SRp20 and CUG-BP1 Modulate Insulin Receptor Exon 11 Alternative Splicing

Abstract

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.In mammals, alternative splicing is a common strategy for creating functional diversities of proteins that have cell and developmentally specific functions. Given the important role for splicing, it is not surprising that a recent estimate has proposed that 50 to 60% of mutations linked to disease affect splicing (21, 43). The majority of human genes undergo alternative pre-mRNA splicing through the use of competing 5Ј or 3Ј splice sites or through alternative inclusion/exclusion of exons in the pre-mRNA. These alternative exons often contain splice sites that diverge from the consensus site, and the presence of cis regulatory elements within the exon and/or the flanking introns determines whether these exons are recognized (18,20,31). These cis elements can have either a positive (enhancer) or a negative (silencer) effect on splicing. Both enhancers and silencers are thought to function through binding to specific trans-acting protein factors (1). Differences in the expression or activities of these trans-acting factors may modulate the recognition of the alternative exon and lead to developmental or tissue-specific differences in splicing. Proteins that bind to specific sequence elements to affect splice site selection include SR proteins, hnRNPs, and other related RNA binding proteins, such as the CELF family, TIA-1, and Raver-1 (11,12,14,25,32). Adding a further layer of regulation, local context, such as RNA secondary structure, may influence the way that binding motifs are recognized by their cognate factors (3, 10, 13).The human insulin receptor (IR) is encoded by a single INSR gene th...

Serine Arginine Splicing Factor 3 Is Involved in Enhanced Splicing of Glucose-6-phosphate Dehydrogenase RNA in Response to Nutrients and Hormones in Liver

“…SRSF3 functions in both alternative splicing and mRNA transport to the cytosol (35,36). Our data suggest that SRSF3 is regulating splicing of G6PD mRNA rather than transport because the loss of SRSF3 decreased the accumulation of spliced RNA (Figs.…”

“…In this regard, members of the hnRNP family of splicing silencing proteins, K, L, and A2/B1, bind to the G6PD regulatory element in the starved state, and this binding is decreased by refeeding (19). Competition between SRSF3 and splicing silencers has been demonstrated in regulation of the alternative splicing of the insulin receptor (36). A similar competition for binding may be part of the regulatory mechanism controlling G6PD splicing in response to nutrients and nutritional status.…”

“…SR proteins enhance the splicing of multiple genes; thus, nutritional status could impact the splicing of many transcripts in addition to G6PD. In this regard, splicing of the insulin receptor mRNA is also regulated by SRSF3, and the resulting isoform switch may be involved in liver development and/or liver regeneration (36). Increased SRSF3 abundance in tumors and cancer cells and identification of multiple SRSF3 targets in differentiating neural cells highlight a much broader role of this protein in organisms (50).…”

RNA-binding protein CUGBP1 regulates insulin secretion via activation of phosphodiesterase 3B in mice

“…Recently, several studies indicate that CUGBP1 may be involved in metabolic disorders. CUGBP1 has been reported to mediate alternative splicing of the insulin receptor [27] and to contribute to insulin resistance [28]. Verma et al showed that CUGBP1 is upregulated in the hearts of diabetic mice [29].…”

Insulin Receptor and its Relationship with Different Forms of Insulin Resistance

“…Such double-stranded stem-loop RNA structures are recognized by trans-acting factors and the whole complex lead to changes in mutual spatial distribution of cis-acting elements in this way forming additional regulatory "possibilities" [18,45,53]. Further studies investigating the alternative splicing of the insulin receptor has led to the identification of ISE and ISS elements in intron 10, as well as to better understanding of its mechanism [133,158]. In addition, SRp20 and SF2/ASF have been shown to modulate exonic enhancer elements (ESE) activity.…”