SRp20 and CUG-BP1 modulate insulin receptor exon 11 alternative splicing.
The insulin receptor (IR) exists as two isoforms, IR-A and IR-B, which result from alternative splicing of exon 11 in the primary transcript. This alternative splicing is cell specific, and the relative proportions of exon 11 isoforms also vary during development, aging, and different disease states. We have previously demonstrated that both intron 10 and exon 11 contain regulatory sequences that affect IR splicing both positively and negatively. In this study, we sought to define the precise sequence elements within exon 11 that control exon recognition and cellular factors that recognize these elements. Using minigenes carrying linker-scanning mutations within exon 11, we detected both exonic splicing enhancer and exonic splicing silencer elements. We identified binding of SRp20 and SF2/ASF to the exonic enhancers and CUG-BP1 to the exonic silencer by RNA affinity chromatography. Overexpression and knockdown studies with hepatoma and embryonic kidney cells demonstrated that SRp20 and SF2/ASF increase exon inclusion but that CUG-BP1 causes exon skipping. We found that CUG-BP1 also binds to an additional intronic splicing silencer, located at the 3' end of intron 10, to promote exon 11 skipping. Thus, we propose that SRp20, SF2/ASF, and CUG-BP1 act antagonistically to regulate IR alternative splicing in vivo and that the relative ratios of SRp20 and SF2/ASF to CUG-BP1 in different cells determine the degree of exon inclusion.
Sen
Supriya
S
VA San Diego Healthcare System, San Diego, California 92161, USA.
Talukdar
Indrani
I
Webster
Nicholas J G
NJ
D017398:Alternative Splicing / Q000235:genetics*; D001483:Base Sequence; D000067879:CELF1 Protein; D002460:Cell Line; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005091:Exons / Q000235:genetics*; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011972:Receptor, Insulin / Q000235:genetics*; D012097:Repressor Proteins / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors; D037941:Silencer Elements, Transcriptional / Q000235:genetics
D000067879:CELF1 Protein; C102843:CELF1 protein, human; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; C491388:SRSF3 protein, human; D000068103:Serine-Arginine Splicing Factors; D011972:Receptor, Insulin