Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1992 Feb 11;20(3):479–485. doi: 10.1093/nar/20.3.479

Human and human-yeast chimeric U6 snRNA genes identify structural elements required for expression in yeast.

R Bordonné 1, C Guthrie 1
PMCID: PMC310411  PMID: 1741282

Abstract

U6 is the most highly conserved spliceosomal snRNA. Previous mutational studies have shown that the majority of essential residues in U6 are located in a region of 35 nucleotides encompassing a conserved hexanucleotide and stem I and stem II of the U4-interaction domain. Although the yeast and human U6 RNAs are 80% identical in this region, the human U6 gene cannot functionally replace the yeast gene in vivo. The human gene is not transcribed when placed in the context of yeast flanking sequences. Transcription of the human gene, but not its function, can be stimulated by the introduction of an A block promoter element in the U6 coding region. Using a set of human-yeast chimeras, we show that the 5' domain and the 3' terminal region of the human U6 gene can each functionally replace the corresponding yeast domains. However, a combination of both domains in a single molecule is lethal. The basis of the inability of the human U6 snRNA to function in yeast cells is discussed.

Full text

PDF
485

Images in this article

Selected References

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

  1. Ares M., Jr U2 RNA from yeast is unexpectedly large and contains homology to vertebrate U4, U5, and U6 small nuclear RNAs. Cell. 1986 Oct 10;47(1):49–59. doi: 10.1016/0092-8674(86)90365-x. [DOI] [PubMed] [Google Scholar]
  2. Bindereif A., Wolff T., Green M. R. Discrete domains of human U6 snRNA required for the assembly of U4/U6 snRNP and splicing complexes. EMBO J. 1990 Jan;9(1):251–255. doi: 10.1002/j.1460-2075.1990.tb08102.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boeke J. D., Trueheart J., Natsoulis G., Fink G. R. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 1987;154:164–175. doi: 10.1016/0076-6879(87)54076-9. [DOI] [PubMed] [Google Scholar]
  4. Bordonné R., Banroques J., Abelson J., Guthrie C. Domains of yeast U4 spliceosomal RNA required for PRP4 protein binding, snRNP-snRNP interactions, and pre-mRNA splicing in vivo. Genes Dev. 1990 Jul;4(7):1185–1196. doi: 10.1101/gad.4.7.1185. [DOI] [PubMed] [Google Scholar]
  5. Brow D. A., Guthrie C. Spliceosomal RNA U6 is remarkably conserved from yeast to mammals. Nature. 1988 Jul 21;334(6179):213–218. doi: 10.1038/334213a0. [DOI] [PubMed] [Google Scholar]
  6. Brow D. A., Guthrie C. Splicing a spliceosomal RNA. Nature. 1989 Jan 5;337(6202):14–15. doi: 10.1038/337014a0. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Carbon P., Murgo S., Ebel J. P., Krol A., Tebb G., Mattaj L. W. A common octamer motif binding protein is involved in the transcription of U6 snRNA by RNA polymerase III and U2 snRNA by RNA polymerase II. Cell. 1987 Oct 9;51(1):71–79. doi: 10.1016/0092-8674(87)90011-0. [DOI] [PubMed] [Google Scholar]
  9. Cheng S. C., Abelson J. Spliceosome assembly in yeast. Genes Dev. 1987 Nov;1(9):1014–1027. doi: 10.1101/gad.1.9.1014. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Datta B., Weiner A. M. Genetic evidence for base pairing between U2 and U6 snRNA in mammalian mRNA splicing. Nature. 1991 Aug 29;352(6338):821–824. doi: 10.1038/352821a0. [DOI] [PubMed] [Google Scholar]
  12. Elledge S. J., Davis R. W. A family of versatile centromeric vectors designed for use in the sectoring-shuffle mutagenesis assay in Saccharomyces cerevisiae. Gene. 1988 Oct 30;70(2):303–312. doi: 10.1016/0378-1119(88)90202-8. [DOI] [PubMed] [Google Scholar]
  13. Fabrizio P., Abelson J. Two domains of yeast U6 small nuclear RNA required for both steps of nuclear precursor messenger RNA splicing. Science. 1990 Oct 19;250(4979):404–409. doi: 10.1126/science.2145630. [DOI] [PubMed] [Google Scholar]
  14. Fabrizio P., McPheeters D. S., Abelson J. In vitro assembly of yeast U6 snRNP: a functional assay. Genes Dev. 1989 Dec;3(12B):2137–2150. doi: 10.1101/gad.3.12b.2137. [DOI] [PubMed] [Google Scholar]
  15. Galli G., Hofstetter H., Birnstiel M. L. Two conserved sequence blocks within eukaryotic tRNA genes are major promoter elements. Nature. 1981 Dec 17;294(5842):626–631. doi: 10.1038/294626a0. [DOI] [PubMed] [Google Scholar]
  16. Geliebter J., Zeff R. A., Melvold R. W., Nathenson S. G. Mitotic recombination in germ cells generated two major histocompatibility complex mutant genes shown to be identical by RNA sequence analysis: Kbm9 and Kbm6. Proc Natl Acad Sci U S A. 1986 May;83(10):3371–3375. doi: 10.1073/pnas.83.10.3371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Goguel V., Liao X. L., Rymond B. C., Rosbash M. U1 snRNP can influence 3'-splice site selection as well as 5'-splice site selection. Genes Dev. 1991 Aug;5(8):1430–1438. doi: 10.1101/gad.5.8.1430. [DOI] [PubMed] [Google Scholar]
  18. Guthrie C. Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein. Science. 1991 Jul 12;253(5016):157–163. doi: 10.1126/science.1853200. [DOI] [PubMed] [Google Scholar]
  19. Guthrie C., Patterson B. Spliceosomal snRNAs. Annu Rev Genet. 1988;22:387–419. doi: 10.1146/annurev.ge.22.120188.002131. [DOI] [PubMed] [Google Scholar]
  20. Hausner T. P., Giglio L. M., Weiner A. M. Evidence for base-pairing between mammalian U2 and U6 small nuclear ribonucleoprotein particles. Genes Dev. 1990 Dec;4(12A):2146–2156. doi: 10.1101/gad.4.12a.2146. [DOI] [PubMed] [Google Scholar]
  21. Igel A. H., Ares M., Jr Internal sequences that distinguish yeast from metazoan U2 snRNA are unnecessary for pre-mRNA splicing. Nature. 1988 Aug 4;334(6181):450–453. doi: 10.1038/334450a0. [DOI] [PubMed] [Google Scholar]
  22. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kleinschmidt A. M., Pederson T., Tani T., Ohshima Y. An intron-containing Schizosaccharomyces pombe U6 RNA gene can be transcribed by human RNA polymerase III. J Mol Biol. 1990 Jan 5;211(1):7–9. doi: 10.1016/0022-2836(90)90005-7. [DOI] [PubMed] [Google Scholar]
  24. Konarska M. M., Sharp P. A. Association of U2, U4, U5, and U6 small nuclear ribonucleoproteins in a spliceosome-type complex in absence of precursor RNA. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5459–5462. doi: 10.1073/pnas.85.15.5459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Konarska M. M., Sharp P. A. Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes. Cell. 1987 Jun 19;49(6):763–774. doi: 10.1016/0092-8674(87)90614-3. [DOI] [PubMed] [Google Scholar]
  26. Kretzner L., Krol A., Rosbash M. Saccharomyces cerevisiae U1 small nuclear RNA secondary structure contains both universal and yeast-specific domains. Proc Natl Acad Sci U S A. 1990 Jan;87(2):851–855. doi: 10.1073/pnas.87.2.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  30. Liao X. L., Kretzner L., Seraphin B., Rosbash M. Universally conserved and yeast-specific U1 snRNA sequences are important but not essential for U1 snRNP function. Genes Dev. 1990 Oct;4(10):1766–1774. doi: 10.1101/gad.4.10.1766. [DOI] [PubMed] [Google Scholar]
  31. Madhani H. D., Bordonné R., Guthrie C. Multiple roles for U6 snRNA in the splicing pathway. Genes Dev. 1990 Dec;4(12B):2264–2277. doi: 10.1101/gad.4.12b.2264. [DOI] [PubMed] [Google Scholar]
  32. McClary J. A., Witney F., Geisselsoder J. Efficient site-directed in vitro mutagenesis using phagemid vectors. Biotechniques. 1989 Mar;7(3):282–289. [PubMed] [Google Scholar]
  33. Miraglia L., Seiwert S., Igel A. H., Ares M., Jr Limited functional equivalence of phylogenetic variation in small nuclear RNA: yeast U2 RNA with altered branchpoint complementarity inhibits splicing and produces a dominant lethal phenotype. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7061–7065. doi: 10.1073/pnas.88.16.7061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Murphy S., Moorefield B., Pieler T. Common mechanisms of promoter recognition by RNA polymerases II and III. Trends Genet. 1989 Apr;5(4):122–126. doi: 10.1016/0168-9525(89)90043-7. [DOI] [PubMed] [Google Scholar]
  35. Pan Z. Q., Prives C. Assembly of functional U1 and U2 human-amphibian hybrid snRNPs in Xenopus laevis oocytes. Science. 1988 Sep 9;241(4871):1328–1331. doi: 10.1126/science.2970672. [DOI] [PubMed] [Google Scholar]
  36. Parry H. D., Mattaj I. W. Positive and negative functional interactions between promoter elements from different classes of RNA polymerase III-transcribed genes. EMBO J. 1990 Apr;9(4):1097–1104. doi: 10.1002/j.1460-2075.1990.tb08215.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Patterson B., Guthrie C. An essential yeast snRNA with a U5-like domain is required for splicing in vivo. Cell. 1987 Jun 5;49(5):613–624. doi: 10.1016/0092-8674(87)90537-x. [DOI] [PubMed] [Google Scholar]
  38. Riedel N., Wolin S., Guthrie C. A subset of yeast snRNA's contains functional binding sites for the highly conserved Sm antigen. Science. 1987 Jan 16;235(4786):328–331. doi: 10.1126/science.2948278. [DOI] [PubMed] [Google Scholar]
  39. Ruby S. W., Abelson J. Pre-mRNA splicing in yeast. Trends Genet. 1991 Mar;7(3):79–85. doi: 10.1016/0168-9525(91)90276-V. [DOI] [PubMed] [Google Scholar]
  40. Shannon K. W., Guthrie C. Suppressors of a U4 snRNA mutation define a novel U6 snRNP protein with RNA-binding motifs. Genes Dev. 1991 May;5(5):773–785. doi: 10.1101/gad.5.5.773. [DOI] [PubMed] [Google Scholar]
  41. Shuster E. O., Guthrie C. Human U2 snRNA can function in pre-mRNA splicing in yeast. Nature. 1990 May 17;345(6272):270–273. doi: 10.1038/345270a0. [DOI] [PubMed] [Google Scholar]
  42. Shuster E. O., Guthrie C. Two conserved domains of yeast U2 snRNA are separated by 945 nonessential nucleotides. Cell. 1988 Oct 7;55(1):41–48. doi: 10.1016/0092-8674(88)90007-4. [DOI] [PubMed] [Google Scholar]
  43. Siliciano P. G., Kivens W. J., Guthrie C. More than half of yeast U1 snRNA is dispensable for growth. Nucleic Acids Res. 1991 Dec 11;19(23):6367–6372. doi: 10.1093/nar/19.23.6367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Singh R., Gupta S., Reddy R. Capping of mammalian U6 small nuclear RNA in vitro is directed by a conserved stem-loop and AUAUAC sequence: conversion of a noncapped RNA into a capped RNA. Mol Cell Biol. 1990 Mar;10(3):939–946. doi: 10.1128/mcb.10.3.939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Singh R., Reddy R. Gamma-monomethyl phosphate: a cap structure in spliceosomal U6 small nuclear RNA. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8280–8283. doi: 10.1073/pnas.86.21.8280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. Tani T., Ohshima Y. mRNA-type introns in U6 small nuclear RNA genes: implications for the catalysis in pre-mRNA splicing. Genes Dev. 1991 Jun;5(6):1022–1031. doi: 10.1101/gad.5.6.1022. [DOI] [PubMed] [Google Scholar]
  48. Tollervey D., Mattaj I. W. Fungal small nuclear ribonucleoproteins share properties with plant and vertebrate U-snRNPs. EMBO J. 1987 Feb;6(2):469–476. doi: 10.1002/j.1460-2075.1987.tb04777.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Vankan P., McGuigan C., Mattaj I. W. Domains of U4 and U6 snRNAs required for snRNP assembly and splicing complementation in Xenopus oocytes. EMBO J. 1990 Oct;9(10):3397–3404. doi: 10.1002/j.1460-2075.1990.tb07541.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wise J. A., Tollervey D., Maloney D., Swerdlow H., Dunn E. J., Guthrie C. Yeast contains small nuclear RNAs encoded by single copy genes. Cell. 1983 Dec;35(3 Pt 2):743–751. doi: 10.1016/0092-8674(83)90107-1. [DOI] [PubMed] [Google Scholar]
  51. Wu J. A., Manley J. L. Base pairing between U2 and U6 snRNAs is necessary for splicing of a mammalian pre-mRNA. Nature. 1991 Aug 29;352(6338):818–821. doi: 10.1038/352818a0. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES