Control of alternative pre-mRNA splicing by distributed pentameric repeats

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Abstract
Multiple copies of the hexamer TGCATG have been shown to regulate fibronectin pre-mRNA alternative splicing. GCATG repeats also are clustered near the regulated calcitonin-specific 3′ splice site in the rat calcitonin/CGRP gene. Specific mutagenesis of these repeats in calcitonin/CGRP pre-mRNA resulted in the loss of calcitonin-specific splicing, suggesting that the native repeats act to enhance alternative exon inclusion. Mutation of subsets of these elements implies that alternative splicing requires a minimum of two repeats, and that the combination of one intronic and one exonic repeat is necessary for optimal cell-specific splicing. However, multimerized intronic repeats inhibited calcitonin-specific splicing in both the wild-type context and in a transcript lacking endogenous repeats. These results suggest that both the number and distribution of repeats may be important features for the regulation of tissue-specific alternative splicing. Further, RNA containing a single repeat bound cell-specific protein complexes, but tissue-specific differences in protein binding were not detected by using multimerized repeats. Together, these data support a novel model for alternative splicing regulation that requires the cell-specific recognition of multiple, distributed sequence elements.