Skip to main content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
J Virol. 1997 Dec; 71(12): 9096–9107.
doi: 10.1128/jvi.71.12.9096-9107.1997
PMCID: PMC230210
PMID: 9371566

Structural, functional, and protein binding analyses of bovine papillomavirus type 1 exonic splicing enhancers.

Z M Zheng, P J He, and C C Baker
Author information Copyright and License information PMC Disclaimer

Abstract

Alternative splicing plays an important role in regulation of bovine papillomavirus type 1 (BPV-1) gene expression. We have recently identified in BPV-1 late pre-mRNAs two purine-rich exonic splicing enhancers (SE1 and SE2) which also stimulate splicing of a Drosophila doublesex (dsx) pre-mRNA containing a suboptimal 3' splice site. In vivo studies now demonstrate that both SE1 and SE2 are required for preferential use of the BPV-1 nucleotide (nt) 3225 3' splice site in nonpermissive cells. Deletion or mutation of either element in a BPV-1 late pre-mRNA switches splicing to the late-specific alternative 3' splice site at nt 3605. To investigate the sequence specificity of these exonic splicing enhancers, various mutant SE1 or SE2 elements were connected to dsx pre-mRNAs and tested for their stimulatory effects on dsx pre-mRNA splicing in vitro. Substitution of U residues for either A or G residues in and around potential ASF/SF2 binding sites in SE1 or SE2 resulted in a significant reduction of splicing enhancer activity. However, the G-to-U substitutions in both enhancers had the largest effect, reducing splicing to near control levels. Further in vitro analyses showed that splicing enhancement by SE2 could be competed with excess unlabeled SE2 RNA, indicating that SE2 activity in HeLa nuclear extracts is mediated by trans-acting factors. UV cross-linking plus immunoprecipitation assays showed that both wild-type SE1 and SE2 RNAs could bind directly to purified HeLa SR proteins SRp30a (ASF/SF2), SRp55, and SRp75. UV cross-linking experiments also identified a 23-kDa protein which binds to SE2 but not SE1. This protein is present in both HeLa nuclear extracts and S100 extracts but absent from SR protein preparations, suggesting that it is not a classical SR protein. Mutant SE elements (containing G- to U-mutations) which had minimal splicing enhancer activity also had very weak binding capacity for these proteins, strongly suggesting that the binding of these proteins is required for splicing enhancer function.

Full Text

The Full Text of this article is available as a PDF (1.8M).

Selected References

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

  • Baker CC, Howley PM. Differential promoter utilization by the bovine papillomavirus in transformed cells and productively infected wart tissues. EMBO J. 1987 Apr;6(4):1027–1035. [PMC free article] [PubMed] [Google Scholar]
  • Barksdale SK, Baker CC. Differentiation-specific expression from the bovine papillomavirus type 1 P2443 and late promoters. J Virol. 1993 Sep;67(9):5605–5616. [PMC free article] [PubMed] [Google Scholar]
  • Barksdale S, Baker CC. Differentiation-specific alternative splicing of bovine papillomavirus late mRNAs. J Virol. 1995 Oct;69(10):6553–6556. [PMC free article] [PubMed] [Google Scholar]
  • Berget SM. Exon recognition in vertebrate splicing. J Biol Chem. 1995 Feb 10;270(6):2411–2414. [PubMed] [Google Scholar]
  • Black DL. Finding splice sites within a wilderness of RNA. RNA. 1995 Oct;1(8):763–771. [PMC free article] [PubMed] [Google Scholar]
  • Bruzik JP, 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. [PMC free article] [PubMed] [Google Scholar]
  • Cáceres JF, Krainer AR. Functional analysis of pre-mRNA splicing factor SF2/ASF structural domains. EMBO J. 1993 Dec;12(12):4715–4726. [PMC free article] [PubMed] [Google Scholar]
  • Colwill K, Pawson T, Andrews B, Prasad J, Manley JL, Bell JC, Duncan PI. The Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intranuclear distribution. EMBO J. 1996 Jan 15;15(2):265–275. [PMC free article] [PubMed] [Google Scholar]
  • Cote GJ, Stolow DT, Peleg S, Berget SM, Gagel RF. 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. [PMC free article] [PubMed] [Google Scholar]
  • Dignam JD, Lebovitz RM, Roeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. [PMC free article] [PubMed] [Google Scholar]
  • Fu XD. The superfamily of arginine/serine-rich splicing factors. RNA. 1995 Sep;1(7):663–680. [PMC free article] [PubMed] [Google Scholar]
  • Fu XD, Maniatis T. Factor required for mammalian spliceosome assembly is localized to discrete regions in the nucleus. Nature. 1990 Feb 1;343(6257):437–441. [PubMed] [Google Scholar]
  • Fu XD, 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. [PMC free article] [PubMed] [Google Scholar]
  • Gaur RK, Valcárcel J, Green MR. Sequential recognition of the pre-mRNA branch point by U2AF65 and a novel spliceosome-associated 28-kDa protein. RNA. 1995 Jun;1(4):407–417. [PMC free article] [PubMed] [Google Scholar]
  • Ge H, Zuo P, Manley JL. Primary structure of the human splicing factor ASF reveals similarities with Drosophila regulators. Cell. 1991 Jul 26;66(2):373–382. [PubMed] [Google Scholar]
  • Gontarek RR, 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. [PMC free article] [PubMed] [Google Scholar]
  • Gui JF, Lane WS, Fu XD. A serine kinase regulates intracellular localization of splicing factors in the cell cycle. Nature. 1994 Jun 23;369(6482):678–682. [PubMed] [Google Scholar]
  • Gui JF, Tronchère H, Chandler SD, Fu XD. Purification and characterization of a kinase specific for the serine- and arginine-rich pre-mRNA splicing factors. Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):10824–10828. [PMC free article] [PubMed] [Google Scholar]
  • Hodges D, Bernstein SI. Genetic and biochemical analysis of alternative RNA splicing. Adv Genet. 1994;31:207–281. [PubMed] [Google Scholar]
  • Jamison SF, Crow A, Garcia-Blanco MA. The spliceosome assembly pathway in mammalian extracts. Mol Cell Biol. 1992 Oct;12(10):4279–4287. [PMC free article] [PubMed] [Google Scholar]
  • Kanopka A, Mühlemann O, Akusjärvi G. Inhibition by SR proteins of splicing of a regulated adenovirus pre-mRNA. Nature. 1996 Jun 6;381(6582):535–538. [PubMed] [Google Scholar]
  • Kohtz JD, Jamison SF, Will CL, Zuo P, Lührmann R, Garcia-Blanco MA, Manley JL. Protein-protein interactions and 5'-splice-site recognition in mammalian mRNA precursors. Nature. 1994 Mar 10;368(6467):119–124. [PubMed] [Google Scholar]
  • Lavigueur A, La Branche H, Kornblihtt AR, 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. [PubMed] [Google Scholar]
  • Lynch KW, Maniatis T. Synergistic interactions between two distinct elements of a regulated splicing enhancer. Genes Dev. 1995 Feb 1;9(3):284–293. [PubMed] [Google Scholar]
  • MacMillan AM, Query CC, Allerson CR, Chen S, Verdine GL, Sharp PA. Dynamic association of proteins with the pre-mRNA branch region. Genes Dev. 1994 Dec 15;8(24):3008–3020. [PubMed] [Google Scholar]
  • Manley JL, Tacke R. SR proteins and splicing control. Genes Dev. 1996 Jul 1;10(13):1569–1579. [PubMed] [Google Scholar]
  • McNally LM, McNally MT. SR protein splicing factors interact with the Rous sarcoma virus negative regulator of splicing element. J Virol. 1996 Feb;70(2):1163–1172. [PMC free article] [PubMed] [Google Scholar]
  • 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. [PubMed] [Google Scholar]
  • Neugebauer KM, Stolk JA, Roth MB. A conserved epitope on a subset of SR proteins defines a larger family of Pre-mRNA splicing factors. J Cell Biol. 1995 May;129(4):899–908. [PMC free article] [PubMed] [Google Scholar]
  • Nielsen DA, Shapiro DJ. Preparation of capped RNA transcripts using T7 RNA polymerase. Nucleic Acids Res. 1986 Jul 25;14(14):5936–5936. [PMC free article] [PubMed] [Google Scholar]
  • Nilsen TW. RNA-RNA interactions in the spliceosome: unraveling the ties that bind. Cell. 1994 Jul 15;78(1):1–4. [PubMed] [Google Scholar]
  • Ramchatesingh J, Zahler AM, Neugebauer KM, Roth MB, Cooper TA. 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. [PMC free article] [PubMed] [Google Scholar]
  • Rossi F, Labourier E, Forné T, Divita G, Derancourt J, Riou JF, Antoine E, Cathala G, Brunel C, Tazi J. Specific phosphorylation of SR proteins by mammalian DNA topoisomerase I. Nature. 1996 May 2;381(6577):80–82. [PubMed] [Google Scholar]
  • Roth MB, Murphy C, Gall JG. A monoclonal antibody that recognizes a phosphorylated epitope stains lampbrush chromosome loops and small granules in the amphibian germinal vesicle. J Cell Biol. 1990 Dec;111(6 Pt 1):2217–2223. [PMC free article] [PubMed] [Google Scholar]
  • Roth MB, Zahler AM, Stolk JA. A conserved family of nuclear phosphoproteins localized to sites of polymerase II transcription. J Cell Biol. 1991 Nov;115(3):587–596. [PMC free article] [PubMed] [Google Scholar]
  • Ruskin B, Zamore PD, Green MR. A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly. Cell. 1988 Jan 29;52(2):207–219. [PubMed] [Google Scholar]
  • Sharp PA. Split genes and RNA splicing. Cell. 1994 Jun 17;77(6):805–815. [PubMed] [Google Scholar]
  • 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. [PMC free article] [PubMed] [Google Scholar]
  • Stamm S, Zhang MQ, Marr TG, Helfman DM. A sequence compilation and comparison of exons that are alternatively spliced in neurons. Nucleic Acids Res. 1994 May 11;22(9):1515–1526. [PMC free article] [PubMed] [Google Scholar]
  • Sun Q, Mayeda A, Hampson RK, Krainer AR, Rottman FM. General splicing factor SF2/ASF promotes alternative splicing by binding to an exonic splicing enhancer. Genes Dev. 1993 Dec;7(12B):2598–2608. [PubMed] [Google Scholar]
  • Tacke R, Manley JL. The human splicing factors ASF/SF2 and SC35 possess distinct, functionally significant RNA binding specificities. EMBO J. 1995 Jul 17;14(14):3540–3551. [PMC free article] [PubMed] [Google Scholar]
  • 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. [PMC free article] [PubMed] [Google Scholar]
  • Tian M, Maniatis T. A splicing enhancer complex controls alternative splicing of doublesex pre-mRNA. Cell. 1993 Jul 16;74(1):105–114. [PubMed] [Google Scholar]
  • Tian M, Maniatis T. A splicing enhancer exhibits both constitutive and regulated activities. Genes Dev. 1994 Jul 15;8(14):1703–1712. [PubMed] [Google Scholar]
  • Valcárcel J, Gaur RK, Singh R, Green MR. 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. [PubMed] [Google Scholar]
  • Valcárcel J, Green MR. The SR protein family: pleiotropic functions in pre-mRNA splicing. Trends Biochem Sci. 1996 Aug;21(8):296–301. [PubMed] [Google Scholar]
  • Watakabe A, Tanaka K, Shimura Y. The role of exon sequences in splice site selection. Genes Dev. 1993 Mar;7(3):407–418. [PubMed] [Google Scholar]
  • Wu JY, Maniatis T. Specific interactions between proteins implicated in splice site selection and regulated alternative splicing. Cell. 1993 Dec 17;75(6):1061–1070. [PubMed] [Google Scholar]
  • Xu R, Teng J, Cooper TA. The cardiac troponin T alternative exon contains a novel purine-rich positive splicing element. Mol Cell Biol. 1993 Jun;13(6):3660–3674. [PMC free article] [PubMed] [Google Scholar]
  • Yeakley JM, Morfin JP, Rosenfeld MG, Fu XD. 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. [PMC free article] [PubMed] [Google Scholar]
  • Zahler AM, Lane WS, Stolk JA, Roth MB. SR proteins: a conserved family of pre-mRNA splicing factors. Genes Dev. 1992 May;6(5):837–847. [PubMed] [Google Scholar]
  • Zamore PD, Green MR. 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. [PMC free article] [PubMed] [Google Scholar]
  • Zheng ZM, He P, Baker CC. Selection of the bovine papillomavirus type 1 nucleotide 3225 3' splice site is regulated through an exonic splicing enhancer and its juxtaposed exonic splicing suppressor. J Virol. 1996 Jul;70(7):4691–4699. [PMC free article] [PubMed] [Google Scholar]
  • 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. [PubMed] [Google Scholar]
Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)
-