Strong RNA splicing enhancers identified by a modified method of cycled selection interact with SR protein

A modified method of cycled selection was used to characterize splicing enh ancers for exon inclusion from a pool of beta -globin-based three exon/two intron pre-mRNAs with a variable number of random nucleotides incorporated in the internal exon. The pre-mRNAs generated by this method contained rand om sequences ranging from 0 to 18 nucleotides in length. This method was us ed to isolate particular splicing enhancer motifs from a previously enriche d pool of extremely diverse enhancers. After four cycles of selection for m RNA containing the internal exon, a distinct enhancer motif (GACGAC...CAGCA G) was highly enriched. This motif served as strong splicing enhancers in a heterogeneous exon. We have shown here that the selected enhancer motif pr omotes exon inclusion through specific interaction with SRp30. We have also shown that although present in many of our selected splicing enhancers con forming to this motif, a typical purine-rich enhancer sequence is dispensab le for either enhancer activity or binding with SRp30.