon by splicing factors and interacting with the poly(A) site to positively affect polyade nylation.