Muscleblind-like 1 (Mbnl1) promotes insulin receptor exon 11 inclusion via binding to a downstream evolutionarily conserved intronic enhancer.
The insulin receptor exists as two isoforms, IR-A and IR-B, which result from alternative splicing of exon 11 in the primary transcript. These two isoforms show a cell-specific distribution, and their relative proportions also vary during development, aging, and in different disease states. We have previously demonstrated that both intron 10 and the alternatively spliced exon 11 contain regulatory sequences that affect insulin receptor splicing both positively and negatively and that these sequences bind the serine/arginine-rich (SR) proteins SRp20 and SF2/ASF and the CELF protein CUG-BP1. In this study, we describe a new intronic splicing element within intron 11 that is highly conserved across species. Using minigenes carrying deletion mutations within intron 11, we demonstrated that this sequence functions as an intronic splicing enhancer. We subsequently used RNA affinity chromatography to identify Mbnl1 as a splicing factor that recognizes this enhancer. By ribonucleoprotein immunoprecipitation, we also established that Mbnl1 binds specifically to the INSR (insulin receptor gene) RNA. Overexpression or knockdown of Mbnl1 in hepatoma and embryonic kidney cells altered the levels of exon 11 inclusion. Finally, we showed that deletion of the intronic enhancer eliminates the ability of Mbnl1 to promote exon inclusion. Collectively, these findings demonstrate a role for Mbnl1 in controlling insulin receptor exon 11 inclusion via binding to a downstream intronic enhancer element.
Medical Research Service, Veterans Affairs San Diego Healthcare System, San Diego, California, USA.
Supriya
S
Sen
Indrani
I
Talukdar
Ying
Y
Liu
Joseph
J
Tam
Sita
S
Reddy
Nicholas J G
NJ
Webster
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004742:Enhancer Elements, Genetic; D019143:Evolution, Molecular; D005091:Exons / Q000235:genetics*; D056945:Hep G2 Cells; D006801:Humans; D007438:Introns / Q000235:genetics*; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D012326:RNA Splicing / Q000235:genetics / Q000502:physiology; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
C493663:MBNL1 protein, human; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D011972:Receptor, Insulin