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. 1996 May;16(5):2325–2331. doi: 10.1128/mcb.16.5.2325

Interactions among SR proteins, an exonic splicing enhancer, and a lentivirus Rev protein regulate alternative splicing.

R R Gontarek 1, D Derse 1
PMCID: PMC231220  PMID: 8628299

Abstract

We examine here the roles of cellular splicing factors and virus regulatory proteins in coordinately regulating alternative splicing of the tat/rev mRNA of equine infectious anemia virus (EIAV). This bicistronic mRNA contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode Rev. In the absence of Rev expression, the four-exon mRNA is synthesized exclusively, but when Rev is expressed, exon 3 is skipped to produce an mRNA that contains only exons 1, 2, and 4. We identify a purine-rich exonic splicing enhancer (ESE) in exon 3 that promotes exon inclusion. Similar to other cellular ESEs that have been identified by other laboratories, the EIAV ESE interacted specifically with SR proteins, a group of serine/arginine-rich splicing factors that function in constitutive and alternative mRNA splicing. Substitution of purines with pyrimidines in the ESE resulted in a switch from exon inclusion to exon skipping in vivo and abolished binding of SR proteins in vitro. Exon skipping was also induced by expression of EIAV Rev. We show that Rev binds to exon 3 RNA in vitro, and while the precise determinants have not been mapped, Rev function in vivo and RNA binding in vitro indicate that the RNA element necessary for Rev responsiveness overlaps or is adjacent to the ESE. We suggest that EIAV Rev promotes exon skipping by interfering with SR protein interactions with RNA or with other splicing factors.

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Selected References

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  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. 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. Bray M., Prasad S., Dubay J. W., Hunter E., Jeang K. T., Rekosh D., Hammarskjöld M. L. A small element from the Mason-Pfizer monkey virus genome makes human immunodeficiency virus type 1 expression and replication Rev-independent. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1256–1260. doi: 10.1073/pnas.91.4.1256. [DOI] [PMC free article] [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. Chang D. D., Sharp P. A. Regulation by HIV Rev depends upon recognition of splice sites. Cell. 1989 Dec 1;59(5):789–795. doi: 10.1016/0092-8674(89)90602-8. [DOI] [PubMed] [Google Scholar]
  6. Cullen B. R. Mechanism of action of regulatory proteins encoded by complex retroviruses. Microbiol Rev. 1992 Sep;56(3):375–394. doi: 10.1128/mr.56.3.375-394.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cáceres J. F., Stamm S., Helfman D. M., Krainer A. R. Regulation of alternative splicing in vivo by overexpression of antagonistic splicing factors. Science. 1994 Sep 16;265(5179):1706–1709. doi: 10.1126/science.8085156. [DOI] [PubMed] [Google Scholar]
  8. Dignam J. D., Lebovitz R. M., Roeder R. G. 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. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. 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]
  12. 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]
  13. Fu X. D. The superfamily of arginine/serine-rich splicing factors. RNA. 1995 Sep;1(7):663–680. [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Gontarek R. R., McNally M. T., Beemon K. Mutation of an RSV intronic element abolishes both U11/U12 snRNP binding and negative regulation of splicing. Genes Dev. 1993 Oct;7(10):1926–1936. doi: 10.1101/gad.7.10.1926. [DOI] [PubMed] [Google Scholar]
  16. Horowitz D. S., Krainer A. R. Mechanisms for selecting 5' splice sites in mammalian pre-mRNA splicing. Trends Genet. 1994 Mar;10(3):100–106. doi: 10.1016/0168-9525(94)90233-x. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Kjems J., Frankel A. D., Sharp P. A. Specific regulation of mRNA splicing in vitro by a peptide from HIV-1 Rev. Cell. 1991 Oct 4;67(1):169–178. doi: 10.1016/0092-8674(91)90580-r. [DOI] [PubMed] [Google Scholar]
  19. Kjems J., Sharp P. A. The basic domain of Rev from human immunodeficiency virus type 1 specifically blocks the entry of U4/U6.U5 small nuclear ribonucleoprotein in spliceosome assembly. J Virol. 1993 Aug;67(8):4769–4776. doi: 10.1128/jvi.67.8.4769-4776.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Krainer A. R., Mayeda A., Kozak D., Binns G. Functional expression of cloned human splicing factor SF2: homology to RNA-binding proteins, U1 70K, and Drosophila splicing regulators. Cell. 1991 Jul 26;66(2):383–394. doi: 10.1016/0092-8674(91)90627-b. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Lu X. B., Heimer J., Rekosh D., Hammarskjöld M. L. U1 small nuclear RNA plays a direct role in the formation of a rev-regulated human immunodeficiency virus env mRNA that remains unspliced. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7598–7602. doi: 10.1073/pnas.87.19.7598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Martarano L., Stephens R., Rice N., Derse D. Equine infectious anemia virus trans-regulatory protein Rev controls viral mRNA stability, accumulation, and alternative splicing. J Virol. 1994 May;68(5):3102–3111. doi: 10.1128/jvi.68.5.3102-3111.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. McNally M. T., Beemon K. Intronic sequences and 3' splice sites control Rous sarcoma virus RNA splicing. J Virol. 1992 Jan;66(1):6–11. doi: 10.1128/jvi.66.1.6-11.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. McNally M. T., Gontarek R. R., Beemon K. Characterization of Rous sarcoma virus intronic sequences that negatively regulate splicing. Virology. 1991 Nov;185(1):99–108. doi: 10.1016/0042-6822(91)90758-4. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. 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]
  33. 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]
  34. 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]
  35. Smith C. W., Patton J. G., Nadal-Ginard B. Alternative splicing in the control of gene expression. Annu Rev Genet. 1989;23:527–577. doi: 10.1146/annurev.ge.23.120189.002523. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Staffa A., Cochrane A. The tat/rev intron of human immunodeficiency virus type 1 is inefficiently spliced because of suboptimal signals in the 3' splice site. J Virol. 1994 May;68(5):3071–3079. doi: 10.1128/jvi.68.5.3071-3079.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Stephens R. M., Derse D., Rice N. R. Cloning and characterization of cDNAs encoding equine infectious anemia virus tat and putative Rev proteins. J Virol. 1990 Aug;64(8):3716–3725. doi: 10.1128/jvi.64.8.3716-3725.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Stephens R. M., Schneider T. D. Features of spliceosome evolution and function inferred from an analysis of the information at human splice sites. J Mol Biol. 1992 Dec 20;228(4):1124–1136. doi: 10.1016/0022-2836(92)90320-j. [DOI] [PubMed] [Google Scholar]
  41. Stoltzfus C. M., Fogarty S. J. Multiple regions in the Rous sarcoma virus src gene intron act in cis to affect the accumulation of unspliced RNA. J Virol. 1989 Apr;63(4):1669–1676. doi: 10.1128/jvi.63.4.1669-1676.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sun Q., Hampson R. K., Rottman F. M. In vitro analysis of bovine growth hormone pre-mRNA alternative splicing. Involvement of exon sequences and trans-acting factor(s). J Biol Chem. 1993 Jul 25;268(21):15659–15666. [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. 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]
  46. 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]
  47. 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]
  48. 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]
  49. 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]
  50. 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]
  51. Zuker M., Stiegler P. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res. 1981 Jan 10;9(1):133–148. doi: 10.1093/nar/9.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. van Oers C. C., Adema G. J., Zandberg H., Moen T. C., Baas P. D. Two different sequence elements within exon 4 are necessary for calcitonin-specific splicing of the human calcitonin/calcitonin gene-related peptide I pre-mRNA. Mol Cell Biol. 1994 Feb;14(2):951–960. doi: 10.1128/mcb.14.2.951. [DOI] [PMC free article] [PubMed] [Google Scholar]

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