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 enhancers 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 random sequences ranging from 0 to 18 nucleotides in length. This method was used to isolate particular splicing enhancer motifs from a previously enriched pool of extremely diverse enhancers. After four cycles of selection for mRNA containing the internal exon, a distinct enhancer motif (GACGAC...CAGCAG) was highly enriched. This motif served as strong splicing enhancers in a heterogeneous exon. We have shown here that the selected enhancer motif promotes exon inclusion through specific interaction with SRp30. We have also shown that although present in many of our selected splicing enhancers conforming to this motif, a typical purine-rich enhancer sequence is dispensable for either enhancer activity or binding with SRp30.
Tian
H
H
Department of Genetics, Center for Genetic and Cellular Therapies, Duke University, Durham, North Carolina 27710, USA. h.tian@cgct.duke.edu
Kole
R
R
D020816:Amino Acid Motifs; D001483:Base Sequence; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004742:Enhancer Elements, Genetic; D004926:Escherichia coli / Q000378:metabolism; D005091:Exons; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D012326:RNA Splicing; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D009687:Nuclear Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors