Identification of Intron and Exon Sequences Involved in Alternative Splicing of Insulin Receptor Pre-mRNA (1998)

The insulin receptor exists as two isoforms, A and B, that result from alternative splicing of exon 11 in the primary transcript. We have shown previously that the alternative splicing is developmentally and hormonally regulated. Consequently, these studies were instigated to identify sequences within the primary RNA transcript that regulate the alternative splicing. Minigenes containing exons 10, 11, and 12 and the intervening introns were constructed and transfected into HepG2 cells, which contain both isoforms of the insulin receptor. The cells were able to splice the minigene transcript to give both A (− exon 11) and B-like (+ exon 11) RNAs. A series of internal deletions within intron 10 were tested for their ability to give A and B RNAs. Intron 10 contained two sequences that modulated exon 11 inclusion; a 48-nucleotide purine-rich sequence at the 5′ end of intron 10 that functions as a splicing enhancer and causes an increase in exon 11 inclusion, and a 43-nucleotide sequence at the 3′ end of intron 10 upstream of the branch point sequence that favors skipping of exon 11. Increasing the length of the polypyrimidine tract at the 3′ end of intron 10 caused exon 11 to be spliced constitutively, indicating that a weak splice site is required for alternative splicing. Finally, point mutations, insertions, and deletions within exon 11 itself were able to regulate inclusion of the exon both positively and negatively. The insulin receptor exists as two isoforms, A and B, that result from alternative splicing of exon 11 in the primary transcript. We have shown previously that the alternative splicing is developmentally and hormonally regulated. Consequently, these studies were instigated to identify sequences within the primary RNA transcript that regulate the alternative splicing. Minigenes containing exons 10, 11, and 12 and the intervening introns were constructed and transfected into HepG2 cells, which contain both isoforms of the insulin receptor. The cells were able to splice the minigene transcript to give both A (− exon 11) and B-like (+ exon 11) RNAs. A series of internal deletions within intron 10 were tested for their ability to give A and B RNAs. Intron 10 contained two sequences that modulated exon 11 inclusion; a 48-nucleotide purine-rich sequence at the 5′ end of intron 10 that functions as a splicing enhancer and causes an increase in exon 11 inclusion, and a 43-nucleotide sequence at the 3′ end of intron 10 upstream of the branch point sequence that favors skipping of exon 11. Increasing the length of the polypyrimidine tract at the 3′ end of intron 10 caused exon 11 to be spliced constitutively, indicating that a weak splice site is required for alternative splicing. Finally, point mutations, insertions, and deletions within exon 11 itself were able to regulate inclusion of the exon both positively and negatively. The human insulin receptor (IR) 1The abbreviations used are: IR, insulin receptor; kb, kilobase pair(s); PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-polymerase chain reaction; BPS, branch point sequence; hnRNP, heterogeneous nuclear ribonucleoprotein. 1The abbreviations used are: IR, insulin receptor; kb, kilobase pair(s); PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-polymerase chain reaction; BPS, branch point sequence; hnRNP, heterogeneous nuclear ribonucleoprotein. is encoded by a single gene that is located on chromosome 19 and composed of 22 exons. The mature IR exists as two isoforms, designated A and B, which result from alternative splicing of the primary transcript (1Ebina Y. Ellis L. Jarnagin K. Edery M. Graf L. Clauser E. Ou J.H. Masiarz F. Kan Y.W. Goldfine I.D. Cell. 1985; 40: 747-758Abstract Full Text PDF PubMed Scopus (958) Google Scholar, 2Seino S. Seino M. Nishi S. Bell G.I. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 114-118Crossref PubMed Scopus (272) Google Scholar, 3Ullrich A. Bell J.R. Chen E.Y. Herrera R. Petruzzelli L.M. Dull T.J. Gray A. Coussens L. Liao Y.C. Tsubokawa M. Nature. 1985; 313: 756-761Crossref PubMed Scopus (1500) Google Scholar). The A isoform lacks exon 11, is expressed ubiquitously, and is the only isoform in lymphocytes, brain, and spleen; the B isoform contains exon 11 and is expressed predominantly in liver, muscle, adipocytes, and kidney (4Mosthaf L. Grako K. Dull T.J. Coussens L. Ullrich A. McClain D.A. EMBO J. 1990; 9: 2409-2413Crossref PubMed Scopus (282) Google Scholar, 5Seino S. Bell G.I. Biochem. Biophys. Res. Commun. 1989; 159: 312-316Crossref PubMed Scopus (252) Google Scholar, 6Moller D.E. Yokota A. Caro J.F. Flier J.S. Mol. Endocrinol. 1989; 3: 1263-1269Crossref PubMed Scopus (257) Google Scholar). Exon 11 is composed of 36 nucleotides that encode a 12-amino acid segment (residues 717–728) of the carboxyl terminus of the α-subunit of IR. A number of investigators have suggested that the isoform ratio could be altered in non-insulin-dependent diabetes mellitus (7Kellerer M. Sesti G. Seffer E. Obermaier-Kusser B. Pongratz D.E. Mosthaf L. Haring H.U. Diabetologia. 1993; 36: 628-632Crossref PubMed Scopus (58) Google Scholar, 8Mosthaf L. Eriksson J. Haring H.U. Groop L. Widen E. Ullrich A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2633-2635Crossref PubMed Scopus (41) Google Scholar, 9Mosthaf L. Vogt B. Haring H.U. Ullrich A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4728-4730Crossref PubMed Scopus (94) Google Scholar, 10Norgren S. Zierath J. Galuska D. Wallberg-Henriksson H. Luthman H. Diabetes. 1993; 42: 675-681Crossref PubMed Google Scholar), but other studies have produced conflicting results (11Anderson C.M. Henry R.R. Knudson P.E. Olefsky J.M. Webster N.J. J. Clin. Endocrinol. Metab. 1993; 76: 1380-1382PubMed Google Scholar, 12Benecke H. Flier J.S. Moller D.E. J. Clin. Invest. 1992; 89: 2066-2070Crossref PubMed Scopus (111) Google Scholar, 13Norgren S. Arner P. Luthman H. J. Clin. Endocrinol. Metab. 1994; 78: 757-762PubMed Google Scholar, 14Hansen T. Bjorbaek C. Vestergaard H. Gronskov K. Bak J.F. Pedersen O. J. Clin. Endocrinol. Metab. 1993; 77: 1500-1505Crossref PubMed Google Scholar). We have found that alterations in isoform ratio in skeletal muscle were associated with hyperinsulinemia rather than diabetes (15Huang Z. Bodkin N.L. Ortmeyer H.K. Zenilman M.E. Webster N.J.G. Hansen B.C. Shuldiner A.R. J. Clin. Endocrinol. Metab. 1996; 81: 1552-1556Crossref PubMed Scopus (16) Google Scholar). Similar results have been found in the rhesus monkey (16Huang Z. Bodkin N.L. Ortmeyer H.K. Hansen B.C. Shuldiner A.R. J. Clin. Invest. 1994; 94: 1289-1296Crossref PubMed Scopus (45) Google Scholar). Along these lines, Sell and co-workers (17Sell S.M. Reese D. Ossowski V.M. J. Biol. Chem. 1994; 269: 30769-30772Abstract Full Text PDF PubMed Google Scholar) have shown that alternative splicing of the IR gene is regulated by insulin in the Fao hepatoma cell line. Furthermore, we have shown that the alternative splicing is hormonally and developmentally regulated in both the HepG2 hepatoma and 3T3-L1 adipocyte cell lines (18Kosaki A. Webster N.J. J. Biol. Chem. 1993; 268: 21990-21996Abstract Full Text PDF PubMed Google Scholar). The changes in splicing were accompanied by increases in insulin sensitivity, as measured by a number of parameters (19Kosaki A. Pillay T.S. Xu L. Webster N.J. J. Biol. Chem. 1995; 270: 20816-20823Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). These data indicate that regulation of the alternative splicing of the IR is important for insulin sensitivity and responsiveness. Splicing of pre-mRNA depends on the presence of relatively short RNA sequence elements, the 3′ splice site, the 5′ splice site, the branch point sequence, and the polypyrimidine tract. In alternative splicing, a given splice site may be selected or ignored depending on the cell type or physiological state. This apparent flexibility of the splicing machinery raises the question of molecular mechanisms involved in selection of certain splice sites over others. A number of factors have been implicated in the choice of alternative splice sites, including RNA secondary structure (20Eperon L.P. Graham I.R. Griffiths A.D. Eperon I.C. Cell. 1988; 54: 393-401Abstract Full Text PDF PubMed Scopus (192) Google Scholar, 21Solnick D. Lee S.I. Mol. Cell. Biol. 1987; 7: 3194-3198Crossref PubMed Scopus (78) Google Scholar, 22Solnick D. Cell. 1985; 43: 667-676Abstract Full Text PDF PubMed Scopus (157) Google Scholar, 23Balvay L. Libri D. Fiszman M.Y. BioEssays. 1993; 15: 165-169Crossref PubMed Scopus (87) Google Scholar), size of the exon (24Sterner D.A. Berget S.M. Mol. Cell. Biol. 1993; 13: 2677-2687Crossref PubMed Scopus (68) Google Scholar, 25Furdon P.J. Kole R. Mol. Cell. Biol. 1988; 8: 860-866Crossref PubMed Scopus (41) Google Scholar), and relative strengths of the competing splice sites (26Nelson K.K. Green M.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6253-6257Crossref PubMed Scopus (72) Google Scholar). Alterations in the splicing pattern of a number of genes have been demonstrated during cellular differentiation (27McKeown M. Annu. Rev. Cell Biol. 1992; 8: 133-155Crossref PubMed Scopus (179) Google Scholar); however, the hormonal regulation of alternative splicing is not as common. We have shown that the alternative splicing of exon 11 of the IR gene is modulated by glucocorticoids in HepG2 cells, and, as mentioned above, insulin modulates splicing in Fao cells (17Sell S.M. Reese D. Ossowski V.M. J. Biol. Chem. 1994; 269: 30769-30772Abstract Full Text PDF PubMed Google Scholar, 18Kosaki A. Webster N.J. J. Biol. Chem. 1993; 268: 21990-21996Abstract Full Text PDF PubMed Google Scholar). Insulin has also been shown to alter the splicing pattern of the COOH terminus of protein kinase C-β in L6 myotubes, and growth factors including epidermal growth factor, platelet-derived growth factor, and basic fibroblast growth factor alter the splicing of the COOH terminus of protein-tyrosine phosphatase 1B in human fibroblasts (28Chalfant C.E. Mischak H. Watson J.E. Winkler B.C. Goodnight J. Farese R.V. Cooper D.R. J. Biol. Chem. 1995; 270: 13326-13332Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 29Shifrin V.I. Neel B.G. J. Biol. Chem. 1993; 268: 25376-25384Abstract Full Text PDF PubMed Google Scholar). Chew and co-workers (30Chew S.L. Lavender P. Clark A.J.L. Ross R.J.M. Endocrinology. 1995; 136: 1939-1944Crossref PubMed Google Scholar) have shown that splicing of the insulin-like growth factor-I mRNA is regulated by growth hormone in HepG2 cells. The molecular mechanisms involved in the hormonal regulation of this process are not understood. A prerequisite for mechanistic studies of hormonal regulation is a knowledge of the RNA sequences involved in the alternative splicing event. To that end, we have identified the regions of intron 10 and exon 11 involved in the alternative splicing of the IR gene. Cell culture reagents were purchased from Life Technologies, Inc., and fetal calf serum was from Gemini Bioproducts (Calabasas, CA). [α-32P]dCTP (3,000 Ci/mmol) was purchased from ICN (Costa Mesa, CA). Taq DNA polymerase (Ampli-Taq) was purchased from Perkin-Elmer. All other chemicals were purchased from Sigma or Fisher. All recombinant DNA manipulations were carried out according to standard protocols. The minigenes were constructed by amplifying regions of the IR gene from HepG2 genomic DNA by PCR using primers containing unique EcoRI, BamHI, HindIII, and BglII restriction sites to allow replacement of segments of the minigene as cassettes. We were not able to obtain the complete intron 11, as it is >8 kb. Consequently, we used the known intronic sequence to amplify the two ends of the intron. Thus, all minigenes contain approximately 180 nucleotides at each end of intron 11. In all plasmids, the minigenes were inserted between EcoRI and BglII sites of pSG5 vector (Stratagene, La Jolla, CA), which contains the SV40 early promoter/enhancer, a rabbit β-globin intron 2, and an SV40 poly(A) signal after the multiple cloning site. All mutants were generated from the complete minigene B, which was composed of five elements (part of exon 10, entire intron 10, exon 11, a deleted intron 11, and part of exon 12). Fig. 1shows schematic diagram of the minigene and the sequence of the introns and exons. Deletions and mutations were verified by dideoxy sequencing. HepG2 cells were maintained routinely in minimum essential medium plus Earle's salts with 10% fetal calf serum at 37 °C under 5% CO2. The cells were plated at a density of ∼2 × 106 cells/well in six-well plates. Medium was changed every 2 days. Minigene plasmids were transfected into HepG2 cells by the calcium phosphate co-precipitation technique. Cells were harvested 48 h later, and total cellular RNA was prepared using RNAzol B (Tel-Test, Inc., Friendswood, TX) according to the manufacturer's protocol. First-strand cDNA was prepared by reverse transcription using 1.0 μg of total RNA in a volume of 20 μl (250 pmol of random hexamer primers, 1 unit of Inhibit-ACE RNase inhibitor (5 Prime → 3 Prime, Inc., Boulder, CO), 200 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.), 50 mm Tris-HCl (pH 8.3), 75 mm KCl, 3 mm MgCl2, 1 mm dithiothreitol, and 1 mm dNTPs) at 42 °C for 1 h. DNA/RNA hybrids were denatured at 95 °C for 2 min. To measure exogenous minigene IR transcripts, the primer pair consisted of oligonucleotides spanning nucleotides 1022–1042 (sense primer, 5′-TAATACGACTCACTATAGGGC-3′) and 1068–1088 (antisense primer, 5′-GCTGCAATAAACAAGTTCTGC-3′), which are unique sequences in the pSG5 vector just upstream and downstream of the cloning sites. To measure endogenous IR transcripts, the primer pair consisted of oligonucleotides spanning nucleotides 2140–2163 (sense primer, 5′-AACCAGAGTGAGTATGAGGATTCG-3′) and 2424–2447 (antisense primer, 5′-TTCTCAAAAGGCCTGTGCTCCTCC-3′) of the IR cDNA (numbering as in Ref. 1Ebina Y. Ellis L. Jarnagin K. Edery M. Graf L. Clauser E. Ou J.H. Masiarz F. Kan Y.W. Goldfine I.D. Cell. 1985; 40: 747-758Abstract Full Text PDF PubMed Scopus (958) Google Scholar), which lie outside the minigene and, thus, are specific for the endogenous gene. Five μl of the cDNA synthesis reaction was used for PCR amplification in a 50-μl final reaction volume (0.5 μmeach oligonucleotide primer, 10 mm Tris-HCl (pH 8.3), 50 mm KCl, 1.5 mm MgCl2, 0.1 mm dNTPs, 2 units of Taq DNA polymerase, and 1 μCi of [α-32P]dCTP). Twenty-five cycles of amplification were performed using a Perkin-Elmer DNA thermal cycler (System 9600). Each cycle consisted of a 30-s denaturation at 94 °C, a 30-s annealing at 55 °C, and a 60-s extension at 72 °C. The number of cycles was optimized to ensure that the amplification lay within the exponential phase. The products of the PCR amplification were resolved by electrophoresis on 8% polyacrylamide gels. The gels were dried and exposed to film at room temperature. The band densities were quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). We have shown previously that the endogenous IR gene in HepG2 cells generates mRNA for both the A and B isoform IR. We used this cell line to investigate which sequences surrounding the alternatively spliced exon 11 are involved in the alternative splicing. A minigene was created from by amplifying each exon and the adjacent intron from the known exon/intron sequences (2Seino S. Seino M. Nishi S. Bell G.I. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 114-118Crossref PubMed Scopus (272) Google Scholar). This minigene contained 110 nucleotides of exon 10, 2.3 kb of intron 10, 36 nucleotides of exon 11, 184 nucleotides of the 5′ end of intron 11, 188 nucleotides of the 3′ end of intron 11, and finally 103 nucleotides of exon 12 (Fig. 2, minigene B). Transfection of minigene B into HepG2 cells gave RNA corresponding to both the A (− exon 11) and B (+ exon 11) isoform splicing patterns in a 40:60 ratio (Fig. 2, panels B and C). The endogenous gene in these cells expressed both isoform RNAs in a 50:50 ratio using the endogenous gene primer pair (data not shown), suggesting that the minigene contained most of the information for correct splicing. A second minigene (minigene A), containing a large internal deletion of 2.0 kb in intron 10 (between positions 3 and 5 in the minigene), caused an increase in the percent of B splicing isoform (Fig. 2, compare minigenes A and B). A further deletion (to position 7) in minigene C caused a complete loss of B isoform splicing suggesting that sequences in this region (between positions 5 and 7) may be important for skipping of exon 11. Two potential branch point sequences (BPS) were identified in the intron upstream of exon 11 (indicated by A in Fig. 2 A). Removal of the upstream potential BPS by deletion to position 9 (minigene D) caused a large (80%) decrease in the amount of B isoform splicing, suggesting that this BPS may be involved in splice site selection. These deletions suggested that the regulatory sequences lie in the region upstream of exon 11. intron 10 was from the 3′ end, and an sequence was found approximately nucleotides upstream of exon 11. A further minigene was created (minigene the of the intron but further the Transfection of minigene into HepG2 cells gave a splicing ratio that was to minigene the sequence to the not splicing. The PCR amplification was performed for cycles in an to identify splice products for these five minigenes (Fig. 2 The spliced products were as as the RNAs that in size to the internal deletions in the RNA was for minigene B as it is to be kb and is not under standard A of was for all minigenes of the size of the internal deletion in intron This PCR was and found to be a splicing intron 10 but containing intron 11. containing intron 10 but intron 11 were Thus, of intron 10 of intron 11 in with the of RNA splicing and that the 3′ splice site of exon 11 is to selection. The increase in B isoform splicing by the internal deletions in minigenes and in Fig. be by alterations in the of the splice sites. this were the deletions have an Consequently, minigenes and were constructed with deletions nucleotides downstream of the 5′ splice site of exon 10 (Fig. position and to the end of minigenes C and (Fig. minigenes and of minigene into HepG2 cells gave B isoform splicing in to minigene A which gave is that of intronic sequences in the 5′ end of intron 10 (between positions 1 and caused a decrease in exon 11 This that this region contains a sequence that inclusion of exon 11. of the sequences between positions 5 and (minigene caused a large increase in the amount of B isoform splicing with the results from minigenes A and C (Fig. The in splice site is for minigenes and than for minigenes A and as a result of the of the upstream enhancer that favors exon 11 These results that sequences between positions 5 and exon of an nucleotides to position 9 (minigene caused complete loss of B isoform splicing. A loss in B isoform splicing was associated with deletion of this region in minigenes C and D. minigene approximately B isoform splicing, suggesting that this deletion the 3′ splice site but is in the presence of the upstream This upstream splicing enhancer was identified by minigenes A and K. of these minigenes contain large internal deletions within intron 10, which the enhancer to the 3′ splice site. is the of this region in the of the This region was deleted in the intron (Fig. minigene of this region caused a decrease in B isoform splicing. Consequently, this region as a splicing enhancer but is it is kb upstream of the splice site minigenes and The enhancer region contains a purine-rich sequence, two deletions were created to the of this purine-rich Minigene lacks the purine-rich sequence, minigene lacks the sequence in this minigene a decrease in B isoform splicing. The purine-rich region is over a of of the have been shown to as splicing in other genes J. Cooper Mol. Cell. Biol. 1995; 15: PubMed Scopus Google Scholar). The sequence in intron 10 not contain but is and may in a The region between positions 5 and that causes exon skipping was as was the enhancer sequence, using minigenes with large internal deletions in intron is the of this region in the of the A minigene was constructed which only the region between positions 5 and (Fig. minigene Transfection of minigene into HepG2 cells gave B isoform RNA with for minigene B with the intron. Thus, of nucleotides from the intron causes an increase in B isoform splicing. The region was further to a 43-nucleotide sequence between positions and (minigene of the nucleotides between positions 5 and on splicing (minigene Removal of nucleotides (minigene or nucleotides to the 3′ splice site of exon 11 (minigene the splice site as be deletions of or nucleotides and caused a in exon 11 inclusion, suggesting that this sequence is for the of the 3′ splice site. internal deletions in intron 10 suggested that of the upstream series of between positions and 9 in the minigene the of the downstream splice site and caused skipping of exon 11 (Fig. minigene all deletions other regulatory regions as have been identified as the branch point nucleotides in but not all of these the in splicing. of the in this region on splicing (Fig. minigene indicating that this sequence be the deletion of the to the splice site (minigene gave exon 11 this result not identify the branch point it is that of these is the of the most of the with the branch point sequence that this could be the branch however, of the branch point in branch point B isoform splicing decrease the region containing the upstream is deleted (minigene is that the deletions may have the of the downstream A deletion from position to 9 between the two of gave a in exon inclusion (minigene The 3′ end of this deletion was 2 nucleotides 5′ to the All of the deletions were constructed by restriction sites. a minigene was constructed with to a site at a position to the deletion mutants (minigene This minigene gave exon inclusion, indicating that of to could result in exon by the of the BPS of nucleotides downstream of the on splicing (minigene The 3′ splice site in this intron contains a single in the of a of in the of the polypyrimidine tract have been shown to have a on RNA splicing M. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). of the to on exon inclusion (minigene the length of the tract to caused the exon to be spliced (minigene Thus, the alternative splicing of exon 11 a weak 3′ splice site and the site the exon to results for other genes J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). To investigate the alternatively spliced exon itself be involved in splice site we mutations into exon 11 in the minigene B. of point mutations in the of the exon caused the exon to be spliced (Fig. minigene point mutations, however, (minigene A deletion of nucleotides or of caused an complete loss of exon inclusion (Fig. minigenes and The point mutations that the exon were tested in with deletions of the region identified (Fig. was with minigenes and that are deleted for the 3′ region (Fig. minigenes and these exon mutations were into minigene which contained a deletion that of the BPS, was an increase in the amount of B isoform splicing. Thus, the BPS is able to in the presence of mutations in the These results indicate that the exon 11 sequences an in the of exon inclusion in both a and The alternative splicing of exon 11 of the IR gene is with the in Fig. The splicing enhancer at the 5′ end of intron 10 favors inclusion of exon 11. This could be to a on the 3′ splice site. as the enhancer is kb upstream of the BPS, the of the enhancer could be on the adjacent 5′ splice site. The site from the the of the enhancer may be to the of the nuclear with the 5′ splice site P.J. 1991; PubMed Scopus Google Scholar). this alternative splicing of the downstream exon is not as this site is used or not the exon is but it may allow the of a splice site. that purine-rich enhancer sequences are known to of splice sites 1995; Google Scholar, J. 1995; Google Scholar, A. La H. A.R. B. 1993; 7: PubMed Scopus (272) Google Scholar, R. J. Cooper Mol. Cell. Biol. 1993; 13: PubMed Scopus Google Scholar, A. A.R. 1993; 7: PubMed Scopus Google Scholar, K. A. Y. Mol. Cell. Biol. 1994; PubMed Scopus Google Scholar, J.F. S. A.R. 1994; PubMed Scopus Google Scholar, A. A.R. Mol. Cell. Biol. 1993; 13: PubMed Scopus Google Scholar). is a of the protein and to splicing enhancer sequences to that identified in the 5′ end of intron of has been shown to inclusion of alternatively spliced exons in the chain B and genes J.F. S. A.R. 1994; PubMed Scopus Google Scholar, A. A.R. Mol. Cell. Biol. 1993; 13: PubMed Scopus Google Scholar). This is to the ability of to the of a splice site, 5′ or over a site. this is by the splicing factor, which favors the of splice sites over The choice of splice sites a between and and are In to the to sequences containing the or G. EMBO J. 1994; 13: PubMed Scopus Google Scholar). The 5′ end of intron 10 also contains the sequence which an site could regulate the alternative splicing is however, deletion of the 5′ end of intron 10 in minigene the potential site but has on the splicing. studies be required to or the regulatory regions that we have identified in the IR gene and splice site selection. the 3′ end of the are two regions that exon is and located in the intron upstream of the secondary structure on the indicate that this region has the potential to a a structure is in is not The of secondary structure in alternative splicing has been L. Libri D. Fiszman M.Y. BioEssays. 1993; 15: 165-169Crossref PubMed Scopus (87) Google Scholar). The polypyrimidine tract at the 3′ splice site is only 9 nucleotides with a of the to give a of 10 not have an on exon however, the length of the polypyrimidine tract to nucleotides the exon Thus, of the exon a splice site. Similar have been with the growth hormone and other genes J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). The second regulatory region is contained within the alternatively spliced exon itself and to have both and the of point mutations caused the exon to be suggesting a for the the other of or deletion of a of caused the exon to be indicating a the point mutations with deletions of the region these regulatory regions could to of the 3′ splice site (Fig. both sequences could be involved in a regulatory region secondary structure the 3′ splice site. studies are to these