IDENTIFICATION OF FUNCTIONAL EXONIC SPLICING ENHANCER MOTIFS RECOGNIZED BY INDIVIDUAL SR PROTEINS

Using an in vitro randomization and functional selection procedure, we have identified three novel classes of exonic splicing enhancers (ESE s) recognized by human SF2/ASF, SRp40, and SRp55, respectively. These ESEs are functional in splicing and are highly specific. For SF2/ASF a nd SRp55, in most cases, only the cognate SR protein can efficiently r ecognize an ESE and activate splicing. In contrast, the SRp40-selected ESEs can function with either SRp40 or SRp55, but not with SF2/ASF. U V cross-linking/competition and immunoprecipitation experiments showed that SR proteins recognize their cognate ESEs in nuclear extract by d irect and specific binding. A motif search algorithm was used to deriv e consensus sequences for ESEs recognized by these SR proteins. Each S R protein yielded a distinct 5- to 7-nucleotide degenerate consensus. These three consensus sequences occur at higher frequencies in exons t han in introns and may thus help define exon-intron boundaries. They o ccur in clusters within regions corresponding to naturally occurring, mapped ESEs. We conclude that a remarkably diverse set of sequences ca n function as ESEs. The degeneracy of these motifs is consistent with the fact that exonic enhancers evolved within extremely diverse protei n coding sequences and are recognized by a small number of SR proteins that bind RNA with limited sequence specificity.