Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1989 Dec 20;8(13):4015–4024. doi: 10.1002/j.1460-2075.1989.tb08584.x

A yeast nucleolar protein related to mammalian fibrillarin is associated with small nucleolar RNA and is essential for viability.

T Schimmang 1, D Tollervey 1, H Kern 1, R Frank 1, E C Hurt 1
PMCID: PMC401576  PMID: 2686980

Abstract

In order to study the structural and functional organization of the eukaryotic nucleolus, we have started to isolate and characterize nucleolar components of the yeast Saccharomyces cerevisiae. We have identified a major 38 kd nucleolar protein (NOP1), which is located within nucleolar structures resembling the dense fibrillar region of mammalian nucleoli. This 38 kd protein is conserved in evolution since affinity-purified antibodies against the yeast protein stain the nucleolus of mammalian cells in indirect immunofluorescence microscopy and the yeast protein is decorated by antibodies directed against human fibrillarin. Affinity-purified antibodies against the yeast NOP1 efficiently precipitate at least seven small nuclear RNAs involved in rRNA maturation. We have cloned the gene encoding the yeast NOP1 protein. Haploid cells carrying a disrupted copy of the gene are not viable, showing that NOP1 is essential for cell growth. The gene codes for a 34.5 kd protein which contains glycine/arginine rich sequence repeats at the amino terminus similar to those found in other nucleolar proteins. This suggests that NOP1 is in association with small nucleolar RNAs, required for rRNA processing and likely to be the homologue of the mammalian fibrillarin.

Full text

PDF
4018

Images in this article

4016Image
on p.4016
4017Image
on p.4017
4018Image
on p.4018
4020Image
on p.4020

Selected References

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

  1. Aris J. P., Blobel G. Identification and characterization of a yeast nucleolar protein that is similar to a rat liver nucleolar protein. J Cell Biol. 1988 Jul;107(1):17–31. doi: 10.1083/jcb.107.1.17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bosserhoff A., Wallach J., Frank R. W. Micropreparative separation of peptides derived from sodium dodecyl sulphate-solubilized proteins. J Chromatogr. 1989 Jun 28;473(1):71–77. doi: 10.1016/s0021-9673(00)91291-3. [DOI] [PubMed] [Google Scholar]
  4. Bourbon H. M., Lapeyre B., Amalric F. Structure of the mouse nucleolin gene. The complete sequence reveals that each RNA binding domain is encoded by two independent exons. J Mol Biol. 1988 Apr 20;200(4):627–638. doi: 10.1016/0022-2836(88)90476-7. [DOI] [PubMed] [Google Scholar]
  5. Bré M. H., Kreis T. E., Karsenti E. Control of microtubule nucleation and stability in Madin-Darby canine kidney cells: the occurrence of noncentrosomal, stable detyrosinated microtubules. J Cell Biol. 1987 Sep;105(3):1283–1296. doi: 10.1083/jcb.105.3.1283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carter C. W., Jr, Kraut J. A proposed model for interaction of polypeptides with RNA. Proc Natl Acad Sci U S A. 1974 Feb;71(2):283–287. doi: 10.1073/pnas.71.2.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chase J. W., Williams K. R. Single-stranded DNA binding proteins required for DNA replication. Annu Rev Biochem. 1986;55:103–136. doi: 10.1146/annurev.bi.55.070186.000535. [DOI] [PubMed] [Google Scholar]
  8. Chou P. Y., Fasman G. D. Empirical predictions of protein conformation. Annu Rev Biochem. 1978;47:251–276. doi: 10.1146/annurev.bi.47.070178.001343. [DOI] [PubMed] [Google Scholar]
  9. Christensen M. E., Fuxa K. P. The nucleolar protein, B-36, contains a glycine and dimethylarginine-rich sequence conserved in several other nuclear RNA-binding proteins. Biochem Biophys Res Commun. 1988 Sep 30;155(3):1278–1283. doi: 10.1016/s0006-291x(88)81279-8. [DOI] [PubMed] [Google Scholar]
  10. Christensen M. E., Moloo J., Swischuk J. L., Schelling M. E. Characterization of the nucleolar protein, B-36, using monoclonal antibodies. Exp Cell Res. 1986 Sep;166(1):77–93. doi: 10.1016/0014-4827(86)90509-4. [DOI] [PubMed] [Google Scholar]
  11. Cobianchi F., SenGupta D. N., Zmudzka B. Z., Wilson S. H. Structure of rodent helix-destabilizing protein revealed by cDNA cloning. J Biol Chem. 1986 Mar 15;261(8):3536–3543. [PubMed] [Google Scholar]
  12. Davis T. N., Thorner J. Isolation of the yeast calmodulin gene using synthetic oligonucleotide probes. Methods Enzymol. 1987;139:248–262. doi: 10.1016/0076-6879(87)39090-1. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Gas N., Escande M. L., Stevens B. J. Immunolocalization of the 100 kDa nucleolar protein during the mitotic cycle in CHO cells. Biol Cell. 1985;53(3):209–218. doi: 10.1111/j.1768-322x.1985.tb00369.x. [DOI] [PubMed] [Google Scholar]
  15. Goessens G. Nucleolar structure. Int Rev Cytol. 1984;87:107–158. doi: 10.1016/s0074-7696(08)62441-9. [DOI] [PubMed] [Google Scholar]
  16. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hughes J. M., Konings D. A., Cesareni G. The yeast homologue of U3 snRNA. EMBO J. 1987 Jul;6(7):2145–2155. doi: 10.1002/j.1460-2075.1987.tb02482.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hurt E. C. A novel nucleoskeletal-like protein located at the nuclear periphery is required for the life cycle of Saccharomyces cerevisiae. EMBO J. 1988 Dec 20;7(13):4323–4334. doi: 10.1002/j.1460-2075.1988.tb03331.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hurt E. C., McDowall A., Schimmang T. Nucleolar and nuclear envelope proteins of the yeast Saccharomyces cerevisiae. Eur J Cell Biol. 1988 Aug;46(3):554–563. [PubMed] [Google Scholar]
  20. Hügle B., Hazan R., Scheer U., Franke W. W. Localization of ribosomal protein S1 in the granular component of the interphase nucleolus and its distribution during mitosis. J Cell Biol. 1985 Mar;100(3):873–886. doi: 10.1083/jcb.100.3.873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hügle B., Scheer U., Franke W. W. Ribocharin: a nuclear Mr 40,000 protein specific to precursor particles of the large ribosomal subunit. Cell. 1985 Jun;41(2):615–627. doi: 10.1016/s0092-8674(85)80034-9. [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. Jong A. Y., Campbell J. L. Isolation of the gene encoding yeast single-stranded nucleic acid binding protein 1. Proc Natl Acad Sci U S A. 1986 Feb;83(4):877–881. doi: 10.1073/pnas.83.4.877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jong A. Y., Clark M. W., Gilbert M., Oehm A., Campbell J. L. Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins. Mol Cell Biol. 1987 Aug;7(8):2947–2955. doi: 10.1128/mcb.7.8.2947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Jordan G. At the heart of the nucleolus. Nature. 1987 Oct 8;329(6139):489–490. doi: 10.1038/329489a0. [DOI] [PubMed] [Google Scholar]
  26. Kilmartin J. V., Wright B., Milstein C. Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line. J Cell Biol. 1982 Jun;93(3):576–582. doi: 10.1083/jcb.93.3.576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lapeyre B., Amalric F., Ghaffari S. H., Rao S. V., Dumbar T. S., Olson M. O. Protein and cDNA sequence of a glycine-rich, dimethylarginine-containing region located near the carboxyl-terminal end of nucleolin (C23 and 100 kDa). J Biol Chem. 1986 Jul 15;261(20):9167–9173. [PubMed] [Google Scholar]
  28. Lapeyre B., Caizergues-Ferrer M., Bouche G., Amalric F. Cloning of cDNA encoding a 100 kDa nucleolar protein (nucleoline) of Chinese hamster ovary cells. Nucleic Acids Res. 1985 Aug 26;13(16):5805–5816. doi: 10.1093/nar/13.16.5805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lischwe M. A., Cook R. G., Ahn Y. S., Yeoman L. C., Busch H. Clustering of glycine and NG,NG-dimethylarginine in nucleolar protein C23. Biochemistry. 1985 Oct 22;24(22):6025–6028. doi: 10.1021/bi00343a001. [DOI] [PubMed] [Google Scholar]
  30. Lischwe M. A., Ochs R. L., Reddy R., Cook R. G., Yeoman L. C., Tan E. M., Reichlin M., Busch H. Purification and partial characterization of a nucleolar scleroderma antigen (Mr = 34,000; pI, 8.5) rich in NG,NG-dimethylarginine. J Biol Chem. 1985 Nov 15;260(26):14304–14310. [PubMed] [Google Scholar]
  31. Mattaj I. W., De Robertis E. M. Nuclear segregation of U2 snRNA requires binding of specific snRNP proteins. Cell. 1985 Jan;40(1):111–118. doi: 10.1016/0092-8674(85)90314-9. [DOI] [PubMed] [Google Scholar]
  32. Nigg E. A. Nuclear function and organization: the potential of immunochemical approaches. Int Rev Cytol. 1988;110:27–92. doi: 10.1016/s0074-7696(08)61847-1. [DOI] [PubMed] [Google Scholar]
  33. Ochs R. L., Lischwe M. A., Shen E., Carroll R. E., Busch H. Nucleologenesis: composition and fate of prenucleolar bodies. Chromosoma. 1985;92(5):330–336. doi: 10.1007/BF00327463. [DOI] [PubMed] [Google Scholar]
  34. Ochs R. L., Lischwe M. A., Spohn W. H., Busch H. Fibrillarin: a new protein of the nucleolus identified by autoimmune sera. Biol Cell. 1985;54(2):123–133. doi: 10.1111/j.1768-322x.1985.tb00387.x. [DOI] [PubMed] [Google Scholar]
  35. Ochs R., Lischwe M., O'Leary P., Busch H. Localization of nucleolar phosphoproteins B23 and C23 during mitosis. Exp Cell Res. 1983 Jun;146(1):139–149. doi: 10.1016/0014-4827(83)90332-4. [DOI] [PubMed] [Google Scholar]
  36. Parker K. A., Steitz J. A. Structural analysis of the human U3 ribonucleoprotein particle reveal a conserved sequence available for base pairing with pre-rRNA. Mol Cell Biol. 1987 Aug;7(8):2899–2913. doi: 10.1128/mcb.7.8.2899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Parker R., Simmons T., Shuster E. O., Siliciano P. G., Guthrie C. Genetic analysis of small nuclear RNAs in Saccharomyces cerevisiae: viable sextuple mutant. Mol Cell Biol. 1988 Aug;8(8):3150–3159. doi: 10.1128/mcb.8.8.3150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Pierron G., Pedron J., Schelling M., Christensen M. Immunoelectron microscopic localization of the nucleolar protein B-36 (fibrillarin) during the cell cycle of Physarum polycephalum. Biol Cell. 1989;65(2):119–126. [PubMed] [Google Scholar]
  39. 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]
  40. Raska I., Reimer G., Jarník M., Kostrouch Z., Raska K., Jr Does the synthesis of ribosomal RNA take place within nucleolar fibrillar centers or dense fibrillar components? Biol Cell. 1989;65(1):79–82. [PubMed] [Google Scholar]
  41. Reimer G., Raska I., Tan E. M., Scheer U. Human autoantibodies: probes for nucleolus structure and function. Virchows Arch B Cell Pathol Incl Mol Pathol. 1987;54(3):131–143. doi: 10.1007/BF02899205. [DOI] [PubMed] [Google Scholar]
  42. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [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. Scheer U., Rose K. M. Localization of RNA polymerase I in interphase cells and mitotic chromosomes by light and electron microscopic immunocytochemistry. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1431–1435. doi: 10.1073/pnas.81.5.1431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Schmidt-Zachmann M. S., Hügle-Dörr B., Franke W. W. A constitutive nucleolar protein identified as a member of the nucleoplasmin family. EMBO J. 1987 Jul;6(7):1881–1890. doi: 10.1002/j.1460-2075.1987.tb02447.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Smitt W. W., Vermeulen C. A., Vlak J. M., Rozijn T. H., Molenaar I. Electron microscopic autoradiographic study of RNA synthesis in yeast nucleus. Exp Cell Res. 1972 Jan;70(1):140–144. doi: 10.1016/0014-4827(72)90191-7. [DOI] [PubMed] [Google Scholar]
  47. Smitt W. W., Vlak J. M., Molenaar I., Rozijn T. H. Nucleolar function of the dense crescent in the yeast nucleus. A biochemical and ultrastructural study. Exp Cell Res. 1973 Aug;80(2):313–321. doi: 10.1016/0014-4827(73)90302-9. [DOI] [PubMed] [Google Scholar]
  48. Spector D. L., Ochs R. L., Busch H. Silver staining, immunofluorescence, and immunoelectron microscopic localization of nucleolar phosphoproteins B23 and C23. Chromosoma. 1984;90(2):139–148. doi: 10.1007/BF00292451. [DOI] [PubMed] [Google Scholar]
  49. Tollervey D. A yeast small nuclear RNA is required for normal processing of pre-ribosomal RNA. EMBO J. 1987 Dec 20;6(13):4169–4175. doi: 10.1002/j.1460-2075.1987.tb02763.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tollervey D., Guthrie C. Deletion of a yeast small nuclear RNA gene impairs growth. EMBO J. 1985 Dec 30;4(13B):3873–3878. doi: 10.1002/j.1460-2075.1985.tb04160.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. 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]
  52. Tollervey D., Wise J. A., Guthrie C. A U4-like small nuclear RNA is dispensable in yeast. Cell. 1983 Dec;35(3 Pt 2):753–762. doi: 10.1016/0092-8674(83)90108-3. [DOI] [PubMed] [Google Scholar]
  53. Tyc K., Steitz J. A. U3, U8 and U13 comprise a new class of mammalian snRNPs localized in the cell nucleolus. EMBO J. 1989 Oct;8(10):3113–3119. doi: 10.1002/j.1460-2075.1989.tb08463.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Warner J. R. Synthesis of ribosomes in Saccharomyces cerevisiae. Microbiol Rev. 1989 Jun;53(2):256–271. doi: 10.1128/mr.53.2.256-271.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. 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]
  56. Zagorski J., Tollervey D., Fournier M. J. Characterization of an SNR gene locus in Saccharomyces cerevisiae that specifies both dispensible and essential small nuclear RNAs. Mol Cell Biol. 1988 Aug;8(8):3282–3290. doi: 10.1128/mcb.8.8.3282. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES