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. 1977 Feb;21(2):683–693. doi: 10.1128/jvi.21.2.683-693.1977

Giant heterogeneous polyadenylic acid on vesicular stomatitis virus mRNA synthesized in vitro in the presence of S-adenosylhomocysteine.

J K Rose, H F Lodish, M L Brock
PMCID: PMC353871  PMID: 189093

Abstract

An in vitro transcription system in which vesicular stomatitis virus (VSV) mRNA species have been synthesized is described. In addition to purified VSV virions, which contain an RNA-dependent RNA polymerase, this system contained a cytoplasmic cell extract that enhanced correct transcription. Gel electrophoretic analysis of the methylated polyadenylic acid [poly(A)]-containing VSV mRNA produced in this system in the presenct of S-adenosylmethionine showed the discrete VSV mRNA species. However, when unmethylated mRNA was synthesized in the presence of S-adenosylhomocysteine, the poly(A)-containing transcripts were large and heterogeneous in molecular weight and did not contain discrete VSV mRNA species. Two-dimensional fingerprint analysis of the methylated and unmethylated products suggested that identical nucleotide sequences were present in the RNAs. Further analysis showed the presence of very large heterogeneous poly(A), 200 to 2,000 nucleotides in lenght, in the unmethylated transcript. Proof that this large poly(A) was covalently linked to the correct VSV mRNA transcripts was obtained by removal of the poly(A) by hybirdization with oligodeoxythymidylic acid and digestion with RNase H. This digestion produced unmethylated VSV mRNA transcripts with the same discrete sizes as the deadenylated RNAs produced from VSV mRNA initially isolated from VSV-infected cells. The results suggest that there is a relationship between methylation at the 5'-end and polyadenylation at the 3'-end of VSV mRNA's. Furthermore, addition of the very large poly(A) does not affect the normal process of sequential transcription of the VSV genome, suggesting that this poly(A) addition is occurring independently of further transcription.

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Selected References

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

  1. Abraham G., Rhodes D. P., Banerjee A. K. The 5' terminal structure of the methylated mRNA synthesized in vitro by vesicular stomatitis virus. Cell. 1975 May;5(1):51–58. doi: 10.1016/0092-8674(75)90091-4. [DOI] [PubMed] [Google Scholar]
  2. Adesnik M., Salditt M., Thomas W., Darnell J. E. Evidence that all messenger RNA molecules (except histone messenger RNA) contain Poly (A) sequences and that the Poly(A) has a nuclear function. J Mol Biol. 1972 Oct 28;71(1):21–30. doi: 10.1016/0022-2836(72)90397-x. [DOI] [PubMed] [Google Scholar]
  3. Ball L. A., White C. N. Order of transcription of genes of vesicular stomatitis virus. Proc Natl Acad Sci U S A. 1976 Feb;73(2):442–446. doi: 10.1073/pnas.73.2.442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baltimore D., Huang A. S., Stampfer M. Ribonucleic acid synthesis of vesicular stomatitis virus, II. An RNA polymerase in the virion. Proc Natl Acad Sci U S A. 1970 Jun;66(2):572–576. doi: 10.1073/pnas.66.2.572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Banerjee A. K., Moyer S. A., Rhodes D. P. Studies on the in vitro adenylation of RNA by vesicular stomatitis virus. Virology. 1974 Oct;61(2):547–558. doi: 10.1016/0042-6822(74)90289-x. [DOI] [PubMed] [Google Scholar]
  6. Banerjee A. K., Rhodes D. P. In vitro synthesis of RNA that contains polyadenylate by virion-associated RNA polymerase of vesicular stomatitis virus. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3566–3570. doi: 10.1073/pnas.70.12.3566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bishop D. H. Complete transcription by the transcriptase of vesicular stomatitis virus. J Virol. 1971 Apr;7(4):486–490. doi: 10.1128/jvi.7.4.486-490.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Both G. W., Moyer S. A., Banerjee A. K. Translation and identification of the mRNA species synthesized in vitro by the virion-associated RNA polymerase of vesicular stomatitis virus. Proc Natl Acad Sci U S A. 1975 Jan;72(1):274–278. doi: 10.1073/pnas.72.1.274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Both G. W., Moyer S. A., Banerjee A. K. Translation and identification of the viral mRNA species isolated from subcellular fractions of vesicular stomatitis virus-infected cells. J Virol. 1975 Apr;15(4):1012–1019. doi: 10.1128/jvi.15.4.1012-1019.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Colonno R. J., Banerjee A. K. A unique RNA species involved in initiation of vesicular stomatitis virus RNA transcription in vitro. Cell. 1976 Jun;8(2):197–204. doi: 10.1016/0092-8674(76)90003-9. [DOI] [PubMed] [Google Scholar]
  11. Darnell J. E., Philipson L., Wall R., Adesnik M. Polyadenylic acid sequences: role in conversion of nuclear RNA into messenger RNA. Science. 1971 Oct 29;174(4008):507–510. doi: 10.1126/science.174.4008.507. [DOI] [PubMed] [Google Scholar]
  12. Duesberg P. H., Vogt P. K. Gel electrophoresis of avian leukosis and sarcoma viral RNA in formamide: comparison with other viral and cellular RNA species. J Virol. 1973 Sep;12(3):594–599. doi: 10.1128/jvi.12.3.594-599.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Edmonds M., Vaughan M. H., Jr, Nakazato H. Polyadenylic acid sequences in the heterogeneous nuclear RNA and rapidly-labeled polyribosomal RNA of HeLa cells: possible evidence for a precursor relationship. Proc Natl Acad Sci U S A. 1971 Jun;68(6):1336–1340. doi: 10.1073/pnas.68.6.1336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ehrenfeld E., Summers D. F. Adenylate-rich sequences in vesicular stomatitis virus messenger ribonucleic acid. J Virol. 1972 Oct;10(4):683–688. doi: 10.1128/jvi.10.4.683-688.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Huang A. S., Baltimore D., Stampfer M. Ribonucleic acid synthesis of vesicular stomatitis virus. 3. Multiple complementary messenger RNA molecules. Virology. 1970 Dec;42(4):946–957. doi: 10.1016/0042-6822(70)90343-0. [DOI] [PubMed] [Google Scholar]
  16. Jelinek W., Adesnik M., Salditt M., Sheiness D., Wall R., Molloy G., Philipson L., Darnell J. E. Further evidence on the nuclear origin and transfer to the cytoplasm of polyadenylic acid sequences in mammalian cell RNA. J Mol Biol. 1973 Apr 15;75(3):515–532. doi: 10.1016/0022-2836(73)90458-0. [DOI] [PubMed] [Google Scholar]
  17. Kerr I. M., Cohen N., Work T. S. Factors controlling amino acid incorporation by ribosomes from krebs II mouse ascites-tumour cells. Biochem J. 1966 Mar;98(3):826–835. doi: 10.1042/bj0980826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Knipe D., Rose J. K., Lodish H. F. Translation of individual species of vesicular stomatitis viral mRNA. J Virol. 1975 Apr;15(4):1004–1011. doi: 10.1128/jvi.15.4.1004-1011.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lee S. Y., Mendecki J., Brawerman G. A polynucleotide segment rich in adenylic acid in the rapidly-labeled polyribosomal RNA component of mouse sarcoma 180 ascites cells. Proc Natl Acad Sci U S A. 1971 Jun;68(6):1331–1335. doi: 10.1073/pnas.68.6.1331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Marshall S., Gillespie D. Poly U tracts absent from viral RNA. Nat New Biol. 1972 Nov 8;240(97):43–45. doi: 10.1038/newbio240043a0. [DOI] [PubMed] [Google Scholar]
  21. Morrison T., Stampfer M., Baltimore D., Lodish H. F. Translation of vesicular stomatitis messenger RNA by extracts from mammalian and plant cells. J Virol. 1974 Jan;13(1):62–72. doi: 10.1128/jvi.13.1.62-72.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Moyer S. A., Abraham G., Adler R., Banerjee A. K. Methylated and blocked 5' termini in vesicular stomatitis virus in vivo mRNAs. Cell. 1975 May;5(1):59–67. doi: 10.1016/0092-8674(75)90092-6. [DOI] [PubMed] [Google Scholar]
  23. Rhodes D. P., Banerjee A. K. 5'-terminal sequence of vesicular stomatitis virus mRNA's synthesized in vitro. J Virol. 1975 Jan;17(1):33–42. doi: 10.1128/jvi.17.1.33-42.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rose J. K. Heterogneeous 5'-terminal structures occur on vesicular stomatitis virus mRNAs. J Biol Chem. 1975 Oct 25;250(20):8098–8104. [PubMed] [Google Scholar]
  25. Rose J. K., Knipe D. Nucleotide sequence complexities, molecular weights, and poly(A) content of the vesicular stomatitis virus mRNA species. J Virol. 1975 Apr;15(4):994–1003. doi: 10.1128/jvi.15.4.994-1003.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shatkin A. J. Animal RNA viruses: genome structure and function. Annu Rev Biochem. 1974;43(0):643–665. doi: 10.1146/annurev.bi.43.070174.003235. [DOI] [PubMed] [Google Scholar]
  27. Sheiness D., Darnell J. E. Polyadenylic acid segment in mRNA becomes shorter with age. Nat New Biol. 1973 Feb 28;241(113):265–268. doi: 10.1038/newbio241265a0. [DOI] [PubMed] [Google Scholar]
  28. Sippel A. E., Stavrianopoulos J. G., Schutz G., Feigelson P. Translational properties of rabbit globin mRNA after specific removal of poly(A) with ribonuclease H. Proc Natl Acad Sci U S A. 1974 Nov;71(11):4635–4639. doi: 10.1073/pnas.71.11.4635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Soria M., Huang A. S. Association of polyadenylic acid with messenger RNA of vesicular stomatitis virus. J Mol Biol. 1973 Jul 5;77(3):449–455. doi: 10.1016/0022-2836(73)90450-6. [DOI] [PubMed] [Google Scholar]
  30. Villarreal L. P., Holland J. J. Synthesis of poly(A) in vitro by purified virions of vesicular stomatitis virus. Nat New Biol. 1973 Nov 7;246(149):17–19. doi: 10.1038/newbio246017a0. [DOI] [PubMed] [Google Scholar]
  31. Vournakis J. N., Efstratiadis A., Kafatos F. C. Electrophoretic patterns of deadenylylated chorion and globin mRNAs. Proc Natl Acad Sci U S A. 1975 Aug;72(8):2959–2963. doi: 10.1073/pnas.72.8.2959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Weiss S. R., Bratt M. A. Polyadenylate sequences on Newcastle disease virus mRNA synthesized in vivo and in vitro. J Virol. 1974 Jun;13(6):1220–1230. doi: 10.1128/jvi.13.6.1220-1230.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]

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