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 (original) (raw)

Abstract

We show that addition of SR proteins to in vitro splicing extracts results in a significant increase in assembly of the earliest prespliceosomal complex E and a corresponding decrease in assembly of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex H. In addition, SR proteins promote formation of the E5' and E3' complexes that assemble on RNAs containing only 5' and 3' splice sites, respectively. We conclude that SR proteins promote the earliest specific recognition of both the 5' and 3' splice sites and are limiting for this function in HeLa nuclear extracts. Using UV cross-linking, we demonstrate specific, splice site-dependent RNA-protein interactions of SR proteins in the E, E5', and E3' complexes. SR proteins do not UV cross-link in the H complex, and conversely, hnRNP cross-linking is largely excluded from the E-type complexes. We also show that a discrete complex resembling the E5' complex assembles on both purine-rich and non-purine-rich exonic splicing enhancers. This complex, which we have designated the Enhancer complex, contains U1 small nuclear RNP (snRNP) and is associated with different SR protein family members, depending on the sequence of the enhancer. We propose that both downstream 5' splice site enhancers and exonic enhancers function by establishing a network of pre-mRNA-protein and protein-protein interactions involving U1 snRNP, SR proteins, and U2AF that is similar to the interactions that bring the 5' and 3' splice sites together in the E complex.

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Selected References

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  1. Abmayr S. M., Reed R., Maniatis T. Identification of a functional mammalian spliceosome containing unspliced pre-mRNA. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7216–7220. doi: 10.1073/pnas.85.19.7216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amrein H., Hedley M. L., Maniatis T. The role of specific protein-RNA and protein-protein interactions in positive and negative control of pre-mRNA splicing by Transformer 2. Cell. 1994 Feb 25;76(4):735–746. doi: 10.1016/0092-8674(94)90512-6. [DOI] [PubMed] [Google Scholar]
  3. Bennett M., Michaud S., Kingston J., Reed R. Protein components specifically associated with prespliceosome and spliceosome complexes. Genes Dev. 1992 Oct;6(10):1986–2000. doi: 10.1101/gad.6.10.1986. [DOI] [PubMed] [Google Scholar]
  4. Bennett M., Piñol-Roma S., Staknis D., Dreyfuss G., Reed R. Differential binding of heterogeneous nuclear ribonucleoproteins to mRNA precursors prior to spliceosome assembly in vitro. Mol Cell Biol. 1992 Jul;12(7):3165–3175. doi: 10.1128/mcb.12.7.3165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chiara M. D., Champion-Arnaud P., Buvoli M., Nadal-Ginard B., Reed R. Specific protein-protein interactions between the essential mammalian spliceosome-associated proteins SAP 61 and SAP 114. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6403–6407. doi: 10.1073/pnas.91.14.6403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cooper T. A., Ordahl C. P. Nucleotide substitutions within the cardiac troponin T alternative exon disrupt pre-mRNA alternative splicing. Nucleic Acids Res. 1989 Oct 11;17(19):7905–7921. doi: 10.1093/nar/17.19.7905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cote G. J., Stolow D. T., Peleg S., Berget S. M., Gagel R. F. Identification of exon sequences and an exon binding protein involved in alternative RNA splicing of calcitonin/CGRP. Nucleic Acids Res. 1992 May 11;20(9):2361–2366. doi: 10.1093/nar/20.9.2361. [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. Dreyfuss G., Matunis M. J., Piñol-Roma S., Burd C. G. hnRNP proteins and the biogenesis of mRNA. Annu Rev Biochem. 1993;62:289–321. doi: 10.1146/annurev.bi.62.070193.001445. [DOI] [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. 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]
  12. Fu X. D., Maniatis T. Factor required for mammalian spliceosome assembly is localized to discrete regions in the nucleus. Nature. 1990 Feb 1;343(6257):437–441. doi: 10.1038/343437a0. [DOI] [PubMed] [Google Scholar]
  13. Fu X. D., Maniatis T. Isolation of a complementary DNA that encodes the mammalian splicing factor SC35. Science. 1992 Apr 24;256(5056):535–538. doi: 10.1126/science.1373910. [DOI] [PubMed] [Google Scholar]
  14. Fu X. D., Maniatis T. The 35-kDa mammalian splicing factor SC35 mediates specific interactions between U1 and U2 small nuclear ribonucleoprotein particles at the 3' splice site. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1725–1729. doi: 10.1073/pnas.89.5.1725. [DOI] [PMC free article] [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. Fu X. D. Specific commitment of different pre-mRNAs to splicing by single SR proteins. Nature. 1993 Sep 2;365(6441):82–85. doi: 10.1038/365082a0. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Ge H., Manley J. L. A protein factor, ASF, controls cell-specific alternative splicing of SV40 early pre-mRNA in vitro. Cell. 1990 Jul 13;62(1):25–34. doi: 10.1016/0092-8674(90)90236-8. [DOI] [PubMed] [Google Scholar]
  19. Gozani O., Patton J. G., Reed R. A novel set of spliceosome-associated proteins and the essential splicing factor PSF bind stably to pre-mRNA prior to catalytic step II of the splicing reaction. EMBO J. 1994 Jul 15;13(14):3356–3367. doi: 10.1002/j.1460-2075.1994.tb06638.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hampson R. K., La Follette L., Rottman F. M. Alternative processing of bovine growth hormone mRNA is influenced by downstream exon sequences. Mol Cell Biol. 1989 Apr;9(4):1604–1610. doi: 10.1128/mcb.9.4.1604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hedley M. L., Maniatis T. Sex-specific splicing and polyadenylation of dsx pre-mRNA requires a sequence that binds specifically to tra-2 protein in vitro. Cell. 1991 May 17;65(4):579–586. doi: 10.1016/0092-8674(91)90090-l. [DOI] [PubMed] [Google Scholar]
  22. Helfman D. M., Ricci W. M., Finn L. A. Alternative splicing of tropomyosin pre-mRNAs in vitro and in vivo. Genes Dev. 1988 Dec;2(12A):1627–1638. doi: 10.1101/gad.2.12a.1627. [DOI] [PubMed] [Google Scholar]
  23. Hoffman B. E., Grabowski P. J. U1 snRNP targets an essential splicing factor, U2AF65, to the 3' splice site by a network of interactions spanning the exon. Genes Dev. 1992 Dec;6(12B):2554–2568. doi: 10.1101/gad.6.12b.2554. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Jamison S. F., Crow A., Garcia-Blanco M. A. The spliceosome assembly pathway in mammalian extracts. Mol Cell Biol. 1992 Oct;12(10):4279–4287. doi: 10.1128/mcb.12.10.4279. [DOI] [PMC free article] [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. Kohtz J. D., Jamison S. F., Will C. L., Zuo P., Lührmann R., Garcia-Blanco M. A., Manley J. L. Protein-protein interactions and 5'-splice-site recognition in mammalian mRNA precursors. Nature. 1994 Mar 10;368(6467):119–124. doi: 10.1038/368119a0. [DOI] [PubMed] [Google Scholar]
  29. Krainer A. R., Conway G. C., Kozak D. Purification and characterization of pre-mRNA splicing factor SF2 from HeLa cells. Genes Dev. 1990 Jul;4(7):1158–1171. doi: 10.1101/gad.4.7.1158. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Kuo H. C., Nasim F. H., Grabowski P. J. Control of alternative splicing by the differential binding of U1 small nuclear ribonucleoprotein particle. Science. 1991 Mar 1;251(4997):1045–1050. doi: 10.1126/science.1825520. [DOI] [PubMed] [Google Scholar]
  32. Lamond A. I., Konarska M. M., Sharp P. A. A mutational analysis of spliceosome assembly: evidence for splice site collaboration during spliceosome formation. Genes Dev. 1987 Aug;1(6):532–543. doi: 10.1101/gad.1.6.532. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Libri D., Goux-Pelletan M., Brody E., Fiszman M. Y. Exon as well as intron sequences are cis-regulating elements for the mutually exclusive alternative splicing of the beta tropomyosin gene. Mol Cell Biol. 1990 Oct;10(10):5036–5046. doi: 10.1128/mcb.10.10.5036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ligtenberg M. J., Gennissen A. M., Vos H. L., Hilkens J. A single nucleotide polymorphism in an exon dictates allele dependent differential splicing of episialin mRNA. Nucleic Acids Res. 1991 Jan 25;19(2):297–301. doi: 10.1093/nar/19.2.297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mardon H. J., Sebastio G., Baralle F. E. A role for exon sequences in alternative splicing of the human fibronectin gene. Nucleic Acids Res. 1987 Oct 12;15(19):7725–7733. doi: 10.1093/nar/15.19.7725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Michaud S., Reed R. A functional association between the 5' and 3' splice site is established in the earliest prespliceosome complex (E) in mammals. Genes Dev. 1993 Jun;7(6):1008–1020. doi: 10.1101/gad.7.6.1008. [DOI] [PubMed] [Google Scholar]
  39. Michaud S., Reed R. An ATP-independent complex commits pre-mRNA to the mammalian spliceosome assembly pathway. Genes Dev. 1991 Dec;5(12B):2534–2546. doi: 10.1101/gad.5.12b.2534. [DOI] [PubMed] [Google Scholar]
  40. Nagoshi R. N., Baker B. S. Regulation of sex-specific RNA splicing at the Drosophila doublesex gene: cis-acting mutations in exon sequences alter sex-specific RNA splicing patterns. Genes Dev. 1990 Jan;4(1):89–97. doi: 10.1101/gad.4.1.89. [DOI] [PubMed] [Google Scholar]
  41. Nelson K. K., Green M. R. Splice site selection and ribonucleoprotein complex assembly during in vitro pre-mRNA splicing. Genes Dev. 1988 Mar;2(3):319–329. doi: 10.1101/gad.2.3.319. [DOI] [PubMed] [Google Scholar]
  42. Reed R., Griffith J., Maniatis T. Purification and visualization of native spliceosomes. Cell. 1988 Jun 17;53(6):949–961. doi: 10.1016/s0092-8674(88)90489-8. [DOI] [PubMed] [Google Scholar]
  43. Reed R., Maniatis T. A role for exon sequences and splice-site proximity in splice-site selection. Cell. 1986 Aug 29;46(5):681–690. doi: 10.1016/0092-8674(86)90343-0. [DOI] [PubMed] [Google Scholar]
  44. Reed R. Protein composition of mammalian spliceosomes assembled in vitro. Proc Natl Acad Sci U S A. 1990 Oct;87(20):8031–8035. doi: 10.1073/pnas.87.20.8031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Reed R. The organization of 3' splice-site sequences in mammalian introns. Genes Dev. 1989 Dec;3(12B):2113–2123. doi: 10.1101/gad.3.12b.2113. [DOI] [PubMed] [Google Scholar]
  46. Ricketts M. H., Simons M. J., Parma J., Mercken L., Dong Q., Vassart G. A nonsense mutation causes hereditary goitre in the Afrikander cattle and unmasks alternative splicing of thyroglobulin transcripts. Proc Natl Acad Sci U S A. 1987 May;84(10):3181–3184. doi: 10.1073/pnas.84.10.3181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. Roth M. B., Zahler A. M., Stolk J. A. A conserved family of nuclear phosphoproteins localized to sites of polymerase II transcription. J Cell Biol. 1991 Nov;115(3):587–596. doi: 10.1083/jcb.115.3.587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Seraphin B., Rosbash M. Identification of functional U1 snRNA-pre-mRNA complexes committed to spliceosome assembly and splicing. Cell. 1989 Oct 20;59(2):349–358. doi: 10.1016/0092-8674(89)90296-1. [DOI] [PubMed] [Google Scholar]
  50. Somasekhar M. B., Mertz J. E. Exon mutations that affect the choice of splice sites used in processing the SV40 late transcripts. Nucleic Acids Res. 1985 Aug 12;13(15):5591–5609. doi: 10.1093/nar/13.15.5591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Staknis D., Reed R. Direct interactions between pre-mRNA and six U2 small nuclear ribonucleoproteins during spliceosome assembly. Mol Cell Biol. 1994 May;14(5):2994–3005. doi: 10.1128/mcb.14.5.2994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]
  53. 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]
  54. Talerico M., Berget S. M. Effect of 5' splice site mutations on splicing of the preceding intron. Mol Cell Biol. 1990 Dec;10(12):6299–6305. doi: 10.1128/mcb.10.12.6299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. 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]
  56. 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]
  57. Tian M., Maniatis T. Positive control of pre-mRNA splicing in vitro. Science. 1992 Apr 10;256(5054):237–240. doi: 10.1126/science.1566072. [DOI] [PubMed] [Google Scholar]
  58. Watakabe A., Sakamoto H., Shimura Y. Repositioning of an alternative exon sequence of mouse IgM pre-mRNA activates splicing of the preceding intron. Gene Expr. 1991;1(3):175–184. [PMC free article] [PubMed] [Google Scholar]
  59. 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]
  60. 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]
  61. 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]
  62. Zahler A. M., Lane W. S., Stolk J. A., Roth M. B. SR proteins: a conserved family of pre-mRNA splicing factors. Genes Dev. 1992 May;6(5):837–847. doi: 10.1101/gad.6.5.837. [DOI] [PubMed] [Google Scholar]
  63. 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]
  64. Zamore P. D., Green M. R. Identification, purification, and biochemical characterization of U2 small nuclear ribonucleoprotein auxiliary factor. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9243–9247. doi: 10.1073/pnas.86.23.9243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. 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]
  66. Zhang M., Zamore P. D., Carmo-Fonseca M., Lamond A. I., Green M. R. Cloning and intracellular localization of the U2 small nuclear ribonucleoprotein auxiliary factor small subunit. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8769–8773. doi: 10.1073/pnas.89.18.8769. [DOI] [PMC free article] [PubMed] [Google Scholar]