Muscleblind-like 1 (Mbnl1) Promotes Insulin Receptor Exon 11 Inclusion via Binding to a Downstream Evolutionarily Conserved Intronic Enhancer

Abstract

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. 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. IntroductionIn mammals, alternative splicing is a common strategy for creating functional diversity in proteins to confer cell and developmentally specific functions. Given its important role, it is not surprising that a recent estimate has proposed that 50–60% of mutations linked to disease affect RNA splicing (1Wang G.S. Cooper T.A. Nat. Rev. Genet. 2007; 8: 749-761Crossref PubMed Scopus (733) Google Scholar, 2López-Bigas N. Audit B. Ouzounis C. Parra G. Guigó R. FEBS Lett. 2005; 579: 1900-1903Crossref PubMed Scopus (261) Google Scholar). The majority of human genes undergo alternative pre-mRNA splicing through the use of competing 5′ or 3′ 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, and the presence of cis regulatory elements within the exon and/or the flanking introns determines whether these exons are recognized (3Krämer A. Annu. Rev. Biochem. 1996; 65: 367-409Crossref PubMed Scopus (617) Google Scholar, 4Rio D.C. Curr. Opin. Cell Biol. 1992; 4: 444-452Crossref PubMed Scopus (26) Google Scholar, 5Lopez A.J. Annu. Rev. Genet. 1998; 32: 279-305Crossref PubMed Scopus (534) Google Scholar). These cis-elements can either have a positive (enhancer) or negative (silencer) effect on splicing. Both enhancers and silencers are thought to function through binding to specific trans-acting protein factors (6Black D.L. Annu. Rev. Biochem. 2003; 72: 291-336Crossref PubMed Scopus (1945) Google Scholar). Differences in the expression or activity 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, 2The abbreviations used are: hnRNPheterogeneous nuclear ribonucleoproteinIRinsulin receptorANOVAanalysis of varianceBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolntnucleotideRTreverse transcriptionsiRNAsmall interfering RNA. and other related RNA-binding proteins such as the CELF family, TIA-1, NOVA1, and A2BP1 (also known as Fox-1) (7Kalsotra A. Xiao X. Ward A.J. Castle J.C. Johnson J.M. Burge C.B. Cooper T.A. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 20333-20338Crossref PubMed Scopus (356) Google Scholar, 8Gromak N. Matlin A.J. Cooper T.A. Smith C.W. RNA. 2003; 9: 443-456Crossref PubMed Scopus (87) Google Scholar, 9Izquierdo J.M. Majós N. Bonnal S. Martínez C. Castelo R. Guigó R. Bilbao D. Valcárcel J. Mol. Cell. 2005; 19: 475-484Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 10Martinez-Contreras R. Cloutier P. Shkreta L. Fisette J.F. Revil T. Chabot B. Adv. Exp. Med. Biol. 2007; 623: 123-147Crossref PubMed Scopus (279) Google Scholar, 11Sanford J.R. Ellis J. Cáceres J.F. Biochem. Soc. Trans. 2005; 33: 443-446Crossref PubMed Scopus (126) Google Scholar, 12Ule J. Jensen K.B. Ruggiu M. Mele A. Ule A. Darnell R.B. Science. 2003; 302: 1212-1215Crossref PubMed Scopus (807) Google Scholar, 13Zhang C. Zhang Z. Castle J. Sun S. Johnson J. Krainer A.R. Zhang M.Q. Genes Dev. 2008; 22: 2550-2563Crossref PubMed Scopus (231) Google Scholar). 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 (14Buratti E. Baralle M. Baralle F.E. Nucleic Acids Res. 2006; 34: 3494-3510Crossref PubMed Scopus (172) Google Scholar, 15Hiller M. Zhang Z. Backofen R. Stamm S. PLoS Genet. 2007; 3: e204Crossref PubMed Scopus (157) Google Scholar, 16Graveley B.R. Cell. 2005; 123: 65-73Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar).The human insulin receptor (IR) is encoded by a single INSR gene (insulin receptor gene) that is located on chromosome 19 and is composed of 22 exons. Transcription of the gene gives rise to two protein isoforms, however, that differ by a 12-amino acid insertion in the hormone-binding domain of the receptor due to alternative splicing of exon 11. This alternative splicing event is restricted to mammals and is not seen in the chicken or frog genes (17Hernández-Sánchez C. Mansilla A. de Pablo F. Zardoya R. Mol. Biol. Evol. 2008; 25: 1043-1053Crossref PubMed Scopus (77) Google Scholar). The IR isoform lacking exon 11 (IR-A) binds both insulin and insulin-like growth factor-II, whereas the exon 11+ isoform (IR-B) only binds insulin (18Frasca F. Pandini G. Scalia P. Sciacca L. Mineo R. Costantino A. Goldfine I.D. Belfiore A. Vigneri R. Mol. Cell. Biol. 1999; 19: 3278-3288Crossref PubMed Scopus (710) Google Scholar). In the adult, the IR-B is expressed predominantly in the insulin-sensitive tissues liver, muscle, adipocytes, and kidney (19Seino S. Bell G.I. Biochem. Biophys. Res. Commun. 1989; 159: 312-316Crossref PubMed Scopus (256) Google Scholar, 20Mosthaf L. Grako K. Dull T.J. Coussens L. Ullrich A. McClain D.A. EMBO J. 1990; 9: 2409-2413Crossref PubMed Scopus (285) Google Scholar, 21Moller D.E. Yokota A. Caro J.F. Flier J.S. Mol. Endocrinol. 1989; 3: 1263-1269Crossref PubMed Scopus (258) Google Scholar) that regulate glucose homeostasis. IR-A is expressed predominantly in fetal cells and the early embryo, including the trophoblast and inner cell mass of the blastocyst (18Frasca F. Pandini G. Scalia P. Sciacca L. Mineo R. Costantino A. Goldfine I.D. Belfiore A. Vigneri R. Mol. Cell. Biol. 1999; 19: 3278-3288Crossref PubMed Scopus (710) Google Scholar, 22Navarrete Santos A. Ramin N. Tonack S. Fischer B. Endocrinology. 2008; 149: 515-524Crossref PubMed Scopus (45) Google Scholar), and there is genetic evidence that the insulin receptor may mediate some of the growth-promoting effects of insulin-like growth factor-II on the mouse embryo (23Louvi A. Accili D. Efstratiadis A. Dev. Biol. 1997; 189: 33-48Crossref PubMed Scopus (319) Google Scholar). Inclusion of IR exon 11 is both developmentally and hormonally regulated and is altered in a number of disease states such as type II diabetes, myotonic dystrophy, aging, and cancer (24Savkur R.S. Philips A.V. Cooper T.A. Nat. Genet. 2001; 29: 40-47Crossref PubMed Scopus (632) Google Scholar, 25Kellerer M. Sesti G. Seffer E. Obermaier-Kusser B. Pongratz D.E. Mosthaf L. Häring H.U. Diabetologia. 1993; 36: 628-632Crossref PubMed Scopus (58) Google Scholar, 26Kosaki A. Webster N.J. J. Biol. Chem. 1993; 268: 21990-21996Abstract Full Text PDF PubMed Google Scholar, 27Mosthaf L. Vogt B. Häring H.U. Ullrich A. Proc. Natl. Acad. Sci. U.S.A. 1991; 88: 4728-4730Crossref PubMed Scopus (94) Google Scholar, 28Norgren S. Zierath J. Galuska D. Wallberg-Henriksson H. Luthman H. Diabetes. 1993; 42: 675-681Crossref PubMed Google Scholar, 29Norgren S. Li L.S. Luthman H. Biochem. Biophys. Res. Commun. 1994; 199: 277-284Crossref PubMed Scopus (34) Google Scholar). The dysregulation of the alternative splicing of the IR may therefore have important consequences for insulin and insulin-like growth factor-II sensitivity and responsiveness. This makes the IR gene an attractive model system for studying the mechanism of alternative splicing, and identification of regulatory sequences and factors that control the IR-B/IR-A ratio is of critical importance for the understanding of the role of IR in different disease states.Myotonic dystrophy type 1 (DM1), one of the most common forms of muscular dystrophy, is associated with muscle hyper-excitability, muscle wasting, cardiac defects, cataracts, smooth muscle disturbances, and insulin resistance. DM1 is caused by an of in the of the protein A.J. D. H. K. T. 1992; Full Text PDF PubMed Scopus Google Scholar, R.S. S. Nat. Genet. 2006; PubMed Scopus Google Scholar). This the proteins, an in the expression of a protein and and to in the splicing of secondary genes in muscle (24Savkur R.S. Philips A.V. Cooper T.A. Nat. Genet. 2001; 29: 40-47Crossref PubMed Scopus (632) Google Scholar, N. Cooper T.A. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar, A.V. Cooper T.A. Science. 1998; PubMed Scopus Google Scholar). is caused by a in the intron of protein pre-mRNA and is also associated with in splicing K. J.F. Science. 2001; PubMed Scopus Google Scholar). splicing of the cardiac is altered to cardiac the is altered muscle hyper-excitability, and the insulin receptor gene is altered to of exon 11 to (24Savkur R.S. Philips A.V. Cooper T.A. Nat. Genet. 2001; 29: 40-47Crossref PubMed Scopus (632) Google Scholar, A. H. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, N. R.S. G. Philips A.V. Cooper T.A. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). The that this the in as it is not seen in other that not insulin have previously intronic splicing enhancers and silencers in the intron 10 of human INSR and have that the CELF protein binds and intronic elements A. J. Webster N.J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Webster N.J. Mol. Cell. Biol. 29: PubMed Scopus Google Scholar). we whether cis-elements in the INSR gene and that the elements not to regulate the we an element in the downstream intron that is conserved across and that this element binds Mbnl1 to promote exon inclusion. of this element eliminates the effect of Mbnl1 in cells that this is the primary binding site of the Mbnl1 to regulate alternative splicing of the INSR also that the relative of Mbnl1 and its the expression of the splice of INSR isoform in a cell or developmental of IR alternative splicing is critical to the insulin receptor isoform ratio and growth factor-II we describe a intronic which is located in the intron downstream of alternatively spliced exon 11. This element by a of and with of sequence This intronic element is conserved across and and as an enhancer in human and of this element and we the ability of Mbnl1 to bind the enhancer in and in We showed that exon 11 inclusion in hepatoma and cells and that the intronic enhancer is for The are different from in S. D. J. Webster N.J. L. S. EMBO J. 2006; 25: PubMed Scopus Google Scholar). Overexpression of Mbnl1 or in the muscle cells not exon 11 knockdown of either Mbnl1 or exon inclusion. is that muscle cells levels of proteins not lead to a further in exon of has that INSR splicing is altered in myotonic dystrophy S. D. J. Webster N.J. L. S. EMBO J. 2006; 25: PubMed Scopus Google Scholar, S. L. S. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, R.S. Philips A.V. Cooper T.A. J.C. J. Genet. Full Text Full Text PDF PubMed Scopus Google Scholar). The proteins are by binding to and in expression of and proteins S. D. J. Webster N.J. L. S. EMBO J. 2006; 25: PubMed Scopus Google Scholar). These in in exon 11 inclusion in the INSR with the binding of to the or of splicing associated with myotonic dystrophy M. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar, J. Proc. Natl. Acad. Sci. U.S.A. 2006; PubMed Scopus Google Scholar). of these have in muscle as this is the site of and gene and proteins are expressed in other Mbnl1 is in muscle and liver, and expression in (7Kalsotra A. Xiao X. Ward A.J. Castle J.C. Johnson J.M. Burge C.B. Cooper T.A. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 20333-20338Crossref PubMed Scopus (356) Google Scholar) have a in and Mbnl1 protein expression in the that the in splicing of This is with the effects of CELF and of proteins on INSR splicing. We have two binding sites for in the INSR one in exon 11 and the other of the sequence in intron 10 S. Webster N.J. Mol. Cell. Biol. 29: PubMed Scopus Google Scholar). Both sites to for the of exon 11 inclusion by as of either element not the We proposed that exon inclusion by RNA secondary and competing for SRp20 In known the for Mbnl1 in the INSR gene evidence for functional effects by Mbnl1 or Mbnl1 is a protein motifs that both RNA and RNA. of the known and binding sites are in of RNA secondary K. Nucleic Acids Res. 2007; PubMed Scopus Google Scholar). Mbnl1 has a for the sequence in the and genes S. S. H. A. S. T. K. M. K. Mol. Genet. 2007; PubMed Scopus Google Scholar). sites downstream of exon 22 of the and Mbnl1 exon 22 S. S. H. A. S. T. K. M. K. Mol. Genet. 2007; PubMed Scopus Google Scholar), the of the sites downstream of the alternatively spliced IR exon 11 is with the effect of Mbnl1 to exon 11 the sites in the and genes are of the alternatively spliced exon N. G. Cooper T.A. EMBO J. PubMed Scopus Google Scholar, K. Nucleic Acids Res. 2007; PubMed Scopus Google Scholar), and Mbnl1 exon in both J. R.B. Nucleic Acids Res. PubMed Scopus Google Scholar) also proposed a model that of an exon is to of the downstream splice whereas Mbnl1 binding downstream of an exon is to of the splice model has proposed for the splicing factor Mele A. Ule J. M. T.A. X. Darnell J.C. Darnell R.B. 2008; PubMed Scopus Google Mbnl1 exon inclusion is In Mbnl1 exon Mbnl1 has to bind to 3′ splice sites and recognition by Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). of alternative splicing in tissues has that Mbnl1 exon as it for the INSR by binding to downstream motifs are downstream of exons that are in muscle and which is with for the these motifs are of exons in J.C. Zhang C. A.V. A. Cooper T.A. Johnson J.M. Nat. Genet. 2008; PubMed Scopus Google Scholar). motifs for the CELF proteins are also downstream of some exons in muscle not These the importance of for the functional effect of the The presence of binding sites in the INSR conserved enhancer element that protein important for Mbnl1 We binding to a in both the human and the INSR other binding sites are is that these sites are also by as a result of protein in the of the enhancer. This the enhancer is a enhancer that the The of recognition of the enhancer is by the that knockdown of either Mbnl1 or exon inclusion in S. D. J. Webster N.J. L. S. EMBO J. 2006; 25: PubMed Scopus Google Scholar). Finally, proteins are not the only splicing factors the intronic enhancer as we have binding sites for and to the relative of these IntroductionIn mammals, alternative splicing is a common strategy for creating functional diversity in proteins to confer cell and developmentally specific functions. Given its important role, it is not surprising that a recent estimate has proposed that 50–60% of mutations linked to disease affect RNA splicing (1Wang G.S. Cooper T.A. Nat. Rev. Genet. 2007; 8: 749-761Crossref PubMed Scopus (733) Google Scholar, 2López-Bigas N. Audit B. Ouzounis C. Parra G. Guigó R. FEBS Lett. 2005; 579: 1900-1903Crossref PubMed Scopus (261) Google Scholar). The majority of human genes undergo alternative pre-mRNA splicing through the use of competing 5′ or 3′ 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, and the presence of cis regulatory elements within the exon and/or the flanking introns determines whether these exons are recognized (3Krämer A. Annu. Rev. Biochem. 1996; 65: 367-409Crossref PubMed Scopus (617) Google Scholar, 4Rio D.C. Curr. Opin. Cell Biol. 1992; 4: 444-452Crossref PubMed Scopus (26) Google Scholar, 5Lopez A.J. Annu. Rev. Genet. 1998; 32: 279-305Crossref PubMed Scopus (534) Google Scholar). These cis-elements can either have a positive (enhancer) or negative (silencer) effect on splicing. Both enhancers and silencers are thought to function through binding to specific trans-acting protein factors (6Black D.L. Annu. Rev. Biochem. 2003; 72: 291-336Crossref PubMed Scopus (1945) Google Scholar). Differences in the expression or activity 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, 2The abbreviations used are: hnRNPheterogeneous nuclear ribonucleoproteinIRinsulin receptorANOVAanalysis of varianceBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolntnucleotideRTreverse transcriptionsiRNAsmall interfering RNA. and other related RNA-binding proteins such as the CELF family, TIA-1, NOVA1, and A2BP1 (also known as Fox-1) (7Kalsotra A. Xiao X. Ward A.J. Castle J.C. Johnson J.M. Burge C.B. Cooper T.A. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 20333-20338Crossref PubMed Scopus (356) Google Scholar, 8Gromak N. Matlin A.J. Cooper T.A. Smith C.W. RNA. 2003; 9: 443-456Crossref PubMed Scopus (87) Google Scholar, 9Izquierdo J.M. Majós N. Bonnal S. Martínez C. Castelo R. Guigó R. Bilbao D. Valcárcel J. Mol. Cell. 2005; 19: 475-484Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 10Martinez-Contreras R. Cloutier P. Shkreta L. Fisette J.F. Revil T. Chabot B. Adv. Exp. Med. Biol. 2007; 623: 123-147Crossref PubMed Scopus (279) Google Scholar, 11Sanford J.R. Ellis J. Cáceres J.F. Biochem. Soc. Trans. 2005; 33: 443-446Crossref PubMed Scopus (126) Google Scholar, 12Ule J. Jensen K.B. Ruggiu M. Mele A. Ule A. Darnell R.B. Science. 2003; 302: 1212-1215Crossref PubMed Scopus (807) Google Scholar, 13Zhang C. Zhang Z. Castle J. Sun S. Johnson J. Krainer A.R. Zhang M.Q. Genes Dev. 2008; 22: 2550-2563Crossref PubMed Scopus (231) Google Scholar). 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 (14Buratti E. Baralle M. Baralle F.E. Nucleic Acids Res. 2006; 34: 3494-3510Crossref PubMed Scopus (172) Google Scholar, 15Hiller M. Zhang Z. Backofen R. Stamm S. PLoS Genet. 2007; 3: e204Crossref PubMed Scopus (157) Google Scholar, 16Graveley B.R. Cell. 2005; 123: 65-73Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar).The human insulin receptor (IR) is encoded by a single INSR gene (insulin receptor gene) that is located on chromosome 19 and is composed of 22 exons. Transcription of the gene gives rise to two protein isoforms, however, that differ by a 12-amino acid insertion in the hormone-binding domain of the receptor due to alternative splicing of exon 11. This alternative splicing event is restricted to mammals and is not seen in the chicken or frog genes (17Hernández-Sánchez C. Mansilla A. de Pablo F. Zardoya R. Mol. Biol. Evol. 2008; 25: 1043-1053Crossref PubMed Scopus (77) Google Scholar). The IR isoform lacking exon 11 (IR-A) binds both insulin and insulin-like growth factor-II, whereas the exon 11+ isoform (IR-B) only binds insulin (18Frasca F. Pandini G. Scalia P. Sciacca L. Mineo R. Costantino A. Goldfine I.D. Belfiore A. Vigneri R. Mol. Cell. Biol. 1999; 19: 3278-3288Crossref PubMed Scopus (710) Google Scholar). In the adult, the IR-B is expressed predominantly in the insulin-sensitive tissues liver, muscle, adipocytes, and kidney (19Seino S. Bell G.I. Biochem. Biophys. Res. Commun. 1989; 159: 312-316Crossref PubMed Scopus (256) Google Scholar, 20Mosthaf L. Grako K. Dull T.J. Coussens L. Ullrich A. McClain D.A. EMBO J. 1990; 9: 2409-2413Crossref PubMed Scopus (285) Google Scholar, 21Moller D.E. Yokota A. Caro J.F. Flier J.S. Mol. Endocrinol. 1989; 3: 1263-1269Crossref PubMed Scopus (258) Google Scholar) that regulate glucose homeostasis. IR-A is expressed predominantly in fetal cells and the early embryo, including the trophoblast and inner cell mass of the blastocyst (18Frasca F. Pandini G. Scalia P. Sciacca L. Mineo R. Costantino A. Goldfine I.D. Belfiore A. Vigneri R. Mol. Cell. Biol. 1999; 19: 3278-3288Crossref PubMed Scopus (710) Google Scholar, 22Navarrete Santos A. Ramin N. Tonack S. Fischer B. Endocrinology. 2008; 149: 515-524Crossref PubMed Scopus (45) Google Scholar), and there is genetic evidence that the insulin receptor may mediate some of the growth-promoting effects of insulin-like growth factor-II on the mouse embryo (23Louvi A. Accili D. Efstratiadis A. Dev. Biol. 1997; 189: 33-48Crossref PubMed Scopus (319) Google Scholar). Inclusion of IR exon 11 is both developmentally and hormonally regulated and is altered in a number of disease states such as type II diabetes, myotonic dystrophy, aging, and cancer (24Savkur R.S. Philips A.V. Cooper T.A. Nat. Genet. 2001; 29: 40-47Crossref PubMed Scopus (632) Google Scholar, 25Kellerer M. Sesti G. Seffer E. Obermaier-Kusser B. Pongratz D.E. Mosthaf L. Häring H.U. Diabetologia. 1993; 36: 628-632Crossref PubMed Scopus (58) Google Scholar, 26Kosaki A. Webster N.J. J. Biol. Chem. 1993; 268: 21990-21996Abstract Full Text PDF PubMed Google Scholar, 27Mosthaf L. Vogt B. Häring H.U. Ullrich A. Proc. Natl. Acad. Sci. U.S.A. 1991; 88: 4728-4730Crossref PubMed Scopus (94) Google Scholar, 28Norgren S. Zierath J. Galuska D. Wallberg-Henriksson H. Luthman H. Diabetes. 1993; 42: 675-681Crossref PubMed Google Scholar, 29Norgren S. Li L.S. Luthman H. Biochem. Biophys. Res. Commun. 1994; 199: 277-284Crossref PubMed Scopus (34) Google Scholar). The dysregulation of the alternative splicing of the IR may therefore have important consequences for insulin and insulin-like growth factor-II sensitivity and responsiveness. This makes the IR gene an attractive model system for studying the mechanism of alternative splicing, and identification of regulatory sequences and factors that control the IR-B/IR-A ratio is of critical importance for the understanding of the role of IR in different disease states.Myotonic dystrophy type 1 (DM1), one of the most common forms of muscular dystrophy, is associated with muscle hyper-excitability, muscle wasting, cardiac defects, cataracts, smooth muscle disturbances, and insulin resistance. DM1 is caused by an of in the of the protein A.J. D. H. K. T. 1992; Full Text PDF PubMed Scopus Google Scholar, R.S. S. Nat. Genet. 2006; PubMed Scopus Google Scholar). This the proteins, an in the expression of a protein and and to in the splicing of secondary genes in muscle (24Savkur R.S. Philips A.V. Cooper T.A. Nat. Genet. 2001; 29: 40-47Crossref PubMed Scopus (632) Google Scholar, N. Cooper T.A. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar, A.V. Cooper T.A. Science. 1998; PubMed Scopus Google Scholar). is caused by a in the intron of protein pre-mRNA and is also associated with in splicing K. J.F. Science. 2001; PubMed Scopus Google Scholar). splicing of the cardiac is altered to cardiac the is altered muscle hyper-excitability, and the insulin receptor gene is altered to of exon 11 to (24Savkur R.S. Philips A.V. Cooper T.A. Nat. Genet. 2001; 29: 40-47Crossref PubMed Scopus (632) Google Scholar, A. H. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, N. R.S. G. Philips A.V. Cooper T.A. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). The that this the in as it is not seen in other that not insulin have previously intronic splicing enhancers and silencers in the intron 10 of human INSR and have that the CELF protein binds and intronic elements A. J. Webster N.J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Webster N.J. Mol. Cell. Biol. 29: PubMed Scopus Google Scholar). we whether cis-elements in the INSR gene and that the elements not to regulate the we an element in the downstream intron that is conserved across and that this element binds Mbnl1 to promote exon inclusion. of this element eliminates the effect of Mbnl1 in cells that this is the primary binding site of the Mbnl1 to regulate alternative splicing of the INSR also that the relative of Mbnl1 and its the expression of the splice of INSR isoform in a cell or developmental