Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1983 Jan;45(1):264–274. doi: 10.1128/jvi.45.1.264-274.1983

Characterization of the 5' termini of purified nascent simian virus 40 late transcripts.

D E Lycan, K J Danna
PMCID: PMC256409  PMID: 6296435

Abstract

The primary transcripts of simian virus 40 are extensively processed in the nuclei of infected monkey cells before they are transported to the cytoplasm as mature mRNAs. To investigate the early steps in this process, in particular, to determine which events occur on nascent chains before the termination of transcription, we have developed a procedure for the purification of nascent viral transcripts. This technique involves the in vitro incorporation of mercurated residues into the growing 3' ends of pre-initiated nascent chains, allowing their specific purification by sulfhydrylcellulose affinity chromatography. We further selected viral specific transcripts by hybridization to simian virus 40 DNA-cellulose. We describe here our analysis of the 5' termini of purified nascent simian virus 40 transcripts. This analysis revealed various cap structures, providing direct evidence that primary viral transcripts are capped before chain completion. The various cap structures exhibited a full range of methylation states. Completely unmethylated GpppA cap cores were identified, as well as caps methylated at the penultimate position only. The presence of GpppAm and GpppmAm caps indicates that, in BSC-1 cells, the penultimate nucleotide can be methylated before 7-methyl-G formation. Furthermore, the proportions of the various intermediates suggest that, in contrast to the viral capping enzymes of vaccinia virus and reovirus, the cellular enzymes methylate in the following order: GpppA leads to GpppAm leads to GpppmAm leads to 7mGpppmAm. In addition to capped ends, we also detected some unprocessed pppA ends. To our knowledge, this is the first time uncapped termini have been identified on RNAs known to be polymerase II products.

Full text

PDF
264

Images in this article

268Image
on p.268
269Image
on p.269
269Image
on p.269
270Image
on p.270
271Image
on p.271

Selected References

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

  1. Babich A., Nevins J. R., Darnell J. E., Jr Early capping of transcripts from the adenovirus major late transcription unit. Nature. 1980 Sep 18;287(5779):246–248. doi: 10.1038/287246a0. [DOI] [PubMed] [Google Scholar]
  2. Batts-Young B., Lodish H. F. Triphosphate residues at the 5' ends of rRNA precursor and 5S RNA from Dictyostelium discoideum. Proc Natl Acad Sci U S A. 1978 Feb;75(2):740–744. doi: 10.1073/pnas.75.2.740. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beyer A. L., Bouton A. H., Miller O. L., Jr Correlation of hnRNP structure and nascent transcript cleavage. Cell. 1981 Oct;26(2 Pt 2):155–165. doi: 10.1016/0092-8674(81)90299-3. [DOI] [PubMed] [Google Scholar]
  4. Birkenmeier E. H., Chiu N., Radonovich M. F., May E., Salzman N. P. Regulation of simian virus 40 early and late gene transcription without viral DNA replication. J Virol. 1979 Mar;29(3):983–989. doi: 10.1128/jvi.29.3.983-989.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Canaani D., Kahana C., Mukamel A., Groner Y. Sequence heterogeneity at the 5' termini of late simian virus 40 19S and 16S mRNAs. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3078–3082. doi: 10.1073/pnas.76.7.3078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chikaraishi D. M., Danna K. J. Simian virus 40 transcriptional complexes incorporate mercurated nucleotides into RNA in vitro. J Virol. 1981 Jan;37(1):511–517. doi: 10.1128/jvi.37.1.511-517.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Contreras R., Fiers W. Initiation of transcription by RNA polymerase II in permeable, SV40-infected or noninfected, CVI cells; evidence for multiple promoters of SV40 late transcription. Nucleic Acids Res. 1981 Jan 24;9(2):215–236. doi: 10.1093/nar/9.2.215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dale R. M., Martin E., Livingston D. C., Ward D. C. Direct covalent mercuration of nucleotides and polynucleotides. Biochemistry. 1975 Jun 3;14(11):2447–2457. doi: 10.1021/bi00682a027. [DOI] [PubMed] [Google Scholar]
  9. Feist P. L., Danna K. J. Sulfhydrylcellulose: a new medium for chromatography of mercurated polynucleotides. Biochemistry. 1981 Jul 21;20(15):4243–4246. doi: 10.1021/bi00518a001. [DOI] [PubMed] [Google Scholar]
  10. Ferdinand F. J., Brown M., Khoury G. Synthesis and characterization of late lytic simian virus 40 RNA from transcriptional complexes. Virology. 1977 May 1;78(1):150–161. doi: 10.1016/0042-6822(77)90087-3. [DOI] [PubMed] [Google Scholar]
  11. Fraser N. W., Sehgal P. B., Darnell J. E., Jr Multiple discrete sites for premature RNA chain termination late in adenovirus-2 infection: enhancement by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2571–2575. doi: 10.1073/pnas.76.6.2571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Furuichi Y., Muthukrishnan S., Tomasz J., Shatkin A. J. Mechanism of formation of reovirus mRNA 5'-terminal blocked and methylated sequence, m7GpppGmpC. J Biol Chem. 1976 Aug 25;251(16):5043–5053. [PubMed] [Google Scholar]
  13. Gariglio P., Mousset S. Isolation and partial characterization of a nuclear RNA polymerase - SV40 DNA complex. FEBS Lett. 1975 Aug 1;56(1):149–155. doi: 10.1016/0014-5793(75)80130-x. [DOI] [PubMed] [Google Scholar]
  14. Gidoni D., Kahana C., Canaani D., Groner Y. Specific in vitro initiation of transcription of simian virus 40 early and late genes occurs at the various cap nucleotides including cytidine. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2174–2178. doi: 10.1073/pnas.78.4.2174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grainger R. M., Maizels N. Dictyostelium ribosomal RNA is processed during transcription. Cell. 1980 Jul;20(3):619–623. doi: 10.1016/0092-8674(80)90308-6. [DOI] [PubMed] [Google Scholar]
  16. Groner Y., Gilboa E., Aviv H. Methylation and capping of RNA polymerase II primary transcripts by HeLa nuclear homogenates. Biochemistry. 1978 Mar 21;17(6):977–982. doi: 10.1021/bi00599a005. [DOI] [PubMed] [Google Scholar]
  17. Hallick R. B., Chelm B. K., Gray P. W., Orozco E. M., Jr Use of aurintricarboxylic acid as an inhibitor of nucleases during nucleic acid isolation. Nucleic Acids Res. 1977 Sep;4(9):3055–3064. doi: 10.1093/nar/4.9.3055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hay N., Skolnik-David H., Aloni Y. Attenuation in the control of SV40 gene expression. Cell. 1982 May;29(1):183–193. doi: 10.1016/0092-8674(82)90102-7. [DOI] [PubMed] [Google Scholar]
  19. Konkel D. A., Ingram V. M. RNA aggregation during sulfhydryl-agarose chromatography of mercurated RNA. Nucleic Acids Res. 1977 Jun;4(6):1979–1988. doi: 10.1093/nar/4.6.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Laub O., Bratosin S., Horowitz M., Aloni Y. The initiation of transcription of SV40 DNA at late time after infection. Virology. 1979 Jan 30;92(2):310–323. doi: 10.1016/0042-6822(79)90136-3. [DOI] [PubMed] [Google Scholar]
  21. Laub O., Jakobovits E. B., Aloni Y. 5,6-dichloro-1-beta-ribofuranosylbenzimidazole enhances premature termination of late transcription of simian virus 40 DNA. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3297–3301. doi: 10.1073/pnas.77.6.3297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Levis R., Penman S. Processing steps and methylation in the formation of the ribosomal RNA of cultured Drosophila cells. J Mol Biol. 1978 May 15;121(2):219–238. doi: 10.1016/s0022-2836(78)80006-0. [DOI] [PubMed] [Google Scholar]
  23. Maxwell I. H., Maxwell F., Hahn W. E. Removal of RNase activity from DNase by affinity chromatography on agarose coupled aminophenylphosphoryl-uridine-2' (3')-phosphate. Nucleic Acids Res. 1977 Jan;4(1):241–246. doi: 10.1093/nar/4.1.241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Moss B., Martin S. A., Ensinger M. J., Boone R. F., Wei C. M. Modification of the 5'-terminals of mRNAs by viral and cellular enzymes. Prog Nucleic Acid Res Mol Biol. 1976;19:63–81. doi: 10.1016/s0079-6603(08)60908-3. [DOI] [PubMed] [Google Scholar]
  25. Noyes B. E., Stark G. R. Nucleic acid hybridization using DNA covalently coupled to cellulose. Cell. 1975 Jul;5(3):301–310. doi: 10.1016/0092-8674(75)90105-1. [DOI] [PubMed] [Google Scholar]
  26. Reeder R. H., Sollner-Webb B., Wahn H. L. Sites of transcription initiation in vivo on Xenopus laevis ribosomal DNA. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5402–5406. doi: 10.1073/pnas.74.12.5402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Salditt-Georgieff M., Harpold M., Chen-Kiang S., Darnell J. E., Jr The addition of 5' cap structures occurs early in hnRNA synthesis and prematurely terminated molecules are capped. Cell. 1980 Jan;19(1):69–78. doi: 10.1016/0092-8674(80)90389-x. [DOI] [PubMed] [Google Scholar]
  28. Schibler U., Perry R. P. The 5'-termini of heterogeneous nuclear RNA: a comparison among molecules of different sizes and ages. Nucleic Acids Res. 1977 Dec;4(12):4133–4149. doi: 10.1093/nar/4.12.4133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sehgal P. B., Derman E., Molloy G. R., Tamm I., Darnell J. E. 5,6-Dichloro-1-Beta-D-ribofuranosylbenzimidazole inhibits initiation of nuclear heterogeneous RNA chains in HeLa cells. Science. 1976 Oct 22;194(4263):431–433. doi: 10.1126/science.982026. [DOI] [PubMed] [Google Scholar]
  30. Shani M., Birkenmeier E., May E., Salzman N. P. Properties of simian virus 40 transcriptional intermediates isolated from nuclei of permissive cells. J Virol. 1977 Jul;23(1):20–28. doi: 10.1128/jvi.23.1.20-28.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Skolnik-David H., Hay N., Aloni Y. Site of premature termination of late transcription of simian virus 40 DNA: enhancement by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole. Proc Natl Acad Sci U S A. 1982 May;79(9):2743–2747. doi: 10.1073/pnas.79.9.2743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Volckaert G., Jou W. M., Fiers W. Analysis of 32P-labeled bacteriophage MS2 RNA by a mini-fingerprinting procedure. Anal Biochem. 1976 May 7;72:433–446. doi: 10.1016/0003-2697(76)90551-0. [DOI] [PubMed] [Google Scholar]
  33. Winicov I., Perry R. P. Synthesis methylation, and capping of nuclear RNA by a subcellular system. Biochemistry. 1976 Nov 16;15(23):5039–5046. doi: 10.1021/bi00668a014. [DOI] [PubMed] [Google Scholar]

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

RESOURCES