A COMPLEX OF NUCLEAR PROTEINS MEDIATES SR PROTEIN-BINDING TO A PURINE-RICH SPLICING ENHANCER

A purine-rich splicing enhancer from a constitutive exon has been show n to shift the alternative splicing of calcitonin/CGRP pre-mRNA in viv o. Here, we demonstrate that the native repetitive GAA sequence compri ses the optimal enhancer element and specifically binds a saturable co mplex of proteins required for general splicing in vitro. This complex contains a 37-kDa protein that directly binds the repetitive GAA sequ ence and SRp40, a member of the SR family of non-snRNP splicing factor s, While purified SR proteins do not stably bind the repetitive GAA el ement, exogenous SR proteins become associated with the GAA element in the presence of nuclear extracts and stimulate GAA-dependent splicing , These results suggest that repetitive GAA sequences enhance splicing by binding a protein complex containing a sequence-specific RNA bindi ng protein and a general splicing activator that, in turn, recruit add itional SR proteins. This type of mechanism resembles the tra/tra-2-de pendent recruitment of SR proteins to the Drosophila doublesex alterna tive splicing regulatory element.