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. 1991 Sep;10(9):2627–2634. doi: 10.1002/j.1460-2075.1991.tb07805.x

Cloning of a yeast U1 snRNP 70K protein homologue: functional conservation of an RNA-binding domain between humans and yeast.

V Smith 1, B G Barrell 1
PMCID: PMC452962  PMID: 1714384

Abstract

We have cloned and sequenced a gene encoding a yeast homologue of the U1 snRNP 70K protein. The gene, SNP1, encodes a protein which has 30% amino acid identity with the human 70K protein and has a predicted molecular weight of 34 kDa. The yeast and human sequences are more closely related to each other than to other (non-U1) RNA-binding proteins, but diverge considerably in their C-terminal portions. In particular, SNP1 lacks the charged carboxy terminus of the human 70K protein. A yeast strain, a alpha 115, was constructed in which one allele of the SNP1 gene contained a 554 bp deletion. Tetrad analysis of a alpha 115 showed that the SNP1 gene is essential for the viability of yeast cells. The complete human 70K gene did not complement snp1, but the lethal snp1 mutation was rescued by plasmids bearing a chimera in which over half the yeast gene was replaced with the homologous region of the human 70K gene, including the RNA-binding domain. These results suggest that SNP1 encodes a functional homologue of the U1 snRNP 70K protein.

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  1. Adam S. A., Nakagawa T., Swanson M. S., Woodruff T. K., Dreyfuss G. mRNA polyadenylate-binding protein: gene isolation and sequencing and identification of a ribonucleoprotein consensus sequence. Mol Cell Biol. 1986 Aug;6(8):2932–2943. doi: 10.1128/mcb.6.8.2932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Bandziulis R. J., Swanson M. S., Dreyfuss G. RNA-binding proteins as developmental regulators. Genes Dev. 1989 Apr;3(4):431–437. doi: 10.1101/gad.3.4.431. [DOI] [PubMed] [Google Scholar]
  4. Bankier A. T., Weston K. M., Barrell B. G. Random cloning and sequencing by the M13/dideoxynucleotide chain termination method. Methods Enzymol. 1987;155:51–93. doi: 10.1016/0076-6879(87)55009-1. [DOI] [PubMed] [Google Scholar]
  5. Banroques J., Abelson J. N. PRP4: a protein of the yeast U4/U6 small nuclear ribonucleoprotein particle. Mol Cell Biol. 1989 Sep;9(9):3710–3719. doi: 10.1128/mcb.9.9.3710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Barton G. J., Sternberg M. J. A strategy for the rapid multiple alignment of protein sequences. Confidence levels from tertiary structure comparisons. J Mol Biol. 1987 Nov 20;198(2):327–337. doi: 10.1016/0022-2836(87)90316-0. [DOI] [PubMed] [Google Scholar]
  7. Beggs J., Lossky M., Anderson G. J. Nuclear pre-mRNA splicing in Saccharomyces cerevisiae. Biochem Soc Symp. 1989;55:69–75. [PubMed] [Google Scholar]
  8. Bjørn S. P., Soltyk A., Beggs J. D., Friesen J. D. PRP4 (RNA4) from Saccharomyces cerevisiae: its gene product is associated with the U4/U6 small nuclear ribonucleoprotein particle. Mol Cell Biol. 1989 Sep;9(9):3698–3709. doi: 10.1128/mcb.9.9.3698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dreyfuss G., Swanson M. S., Piñol-Roma S. Heterogeneous nuclear ribonucleoprotein particles and the pathway of mRNA formation. Trends Biochem Sci. 1988 Mar;13(3):86–91. doi: 10.1016/0968-0004(88)90046-1. [DOI] [PubMed] [Google Scholar]
  10. Etzerodt M., Vignali R., Ciliberto G., Scherly D., Mattaj I. W., Philipson L. Structure and expression of a Xenopus gene encoding an snRNP protein (U1 70K). EMBO J. 1988 Dec 20;7(13):4311–4321. doi: 10.1002/j.1460-2075.1988.tb03330.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  12. George D. G., Barker W. C., Hunt L. T. The protein identification resource (PIR). Nucleic Acids Res. 1986 Jan 10;14(1):11–15. doi: 10.1093/nar/14.1.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Hinterberger M., Pettersson I., Steitz J. A. Isolation of small nuclear ribonucleoproteins containing U1, U2, U4, U5, and U6 RNAs. J Biol Chem. 1983 Feb 25;258(4):2604–2613. [PubMed] [Google Scholar]
  15. Hornig H., Fischer U., Costas M., Rauh A., Lührmann R. Analysis of genomic clones of the murine U1RNA-associated 70-kDa protein reveals a high evolutionary conservation of the protein between human and mouse. Eur J Biochem. 1989 Jun 1;182(1):45–50. doi: 10.1111/j.1432-1033.1989.tb14798.x. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Kishimoto A., Nishiyama K., Nakanishi H., Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y. Studies on the phosphorylation of myelin basic protein by protein kinase C and adenosine 3':5'-monophosphate-dependent protein kinase. J Biol Chem. 1985 Oct 15;260(23):12492–12499. [PubMed] [Google Scholar]
  18. 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]
  19. Kretzner L., Rymond B. C., Rosbash M. S. cerevisiae U1 RNA is large and has limited primary sequence homology to metazoan U1 snRNA. Cell. 1987 Aug 14;50(4):593–602. doi: 10.1016/0092-8674(87)90032-8. [DOI] [PubMed] [Google Scholar]
  20. Krämer A., Keller W., Appel B., Lührmann R. The 5' terminus of the RNA moiety of U1 small nuclear ribonucleoprotein particles is required for the splicing of messenger RNA precursors. Cell. 1984 Aug;38(1):299–307. doi: 10.1016/0092-8674(84)90551-8. [DOI] [PubMed] [Google Scholar]
  21. Kuenzel E. A., Mulligan J. A., Sommercorn J., Krebs E. G. Substrate specificity determinants for casein kinase II as deduced from studies with synthetic peptides. J Biol Chem. 1987 Jul 5;262(19):9136–9140. [PubMed] [Google Scholar]
  22. Langford C. J., Gallwitz D. Evidence for an intron-contained sequence required for the splicing of yeast RNA polymerase II transcripts. Cell. 1983 Jun;33(2):519–527. doi: 10.1016/0092-8674(83)90433-6. [DOI] [PubMed] [Google Scholar]
  23. Langford C. J., Klinz F. J., Donath C., Gallwitz D. Point mutations identify the conserved, intron-contained TACTAAC box as an essential splicing signal sequence in yeast. Cell. 1984 Mar;36(3):645–653. doi: 10.1016/0092-8674(84)90344-1. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Lossky M., Anderson G. J., Jackson S. P., Beggs J. Identification of a yeast snRNP protein and detection of snRNP-snRNP interactions. Cell. 1987 Dec 24;51(6):1019–1026. doi: 10.1016/0092-8674(87)90588-5. [DOI] [PubMed] [Google Scholar]
  26. Mancebo R., Lo P. C., Mount S. M. Structure and expression of the Drosophila melanogaster gene for the U1 small nuclear ribonucleoprotein particle 70K protein. Mol Cell Biol. 1990 Jun;10(6):2492–2502. doi: 10.1128/mcb.10.6.2492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Maniatis T., Reed R. The role of small nuclear ribonucleoprotein particles in pre-mRNA splicing. Nature. 1987 Feb 19;325(6106):673–678. doi: 10.1038/325673a0. [DOI] [PubMed] [Google Scholar]
  28. Marin O., Meggio F., Marchiori F., Borin G., Pinna L. A. Site specificity of casein kinase-2 (TS) from rat liver cytosol. A study with model peptide substrates. Eur J Biochem. 1986 Oct 15;160(2):239–244. doi: 10.1111/j.1432-1033.1986.tb09962.x. [DOI] [PubMed] [Google Scholar]
  29. Mortimer R. K., Schild D., Contopoulou C. R., Kans J. A. Genetic and physical maps of Saccharomyces cerevisiae. Methods Enzymol. 1991;194:827–863. doi: 10.1016/0076-6879(91)94060-p. [DOI] [PubMed] [Google Scholar]
  30. Mount S. M., Pettersson I., Hinterberger M., Karmas A., Steitz J. A. The U1 small nuclear RNA-protein complex selectively binds a 5' splice site in vitro. Cell. 1983 Jun;33(2):509–518. doi: 10.1016/0092-8674(83)90432-4. [DOI] [PubMed] [Google Scholar]
  31. Nagai K., Oubridge C., Jessen T. H., Li J., Evans P. R. Crystal structure of the RNA-binding domain of the U1 small nuclear ribonucleoprotein A. Nature. 1990 Dec 6;348(6301):515–520. doi: 10.1038/348515a0. [DOI] [PubMed] [Google Scholar]
  32. Olson M. V., Dutchik J. E., Graham M. Y., Brodeur G. M., Helms C., Frank M., MacCollin M., Scheinman R., Frank T. Random-clone strategy for genomic restriction mapping in yeast. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7826–7830. doi: 10.1073/pnas.83.20.7826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Patton J. R., Pederson T. The Mr 70,000 protein of the U1 small nuclear ribonucleoprotein particle binds to the 5' stem-loop of U1 RNA and interacts with Sm domain proteins. Proc Natl Acad Sci U S A. 1988 Feb;85(3):747–751. doi: 10.1073/pnas.85.3.747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pettersson I., Hinterberger M., Mimori T., Gottlieb E., Steitz J. A. The structure of mammalian small nuclear ribonucleoproteins. Identification of multiple protein components reactive with anti-(U1)ribonucleoprotein and anti-Sm autoantibodies. J Biol Chem. 1984 May 10;259(9):5907–5914. [PubMed] [Google Scholar]
  36. Query C. C., Bentley R. C., Keene J. D. A common RNA recognition motif identified within a defined U1 RNA binding domain of the 70K U1 snRNP protein. Cell. 1989 Apr 7;57(1):89–101. doi: 10.1016/0092-8674(89)90175-x. [DOI] [PubMed] [Google Scholar]
  37. Query C. C., Bentley R. C., Keene J. D. A specific 31-nucleotide domain of U1 RNA directly interacts with the 70K small nuclear ribonucleoprotein component. Mol Cell Biol. 1989 Nov;9(11):4872–4881. doi: 10.1128/mcb.9.11.4872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Riedel N., Wise J. A., Swerdlow H., Mak A., Guthrie C. Small nuclear RNAs from Saccharomyces cerevisiae: unexpected diversity in abundance, size, and molecular complexity. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8097–8101. doi: 10.1073/pnas.83.21.8097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ruby S. W., Abelson J. An early hierarchic role of U1 small nuclear ribonucleoprotein in spliceosome assembly. Science. 1988 Nov 18;242(4881):1028–1035. doi: 10.1126/science.2973660. [DOI] [PubMed] [Google Scholar]
  40. Sachs A. B., Bond M. W., Kornberg R. D. A single gene from yeast for both nuclear and cytoplasmic polyadenylate-binding proteins: domain structure and expression. Cell. 1986 Jun 20;45(6):827–835. doi: 10.1016/0092-8674(86)90557-x. [DOI] [PubMed] [Google Scholar]
  41. Sachs A. B., Davis R. W., Kornberg R. D. A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability. Mol Cell Biol. 1987 Sep;7(9):3268–3276. doi: 10.1128/mcb.7.9.3268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  43. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Seraphin B., Rosbash M. Identification of functional U1 snRNA-pre-mRNA complexes committed to spliceosome assembly and splicing. Cell. 1989 Oct 20;59(2):349–358. doi: 10.1016/0092-8674(89)90296-1. [DOI] [PubMed] [Google Scholar]
  45. Sharp P. A. Splicing of messenger RNA precursors. Science. 1987 Feb 13;235(4790):766–771. doi: 10.1126/science.3544217. [DOI] [PubMed] [Google Scholar]
  46. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Siliciano P. G., Guthrie C. 5' splice site selection in yeast: genetic alterations in base-pairing with U1 reveal additional requirements. Genes Dev. 1988 Oct;2(10):1258–1267. doi: 10.1101/gad.2.10.1258. [DOI] [PubMed] [Google Scholar]
  48. Siliciano P. G., Jones M. H., Guthrie C. Saccharomyces cerevisiae has a U1-like small nuclear RNA with unexpected properties. Science. 1987 Sep 18;237(4821):1484–1487. doi: 10.1126/science.3306922. [DOI] [PubMed] [Google Scholar]
  49. Smith V., Brown C. M., Bankier A. T., Barrell B. G. Semiautomated preparation of DNA templates for large-scale sequencing projects. DNA Seq. 1990;1(1):73–78. doi: 10.3109/10425179009041349. [DOI] [PubMed] [Google Scholar]
  50. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  51. Spritz R. A., Strunk K., Surowy C. S., Hoch S. O., Barton D. E., Francke U. The human U1-70K snRNP protein: cDNA cloning, chromosomal localization, expression, alternative splicing and RNA-binding. Nucleic Acids Res. 1987 Dec 23;15(24):10373–10391. doi: 10.1093/nar/15.24.10373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Spritz R. A., Strunk K., Surowy C. S., Mohrenweiser H. W. Human U1-70K ribonucleoprotein antigen gene: organization, nucleotide sequence, and mapping to locus 19q13.3. Genomics. 1990 Oct;8(2):371–379. doi: 10.1016/0888-7543(90)90295-6. [DOI] [PubMed] [Google Scholar]
  53. Staden R. Automation of the computer handling of gel reading data produced by the shotgun method of DNA sequencing. Nucleic Acids Res. 1982 Aug 11;10(15):4731–4751. doi: 10.1093/nar/10.15.4731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Stockwell P. A. HOMED: a homologous sequence editor. Trends Biochem Sci. 1988 Aug;13(8):322–324. doi: 10.1016/0968-0004(88)90130-2. [DOI] [PubMed] [Google Scholar]
  55. Struhl K., Stinchcomb D. T., Scherer S., Davis R. W. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1035–1039. doi: 10.1073/pnas.76.3.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Surowy C. S., van Santen V. L., Scheib-Wixted S. M., Spritz R. A. Direct, sequence-specific binding of the human U1-70K ribonucleoprotein antigen protein to loop I of U1 small nuclear RNA. Mol Cell Biol. 1989 Oct;9(10):4179–4186. doi: 10.1128/mcb.9.10.4179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Séraphin B., Kretzner L., Rosbash M. A U1 snRNA:pre-mRNA base pairing interaction is required early in yeast spliceosome assembly but does not uniquely define the 5' cleavage site. EMBO J. 1988 Aug;7(8):2533–2538. doi: 10.1002/j.1460-2075.1988.tb03101.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Taylor W. R. Identification of protein sequence homology by consensus template alignment. J Mol Biol. 1986 Mar 20;188(2):233–258. doi: 10.1016/0022-2836(86)90308-6. [DOI] [PubMed] [Google Scholar]
  59. Teem J. L., Abovich N., Kaufer N. F., Schwindinger W. F., Warner J. R., Levy A., Woolford J., Leer R. J., van Raamsdonk-Duin M. M., Mager W. H. A comparison of yeast ribosomal protein gene DNA sequences. Nucleic Acids Res. 1984 Nov 26;12(22):8295–8312. doi: 10.1093/nar/12.22.8295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Theissen H., Etzerodt M., Reuter R., Schneider C., Lottspeich F., Argos P., Lührmann R., Philipson L. Cloning of the human cDNA for the U1 RNA-associated 70K protein. EMBO J. 1986 Dec 1;5(12):3209–3217. doi: 10.1002/j.1460-2075.1986.tb04631.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Thompson-Jäger S., Domdey H. Yeast pre-mRNA splicing requires a minimum distance between the 5' splice site and the internal branch acceptor site. Mol Cell Biol. 1987 Nov;7(11):4010–4016. doi: 10.1128/mcb.7.11.4010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. 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]
  63. White P. J., Billings P. B., Hoch S. O. Assays for the Sm and RNP autoantigens: the requirement for RNA and influence of the tissue source. J Immunol. 1982 Jun;128(6):2751–2756. [PubMed] [Google Scholar]
  64. Woodgett J. R., Gould K. L., Hunter T. Substrate specificity of protein kinase C. Use of synthetic peptides corresponding to physiological sites as probes for substrate recognition requirements. Eur J Biochem. 1986 Nov 17;161(1):177–184. doi: 10.1111/j.1432-1033.1986.tb10139.x. [DOI] [PubMed] [Google Scholar]
  65. Zhuang Y., Weiner A. M. A compensatory base change in U1 snRNA suppresses a 5' splice site mutation. Cell. 1986 Sep 12;46(6):827–835. doi: 10.1016/0092-8674(86)90064-4. [DOI] [PubMed] [Google Scholar]

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