Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1992 Jan;66(1):6–11. doi: 10.1128/jvi.66.1.6-11.1992

Intronic sequences and 3' splice sites control Rous sarcoma virus RNA splicing.

M T McNally 1, K Beemon 1
PMCID: PMC238253  PMID: 1309264

Abstract

cis-acting sequences of Rous sarcoma virus (RSV) RNA involved in control of the incomplete splicing that is part of the retroviral life cycle have been studied. The 5' and two alternative 3' splice sites, as well as negative regulator of splicing element in the intron, have been introduced into chimeric constructs, and their responsive roles in splicing inhibition have been evaluated by transient transfection experiments. Although the RSV 5' splice site was used efficiently in these assays, substrates containing either the RSV env or the RSV src 3' splice site were not spliced completely, resulting in 40 to 50% unspliced RNA. Addition of the negative regulator of splicing element to substrates containing RSV 3' splice sites resulted in greater inhibition of splicing (70 to 80% unspliced RNA), suggesting that the two elements function independently and additively. Deletion of sequences more than 70 nucleotides upstream of the src 3' splice site resulted in efficient splicing at this site, suggesting that inefficient usage is not inherent in this splice site but is instead due to to sequences upstream of it. Insertion of these upstream sequences into the intron of a heterologous pre-mRNA resulted in partial inhibition of its splicing. In addition, secondary structure interactions were predicted to occur between the src 3' splice site and the inhibitory sequences upstream of it. Thus, RSV splicing control involves both intronic sequences and 3' splice sites, with different mechanisms involved in the underutilization of the env and src splice acceptor sites.

Full text

PDF
6

Images in this article

8Image
on p.8
9Image
on p.9
9Image
on p.9

Selected References

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

  1. Arrigo S., Beemon K. Regulation of Rous sarcoma virus RNA splicing and stability. Mol Cell Biol. 1988 Nov;8(11):4858–4867. doi: 10.1128/mcb.8.11.4858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barker G. F., Beemon K. Nonsense codons within the Rous sarcoma virus gag gene decrease the stability of unspliced viral RNA. Mol Cell Biol. 1991 May;11(5):2760–2768. doi: 10.1128/mcb.11.5.2760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berberich S. L., Stoltzfus C. M. Mutations in the regions of the Rous sarcoma virus 3' splice sites: implications for regulation of alternative splicing. J Virol. 1991 May;65(5):2640–2646. doi: 10.1128/jvi.65.5.2640-2646.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  5. Clouet d'Orval B., d'Aubenton Carafa Y., Sirand-Pugnet P., Gallego M., Brody E., Marie J. RNA secondary structure repression of a muscle-specific exon in HeLa cell nuclear extracts. Science. 1991 Jun 28;252(5014):1823–1828. doi: 10.1126/science.2063195. [DOI] [PubMed] [Google Scholar]
  6. Cobrinik D., Katz R., Terry R., Skalka A. M., Leis J. Avian sarcoma and leukosis virus pol-endonuclease recognition of the tandem long terminal repeat junction: minimum site required for cleavage is also required for viral growth. J Virol. 1987 Jun;61(6):1999–2008. doi: 10.1128/jvi.61.6.1999-2008.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Green M. R. Pre-mRNA splicing. Annu Rev Genet. 1986;20:671–708. doi: 10.1146/annurev.ge.20.120186.003323. [DOI] [PubMed] [Google Scholar]
  11. Jones T. R., Cole M. D. Rapid cytoplasmic turnover of c-myc mRNA: requirement of the 3' untranslated sequences. Mol Cell Biol. 1987 Dec;7(12):4513–4521. doi: 10.1128/mcb.7.12.4513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Katz R. A., Kotler M., Skalka A. M. cis-acting intron mutations that affect the efficiency of avian retroviral RNA splicing: implication for mechanisms of control. J Virol. 1988 Aug;62(8):2686–2695. doi: 10.1128/jvi.62.8.2686-2695.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Libri D., Piseri A., Fiszman M. Y. Tissue-specific splicing in vivo of the beta-tropomyosin gene: dependence on an RNA secondary structure. Science. 1991 Jun 28;252(5014):1842–1845. doi: 10.1126/science.2063196. [DOI] [PubMed] [Google Scholar]
  15. Linial M. Creation of a processed pseudogene by retroviral infection. Cell. 1987 Apr 10;49(1):93–102. doi: 10.1016/0092-8674(87)90759-8. [DOI] [PubMed] [Google Scholar]
  16. Lopata M. A., Cleveland D. W., Sollner-Webb B. High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment. Nucleic Acids Res. 1984 Jul 25;12(14):5707–5717. doi: 10.1093/nar/12.14.5707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Schwartz D. E., Tizard R., Gilbert W. Nucleotide sequence of Rous sarcoma virus. Cell. 1983 Mar;32(3):853–869. doi: 10.1016/0092-8674(83)90071-5. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Stoltzfus C. M. Synthesis and processing of avian sarcoma retrovirus RNA. Adv Virus Res. 1988;35:1–38. doi: 10.1016/s0065-3527(08)60707-1. [DOI] [PubMed] [Google Scholar]
  23. Varmus H. Regulation of HIV and HTLV gene expression. Genes Dev. 1988 Sep;2(9):1055–1062. doi: 10.1101/gad.2.9.1055. [DOI] [PubMed] [Google Scholar]
  24. 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]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES