SRp20 and hnRNP A1 binding to IR exon 11 modulate its alternative splicing

Abstract

Alternative splicing of the insulin receptor (IR) transcripts generates two isoforms: isoform A (IR‐A), which lacks 36 bp exon 11, and isoform B (IR‐B), which includes exon 11. The IR‐B and IR‐A isoforms show a cell type specific distribution with their relative proportion varying during development, aging and different disease states. Normally, IR‐B predominates in insulin‐sensitive tissues such as liver, muscle, adipocytes and kidney whereas, various pathological conditions mostly express IR‐A isoform. Also, IR‐B has greater insulin responsiveness than does IR‐A. Recent studies demonstrate that IR gene also exhibits metabolic and hormonal regulation of splicing, although the regulatory factors are mostly unknown. Our lab has previously demonstrated that both exon 11 and intron 10 contain regulatory sequences that affect IR alternative splicing both positively and negatively. Here, we focus on the role of specific cis acting regulatory elements within exon 11 of the IR gene. Fine mapping of cis acting elements within exon has been carried out by six linker scanning mutations (LS1‐LS6) throughout the exon 11. Results indicate the presence of an exonic splicing enhancer (ESE) in the first 12 nucleotides and an exonic splicing silencer (ESS) within the central 12 nucleotides of exon 11. Most naturally occurring ESEs have been shown to bind specific SR proteins, whereas the best characterized ESSs are bound by particular hnRNP proteins. Sequence analysis of ESE in IR exon 11 revealed that it contains a (CUCUUC) consensus binding sequence for SR protein, SRp20. The ESS contains a sequence (CAGGCA) that partially matches the previously identified high‐affinity binding sequence for hnRNP A1 (UAGGGA). Co‐transfection in HepG2 cells of IR minigene B and several SR and hnRNP protein expression vectors reveals that hnRNP A1 is a negative regulator, whereas SRp20 is a positive regulator for IR exon 11 inclusion. Co‐transfection of the deletion and linker scanning mutants of exon 11 with SRp20 or hnRNP A1 confirmed the binding location of these proteins in IR exon 11. To establish in‐vitro binding of SRp20 and hnRNP A1 in ESE and ESS respectively, an RNA affinity assay was performed using RNA templates corresponding to wild type and linker scanning mutants in exon 11, coupled to adipic acid beads and incubated with HeLa cell nuclear extract. By western blot analysis of the eluted proteins, we have established that ESE binds SRp20 whereas the ESS binds hnRNP A1. Co‐transfection of minigene B with siRNA against hnRNP A1 increased whereas siRNA against SRp20 decreased the inclusion of exon 11. Purification of these regulatory proteins for in‐vitro splicing assay as well as UV cross linked competition assay using radio‐labeled probes is currently underway. These results demonstrate for the first time that SRp20 and hnRNP A1 play an antagonistic regulatory role in IR alternative splicing via binding to juxtaposed exonic elements.