Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Jul;15(7):3597–3607. doi: 10.1128/mcb.15.7.3597

Functional mRNA can be generated by RNA polymerase III.

S Gunnery 1, M B Mathews 1
PMCID: PMC230597  PMID: 7791767

Abstract

Eukaryotic cellular mRNA is believed to be synthesized exclusively by RNA polymerase II (pol II), whereas pol I produces long rRNAs and pol III produces 5S rRNA, tRNA, and other small RNAs. To determine whether this functional differentiation is obligatory, we examined the translational potential of an artificial pol III transcript. The coding region of the human immunodeficiency virus type 1 tat gene was placed under the control of a strong pol III promoter from the adenovirus type 2 VA RNAI gene. The resultant chimera, pVA-Tat, was transcribed accurately in vivo and in vitro and gave rise to Tat protein, which transactivated a human immunodeficiency virus-driven chloramphenicol acetyltransferase reporter construct in transfected HeLa cells. pol III-specific mutations down-regulated VA-Tat RNA production in vivo and in vitro and dramatically reduced chloramphenicol acetyltransferase transactivation. As expected for a pol III transcript, VA-Tat RNA was not detectably capped at its 5' end or polyadenylated at its 3' end, but, like mRNA, it was associated with polysomes in a salt-stable manner. Mutational analysis of a short open reading frame upstream of the Tat-coding sequence implicates scanning in the initiation of VA-Tat RNA translation despite the absence of a cap. In comparison with tat mRNA generated by pol II, VA-Tat RNA was present on smaller polysomes and was apparently translated less efficiently, which is consistent with a relatively low initiation rate. Evidently, human cells are capable of utilizing pol III transcripts as functional mRNAs, and neither a cap nor a poly(A) tail is essential for translation, although they may be stimulatory. These findings raise the possibility that some cellular mRNAs are made by pol I or pol III.

Full Text

The Full Text of this article is available as a PDF (536.6 KB).

Selected References

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

  1. Anderson C. W., Lewis J. B., Atkins J. F., Gesteland R. F. Cell-free synthesis of adenovirus 2 proteins programmed by fractionated messenger RNA: a comparison of polypeptide products and messenger RNA lengths. Proc Natl Acad Sci U S A. 1974 Jul;71(7):2756–2760. doi: 10.1073/pnas.71.7.2756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ballantine J. E., Woodland H. R. Polyadenylation of histone mRNA in Xenopus oocytes and embryos. FEBS Lett. 1985 Jan 28;180(2):224–228. doi: 10.1016/0014-5793(85)81075-9. [DOI] [PubMed] [Google Scholar]
  3. Bentley D. L., Brown W. L., Groudine M. Accurate, TATA box-dependent polymerase III transcription from promoters of the c-myc gene in injected Xenopus oocytes. Genes Dev. 1989 Aug;3(8):1179–1189. doi: 10.1101/gad.3.8.1179. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Bruenn J., Keitz B. The 5' ends of yeast killer factor RNAs are pppGp. Nucleic Acids Res. 1976 Oct;3(10):2427–2436. doi: 10.1093/nar/3.10.2427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Caras I. W., Davitz M. A., Rhee L., Weddell G., Martin D. W., Jr, Nussenzweig V. Cloning of decay-accelerating factor suggests novel use of splicing to generate two proteins. Nature. 1987 Feb 5;325(6104):545–549. doi: 10.1038/325545a0. [DOI] [PubMed] [Google Scholar]
  7. Carlson D. P., Ross J. alpha-Amanitin-insensitive transcription of mouse beta major-globin 5'-flanking and structural gene sequences correlates with mRNA expression. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7782–7786. doi: 10.1073/pnas.81.24.7782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chung J., Sussman D. J., Zeller R., Leder P. The c-myc gene encodes superimposed RNA polymerase II and III promoters. Cell. 1987 Dec 24;51(6):1001–1008. doi: 10.1016/0092-8674(87)90586-1. [DOI] [PubMed] [Google Scholar]
  9. Cullen B. R. Trans-activation of human immunodeficiency virus occurs via a bimodal mechanism. Cell. 1986 Sep 26;46(7):973–982. doi: 10.1016/0092-8674(86)90696-3. [DOI] [PubMed] [Google Scholar]
  10. Decker C. J., Parker R. A turnover pathway for both stable and unstable mRNAs in yeast: evidence for a requirement for deadenylation. Genes Dev. 1993 Aug;7(8):1632–1643. doi: 10.1101/gad.7.8.1632. [DOI] [PubMed] [Google Scholar]
  11. Descombes P., Schibler U. A liver-enriched transcriptional activator protein, LAP, and a transcriptional inhibitory protein, LIP, are translated from the same mRNA. Cell. 1991 Nov 1;67(3):569–579. doi: 10.1016/0092-8674(91)90531-3. [DOI] [PubMed] [Google Scholar]
  12. England T. E., Uhlenbeck O. C. 3'-terminal labelling of RNA with T4 RNA ligase. Nature. 1978 Oct 12;275(5680):560–561. doi: 10.1038/275560a0. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Galili G., Kawata E. E., Smith L. D., Larkins B. A. Role of the 3'-poly(A) sequence in translational regulation of mRNAs in Xenopus laevis oocytes. J Biol Chem. 1988 Apr 25;263(12):5764–5770. [PubMed] [Google Scholar]
  15. Gallie D. R., Walbot V. RNA pseudoknot domain of tobacco mosaic virus can functionally substitute for a poly(A) tail in plant and animal cells. Genes Dev. 1990 Jul;4(7):1149–1157. doi: 10.1101/gad.4.7.1149. [DOI] [PubMed] [Google Scholar]
  16. Geballe A. P., Morris D. R. Initiation codons within 5'-leaders of mRNAs as regulators of translation. Trends Biochem Sci. 1994 Apr;19(4):159–164. doi: 10.1016/0968-0004(94)90277-1. [DOI] [PubMed] [Google Scholar]
  17. Greer C. L., Peebles C. L., Gegenheimer P., Abelson J. Mechanism of action of a yeast RNA ligase in tRNA splicing. Cell. 1983 Feb;32(2):537–546. doi: 10.1016/0092-8674(83)90473-7. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Gunnery S., Green S. R., Mathews M. B. Tat-responsive region RNA of human immunodeficiency virus type 1 stimulates protein synthesis in vivo and in vitro: relationship between structure and function. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11557–11561. doi: 10.1073/pnas.89.23.11557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Herbomel P., Bourachot B., Yaniv M. Two distinct enhancers with different cell specificities coexist in the regulatory region of polyoma. Cell. 1984 Dec;39(3 Pt 2):653–662. doi: 10.1016/0092-8674(84)90472-0. [DOI] [PubMed] [Google Scholar]
  21. Hernandez N. TBP, a universal eukaryotic transcription factor? Genes Dev. 1993 Jul;7(7B):1291–1308. doi: 10.1101/gad.7.7b.1291. [DOI] [PubMed] [Google Scholar]
  22. Herrmann C. H., Dery C. V., Mathews M. B. Transactivation of host and viral genes by the adenovirus E1B 19K tumor antigen. Oncogene. 1987;2(1):25–35. [PubMed] [Google Scholar]
  23. Herrmann C. H., Mathews M. B. The adenovirus E1B 19-kilodalton protein stimulates gene expression by increasing DNA levels. Mol Cell Biol. 1989 Dec;9(12):5412–5423. doi: 10.1128/mcb.9.12.5412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Huang W., Pruzan R., Flint S. J. In vivo transcription from the adenovirus E2 early promoter by RNA polymerase III. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1265–1269. doi: 10.1073/pnas.91.4.1265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Jackson R. J., Standart N. Do the poly(A) tail and 3' untranslated region control mRNA translation? Cell. 1990 Jul 13;62(1):15–24. doi: 10.1016/0092-8674(90)90235-7. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Kozak M. An analysis of vertebrate mRNA sequences: intimations of translational control. J Cell Biol. 1991 Nov;115(4):887–903. doi: 10.1083/jcb.115.4.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kozak M. Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes. Mol Cell Biol. 1987 Oct;7(10):3438–3445. doi: 10.1128/mcb.7.10.3438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kozak M. The scanning model for translation: an update. J Cell Biol. 1989 Feb;108(2):229–241. doi: 10.1083/jcb.108.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Kramerov D. A., Tillib S. V., Shumyatsky G. P., Georgiev G. P. The most abundant nascent poly(A) + RNAs are transcribed by RNA polymerase III in murine tumor cells. Nucleic Acids Res. 1990 Aug 11;18(15):4499–4506. doi: 10.1093/nar/18.15.4499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Krayev A. S., Markusheva T. V., Kramerov D. A., Ryskov A. P., Skryabin K. G., Bayev A. A., Georgiev G. P. Ubiquitous transposon-like repeats B1 and B2 of the mouse genome: B2 sequencing. Nucleic Acids Res. 1982 Dec 11;10(23):7461–7475. doi: 10.1093/nar/10.23.7461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Laspia M. F., Rice A. P., Mathews M. B. HIV-1 Tat protein increases transcriptional initiation and stabilizes elongation. Cell. 1989 Oct 20;59(2):283–292. doi: 10.1016/0092-8674(89)90290-0. [DOI] [PubMed] [Google Scholar]
  34. Lewis E. D., Manley J. L. Polyadenylylation of an mRNA precursor occurs independently of transcription by RNA polymerase II in vivo. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8555–8559. doi: 10.1073/pnas.83.22.8555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lobo S. M., Hernandez N. A 7 bp mutation converts a human RNA polymerase II snRNA promoter into an RNA polymerase III promoter. Cell. 1989 Jul 14;58(1):55–67. doi: 10.1016/0092-8674(89)90402-9. [DOI] [PubMed] [Google Scholar]
  36. Lopata M. A., Cleveland D. W., Sollner-Webb B. RNA polymerase specificity of mRNA production and enhancer action. Proc Natl Acad Sci U S A. 1986 Sep;83(18):6677–6681. doi: 10.1073/pnas.83.18.6677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Margalit H., Nadir E., Ben-Sasson S. A. A complete Alu element within the coding sequence of a central gene. Cell. 1994 Jul 29;78(2):173–174. doi: 10.1016/0092-8674(94)90287-9. [DOI] [PubMed] [Google Scholar]
  38. Mathews M. B., Shenk T. Adenovirus virus-associated RNA and translation control. J Virol. 1991 Nov;65(11):5657–5662. doi: 10.1128/jvi.65.11.5657-5662.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Mellits K. H., Kostura M., Mathews M. B. Interaction of adenovirus VA RNAl with the protein kinase DAI: nonequivalence of binding and function. Cell. 1990 Jun 1;61(5):843–852. doi: 10.1016/0092-8674(90)90194-j. [DOI] [PubMed] [Google Scholar]
  40. Mellits K. H., Mathews M. B. Effects of mutations in stem and loop regions on the structure and function of adenovirus VA RNAI. EMBO J. 1988 Sep;7(9):2849–2859. doi: 10.1002/j.1460-2075.1988.tb03141.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Mercer J. F., Wake S. A. An analysis of the rate of metallothionein mRNA poly(A)-shortening using RNA blot hybridization. Nucleic Acids Res. 1985 Nov 25;13(22):7929–7943. doi: 10.1093/nar/13.22.7929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Moss B. 5' end labeling of RNA with capping and methylating enzymes. Gene Amplif Anal. 1981;2:253–266. [PubMed] [Google Scholar]
  43. Munroe D., Jacobson A. mRNA poly(A) tail, a 3' enhancer of translational initiation. Mol Cell Biol. 1990 Jul;10(7):3441–3455. doi: 10.1128/mcb.10.7.3441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Nassal M., Junker-Niepmann M., Schaller H. Translational inactivation of RNA function: discrimination against a subset of genomic transcripts during HBV nucleocapsid assembly. Cell. 1990 Dec 21;63(6):1357–1363. doi: 10.1016/0092-8674(90)90431-d. [DOI] [PubMed] [Google Scholar]
  45. Peabody D. S., Subramani S., Berg P. Effect of upstream reading frames on translation efficiency in simian virus 40 recombinants. Mol Cell Biol. 1986 Jul;6(7):2704–2711. doi: 10.1128/mcb.6.7.2704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Piras G., Kashanchi F., Radonovich M. F., Duvall J. F., Brady J. N. Transcription of the human T-cell lymphotropic virus type I promoter by an alpha-amanitin-resistant polymerase. J Virol. 1994 Oct;68(10):6170–6179. doi: 10.1128/jvi.68.10.6170-6179.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Proweller A., Butler S. Efficient translation of poly(A)-deficient mRNAs in Saccharomyces cerevisiae. Genes Dev. 1994 Nov 1;8(21):2629–2640. doi: 10.1101/gad.8.21.2629. [DOI] [PubMed] [Google Scholar]
  48. Pruzan R., Chatterjee P. K., Flint S. J. Specific transcription from the adenovirus E2E promoter by RNA polymerase III requires a subpopulation of TFIID. Nucleic Acids Res. 1992 Nov 11;20(21):5705–5712. doi: 10.1093/nar/20.21.5705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Rasmussen E. B., Lis J. T. In vivo transcriptional pausing and cap formation on three Drosophila heat shock genes. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):7923–7927. doi: 10.1073/pnas.90.17.7923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Rohan R. M., Ketner G. A comprehensive collection of point mutations in the internal promoter of the adenoviral VAI gene. J Biol Chem. 1987 Jun 25;262(18):8500–8507. [PubMed] [Google Scholar]
  51. 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]
  52. Rosen C. A., Sodroski J. G., Haseltine W. A. The location of cis-acting regulatory sequences in the human T cell lymphotropic virus type III (HTLV-III/LAV) long terminal repeat. Cell. 1985 Jul;41(3):813–823. doi: 10.1016/s0092-8674(85)80062-3. [DOI] [PubMed] [Google Scholar]
  53. Sachs A. B., Davis R. W. The poly(A) binding protein is required for poly(A) shortening and 60S ribosomal subunit-dependent translation initiation. Cell. 1989 Sep 8;58(5):857–867. doi: 10.1016/0092-8674(89)90938-0. [DOI] [PubMed] [Google Scholar]
  54. 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]
  55. Sharma S., Mehta S., Morgan J., Maizel A. Molecular cloning and expression of a human B-cell growth factor gene in Escherichia coli. Science. 1987 Mar 20;235(4795):1489–1492. doi: 10.1126/science.3547651. [DOI] [PubMed] [Google Scholar]
  56. 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]
  57. Singh K., Carey M., Saragosti S., Botchan M. Expression of enhanced levels of small RNA polymerase III transcripts encoded by the B2 repeats in simian virus 40-transformed mouse cells. Nature. 1985 Apr 11;314(6011):553–556. doi: 10.1038/314553a0. [DOI] [PubMed] [Google Scholar]
  58. Sisodia S. S., Sollner-Webb B., Cleveland D. W. Specificity of RNA maturation pathways: RNAs transcribed by RNA polymerase III are not substrates for splicing or polyadenylation. Mol Cell Biol. 1987 Oct;7(10):3602–3612. doi: 10.1128/mcb.7.10.3602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. 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]
  60. Sonenberg N. Remarks on the mechanism of ribosome binding to eukaryotic mRNAs. Gene Expr. 1993;3(3):317–323. [PMC free article] [PubMed] [Google Scholar]
  61. Spaete R. R., Mocarski E. S. Regulation of cytomegalovirus gene expression: alpha and beta promoters are trans activated by viral functions in permissive human fibroblasts. J Virol. 1985 Oct;56(1):135–143. doi: 10.1128/jvi.56.1.135-143.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Spector D. L. Nuclear organization of pre-mRNA processing. Curr Opin Cell Biol. 1993 Jun;5(3):442–447. doi: 10.1016/0955-0674(93)90009-f. [DOI] [PubMed] [Google Scholar]
  63. Tanaka M., Herr W. Differential transcriptional activation by Oct-1 and Oct-2: interdependent activation domains induce Oct-2 phosphorylation. Cell. 1990 Feb 9;60(3):375–386. doi: 10.1016/0092-8674(90)90589-7. [DOI] [PubMed] [Google Scholar]
  64. Terns M. P., Lund E., Dahlberg J. E. 3'-end-dependent formation of U6 small nuclear ribonucleoprotein particles in Xenopus laevis oocyte nuclei. Mol Cell Biol. 1992 Jul;12(7):3032–3040. doi: 10.1128/mcb.12.7.3032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Widner W. R., Wickner R. B. Evidence that the SKI antiviral system of Saccharomyces cerevisiae acts by blocking expression of viral mRNA. Mol Cell Biol. 1993 Jul;13(7):4331–4341. doi: 10.1128/mcb.13.7.4331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Zietkiewicz E., Makałowski W., Mitchell G. A., Labuda D. Phylogenetic analysis of a reported complementary DNA sequence. Science. 1994 Aug 19;265(5175):1110–1111. doi: 10.1126/science.8066452. [DOI] [PubMed] [Google Scholar]
  67. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES