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. 1997 Jun;8(6):1051–1061. doi: 10.1091/mbc.8.6.1051

An unusual fibrillarin gene and protein: structure and functional implications.

E David 1, J B McNeil 1, V Basile 1, R E Pearlman 1
PMCID: PMC305713  PMID: 9201715

Abstract

The diploid germinal nucleus of the ciliated protozoan Tetrahymena thermophila is unusual among eukaryotes in that it encodes a single copy of the gene for rRNA allowing identification of cis-acting mutations in rDNA affecting rRNA structure, function, and processing. The generally conserved nucleolar protein fibrillarin has been characterized from a number of systems and is involved in pre-rRNA processing. We have demonstrated that Tetrahymena has fibrillarin and have analyzed the cDNA and the genomic DNA encoding this protein. The derived amino acid sequence of the N-terminal region of Tetrahymena fibrillarin shows little similarity with the generally highly conserved glycine/arginine-rich N-terminal domain of other eukaryotic fibrillarins. The remainder of the amino acid sequence of the molecule is more conserved. Polyclonal antibodies generated against the full-length Tetrahymena fibrillarin expressed in bacteria recognize a protein of M(r) approximately 32,000 in whole-cell or nucleolar preparations. Immunocytochemistry localizes fibrillarin to nucleoli in the somatic macronuclei of vegetative cells. Transformation experiments demonstrate that fibrillarin is an essential protein in Tetrahymena. The Tetrahymena fibrillarin is expressed but does not complement a NOP1 null mutation when transformed into the yeast Saccharomyces cerevisiae, indicating less functional conservation among fibrillarins than previously suggested.

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Selected References

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  1. Aris J. P., Blobel G. cDNA cloning and sequencing of human fibrillarin, a conserved nucleolar protein recognized by autoimmune antisera. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):931–935. doi: 10.1073/pnas.88.3.931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Balakin A. G., Smith L., Fournier M. J. The RNA world of the nucleolus: two major families of small RNAs defined by different box elements with related functions. Cell. 1996 Sep 6;86(5):823–834. doi: 10.1016/s0092-8674(00)80156-7. [DOI] [PubMed] [Google Scholar]
  3. Baserga S. J., Yang X. D., Steitz J. A. An intact Box C sequence in the U3 snRNA is required for binding of fibrillarin, the protein common to the major family of nucleolar snRNPs. EMBO J. 1991 Sep;10(9):2645–2651. doi: 10.1002/j.1460-2075.1991.tb07807.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cappai R., Osborn A. H., Handman E. Cloning and sequence of a Leishmania major homologue to the fibrillarin gene. Mol Biochem Parasitol. 1994 Apr;64(2):353–355. doi: 10.1016/0166-6851(94)00047-6. [DOI] [PubMed] [Google Scholar]
  5. Cassidy-Hanley D., Bowen J., Lee J. H., Cole E., VerPlank L. A., Gaertig J., Gorovsky M. A., Bruns P. J. Germline and somatic transformation of mating Tetrahymena thermophila by particle bombardment. Genetics. 1997 May;146(1):135–147. doi: 10.1093/genetics/146.1.135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen D. C., Yang B. C., Kuo T. T. One-step transformation of yeast in stationary phase. Curr Genet. 1992 Jan;21(1):83–84. doi: 10.1007/BF00318659. [DOI] [PubMed] [Google Scholar]
  7. Dente L., Cesareni G., Cortese R. pEMBL: a new family of single stranded plasmids. Nucleic Acids Res. 1983 Mar 25;11(6):1645–1655. doi: 10.1093/nar/11.6.1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dobson M. J., Pearlman R. E., Karaiskakis A., Spyropoulos B., Moens P. B. Synaptonemal complex proteins: occurrence, epitope mapping and chromosome disjunction. J Cell Sci. 1994 Oct;107(Pt 10):2749–2760. doi: 10.1242/jcs.107.10.2749. [DOI] [PubMed] [Google Scholar]
  9. Don R. H., Cox P. T., Wainwright B. J., Baker K., Mattick J. S. 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 1991 Jul 25;19(14):4008–4008. doi: 10.1093/nar/19.14.4008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Engberg J. The ribosomal RNA genes of Tetrahymena: structure and function. Eur J Cell Biol. 1985 Jan;36(1):133–151. [PubMed] [Google Scholar]
  11. Gaertig J., Gu L., Hai B., Gorovsky M. A. High frequency vector-mediated transformation and gene replacement in Tetrahymena. Nucleic Acids Res. 1994 Dec 11;22(24):5391–5398. doi: 10.1093/nar/22.24.5391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ghisolfi L., Joseph G., Amalric F., Erard M. The glycine-rich domain of nucleolin has an unusual supersecondary structure responsible for its RNA-helix-destabilizing properties. J Biol Chem. 1992 Feb 15;267(5):2955–2959. [PubMed] [Google Scholar]
  13. Girard J. P., Feliu J., Caizergues-Ferrer M., Lapeyre B. Study of multiple fibrillarin mRNAs reveals that 3' end formation in Schizosaccharomyces pombe is sensitive to cold shock. Nucleic Acids Res. 1993 Apr 25;21(8):1881–1887. doi: 10.1093/nar/21.8.1881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Girard J. P., Lehtonen H., Caizergues-Ferrer M., Amalric F., Tollervey D., Lapeyre B. GAR1 is an essential small nucleolar RNP protein required for pre-rRNA processing in yeast. EMBO J. 1992 Feb;11(2):673–682. doi: 10.1002/j.1460-2075.1992.tb05099.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Girard J. P., Lehtonen H., Caizergues-Ferrer M., Amalric F., Tollervey D., Lapeyre B. GAR1 is an essential small nucleolar RNP protein required for pre-rRNA processing in yeast. EMBO J. 1992 Feb;11(2):673–682. doi: 10.1002/j.1460-2075.1992.tb05099.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Golding G. B., Tsao N., Pearlman R. E. Evidence for intron capture: an unusual path for the evolution of proteins. Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7506–7509. doi: 10.1073/pnas.91.16.7506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gorovsky M. A., Yao M. C., Keevert J. B., Pleger G. L. Isolation of micro- and macronuclei of Tetrahymena pyriformis. Methods Cell Biol. 1975;9(0):311–327. doi: 10.1016/s0091-679x(08)60080-1. [DOI] [PubMed] [Google Scholar]
  18. Heinonen T. Y., Pearlman R. E. A germ line-specific sequence element in an intron in Tetrahymena thermophila. J Biol Chem. 1994 Jul 1;269(26):17428–17433. [PubMed] [Google Scholar]
  19. Henríquez R., Blobel G., Aris J. P. Isolation and sequencing of NOP1. A yeast gene encoding a nucleolar protein homologous to a human autoimmune antigen. J Biol Chem. 1990 Feb 5;265(4):2209–2215. [PubMed] [Google Scholar]
  20. Higashinakagawa T., Narushima-Iio M., Saiga H., Kondo S., Mita T. Properties of isolated extrachromosomal nucleoli from Tetrahymena pyriformis. Chromosoma. 1992 Apr;101(7):413–419. doi: 10.1007/BF00582835. [DOI] [PubMed] [Google Scholar]
  21. Higgins D. G., Sharp P. M. Fast and sensitive multiple sequence alignments on a microcomputer. Comput Appl Biosci. 1989 Apr;5(2):151–153. doi: 10.1093/bioinformatics/5.2.151. [DOI] [PubMed] [Google Scholar]
  22. Hozák P. Catching RNA polymerase I in Flagranti: ribosomal genes are transcribed in the dense fibrillar component of the nucleolus. Exp Cell Res. 1995 Feb;216(2):285–289. doi: 10.1006/excr.1995.1036. [DOI] [PubMed] [Google Scholar]
  23. Hughes J. M., Ares M., Jr Depletion of U3 small nucleolar RNA inhibits cleavage in the 5' external transcribed spacer of yeast pre-ribosomal RNA and impairs formation of 18S ribosomal RNA. EMBO J. 1991 Dec;10(13):4231–4239. doi: 10.1002/j.1460-2075.1991.tb05001.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Jansen R. P., Hurt E. C., Kern H., Lehtonen H., Carmo-Fonseca M., Lapeyre B., Tollervey D. Evolutionary conservation of the human nucleolar protein fibrillarin and its functional expression in yeast. J Cell Biol. 1991 May;113(4):715–729. doi: 10.1083/jcb.113.4.715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Kass S., Tyc K., Steitz J. A., Sollner-Webb B. The U3 small nucleolar ribonucleoprotein functions in the first step of preribosomal RNA processing. Cell. 1990 Mar 23;60(6):897–908. doi: 10.1016/0092-8674(90)90338-f. [DOI] [PubMed] [Google Scholar]
  29. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  30. Lapeyre B., Bourbon H., Amalric F. Nucleolin, the major nucleolar protein of growing eukaryotic cells: an unusual protein structure revealed by the nucleotide sequence. Proc Natl Acad Sci U S A. 1987 Mar;84(6):1472–1476. doi: 10.1073/pnas.84.6.1472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lapeyre B., Mariottini P., Mathieu C., Ferrer P., Amaldi F., Amalric F., Caizergues-Ferrer M. Molecular cloning of Xenopus fibrillarin, a conserved U3 small nuclear ribonucleoprotein recognized by antisera from humans with autoimmune disease. Mol Cell Biol. 1990 Jan;10(1):430–434. doi: 10.1128/mcb.10.1.430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lee W. C., Xue Z. X., Mélèse T. The NSR1 gene encodes a protein that specifically binds nuclear localization sequences and has two RNA recognition motifs. J Cell Biol. 1991 Apr;113(1):1–12. doi: 10.1083/jcb.113.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Maxwell E. S., Fournier M. J. The small nucleolar RNAs. Annu Rev Biochem. 1995;64:897–934. doi: 10.1146/annurev.bi.64.070195.004341. [DOI] [PubMed] [Google Scholar]
  35. Najbauer J., Johnson B. A., Young A. L., Aswad D. W. Peptides with sequences similar to glycine, arginine-rich motifs in proteins interacting with RNA are efficiently recognized by methyltransferase(s) modifying arginine in numerous proteins. J Biol Chem. 1993 May 15;268(14):10501–10509. [PubMed] [Google Scholar]
  36. Nilsson J. R., Leick V. Nucleolar organization and ribosome formation in Tetrahymena pyriformis GL. Exp Cell Res. 1970 Jun;60(3):361–372. doi: 10.1016/0014-4827(70)90529-x. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. 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]
  39. Orum H., Nielsen H., Engberg J. Sequence and proposed secondary structure of the Tetrahymena thermophila U3-snRNA. Nucleic Acids Res. 1993 May 25;21(10):2511–2511. doi: 10.1093/nar/21.10.2511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Potter S., Durovic P., Dennis P. P. Ribosomal RNA precursor processing by a eukaryotic U3 small nucleolar RNA-like molecule in an archaeon. Science. 1995 May 19;268(5213):1056–1060. doi: 10.1126/science.7538698. [DOI] [PubMed] [Google Scholar]
  42. Prescott D. M. The DNA of ciliated protozoa. Microbiol Rev. 1994 Jun;58(2):233–267. doi: 10.1128/mr.58.2.233-267.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Raska I., Dundr M., Koberna K., Melcák I., Risueño M. C., Török I. Does the synthesis of ribosomal RNA take place within nucleolar fibrillar centers or dense fibrillar components? A critical appraisal. J Struct Biol. 1995 Jan-Feb;114(1):1–22. doi: 10.1006/jsbi.1995.1001. [DOI] [PubMed] [Google Scholar]
  44. Russell I. D., Tollervey D. NOP3 is an essential yeast protein which is required for pre-rRNA processing. J Cell Biol. 1992 Nov;119(4):737–747. doi: 10.1083/jcb.119.4.737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Saitou N., Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987 Jul;4(4):406–425. doi: 10.1093/oxfordjournals.molbev.a040454. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. 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]
  48. Schimmang T., Tollervey D., Kern H., Frank R., Hurt E. C. A yeast nucleolar protein related to mammalian fibrillarin is associated with small nucleolar RNA and is essential for viability. EMBO J. 1989 Dec 20;8(13):4015–4024. doi: 10.1002/j.1460-2075.1989.tb08584.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. 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]
  50. Shen X., Yu L., Weir J. W., Gorovsky M. A. Linker histones are not essential and affect chromatin condensation in vivo. Cell. 1995 Jul 14;82(1):47–56. doi: 10.1016/0092-8674(95)90051-9. [DOI] [PubMed] [Google Scholar]
  51. 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]
  52. Stargell L. A., Gorovsky M. A. TATA-binding protein and nuclear differentiation in Tetrahymena thermophila. Mol Cell Biol. 1994 Jan;14(1):723–734. doi: 10.1128/mcb.14.1.723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Takemasa T., Takagi T., Kobayashi T., Konishi K., Watanabe Y. The third calmodulin family protein in Tetrahymena. Cloning of the cDNA for Tetrahymena calcium-binding protein of 23 kDa (TCBP-23). J Biol Chem. 1990 Feb 15;265(5):2514–2517. [PubMed] [Google Scholar]
  54. Tollervey D., Lehtonen H., Carmo-Fonseca M., Hurt E. C. The small nucleolar RNP protein NOP1 (fibrillarin) is required for pre-rRNA processing in yeast. EMBO J. 1991 Mar;10(3):573–583. doi: 10.1002/j.1460-2075.1991.tb07984.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Tourancheau A. B., Tsao N., Klobutcher L. A., Pearlman R. E., Adoutte A. Genetic code deviations in the ciliates: evidence for multiple and independent events. EMBO J. 1995 Jul 3;14(13):3262–3267. doi: 10.1002/j.1460-2075.1995.tb07329.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Turley S. J., Tan E. M., Pollard K. M. Molecular cloning and sequence analysis of U3 snoRNA-associated mouse fibrillarin. Biochim Biophys Acta. 1993 Oct 19;1216(1):119–122. doi: 10.1016/0167-4781(93)90046-g. [DOI] [PubMed] [Google Scholar]
  57. 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]
  58. Vernet T., Dignard D., Thomas D. Y. A family of yeast expression vectors containing the phage f1 intergenic region. Gene. 1987;52(2-3):225–233. doi: 10.1016/0378-1119(87)90049-7. [DOI] [PubMed] [Google Scholar]
  59. Vohra G. B., Golding G. B., Tsao N., Pearlman R. E. A phylogenetic analysis based on the gene encoding phosphoglycerate kinase. J Mol Evol. 1992 May;34(5):383–395. doi: 10.1007/BF00162995. [DOI] [PubMed] [Google Scholar]
  60. Wenkert D., Allis C. D. Timing of the appearance of macronuclear-specific histone variant hv1 and gene expression in developing new macronuclei of Tetrahymena thermophila. J Cell Biol. 1984 Jun;98(6):2107–2117. doi: 10.1083/jcb.98.6.2107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Yang C. H., Lambie E. J., Hardin J., Craft J., Snyder M. Higher order structure is present in the yeast nucleus: autoantibody probes demonstrate that the nucleolus lies opposite the spindle pole body. Chromosoma. 1989 Aug;98(2):123–128. doi: 10.1007/BF00291048. [DOI] [PubMed] [Google Scholar]
  62. Yao M. C., Gall J. G. A single integrated gene for ribosomal RNA in a eucaryote, Tetrahymena pyriformis. Cell. 1977 Sep;12(1):121–132. doi: 10.1016/0092-8674(77)90190-8. [DOI] [PubMed] [Google Scholar]
  63. Yao M. C. Programmed DNA deletions in Tetrahymena: mechanisms and implications. Trends Genet. 1996 Jan;12(1):26–30. doi: 10.1016/0168-9525(96)81385-0. [DOI] [PubMed] [Google Scholar]

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