Skip to main content
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Aug;15(8):3979–3988. doi: 10.1128/mcb.15.8.3979

A 32-nucleotide exon-splicing enhancer regulates usage of competing 5' splice sites in a differential internal exon.

M B Humphrey 1, J Bryan 1, T A Cooper 1, S M Berget 1
PMCID: PMC230637  PMID: 7623794

Abstract

Large alternatively spliced internal exons are uncommon in vertebrate genes, and the mechanisms governing their usage are unknown. In this report, we examined alternative splicing of a 1-kb internal exon from the human caldesmon gene containing two regulated 5' splice sites that are 687 nucleotides apart. In cell lines normally splicing caldesmon RNA via utilization of the exon-internal 5' splice site, inclusion of the differential exon required a long purine-rich sequence located between the two competing 5' splice sites. This element consisted of four identical 32-nucleotide purine-rich repeats that resemble exon-splicing enhancers (ESE) identified in other genes. One 32-nucleotide repeat supported exon inclusion, repressed usage of the terminal 5' splice site, and functioned in a heterologous exon dependent on exon enhancers for inclusion, indicating that the caldesmon purine-rich sequence can be classified as an ESE. The ESE was required for utilization of the internal 5' splice site only in the presence of the competing 5' splice site and had no effect when placed downstream of the terminal 5' splice site. In the absence of the internal 5' splice site, the ESE activated a normally silent cryptic 5' splice site near the natural internal 5' splice site, indicating that the ESE stimulates upstream 5' splice site selection. We propose that the caldesmon ESE functions to regulate competition between two 5' splice sites within a differential internal exon.

Full Text

The Full Text of this article is available as a PDF (501.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amendt B. A., Hesslein D., Chang L. J., Stoltzfus C. M. Presence of negative and positive cis-acting RNA splicing elements within and flanking the first tat coding exon of human immunodeficiency virus type 1. Mol Cell Biol. 1994 Jun;14(6):3960–3970. doi: 10.1128/mcb.14.6.3960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bretscher A., Lynch W. Identification and localization of immunoreactive forms of caldesmon in smooth and nonmuscle cells: a comparison with the distributions of tropomyosin and alpha-actinin. J Cell Biol. 1985 May;100(5):1656–1663. doi: 10.1083/jcb.100.5.1656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brunak S., Engelbrecht J., Knudsen S. Prediction of human mRNA donor and acceptor sites from the DNA sequence. J Mol Biol. 1991 Jul 5;220(1):49–65. doi: 10.1016/0022-2836(91)90380-o. [DOI] [PubMed] [Google Scholar]
  4. Caputi M., Casari G., Guenzi S., Tagliabue R., Sidoli A., Melo C. A., Baralle F. E. A novel bipartite splicing enhancer modulates the differential processing of the human fibronectin EDA exon. Nucleic Acids Res. 1994 Mar 25;22(6):1018–1022. doi: 10.1093/nar/22.6.1018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen I. T., Chasin L. A. Direct selection for mutations affecting specific splice sites in a hamster dihydrofolate reductase minigene. Mol Cell Biol. 1993 Jan;13(1):289–300. doi: 10.1128/mcb.13.1.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dingus J., Hwo S., Bryan J. Identification by monoclonal antibodies and characterization of human platelet caldesmon. J Cell Biol. 1986 May;102(5):1748–1757. doi: 10.1083/jcb.102.5.1748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dirksen W. P., Hampson R. K., Sun Q., Rottman F. M. A purine-rich exon sequence enhances alternative splicing of bovine growth hormone pre-mRNA. J Biol Chem. 1994 Mar 4;269(9):6431–6436. [PubMed] [Google Scholar]
  9. Dominski Z., Kole R. Selection of splice sites in pre-mRNAs with short internal exons. Mol Cell Biol. 1991 Dec;11(12):6075–6083. doi: 10.1128/mcb.11.12.6075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Eperon I. C., Ireland D. C., Smith R. A., Mayeda A., Krainer A. R. Pathways for selection of 5' splice sites by U1 snRNPs and SF2/ASF. EMBO J. 1993 Sep;12(9):3607–3617. doi: 10.1002/j.1460-2075.1993.tb06034.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Eperon L. P., Graham I. R., Griffiths A. D., Eperon I. C. Effects of RNA secondary structure on alternative splicing of pre-mRNA: is folding limited to a region behind the transcribing RNA polymerase? Cell. 1988 Jul 29;54(3):393–401. doi: 10.1016/0092-8674(88)90202-4. [DOI] [PubMed] [Google Scholar]
  12. Estes P. A., Cooke N. E., Liebhaber S. A. A native RNA secondary structure controls alternative splice-site selection and generates two human growth hormone isoforms. J Biol Chem. 1992 Jul 25;267(21):14902–14908. [PubMed] [Google Scholar]
  13. Frid M. G., Shekhonin B. V., Koteliansky V. E., Glukhova M. A. Phenotypic changes of human smooth muscle cells during development: late expression of heavy caldesmon and calponin. Dev Biol. 1992 Oct;153(2):185–193. doi: 10.1016/0012-1606(92)90104-o. [DOI] [PubMed] [Google Scholar]
  14. Fu X. D., Katz R. A., Skalka A. M., Maniatis T. The role of branchpoint and 3'-exon sequences in the control of balanced splicing of avian retrovirus RNA. Genes Dev. 1991 Feb;5(2):211–220. doi: 10.1101/gad.5.2.211. [DOI] [PubMed] [Google Scholar]
  15. Fu X. D., Mayeda A., Maniatis T., Krainer A. R. General splicing factors SF2 and SC35 have equivalent activities in vitro, and both affect alternative 5' and 3' splice site selection. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11224–11228. doi: 10.1073/pnas.89.23.11224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Furdon P. J., Kole R. The length of the downstream exon and the substitution of specific sequences affect pre-mRNA splicing in vitro. Mol Cell Biol. 1988 Feb;8(2):860–866. doi: 10.1128/mcb.8.2.860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Glukhova M. A., Frid M. G., Koteliansky V. E. Developmental changes in expression of contractile and cytoskeletal proteins in human aortic smooth muscle. J Biol Chem. 1990 Aug 5;265(22):13042–13046. [PubMed] [Google Scholar]
  18. Glukhova M. A., Kabakov A. E., Frid M. G., Ornatsky O. I., Belkin A. M., Mukhin D. N., Orekhov A. N., Koteliansky V. E., Smirnov V. N. Modulation of human aorta smooth muscle cell phenotype: a study of muscle-specific variants of vinculin, caldesmon, and actin expression. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9542–9546. doi: 10.1073/pnas.85.24.9542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Glukhova M. A., Kabakov A. E., Ornatsky O. I., Vasilevskaya T. D., Koteliansky V. E., Smirnov V. N. Immunoreactive forms of caldesmon in cultivated human vascular smooth muscle cells. FEBS Lett. 1987 Jun 29;218(2):292–294. doi: 10.1016/0014-5793(87)81064-5. [DOI] [PubMed] [Google Scholar]
  20. Haruna M., Hayashi K., Yano H., Takeuchi O., Sobue K. Common structural and expressional properties of vertebrate caldesmon genes. Biochem Biophys Res Commun. 1993 Nov 30;197(1):145–153. doi: 10.1006/bbrc.1993.2453. [DOI] [PubMed] [Google Scholar]
  21. Hawkins J. D. A survey on intron and exon lengths. Nucleic Acids Res. 1988 Nov 11;16(21):9893–9908. doi: 10.1093/nar/16.21.9893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hayashi K., Yano H., Hashida T., Takeuchi R., Takeda O., Asada K., Takahashi E., Kato I., Sobue K. Genomic structure of the human caldesmon gene. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):12122–12126. doi: 10.1073/pnas.89.24.12122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hoshijima K., Inoue K., Higuchi I., Sakamoto H., Shimura Y. Control of doublesex alternative splicing by transformer and transformer-2 in Drosophila. Science. 1991 May 10;252(5007):833–836. doi: 10.1126/science.1902987. [DOI] [PubMed] [Google Scholar]
  24. Humphrey M. B., Herrera-Sosa H., Gonzalez G., Lee R., Bryan J. Cloning of cDNAs encoding human caldesmons. Gene. 1992 Mar 15;112(2):197–204. doi: 10.1016/0378-1119(92)90376-z. [DOI] [PubMed] [Google Scholar]
  25. Kakiuchi R., Inui M., Morimoto K., Kanda K., Sobue K., Kakiuchi S. Caldesmon, a calmodulin-binding, F actin-interacting protein, is present in aorta, uterus and platelets. FEBS Lett. 1983 Apr 18;154(2):351–356. doi: 10.1016/0014-5793(83)80181-1. [DOI] [PubMed] [Google Scholar]
  26. Kakizuka A., Ingi T., Murai T., Nakanishi S. A set of U1 snRNA-complementary sequences involved in governing alternative RNA splicing of the kininogen genes. J Biol Chem. 1990 Jun 15;265(17):10102–10108. [PubMed] [Google Scholar]
  27. Katz R. A., Skalka A. M. Control of retroviral RNA splicing through maintenance of suboptimal processing signals. Mol Cell Biol. 1990 Feb;10(2):696–704. doi: 10.1128/mcb.10.2.696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kitamura N., Kitagawa H., Fukushima D., Takagaki Y., Miyata T., Nakanishi S. Structural organization of the human kininogen gene and a model for its evolution. J Biol Chem. 1985 Jul 15;260(14):8610–8617. [PubMed] [Google Scholar]
  29. Krainer A. R., Conway G. C., Kozak D. The essential pre-mRNA splicing factor SF2 influences 5' splice site selection by activating proximal sites. Cell. 1990 Jul 13;62(1):35–42. doi: 10.1016/0092-8674(90)90237-9. [DOI] [PubMed] [Google Scholar]
  30. Lavigueur A., La Branche H., Kornblihtt A. R., Chabot B. A splicing enhancer in the human fibronectin alternate ED1 exon interacts with SR proteins and stimulates U2 snRNP binding. Genes Dev. 1993 Dec;7(12A):2405–2417. doi: 10.1101/gad.7.12a.2405. [DOI] [PubMed] [Google Scholar]
  31. Libri D., Piseri A., Fiszman M. Y. Tissue-specific splicing in vivo of the beta-tropomyosin gene: dependence on an RNA secondary structure. Science. 1991 Jun 28;252(5014):1842–1845. doi: 10.1126/science.2063196. [DOI] [PubMed] [Google Scholar]
  32. Litchfield D. W., Ball E. H. Phosphorylation of caldesmon77 by protein kinase C in vitro and in intact human platelets. J Biol Chem. 1987 Jun 15;262(17):8056–8060. [PubMed] [Google Scholar]
  33. Mar J. H., Antin P. B., Cooper T. A., Ordahl C. P. Analysis of the upstream regions governing expression of the chicken cardiac troponin T gene in embryonic cardiac and skeletal muscle cells. J Cell Biol. 1988 Aug;107(2):573–585. doi: 10.1083/jcb.107.2.573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Marston S. B., Lehman W. Caldesmon is a Ca2+-regulatory component of native smooth-muscle thin filaments. Biochem J. 1985 Nov 1;231(3):517–522. doi: 10.1042/bj2310517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Marston S. B., Redwood C. S. The molecular anatomy of caldesmon. Biochem J. 1991 Oct 1;279(Pt 1):1–16. doi: 10.1042/bj2790001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Marston S. Calcium ion-dependent regulation of uterine smooth muscle thin filaments by caldesmon. Am J Obstet Gynecol. 1989 Jan;160(1):252–257. doi: 10.1016/0002-9378(89)90131-2. [DOI] [PubMed] [Google Scholar]
  37. Mayeda A., Helfman D. M., Krainer A. R. Modulation of exon skipping and inclusion by heterogeneous nuclear ribonucleoprotein A1 and pre-mRNA splicing factor SF2/ASF. Mol Cell Biol. 1993 May;13(5):2993–3001. doi: 10.1128/mcb.13.5.2993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Mayeda A., Krainer A. R. Regulation of alternative pre-mRNA splicing by hnRNP A1 and splicing factor SF2. Cell. 1992 Jan 24;68(2):365–375. doi: 10.1016/0092-8674(92)90477-t. [DOI] [PubMed] [Google Scholar]
  39. Nelson K. K., Green M. R. Mechanism for cryptic splice site activation during pre-mRNA splicing. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6253–6257. doi: 10.1073/pnas.87.16.6253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Ohshima Y., Gotoh Y. Signals for the selection of a splice site in pre-mRNA. Computer analysis of splice junction sequences and like sequences. J Mol Biol. 1987 May 20;195(2):247–259. doi: 10.1016/0022-2836(87)90647-4. [DOI] [PubMed] [Google Scholar]
  41. Owada M. K., Hakura A., Iida K., Yahara I., Sobue K., Kakiuchi S. Occurrence of caldesmon (a calmodulin-binding protein) in cultured cells: comparison of normal and transformed cells. Proc Natl Acad Sci U S A. 1984 May;81(10):3133–3137. doi: 10.1073/pnas.81.10.3133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Robberson B. L., Cote G. J., Berget S. M. Exon definition may facilitate splice site selection in RNAs with multiple exons. Mol Cell Biol. 1990 Jan;10(1):84–94. doi: 10.1128/mcb.10.1.84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sobue K., Muramoto Y., Fujita M., Kakiuchi S. Purification of a calmodulin-binding protein from chicken gizzard that interacts with F-actin. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5652–5655. doi: 10.1073/pnas.78.9.5652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sobue K., Sellers J. R. Caldesmon, a novel regulatory protein in smooth muscle and nonmuscle actomyosin systems. J Biol Chem. 1991 Jul 5;266(19):12115–12118. [PubMed] [Google Scholar]
  45. Solnick D. Alternative splicing caused by RNA secondary structure. Cell. 1985 Dec;43(3 Pt 2):667–676. doi: 10.1016/0092-8674(85)90239-9. [DOI] [PubMed] [Google Scholar]
  46. Solnick D., Lee S. I. Amount of RNA secondary structure required to induce an alternative splice. Mol Cell Biol. 1987 Sep;7(9):3194–3198. doi: 10.1128/mcb.7.9.3194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Staknis D., Reed R. SR proteins promote the first specific recognition of Pre-mRNA and are present together with the U1 small nuclear ribonucleoprotein particle in a general splicing enhancer complex. Mol Cell Biol. 1994 Nov;14(11):7670–7682. doi: 10.1128/mcb.14.11.7670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sterner D. A., Berget S. M. In vivo recognition of a vertebrate mini-exon as an exon-intron-exon unit. Mol Cell Biol. 1993 May;13(5):2677–2687. doi: 10.1128/mcb.13.5.2677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Streuli M., Saito H. Regulation of tissue-specific alternative splicing: exon-specific cis-elements govern the splicing of leukocyte common antigen pre-mRNA. EMBO J. 1989 Mar;8(3):787–796. doi: 10.1002/j.1460-2075.1989.tb03439.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sun Q., Mayeda A., Hampson R. K., Krainer A. R., Rottman F. M. General splicing factor SF2/ASF promotes alternative splicing by binding to an exonic splicing enhancer. Genes Dev. 1993 Dec;7(12B):2598–2608. doi: 10.1101/gad.7.12b.2598. [DOI] [PubMed] [Google Scholar]
  51. Takagaki Y., Kitamura N., Nakanishi S. Cloning and sequence analysis of cDNAs for human high molecular weight and low molecular weight prekininogens. Primary structures of two human prekininogens. J Biol Chem. 1985 Jul 15;260(14):8601–8609. [PubMed] [Google Scholar]
  52. Tanaka K., Watakabe A., Shimura Y. Polypurine sequences within a downstream exon function as a splicing enhancer. Mol Cell Biol. 1994 Feb;14(2):1347–1354. doi: 10.1128/mcb.14.2.1347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Tian M., Maniatis T. A splicing enhancer complex controls alternative splicing of doublesex pre-mRNA. Cell. 1993 Jul 16;74(1):105–114. doi: 10.1016/0092-8674(93)90298-5. [DOI] [PubMed] [Google Scholar]
  54. Tian M., Maniatis T. A splicing enhancer exhibits both constitutive and regulated activities. Genes Dev. 1994 Jul 15;8(14):1703–1712. doi: 10.1101/gad.8.14.1703. [DOI] [PubMed] [Google Scholar]
  55. Tsukahara T., Casciato C., Helfman D. M. Alternative splicing of beta-tropomyosin pre-mRNA: multiple cis-elements can contribute to the use of the 5'- and 3'-splice sites of the nonmuscle/smooth muscle exon 6. Nucleic Acids Res. 1994 Jun 25;22(12):2318–2325. doi: 10.1093/nar/22.12.2318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Ueki N., Sobue K., Kanda K., Hada T., Higashino K. Expression of high and low molecular weight caldesmons during phenotypic modulation of smooth muscle cells. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9049–9053. doi: 10.1073/pnas.84.24.9049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Wakamatsu N., Kobayashi H., Miyatake T., Tsuji S. A novel exon mutation in the human beta-hexosaminidase beta subunit gene affects 3' splice site selection. J Biol Chem. 1992 Feb 5;267(4):2406–2413. [PubMed] [Google Scholar]
  58. Wang C. L., Chalovich J. M., Graceffa P., Lu R. C., Mabuchi K., Stafford W. F. A long helix from the central region of smooth muscle caldesmon. J Biol Chem. 1991 Jul 25;266(21):13958–13963. [PMC free article] [PubMed] [Google Scholar]
  59. Watakabe A., Inoue K., Sakamoto H., Shimura Y. A secondary structure at the 3' splice site affects the in vitro splicing reaction of mouse immunoglobulin mu chain pre-mRNAs. Nucleic Acids Res. 1989 Oct 25;17(20):8159–8169. doi: 10.1093/nar/17.20.8159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Watakabe A., Tanaka K., Shimura Y. The role of exon sequences in splice site selection. Genes Dev. 1993 Mar;7(3):407–418. doi: 10.1101/gad.7.3.407. [DOI] [PubMed] [Google Scholar]
  61. Wu J. Y., Maniatis T. Specific interactions between proteins implicated in splice site selection and regulated alternative splicing. Cell. 1993 Dec 17;75(6):1061–1070. doi: 10.1016/0092-8674(93)90316-i. [DOI] [PubMed] [Google Scholar]
  62. Xu R., Teng J., Cooper T. A. The cardiac troponin T alternative exon contains a novel purine-rich positive splicing element. Mol Cell Biol. 1993 Jun;13(6):3660–3674. doi: 10.1128/mcb.13.6.3660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Yamashiro S., Yamakita Y., Hosoya H., Matsumura F. Phosphorylation of non-muscle caldesmon by p34cdc2 kinase during mitosis. Nature. 1991 Jan 10;349(6305):169–172. doi: 10.1038/349169a0. [DOI] [PubMed] [Google Scholar]
  64. Yang X., Bani M. R., Lu S. J., Rowan S., Ben-David Y., Chabot B. The A1 and A1B proteins of heterogeneous nuclear ribonucleoparticles modulate 5' splice site selection in vivo. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):6924–6928. doi: 10.1073/pnas.91.15.6924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Yano H., Hayashi K., Haruna M., Sobue K. Identification of two distinct promoters in the chicken caldesmon gene. Biochem Biophys Res Commun. 1994 Jun 15;201(2):618–626. doi: 10.1006/bbrc.1994.1746. [DOI] [PubMed] [Google Scholar]
  66. Zahler A. M., Neugebauer K. M., Lane W. S., Roth M. B. Distinct functions of SR proteins in alternative pre-mRNA splicing. Science. 1993 Apr 9;260(5105):219–222. doi: 10.1126/science.8385799. [DOI] [PubMed] [Google Scholar]
  67. Zamore P. D., Patton J. G., Green M. R. Cloning and domain structure of the mammalian splicing factor U2AF. Nature. 1992 Feb 13;355(6361):609–614. doi: 10.1038/355609a0. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES