AN INTRON ENHANCER RECOGNIZED BY SPLICING FACTORS ACTIVATES POLYADENYLATION

Alternative processing of the pre-messenger RNA encoding calcitonin/ca lcitonin gene-related peptide (CT/CGRP) involves alternative inclusion of a 3'-terminal exon (exon 4) embedded within a six exon primary tra nscript. Expression of CT/CGRP in transgenic mice indicates that inclu sion of exon 4 occurs in a wide variety of tissues, suggesting that th e factors responsible for exon 4 inclusion are widely distributed. Inc lusion of exon 4 requires an enhancer sequence located within the intr on downstream of the poly(A) site of exon 4. Here we show that the int ron enhancer activated in vitro polyadenylation cleavage of precursor RNAs containing the CT/CGRP exon 4 poly(A) site or heterologous poly(A ) sites. To our knowledge this is the first example of an intron-locat ed enhancer that facilitates polyadenylation. Within the enhancer sequ ence is a 5' splice site sequence immediately preceded by a pyrimidine tract. This 5' splice site sequence was required for enhanced polyade nylation and was recognized by both U1 small nuclear ribonucleoprotein s (snRNPs) and alternative splicing factor/splicing factor 2 (ASF/SF2) . Enhancement of polyadenylation required U1 RNA, suggesting that the 5' splice site sequence within the enhancer mediates enhancement via i nteraction with factors normally associated with functional 5' splice sites. Mutation of the polypyrimidine track of the enhancer also inhib ited in vitro polyadenylation cleavage. Oligonucleotide competitions a nd UV cross-linking indicated that the enhancer pyrimidine track binds the polypyrimidine tract binding protein (PTB), but not U2 snRNP auxi liary factor (U2AF), and that binding of PTB was required for maximal enhancer-mediated polyadenylation. These results suggest that the enha ncer binds known splicing factors, and that binding of these factors a ctivates polyadenylation cleavage. Furthermore, these results suggest that regulation of alternative processing of CT/CGRP could occur at th e level of polyadenylation, rather than splicing.