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. 1986 Dec 22;14(24):9911–9926. doi: 10.1093/nar/14.24.9911

Alternative splicing of SV40 early pre-mRNA in vitro.

V L van Santen, R A Spritz
PMCID: PMC341343  PMID: 3027668

Abstract

Simian virus 40 (SV40) early pre-mRNA is spliced using either of two alternative 5' splice sites and a common 3' splice site to produce two mRNAs that encode the T and t antigens. We have studied alternative splicing of SV40 early pre-mRNA in vitro using a HeLa cell nuclear extract. Synthetic SV40 early transcripts are processed to T and t antigen mRNAs in vitro. As in SV40-infected cells in vivo, cleavage at the T antigen 5' splice site is more efficient than cleavage at the t antigen 5' splice site in vitro, although both of these 5' splice sites are utilized relatively inefficiently in vitro. The ratio of cleavage at the T and t antigen 5' splice sites is not changed significantly by a number of alterations in the conditions under which the in vitro splicing reactions are carried out.

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Selected References

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  1. Alwine J. C., Khoury G. Control of simian virus 40 gene expression at the levels of RNA synthesis and processing: thermally induced changes in the ratio of the simian virus 40 early mRNA's and proteins. J Virol. 1980 Jul;35(1):157–164. doi: 10.1128/jvi.35.1.157-164.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berger S. L., Birkenmeier C. S. Inhibition of intractable nucleases with ribonucleoside--vanadyl complexes: isolation of messenger ribonucleic acid from resting lymphocytes. Biochemistry. 1979 Nov 13;18(23):5143–5149. doi: 10.1021/bi00590a018. [DOI] [PubMed] [Google Scholar]
  3. Black D. L., Chabot B., Steitz J. A. U2 as well as U1 small nuclear ribonucleoproteins are involved in premessenger RNA splicing. Cell. 1985 Oct;42(3):737–750. doi: 10.1016/0092-8674(85)90270-3. [DOI] [PubMed] [Google Scholar]
  4. Deutscher S. L., Bhat B. M., Pursley M. H., Cladaras C., Wold W. S. Novel deletion mutants that enhance a distant upstream 5' splice in the E3 transcription unit of adenovirus 2. Nucleic Acids Res. 1985 Aug 26;13(16):5771–5788. doi: 10.1093/nar/13.16.5771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Fradin A., Jove R., Hemenway C., Keiser H. D., Manley J. L., Prives C. Splicing pathways of SV40 mRNAs in X. laevis oocytes differ in their requirements for snRNPs. Cell. 1984 Jul;37(3):927–936. doi: 10.1016/0092-8674(84)90427-6. [DOI] [PubMed] [Google Scholar]
  7. Fromm M., Berg P. Deletion mapping of DNA regions required for SV40 early region promoter function in vivo. J Mol Appl Genet. 1982;1(5):457–481. [PubMed] [Google Scholar]
  8. Ghosh P. K., Lebowitz P. Simian virus 40 early mRNA's contain multiple 5' termini upstream and downstream from a Hogness-Goldberg sequence; a shift in 5' termini during the lytic cycle is mediated by large T antigen. J Virol. 1981 Oct;40(1):224–240. doi: 10.1128/jvi.40.1.224-240.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ghosh P. K., Roy P., Barkan A., Mertz J. E., Weissman S. M., Lebowitz P. Unspliced functional late 19S mRNAs containing intervening sequences are produced by a late leader mutant of simian virus 40. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1386–1390. doi: 10.1073/pnas.78.3.1386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  11. Hansen U., Tenen D. G., Livingston D. M., Sharp P. A. T antigen repression of SV40 early transcription from two promoters. Cell. 1981 Dec;27(3 Pt 2):603–613. doi: 10.1016/0092-8674(81)90402-5. [DOI] [PubMed] [Google Scholar]
  12. Hardy S. F., Grabowski P. J., Padgett R. A., Sharp P. A. Cofactor requirements of splicing of purified messenger RNA precursors. Nature. 1984 Mar 22;308(5957):375–377. doi: 10.1038/308375a0. [DOI] [PubMed] [Google Scholar]
  13. Hernandez N., Keller W. Splicing of in vitro synthesized messenger RNA precursors in HeLa cell extracts. Cell. 1983 Nov;35(1):89–99. doi: 10.1016/0092-8674(83)90211-8. [DOI] [PubMed] [Google Scholar]
  14. Khoury G., Alwine J., Goldman N., Gruss P., Jay G. New chimeric splice junction in adenovirus type 2-simian virus 40 hybrid viral mRNA. J Virol. 1980 Oct;36(1):143–151. doi: 10.1128/jvi.36.1.143-151.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Khoury G., Gruss P., Dhar R., Lai C. J. Processing and expression of early SV40 mRNA: a role for RNA conformation in splicing. Cell. 1979 Sep;18(1):85–92. doi: 10.1016/0092-8674(79)90356-8. [DOI] [PubMed] [Google Scholar]
  16. Krainer A. R., Maniatis T., Ruskin B., Green M. R. Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro. Cell. 1984 Apr;36(4):993–1005. doi: 10.1016/0092-8674(84)90049-7. [DOI] [PubMed] [Google Scholar]
  17. Lang K. M., van Santen V. L., Spritz R. A. The two intervening sequences of human beta- and gamma-globin pre-mRNAs are excised in a preferred temporal order in vitro. EMBO J. 1985 Aug;4(8):1991–1996. doi: 10.1002/j.1460-2075.1985.tb03882.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Mohr S. C., Thach R. E. Application of ribonuclease T1 to the synthesis of oligoribonucleotides of defined base sequence. J Biol Chem. 1969 Dec 25;244(24):6566–6576. [PubMed] [Google Scholar]
  20. Noble J. C., Prives C., Manley J. L. In vitro splicing of simian virus 40 early pre mRNA. Nucleic Acids Res. 1986 Feb 11;14(3):1219–1235. doi: 10.1093/nar/14.3.1219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Padgett R. A., Konarska M. M., Grabowski P. J., Hardy S. F., Sharp P. A. Lariat RNA's as intermediates and products in the splicing of messenger RNA precursors. Science. 1984 Aug 31;225(4665):898–903. doi: 10.1126/science.6206566. [DOI] [PubMed] [Google Scholar]
  22. Padgett R. A., Mount S. M., Steitz J. A., Sharp P. A. Splicing of messenger RNA precursors is inhibited by antisera to small nuclear ribonucleoprotein. Cell. 1983 Nov;35(1):101–107. doi: 10.1016/0092-8674(83)90212-x. [DOI] [PubMed] [Google Scholar]
  23. Reddy V. B., Ghosh P. K., Lebowitz P., Piatak M., Weissman S. M. Simian virus 40 early mRNA's. I. Genomic localization of 3' and 5' termini and two major splices in mRNA from transformed and lytically infected cells. J Virol. 1979 Apr;30(1):279–296. doi: 10.1128/jvi.30.1.279-296.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ross J. A precursor of globin messenger RNA. J Mol Biol. 1976 Sep 15;106(2):403–420. doi: 10.1016/0022-2836(76)90093-0. [DOI] [PubMed] [Google Scholar]
  25. Ruskin B., Krainer A. R., Maniatis T., Green M. R. Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro. Cell. 1984 Aug;38(1):317–331. doi: 10.1016/0092-8674(84)90553-1. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. White R. T., Berg P., Villarreal L. P. Simian virus 40-rabbit beta-globin recombinants lacking late mRNA splice sites express cytoplasmic RNAs with altered structures. J Virol. 1982 Apr;42(1):262–274. doi: 10.1128/jvi.42.1.262-274.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wickens M., Stephenson P. Role of the conserved AAUAAA sequence: four AAUAAA point mutants prevent messenger RNA 3' end formation. Science. 1984 Nov 30;226(4678):1045–1051. doi: 10.1126/science.6208611. [DOI] [PubMed] [Google Scholar]
  30. Yang V. W., Lerner M. R., Steitz J. A., Flint S. J. A small nuclear ribonucleoprotein is required for splicing of adenoviral early RNA sequences. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1371–1375. doi: 10.1073/pnas.78.3.1371. [DOI] [PMC free article] [PubMed] [Google Scholar]

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