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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1986 Nov;83(22):8555–8559. doi: 10.1073/pnas.83.22.8555

Polyadenylylation of an mRNA precursor occurs independently of transcription by RNA polymerase II in vivo.

E D Lewis, J L Manley
PMCID: PMC386969  PMID: 2877459

Abstract

Most eukaryotic messenger RNAs are transcribed as precursor molecules that must be processed by capping, splicing, 3' cleavage, and polyadenylylation to yield mature mRNAs. An important, unresolved issue is whether any of these reactions are linked either to transcription by RNA polymerase II or to each other. To address one aspect of this question, we constructed a chimeric gene containing an RNA polymerase III promoter (the adenovirus VAI promoter) fused to the body and 3'-flanking sequences of a protein-coding gene (the herpesvirus tk gene). Here we show that this hybrid gene was transcribed from the RNA polymerase III promoter following transfection of human 293 cells and that the transcripts produced were stable and efficiently transported to the cytoplasm. Although a significant proportion of the transcripts were prematurely terminated at specific sites within the gene, a high percentage of the full-length RNA was accurately cleaved and polyadenylylated. These results demonstrate that cleavage and polyadenylylation of mRNA precursors are not obligatorily coupled to transcription by RNA polymerase II in vivo.

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Selected References

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  1. Aiello L., Guilfoyle R., Huebner K., Weinmann R. Adenovirus 5 DNA sequences present and RNA sequences transcribed in transformed human embryo kidney cells (HEK-Ad-5 or 293). Virology. 1979 Apr 30;94(2):460–469. doi: 10.1016/0042-6822(79)90476-8. [DOI] [PubMed] [Google Scholar]
  2. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Berk A. J., Sharp P. A. Spliced early mRNAs of simian virus 40. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1274–1278. doi: 10.1073/pnas.75.3.1274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bogenhagen D. F., Brown D. D. Nucleotide sequences in Xenopus 5S DNA required for transcription termination. Cell. 1981 Apr;24(1):261–270. doi: 10.1016/0092-8674(81)90522-5. [DOI] [PubMed] [Google Scholar]
  6. Carmichael G. G., McMaster G. K. The analysis of nucleic acids in gels using glyoxal and acridine orange. Methods Enzymol. 1980;65(1):380–391. doi: 10.1016/s0076-6879(80)65049-6. [DOI] [PubMed] [Google Scholar]
  7. Coppola J. A., Field A. S., Luse D. S. Promoter-proximal pausing by RNA polymerase II in vitro: transcripts shorter than 20 nucleotides are not capped. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1251–1255. doi: 10.1073/pnas.80.5.1251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Edery I., Sonenberg N. Cap-dependent RNA splicing in a HeLa nuclear extract. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7590–7594. doi: 10.1073/pnas.82.22.7590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fowlkes D. M., Shenk T. Transcriptional control regions of the adenovirus VAI RNA gene. Cell. 1980 Nov;22(2 Pt 2):405–413. doi: 10.1016/0092-8674(80)90351-7. [DOI] [PubMed] [Google Scholar]
  10. Francoeur A. M., Mathews M. B. Interaction between VA RNA and the lupus antigen La: formation of a ribonucleoprotein particle in vitro. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6772–6776. doi: 10.1073/pnas.79.22.6772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grabowski P. J., Seiler S. R., Sharp P. A. A multicomponent complex is involved in the splicing of messenger RNA precursors. Cell. 1985 Aug;42(1):345–353. doi: 10.1016/s0092-8674(85)80130-6. [DOI] [PubMed] [Google Scholar]
  12. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  13. Green M. R., Maniatis T., Melton D. A. Human beta-globin pre-mRNA synthesized in vitro is accurately spliced in Xenopus oocyte nuclei. Cell. 1983 Mar;32(3):681–694. doi: 10.1016/0092-8674(83)90054-5. [DOI] [PubMed] [Google Scholar]
  14. Guilfoyle R., Weinmann R. Control region for adenovirus VA RNA transcription. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3378–3382. doi: 10.1073/pnas.78.6.3378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hoeffler W. K., Roeder R. G. Enhancement of RNA polymerase III transcription by the E1A gene product of adenovirus. Cell. 1985 Jul;41(3):955–963. doi: 10.1016/s0092-8674(85)80076-3. [DOI] [PubMed] [Google Scholar]
  16. Hu S. L., Manley J. L. DNA sequence required for initiation of transcription in vitro from the major late promoter of adenovirus 2. Proc Natl Acad Sci U S A. 1981 Feb;78(2):820–824. doi: 10.1073/pnas.78.2.820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jove R., Manley J. L. In vitro transcription from the adenovirus 2 major late promoter utilizing templates truncated at promoter-proximal sites. J Biol Chem. 1984 Jul 10;259(13):8513–8521. [PubMed] [Google Scholar]
  18. Jove R., Manley J. L. Transcription initiation by RNA polymerase II is inhibited by S-adenosylhomocysteine. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5842–5846. doi: 10.1073/pnas.79.19.5842. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Konarska M. M., Padgett R. A., Sharp P. A. Recognition of cap structure in splicing in vitro of mRNA precursors. Cell. 1984 Oct;38(3):731–736. doi: 10.1016/0092-8674(84)90268-x. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Lewis E. D., Manley J. L. Control of adenovirus late promoter expression in two human cell lines. Mol Cell Biol. 1985 Sep;5(9):2433–2442. doi: 10.1128/mcb.5.9.2433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lewis E. D., Manley J. L. Repression of simian virus 40 early transcription by viral DNA replication in human 293 cells. Nature. 1985 Sep 12;317(6033):172–175. doi: 10.1038/317172a0. [DOI] [PubMed] [Google Scholar]
  23. Manley J. L. Analysis of the expression of genes encoding animal mRNA by in vitro techniques. Prog Nucleic Acid Res Mol Biol. 1983;30:195–244. doi: 10.1016/s0079-6603(08)60687-x. [DOI] [PubMed] [Google Scholar]
  24. Manley J. L., Sharp P. A., Gefter M. L. RNA synthesis in isolated nuclei: in vitro initiation of adenovirus 2 major late mRNA precursor. Proc Natl Acad Sci U S A. 1979 Jan;76(1):160–164. doi: 10.1073/pnas.76.1.160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. McKnight S. L., Gavis E. R. Expression of the herpes thymidine kinase gene in Xenopus laevis oocytes: an assay for the study of deletion mutants constructed in vitro. Nucleic Acids Res. 1980 Dec 20;8(24):5931–5948. doi: 10.1093/nar/8.24.5931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Moore C. L., Sharp P. A. Accurate cleavage and polyadenylation of exogenous RNA substrate. Cell. 1985 Jul;41(3):845–855. doi: 10.1016/s0092-8674(85)80065-9. [DOI] [PubMed] [Google Scholar]
  28. Moore C. L., Sharp P. A. Site-specific polyadenylation in a cell-free reaction. Cell. 1984 Mar;36(3):581–591. doi: 10.1016/0092-8674(84)90337-4. [DOI] [PubMed] [Google Scholar]
  29. Nevins J. R., Darnell J. E., Jr Steps in the processing of Ad2 mRNA: poly(A)+ nuclear sequences are conserved and poly(A) addition precedes splicing. Cell. 1978 Dec;15(4):1477–1493. doi: 10.1016/0092-8674(78)90071-5. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Samuels M., Fire A., Sharp P. A. Dinucleotide priming of transcription mediated by RNA polymerase II. J Biol Chem. 1984 Feb 25;259(4):2517–2525. [PubMed] [Google Scholar]
  34. Shatkin A. J. Capping of eucaryotic mRNAs. Cell. 1976 Dec;9(4 Pt 2):645–653. doi: 10.1016/0092-8674(76)90128-8. [DOI] [PubMed] [Google Scholar]
  35. Smale S. T., Tjian R. Transcription of herpes simplex virus tk sequences under the control of wild-type and mutant human RNA polymerase I promoters. Mol Cell Biol. 1985 Feb;5(2):352–362. doi: 10.1128/mcb.5.2.352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Stefano J. E. Purified lupus antigen La recognizes an oligouridylate stretch common to the 3' termini of RNA polymerase III transcripts. Cell. 1984 Jan;36(1):145–154. doi: 10.1016/0092-8674(84)90083-7. [DOI] [PubMed] [Google Scholar]
  37. Vennström B., Pettersson U., Philipson L. Two initiation sites for adenovirus 5.5S RNA. Nucleic Acids Res. 1978 Jan;5(1):195–204. doi: 10.1093/nar/5.1.195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Weinmann R., Brendler T. G., Raskas H. J., Roeder R. G. Low molecular weight viral RNAs transcribed by RNA polymerase III during adenovirus 2 infection. Cell. 1976 Apr;7(4):557–566. doi: 10.1016/0092-8674(76)90206-3. [DOI] [PubMed] [Google Scholar]
  39. Wigler M., Pellicer A., Silverstein S., Axel R., Urlaub G., Chasin L. DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1373–1376. doi: 10.1073/pnas.76.3.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]

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