Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1994 Aug;14(8):5450–5457. doi: 10.1128/mcb.14.8.5450

Transcription of a variant human U6 small nuclear RNA gene is controlled by a novel, internal RNA polymerase III promoter.

J W Tichelaar 1, B Knerer 1, A Vrabel 1, E D Wieben 1
PMCID: PMC359064  PMID: 8035822

Abstract

Promoter elements in the 5' flanking regions of vertebrate U6 RNA genes have been shown to be both necessary and sufficient for transcription by RNA polymerase III. We have recently isolated and characterized a variant human U6 gene (87U6) that can be transcribed by RNA polymerase III in vitro in the absence of any natural 5' or 3' flanking sequences. This gene contains 10 nucleotide differences from the previously characterized human U6 gene (wtU6) within the coding region but has no homology to wtU6 in the upstream promoter region. By constructing chimeras between these two genes, we have shown that mutation of as few as two nucleotides in the coding region of the human U6 RNA gene is sufficient to create an internal promoter that is functional in vitro. A T-to-C transition at position 57 and a single T deletion at position 52 produce an internal U6 promoter that is nearly as active in vitro as the external U6 polymerase III promoter utilized by wtU6. Neither of these residues is absolutely conserved during evolution, and both of these nucleotide changes occur within the previously noted A box homology. Deletion and linker scanning mutations within the coding region of this variant U6 gene suggest that, in addition to the central region including bp 52 and 57, sequences at the extreme 5' end of the gene are critical for efficient transcription. In contrast, flanking sequences have a minor effect on transcriptional efficiency. This arrangement is unique among internal RNA polymerase III promoters and may indicate unique regulation of this gene.

Full text

PDF
5450

Images in this article

5453Image
on p.5453
5453Image
on p.5453
5454Image
on p.5454
5455Image
on p.5455
5455Image
on p.5455
5455Image
on p.5455
5456Image
on p.5456

Selected References

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

  1. Arnold G. J., Gross H. J. Unrelated leader sequences can efficiently promote human tRNA gene transcription. Gene. 1987;51(2-3):237–246. doi: 10.1016/0378-1119(87)90312-x. [DOI] [PubMed] [Google Scholar]
  2. Bark C., Weller P., Zabielski J., Janson L., Pettersson U. A distant enhancer element is required for polymerase III transcription of a U6 RNA gene. Nature. 1987 Jul 23;328(6128):356–359. doi: 10.1038/328356a0. [DOI] [PubMed] [Google Scholar]
  3. Bogenhagen D. F., Sakonju S., Brown D. D. A control region in the center of the 5S RNA gene directs specific initiation of transcription: II. The 3' border of the region. Cell. 1980 Jan;19(1):27–35. doi: 10.1016/0092-8674(80)90385-2. [DOI] [PubMed] [Google Scholar]
  4. Bordonné R., Guthrie C. Human and human-yeast chimeric U6 snRNA genes identify structural elements required for expression in yeast. Nucleic Acids Res. 1992 Feb 11;20(3):479–485. doi: 10.1093/nar/20.3.479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bredow S., Kleinert H., Benecke B. J. Sequence and factor requirements for faithful in vitro transcription of human 7SL DNA. Gene. 1990 Feb 14;86(2):217–225. doi: 10.1016/0378-1119(90)90282-v. [DOI] [PubMed] [Google Scholar]
  6. Brow D. A., Guthrie C. Transcription of a yeast U6 snRNA gene requires a polymerase III promoter element in a novel position. Genes Dev. 1990 Aug;4(8):1345–1356. doi: 10.1101/gad.4.8.1345. [DOI] [PubMed] [Google Scholar]
  7. Burnol A. F., Margottin F., Schultz P., Marsolier M. C., Oudet P., Sentenac A. Basal promoter and enhancer element of yeast U6 snRNA gene. J Mol Biol. 1993 Oct 20;233(4):644–658. doi: 10.1006/jmbi.1993.1542. [DOI] [PubMed] [Google Scholar]
  8. Das G., Henning D., Wright D., Reddy R. Upstream regulatory elements are necessary and sufficient for transcription of a U6 RNA gene by RNA polymerase III. EMBO J. 1988 Feb;7(2):503–512. doi: 10.1002/j.1460-2075.1988.tb02838.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eschenlauer J. B., Kaiser M. W., Gerlach V. L., Brow D. A. Architecture of a yeast U6 RNA gene promoter. Mol Cell Biol. 1993 May;13(5):3015–3026. doi: 10.1128/mcb.13.5.3015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fantoni A., Dare A. O., Tschudi C. RNA polymerase III-mediated transcription of the trypanosome U2 small nuclear RNA gene is controlled by both intragenic and extragenic regulatory elements. Mol Cell Biol. 1994 Mar;14(3):2021–2028. doi: 10.1128/mcb.14.3.2021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Frendewey D., Barta I., Gillespie M., Potashkin J. Schizosaccharomyces U6 genes have a sequence within their introns that matches the B box consensus of tRNA internal promoters. Nucleic Acids Res. 1990 Apr 25;18(8):2025–2032. doi: 10.1093/nar/18.8.2025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Garcia A. D., O'Connell A. M., Sharp S. J. Formation of an active transcription complex in the Drosophila melanogaster 5S RNA gene is dependent on an upstream region. Mol Cell Biol. 1987 Jun;7(6):2046–2051. doi: 10.1128/mcb.7.6.2046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Geiduschek E. P., Tocchini-Valentini G. P. Transcription by RNA polymerase III. Annu Rev Biochem. 1988;57:873–914. doi: 10.1146/annurev.bi.57.070188.004301. [DOI] [PubMed] [Google Scholar]
  14. Hayashi K. Organization of sequences related to U6 RNA in the human genome. Nucleic Acids Res. 1981 Jul 24;9(14):3379–3388. doi: 10.1093/nar/9.14.3379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Horton R. M., Ho S. N., Pullen J. K., Hunt H. D., Cai Z., Pease L. R. Gene splicing by overlap extension. Methods Enzymol. 1993;217:270–279. doi: 10.1016/0076-6879(93)17067-f. [DOI] [PubMed] [Google Scholar]
  16. Itoh H., Toyama R., Kozasa T., Tsukamoto T., Matsuoka M., Kaziro Y. Presence of three distinct molecular species of Gi protein alpha subunit. Structure of rat cDNAs and human genomic DNAs. J Biol Chem. 1988 May 15;263(14):6656–6664. [PubMed] [Google Scholar]
  17. Kleinert H., Gladen A., Geisler M., Benecke B. J. Differential regulation of transcription of human 7 S K and 7 S L RNA genes. J Biol Chem. 1988 Aug 15;263(23):11511–11515. [PubMed] [Google Scholar]
  18. Krol A., Carbon P., Ebel J. P., Appel B. Xenopus tropicalis U6 snRNA genes transcribed by Pol III contain the upstream promoter elements used by Pol II dependent U snRNA genes. Nucleic Acids Res. 1987 Mar 25;15(6):2463–2478. doi: 10.1093/nar/15.6.2463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kunkel G. R., Maser R. L., Calvet J. P., Pederson T. U6 small nuclear RNA is transcribed by RNA polymerase III. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8575–8579. doi: 10.1073/pnas.83.22.8575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kunkel G. R., Pederson T. Transcription of a human U6 small nuclear RNA gene in vivo withstands deletion of intragenic sequences but not of an upstream TATATA box. Nucleic Acids Res. 1989 Sep 25;17(18):7371–7379. doi: 10.1093/nar/17.18.7371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kunkel G. R., Pederson T. Upstream elements required for efficient transcription of a human U6 RNA gene resemble those of U1 and U2 genes even though a different polymerase is used. Genes Dev. 1988 Feb;2(2):196–204. doi: 10.1101/gad.2.2.196. [DOI] [PubMed] [Google Scholar]
  22. Kunkel G. R. RNA polymerase III transcription of genes that lack internal control regions. Biochim Biophys Acta. 1991 Jan 17;1088(1):1–9. doi: 10.1016/0167-4781(91)90146-d. [DOI] [PubMed] [Google Scholar]
  23. Lobo S. M., Ifill S., Hernandez N. cis-acting elements required for RNA polymerase II and III transcription in the human U2 and U6 snRNA promoters. Nucleic Acids Res. 1990 May 25;18(10):2891–2899. doi: 10.1093/nar/18.10.2891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lobo S. M., Lister J., Sullivan M. L., Hernandez N. The cloned RNA polymerase II transcription factor IID selects RNA polymerase III to transcribe the human U6 gene in vitro. Genes Dev. 1991 Aug;5(8):1477–1489. doi: 10.1101/gad.5.8.1477. [DOI] [PubMed] [Google Scholar]
  25. Lund E. Heterogeneity of human U1 snRNAs. Nucleic Acids Res. 1988 Jul 11;16(13):5813–5826. doi: 10.1093/nar/16.13.5813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Morry M. J., Harding J. D. Modulation of transcriptional activity and stable complex formation by 5'-flanking regions of mouse tRNAHis genes. Mol Cell Biol. 1986 Jan;6(1):105–115. doi: 10.1128/mcb.6.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Patton J. G., Wieben E. D. U1 precursors: variant 3' flanking sequences are transcribed in human cells. J Cell Biol. 1987 Feb;104(2):175–182. doi: 10.1083/jcb.104.2.175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Reddy R., Henning D., Das G., Harless M., Wright D. The capped U6 small nuclear RNA is transcribed by RNA polymerase III. J Biol Chem. 1987 Jan 5;262(1):75–81. [PubMed] [Google Scholar]
  29. Sakonju S., Bogenhagen D. F., Brown D. D. A control region in the center of the 5S RNA gene directs specific initiation of transcription: I. The 5' border of the region. Cell. 1980 Jan;19(1):13–25. doi: 10.1016/0092-8674(80)90384-0. [DOI] [PubMed] [Google Scholar]
  30. Sontheimer E. J., Steitz J. A. Three novel functional variants of human U5 small nuclear RNA. Mol Cell Biol. 1992 Feb;12(2):734–746. doi: 10.1128/mcb.12.2.734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sprague K. U., Larson D., Morton D. 5' flanking sequence signals are required for activity of silkworm alanine tRNA genes in homologous in vitro transcription systems. Cell. 1980 Nov;22(1 Pt 1):171–178. doi: 10.1016/0092-8674(80)90165-8. [DOI] [PubMed] [Google Scholar]
  32. Tani T., Ohshima Y. The gene for the U6 small nuclear RNA in fission yeast has an intron. Nature. 1989 Jan 5;337(6202):87–90. doi: 10.1038/337087a0. [DOI] [PubMed] [Google Scholar]
  33. Theissen H., Rinke J., Traver C. N., Lührmann R., Appel B. Novel structure of a human U6 snRNA pseudogene. Gene. 1985;36(1-2):195–199. doi: 10.1016/0378-1119(85)90086-1. [DOI] [PubMed] [Google Scholar]
  34. Ullu E., Weiner A. M. Upstream sequences modulate the internal promoter of the human 7SL RNA gene. 1985 Nov 28-Dec 4Nature. 318(6044):371–374. doi: 10.1038/318371a0. [DOI] [PubMed] [Google Scholar]
  35. Weil P. A., Segall J., Harris B., Ng S. Y., Roeder R. G. Faithful transcription of eukaryotic genes by RNA polymerase III in systems reconstituted with purified DNA templates. J Biol Chem. 1979 Jul 10;254(13):6163–6173. [PubMed] [Google Scholar]
  36. Wieben E. D., Rohleder A. M., Nenninger J. M., Pederson T. cDNA cloning of a human autoimmune nuclear ribonucleoprotein antigen. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7914–7918. doi: 10.1073/pnas.82.23.7914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wieben E. D., Vrabel A. M., Holicky E. L., Klisak I., Sparkes R. S., Stanford D. R. A U6 snRNA gene with an internal promoter is juxtaposed to an snRNP protein sequence within an intron of a human G protein gene. Nucleic Acids Res. 1991 Jun 11;19(11):2869–2874. doi: 10.1093/nar/19.11.2869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Willis I. M. RNA polymerase III. Genes, factors and transcriptional specificity. Eur J Biochem. 1993 Feb 15;212(1):1–11. doi: 10.1111/j.1432-1033.1993.tb17626.x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES