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EMBO J. Sep 1991; 10(9): 2645–2651.
PMCID: PMC452965

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.

Abstract

The mammalian U3 snRNP is one member of a recently described family of nucleolar snRNPs which also includes U8, U13, U14, X and Y. All of these snRNPs are immunoprecipitable by anti-fibrillarin autoantibodies, suggesting the existence of a common binding site for the 34 kDa fibrillarin (Fb) protein. Two short nucleotide sequences, called Boxes C and D, present in each of these RNAs are the most likely sites for fibrillarin binding. We have developed a HeLa in vitro assembly system for binding of fibrillarin to human U3 snRNA. Reconstitution of the input RNA is specific in our assay since four of the other nucleolar small RNAs (U8, U13, X and Y) which have Boxes C and D become immunoprecipitable by anti-fibrillarin whereas two RNAs which lack these sequences (5S and 5.8S) do not. Deletion analyses of the U3 snRNA demonstrate that the presence of Box C but not Box D is required for fibrillarin binding. Moreover, seven single or double site-specific mutations in the U3 Box C abolish binding. The role of the Box C-fibrillarin interaction in the biogenesis of the Fb snRNPs is discussed.

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Selected References

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  • Dignam JD, Lebovitz RM, Roeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. [PMC free article] [PubMed]
  • Feeney RJ, Sauterer RA, Feeney JL, Zieve GW. Cytoplasmic assembly and nuclear accumulation of mature small nuclear ribonucleoprotein particles. J Biol Chem. 1989 Apr 5;264(10):5776–5783. [PubMed]
  • Fischer U, Lührmann R. An essential signaling role for the m3G cap in the transport of U1 snRNP to the nucleus. Science. 1990 Aug 17;249(4970):786–790. [PubMed]
  • Fisher DE, Conner GE, Reeves WH, Wisniewolski R, Blobel G. Small nuclear ribonucleoprotein particle assembly in vivo: demonstration of a 6S RNA-free core precursor and posttranslational modification. Cell. 1985 Oct;42(3):751–758. [PubMed]
  • Hamm J, Darzynkiewicz E, Tahara SM, Mattaj IW. The trimethylguanosine cap structure of U1 snRNA is a component of a bipartite nuclear targeting signal. Cell. 1990 Aug 10;62(3):569–577. [PubMed]
  • Hamm J, Dathan NA, Scherly D, Mattaj IW. Multiple domains of U1 snRNA, including U1 specific protein binding sites, are required for splicing. EMBO J. 1990 Apr;9(4):1237–1244. [PMC free article] [PubMed]
  • Hamm J, Kazmaier M, Mattaj IW. In vitro assembly of U1 snRNPs. EMBO J. 1987 Nov;6(11):3479–3485. [PMC free article] [PubMed]
  • Hamm J, van Santen VL, Spritz RA, Mattaj IW. Loop I of U1 small nuclear RNA is the only essential RNA sequence for binding of specific U1 small nuclear ribonucleoprotein particle proteins. Mol Cell Biol. 1988 Nov;8(11):4787–4791. [PMC free article] [PubMed]
  • Heintz N, Roeder RG. Transcription of human histone genes in extracts from synchronized HeLa cells. Proc Natl Acad Sci U S A. 1984 May;81(9):2713–2717. [PMC free article] [PubMed]
  • Henríquez R, Blobel G, Aris JP. 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]
  • Hernandez N, Weiner AM. Formation of the 3' end of U1 snRNA requires compatible snRNA promoter elements. Cell. 1986 Oct 24;47(2):249–258. [PubMed]
  • Hughes JM, Konings DA, Cesareni G. The yeast homologue of U3 snRNA. EMBO J. 1987 Jul;6(7):2145–2155. [PMC free article] [PubMed]
  • Jansen RP, Hurt EC, 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. [PMC free article] [PubMed]
  • Jarmolowski A, Zagorski J, Li HV, Fournier MJ. Identification of essential elements in U14 RNA of Saccharomyces cerevisiae. EMBO J. 1990 Dec;9(13):4503–4509. [PMC free article] [PubMed]
  • Jeppesen C, Stebbins-Boaz B, Gerbi SA. Nucleotide sequence determination and secondary structure of Xenopus U3 snRNA. Nucleic Acids Res. 1988 Mar 25;16(5):2127–2148. [PMC free article] [PubMed]
  • Jones MH, Guthrie C. Unexpected flexibility in an evolutionarily conserved protein-RNA interaction: genetic analysis of the Sm binding site. EMBO J. 1990 Aug;9(8):2555–2561. [PMC free article] [PubMed]
  • Kass S, Tyc K, Steitz JA, 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. [PubMed]
  • Kunkel TA, Roberts JD, Zakour RA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Liautard JP, Sri-Widada J, Brunel C, Jeanteur P. Structural organization of ribonucleoproteins containing small nuclear RNAs from HeLa cells. Proteins interact closely with a similar structural domain of U1, U2, U4 and U5 small nuclear RNAs. J Mol Biol. 1982 Dec 15;162(3):623–643. [PubMed]
  • Lischwe MA, Ochs RL, Reddy R, Cook RG, Yeoman LC, Tan EM, 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]
  • Liu J, Maxwell ES. Mouse U14 snRNA is encoded in an intron of the mouse cognate hsc70 heat shock gene. Nucleic Acids Res. 1990 Nov 25;18(22):6565–6571. [PMC free article] [PubMed]
  • Lutz-Freyermuth C, Keene JD, Lutz-Reyermuth C. The U1 RNA-binding site of the U1 small nuclear ribonucleoprotein (snRNP)-associated A protein suggests a similarity with U2 snRNPs. Mol Cell Biol. 1989 Jul;9(7):2975–2982. [PMC free article] [PubMed]
  • Manley JL, Fire A, Cano A, Sharp PA, Gefter ML. DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3855–3859. [PMC free article] [PubMed]
  • Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K, Green MR. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. [PMC free article] [PubMed]
  • Nakamura T, Prestayko AW, Busch H. Studies on nucleolar 4 to 6 S ribonucleic acid of Novikoff hepatoma cells. J Biol Chem. 1968 Apr 10;243(7):1368–1375. [PubMed]
  • Neuman de Vegvar HE, Dahlberg JE. Nucleocytoplasmic transport and processing of small nuclear RNA precursors. Mol Cell Biol. 1990 Jul;10(7):3365–3375. [PMC free article] [PubMed]
  • Parker KA, Steitz JA. 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. [PMC free article] [PubMed]
  • Patton JR, 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. [PMC free article] [PubMed]
  • Patton JR, Patterson RJ, Pederson T. Reconstitution of the U1 small nuclear ribonucleoprotein particle. Mol Cell Biol. 1987 Nov;7(11):4030–4037. [PMC free article] [PubMed]
  • Prestayko AW, Tonato M, Busch H. Low molecular weight RNA associated with 28 s nucleolar RNA. J Mol Biol. 1970 Feb 14;47(3):505–515. [PubMed]
  • Query CC, Bentley RC, Keene JD. 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. [PMC free article] [PubMed]
  • Savino R, Gerbi SA. In vivo disruption of Xenopus U3 snRNA affects ribosomal RNA processing. EMBO J. 1990 Jul;9(7):2299–2308. [PMC free article] [PubMed]
  • Scherly D, Boelens W, van Venrooij WJ, Dathan NA, Hamm J, Mattaj IW. Identification of the RNA binding segment of human U1 A protein and definition of its binding site on U1 snRNA. EMBO J. 1989 Dec 20;8(13):4163–4170. [PMC free article] [PubMed]
  • Scherly D, Boelens W, Dathan NA, van Venrooij WJ, Mattaj IW. Major determinants of the specificity of interaction between small nuclear ribonucleoproteins U1A and U2B'' and their cognate RNAs. Nature. 1990 Jun 7;345(6275):502–506. [PubMed]
  • Schimmang T, Tollervey D, Kern H, Frank R, Hurt EC. 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. [PMC free article] [PubMed]
  • Spritz RA, Strunk K, Surowy CS, Hoch SO, Barton DE, 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. [PMC free article] [PubMed]
  • Steitz JA. Immunoprecipitation of ribonucleoproteins using autoantibodies. Methods Enzymol. 1989;180:468–481. [PubMed]
  • Surowy CS, van Santen VL, Scheib-Wixted SM, Spritz RA. 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. [PMC free article] [PubMed]
  • Toczyski DP, Steitz JA. EAP, a highly conserved cellular protein associated with Epstein-Barr virus small RNAs (EBERs). EMBO J. 1991 Feb;10(2):459–466. [PMC free article] [PubMed]
  • Tyc K, Steitz JA. U3, U8 and U13 comprise a new class of mammalian snRNPs localized in the cell nucleolus. EMBO J. 1989 Oct;8(10):3113–3119. [PMC free article] [PubMed]
  • Weinberg RA, Penman S. Small molecular weight monodisperse nuclear RNA. J Mol Biol. 1968 Dec;38(3):289–304. [PubMed]
  • Wise JA, Weiner AM. Dictyostelium small nuclear RNA D2 is homologous to rat nucleolar RNA U3 and is encoded by a dispersed multigene family. Cell. 1980 Nov;22(1 Pt 1):109–118. [PubMed]
  • Yuo CY, Weiner AM. Genetic analysis of the role of human U1 snRNA in mRNA splicing: I. Effect of mutations in the highly conserved stem-loop I of U1. Genes Dev. 1989 May;3(5):697–707. [PubMed]
  • Zieve GW, Sauterer RA. Cell biology of the snRNP particles. Crit Rev Biochem Mol Biol. 1990;25(1):1–46. [PubMed]
  • Zoller MJ, Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. [PubMed]

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