Skip to main content
PMC home page
RNA logoLink to RNA
. 1998 Dec;4(12):1664–1673. doi: 10.1017/s1355838298981432

Purine-rich enhancers function in the AT-AC pre-mRNA splicing pathway and do so independently of intact U1 snRNP.

Q Wu 1, A R Krainer 1
PMCID: PMC1369733  PMID: 9848661

Abstract

A rare class of introns in higher eukaryotes is processed by the recently discovered AT-AC spliceosome. AT-AC introns are processed inefficiently in vitro, but the reaction is stimulated by exon-definition interactions involving binding of U1 snRNP to the 5' splice site of the downstream conventional intron. We report that purine-rich exonic splicing enhancers also strongly stimulate sodium channel AT-AC splicing. Intact U2, U4, or U6 snRNAs are not required for enhancer function or for exon definition. Enhancer function is independent of U1 snRNP, showing that splicing stimulation by a downstream 5' splice site and by an exonic enhancer differ mechanistically.

Full Text

The Full Text of this article is available as a PDF (785.1 KB).

Selected References

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

  1. Achsel T., Shimura Y. Factors involved in the activation of pre-mRNA splicing from downstream splicing enhancers. J Biochem. 1996 Jul;120(1):53–60. doi: 10.1093/oxfordjournals.jbchem.a021393. [DOI] [PubMed] [Google Scholar]
  2. Amendt B. A., Si Z. H., Stoltzfus C. M. Presence of exon splicing silencers within human immunodeficiency virus type 1 tat exon 2 and tat-rev exon 3: evidence for inhibition mediated by cellular factors. Mol Cell Biol. 1995 Aug;15(8):4606–4615. doi: 10.1128/mcb.15.8.4606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barabino S. M., Blencowe B. J., Ryder U., Sproat B. S., Lamond A. I. Targeted snRNP depletion reveals an additional role for mammalian U1 snRNP in spliceosome assembly. Cell. 1990 Oct 19;63(2):293–302. doi: 10.1016/0092-8674(90)90162-8. [DOI] [PubMed] [Google Scholar]
  4. Berget S. M. Exon recognition in vertebrate splicing. J Biol Chem. 1995 Feb 10;270(6):2411–2414. doi: 10.1074/jbc.270.6.2411. [DOI] [PubMed] [Google Scholar]
  5. Black D. L. Finding splice sites within a wilderness of RNA. RNA. 1995 Oct;1(8):763–771. [PMC free article] [PubMed] [Google Scholar]
  6. Bruzik J. P., Maniatis T. Enhancer-dependent interaction between 5' and 3' splice sites in trans. Proc Natl Acad Sci U S A. 1995 Jul 18;92(15):7056–7059. doi: 10.1073/pnas.92.15.7056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bruzik J. P., Steitz J. A. Spliced leader RNA sequences can substitute for the essential 5' end of U1 RNA during splicing in a mammalian in vitro system. Cell. 1990 Sep 7;62(5):889–899. doi: 10.1016/0092-8674(90)90264-f. [DOI] [PubMed] [Google Scholar]
  8. Chiara M. D., Reed R. A two-step mechanism for 5' and 3' splice-site pairing. Nature. 1995 Jun 8;375(6531):510–513. doi: 10.1038/375510a0. [DOI] [PubMed] [Google Scholar]
  9. Coulter L. R., Landree M. A., Cooper T. A. Identification of a new class of exonic splicing enhancers by in vivo selection. Mol Cell Biol. 1997 Apr;17(4):2143–2150. doi: 10.1128/mcb.17.4.2143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Crispino J. D., Blencowe B. J., Sharp P. A. Complementation by SR proteins of pre-mRNA splicing reactions depleted of U1 snRNP. Science. 1994 Sep 23;265(5180):1866–1869. doi: 10.1126/science.8091213. [DOI] [PubMed] [Google Scholar]
  11. Crispino J. D., Sharp P. A. A U6 snRNA:pre-mRNA interaction can be rate-limiting for U1-independent splicing. Genes Dev. 1995 Sep 15;9(18):2314–2323. doi: 10.1101/gad.9.18.2314. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Furth P. A., Choe W. T., Rex J. H., Byrne J. C., Baker C. C. Sequences homologous to 5' splice sites are required for the inhibitory activity of papillomavirus late 3' untranslated regions. Mol Cell Biol. 1994 Aug;14(8):5278–5289. doi: 10.1128/mcb.14.8.5278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gontarek R. R., Derse D. Interactions among SR proteins, an exonic splicing enhancer, and a lentivirus Rev protein regulate alternative splicing. Mol Cell Biol. 1996 May;16(5):2325–2331. doi: 10.1128/mcb.16.5.2325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hall S. L., Padgett R. A. Conserved sequences in a class of rare eukaryotic nuclear introns with non-consensus splice sites. J Mol Biol. 1994 Jun 10;239(3):357–365. doi: 10.1006/jmbi.1994.1377. [DOI] [PubMed] [Google Scholar]
  17. Hall S. L., Padgett R. A. Requirement of U12 snRNA for in vivo splicing of a minor class of eukaryotic nuclear pre-mRNA introns. Science. 1996 Mar 22;271(5256):1716–1718. doi: 10.1126/science.271.5256.1716. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Hertel K. J., Lynch K. W., Maniatis T. Common themes in the function of transcription and splicing enhancers. Curr Opin Cell Biol. 1997 Jun;9(3):350–357. doi: 10.1016/s0955-0674(97)80007-5. [DOI] [PubMed] [Google Scholar]
  20. Humphrey M. B., Bryan J., Cooper T. A., Berget S. M. A 32-nucleotide exon-splicing enhancer regulates usage of competing 5' splice sites in a differential internal exon. Mol Cell Biol. 1995 Aug;15(8):3979–3988. doi: 10.1128/mcb.15.8.3979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hwang D. Y., Cohen J. B. U1 snRNA promotes the selection of nearby 5' splice sites by U6 snRNA in mammalian cells. Genes Dev. 1996 Feb 1;10(3):338–350. doi: 10.1101/gad.10.3.338. [DOI] [PubMed] [Google Scholar]
  22. Incorvaia R., Padgett R. A. Base pairing with U6atac snRNA is required for 5' splice site activation of U12-dependent introns in vivo. RNA. 1998 Jun;4(6):709–718. doi: 10.1017/s1355838298980207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jackson I. J. A reappraisal of non-consensus mRNA splice sites. Nucleic Acids Res. 1991 Jul 25;19(14):3795–3798. doi: 10.1093/nar/19.14.3795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jensen A. C. Ledelse gennem Målsaetning i sundhedssektoren: større tilfredshed kan opnåes gennem personale udvikling. Sygeplejersken. 1976 May 5;76(17):13-7, 19. [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Kolossova I., Padgett R. A. U11 snRNA interacts in vivo with the 5' splice site of U12-dependent (AU-AC) pre-mRNA introns. RNA. 1997 Mar;3(3):227–233. [PMC free article] [PubMed] [Google Scholar]
  28. Konarska M. M., Sharp P. A. Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes. Cell. 1987 Jun 19;49(6):763–774. doi: 10.1016/0092-8674(87)90614-3. [DOI] [PubMed] [Google Scholar]
  29. 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]
  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. Lerner M. R., Boyle J. A., Mount S. M., Wolin S. L., Steitz J. A. Are snRNPs involved in splicing? Nature. 1980 Jan 10;283(5743):220–224. doi: 10.1038/283220a0. [DOI] [PubMed] [Google Scholar]
  32. Liu H. X., Zhang M., Krainer A. R. Identification of functional exonic splicing enhancer motifs recognized by individual SR proteins. Genes Dev. 1998 Jul 1;12(13):1998–2012. doi: 10.1101/gad.12.13.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lou H., Gagel R. F., Berget S. M. An intron enhancer recognized by splicing factors activates polyadenylation. Genes Dev. 1996 Jan 15;10(2):208–219. doi: 10.1101/gad.10.2.208. [DOI] [PubMed] [Google Scholar]
  34. Lynch K. W., Maniatis T. Synergistic interactions between two distinct elements of a regulated splicing enhancer. Genes Dev. 1995 Feb 1;9(3):284–293. doi: 10.1101/gad.9.3.284. [DOI] [PubMed] [Google Scholar]
  35. Manley J. L., Tacke R. SR proteins and splicing control. Genes Dev. 1996 Jul 1;10(13):1569–1579. doi: 10.1101/gad.10.13.1569. [DOI] [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. Nilsen T. W. A parallel spliceosome. Science. 1996 Sep 27;273(5283):1813–1813. doi: 10.1126/science.273.5283.1813. [DOI] [PubMed] [Google Scholar]
  38. Ramchatesingh J., Zahler A. M., Neugebauer K. M., Roth M. B., Cooper T. A. A subset of SR proteins activates splicing of the cardiac troponin T alternative exon by direct interactions with an exonic enhancer. Mol Cell Biol. 1995 Sep;15(9):4898–4907. doi: 10.1128/mcb.15.9.4898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Seiwert S. D., Steitz J. A. Uncoupling two functions of the U1 small nuclear ribonucleoprotein particle during in vitro splicing. Mol Cell Biol. 1993 Jun;13(6):3135–3145. doi: 10.1128/mcb.13.6.3135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sharp P. A., Burge C. B. Classification of introns: U2-type or U12-type. Cell. 1997 Dec 26;91(7):875–879. doi: 10.1016/s0092-8674(00)80479-1. [DOI] [PubMed] [Google Scholar]
  42. Staffa A., Cochrane A. Identification of positive and negative splicing regulatory elements within the terminal tat-rev exon of human immunodeficiency virus type 1. Mol Cell Biol. 1995 Aug;15(8):4597–4605. doi: 10.1128/mcb.15.8.4597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Stark J. M., Bazett-Jones D. P., Herfort M., Roth M. B. SR proteins are sufficient for exon bridging across an intron. Proc Natl Acad Sci U S A. 1998 Mar 3;95(5):2163–2168. doi: 10.1073/pnas.95.5.2163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Steingrimsdottir H., Rowley G., Dorado G., Cole J., Lehmann A. R. Mutations which alter splicing in the human hypoxanthine-guanine phosphoribosyltransferase gene. Nucleic Acids Res. 1992 Mar 25;20(6):1201–1208. doi: 10.1093/nar/20.6.1201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. Tacke R., Manley J. L. The human splicing factors ASF/SF2 and SC35 possess distinct, functionally significant RNA binding specificities. EMBO J. 1995 Jul 17;14(14):3540–3551. doi: 10.1002/j.1460-2075.1995.tb07360.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. Tarn W. Y., Steitz J. A. A novel spliceosome containing U11, U12, and U5 snRNPs excises a minor class (AT-AC) intron in vitro. Cell. 1996 Mar 8;84(5):801–811. doi: 10.1016/s0092-8674(00)81057-0. [DOI] [PubMed] [Google Scholar]
  50. Tarn W. Y., Steitz J. A. Highly diverged U4 and U6 small nuclear RNAs required for splicing rare AT-AC introns. Science. 1996 Sep 27;273(5283):1824–1832. doi: 10.1126/science.273.5283.1824. [DOI] [PubMed] [Google Scholar]
  51. Tarn W. Y., Steitz J. A. Pre-mRNA splicing: the discovery of a new spliceosome doubles the challenge. Trends Biochem Sci. 1997 Apr;22(4):132–137. doi: 10.1016/s0968-0004(97)01018-9. [DOI] [PubMed] [Google Scholar]
  52. Tarn W. Y., Steitz J. A. SR proteins can compensate for the loss of U1 snRNP functions in vitro. Genes Dev. 1994 Nov 15;8(22):2704–2717. doi: 10.1101/gad.8.22.2704. [DOI] [PubMed] [Google Scholar]
  53. Tian H., Kole R. Selection of novel exon recognition elements from a pool of random sequences. Mol Cell Biol. 1995 Nov;15(11):6291–6298. doi: 10.1128/mcb.15.11.6291. [DOI] [PMC free article] [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. Valcárcel J., Gaur R. K., Singh R., Green M. R. Interaction of U2AF65 RS region with pre-mRNA branch point and promotion of base pairing with U2 snRNA [corrected]. Science. 1996 Sep 20;273(5282):1706–1709. doi: 10.1126/science.273.5282.1706. [DOI] [PubMed] [Google Scholar]
  56. Wang Z., Hoffmann H. M., Grabowski P. J. Intrinsic U2AF binding is modulated by exon enhancer signals in parallel with changes in splicing activity. RNA. 1995 Mar;1(1):21–35. [PMC free article] [PubMed] [Google Scholar]
  57. Wassarman D. A., Steitz J. A. A base-pairing interaction between U2 and U6 small nuclear RNAs occurs in > 150S complexes in HeLa cell extracts: implications for the spliceosome assembly pathway. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7139–7143. doi: 10.1073/pnas.90.15.7139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wassarman K. M., Steitz J. A. Association with terminal exons in pre-mRNAs: a new role for the U1 snRNP? Genes Dev. 1993 Apr;7(4):647–659. doi: 10.1101/gad.7.4.647. [DOI] [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 Q., Krainer A. R. Splicing of a divergent subclass of AT-AC introns requires the major spliceosomal snRNAs. RNA. 1997 Jun;3(6):586–601. [PMC free article] [PubMed] [Google Scholar]
  61. Wu Q., Krainer A. R. U1-mediated exon definition interactions between AT-AC and GT-AG introns. Science. 1996 Nov 8;274(5289):1005–1008. doi: 10.1126/science.274.5289.1005. [DOI] [PubMed] [Google Scholar]
  62. Yeakley J. M., Hedjran F., Morfin J. P., Merillat N., Rosenfeld M. G., Emeson R. B. Control of calcitonin/calcitonin gene-related peptide pre-mRNA processing by constitutive intron and exon elements. Mol Cell Biol. 1993 Oct;13(10):5999–6011. doi: 10.1128/mcb.13.10.5999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Yeakley J. M., Morfin J. P., Rosenfeld M. G., Fu X. D. A complex of nuclear proteins mediates SR protein binding to a purine-rich splicing enhancer. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7582–7587. doi: 10.1073/pnas.93.15.7582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Yu Y. T., Steitz J. A. Site-specific crosslinking of mammalian U11 and u6atac to the 5' splice site of an AT-AC intron. Proc Natl Acad Sci U S A. 1997 Jun 10;94(12):6030–6035. doi: 10.1073/pnas.94.12.6030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Zahler A. M., Roth M. B. Distinct functions of SR proteins in recruitment of U1 small nuclear ribonucleoprotein to alternative 5' splice sites. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2642–2646. doi: 10.1073/pnas.92.7.2642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Zuo P., Maniatis T. The splicing factor U2AF35 mediates critical protein-protein interactions in constitutive and enhancer-dependent splicing. Genes Dev. 1996 Jun 1;10(11):1356–1368. doi: 10.1101/gad.10.11.1356. [DOI] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES