CONTROL OF ALTERNATIVE PRE-MESSENGER-RNA SPLICING BY DISTRIBUTED PENTAMERIC REPEATS

Multiple copies of the hexamer TGCATG have been shown to regulate fibr onectin pre-mRNA alternative splicing, GCATG repeats also are clustere d near the regulated calcitonin-specific 3' splice site in the rat cal citonin/CGRP gene, Specific mutagenesis of these repeats in calcitonin /CGRP pre-mRNA resulted in the loss of calcitonin-specific splicing, s uggesting that the native repeats act to enhance alternative exon incl usion, Mutation of subsets of these elements implies that alternative splicing requires a minimum of two repeats, and that the combination o f one intronic and one exonic repeat is necessary for optimal cell-spe cific splicing, However, multimerized intronic repeats inhibited calci tonin-specific splicing in both the wild-type context and in a transcr ipt lacking endogenous repeats. These results suggest that both the nu mber and distribution of repeats may be important features for the reg ulation of tissue-specific alternative splicing, Further, RNA containi ng a single repeat bound cell-specific protein complexes, but tissue-s pecific differences in protein binding were not detected by using mult imerized repeats. Together, these data support a novel model for alter native splicing regulation that requires the cell specific recognition of multiple, distributed sequence elements.