Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Human Fibrillarin Forms a Sub-complex with Splicing Factor 2-associated p32, Protein Arginine Methyltransferases, and Tubulins α3 and β1 That Is Independent of Its Association with Preribosomal Ribonucleoprotein Complexes

2004; Elsevier BV; Volume: 279; Issue: 3 Linguagem: Inglês

10.1074/jbc.m305604200

ISSN

1083-351X

Autores

Mitsuaki Yanagida, Toshiya Hayano, Yoshio Yamauchi, Takashi Shinkawa, Tohru Natsume, Toshiaki Isobe, Nobuhiro Takahashi,

Tópico(s)

Epigenetics and DNA Methylation

Resumo

Fibrillarin (FIB, Nop1p in yeast) is an RNA methyltransferase found not only in the fibrillar region of the nucleolus but also in Cajal bodies. FIB is essential for efficient processing of preribosomal RNA during ribosome biogenesis, although its precise function in this process and its role in Cajal bodies remain uncertain. Here, we demonstrate that the human FIB N-terminal glycine- and arginine-rich domain (residues 1–77) and its spacer region 1 (78–132) interact with splicing factor 2-associated p32 (SF2A-p32) and that the FIB methyltransferase-like domain (133–321) interacts with protein-arginine methyltransferase 5 (PRMT5, Janus kinase-binding protein 1). We also show that these proteins associate with several additional proteins, including PRMT1, tubulin α3, and tubulin β1 to form a sub-complex that is principally independent of the association of FIB with preribosomal ribonucleoprotein complexes that co-immunoprecipitate with the sub-complex in human cells expressing FLAG-tagged FIB. Based on the physical association of FIB with SF2A-p32 and PRMTs, as well as the other reported results, we propose that FIB may coordinate both RNA and protein methylation during the processes of ribosome biogenesis in the nucleolus and RNA editing such as small nuclear (nucleolar) ribonucleoprotein biogenesis in Cajal bodies. Fibrillarin (FIB, Nop1p in yeast) is an RNA methyltransferase found not only in the fibrillar region of the nucleolus but also in Cajal bodies. FIB is essential for efficient processing of preribosomal RNA during ribosome biogenesis, although its precise function in this process and its role in Cajal bodies remain uncertain. Here, we demonstrate that the human FIB N-terminal glycine- and arginine-rich domain (residues 1–77) and its spacer region 1 (78–132) interact with splicing factor 2-associated p32 (SF2A-p32) and that the FIB methyltransferase-like domain (133–321) interacts with protein-arginine methyltransferase 5 (PRMT5, Janus kinase-binding protein 1). We also show that these proteins associate with several additional proteins, including PRMT1, tubulin α3, and tubulin β1 to form a sub-complex that is principally independent of the association of FIB with preribosomal ribonucleoprotein complexes that co-immunoprecipitate with the sub-complex in human cells expressing FLAG-tagged FIB. Based on the physical association of FIB with SF2A-p32 and PRMTs, as well as the other reported results, we propose that FIB may coordinate both RNA and protein methylation during the processes of ribosome biogenesis in the nucleolus and RNA editing such as small nuclear (nucleolar) ribonucleoprotein biogenesis in Cajal bodies. Fibrillarin (FIB) 1The abbreviations used are: FIB, fibrillarin; snRNP, small nuclear ribonucleoprotein; snoRNP, small nucleolar ribonucleoprotein; LC, liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectrometry; PBS, phosphate-buffered saline; pre-rRNP, preribosomal ribonucleoprotein; RNase, ribonuclease; RNP, ribonucleoprotein; rRNA, ribosomal RNA; SMN, survival motor neuron; RBD, RNA-binding domain; NLS, nucleolar localization signal; NS, SV40 nuclear localization signal.1The abbreviations used are: FIB, fibrillarin; snRNP, small nuclear ribonucleoprotein; snoRNP, small nucleolar ribonucleoprotein; LC, liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectrometry; PBS, phosphate-buffered saline; pre-rRNP, preribosomal ribonucleoprotein; RNase, ribonuclease; RNP, ribonucleoprotein; rRNA, ribosomal RNA; SMN, survival motor neuron; RBD, RNA-binding domain; NLS, nucleolar localization signal; NS, SV40 nuclear localization signal. is the most abundant protein in the fibrillar regions of the nucleolus where ribosomal RNA transcription and early preribosomal RNA (pre-rRNA) processing take place (1.Warner J.R. Curr. Opin. Cell Biol. 1990; 2: 521-527Crossref PubMed Scopus (124) Google Scholar, 2.Eichler D.C. Craig N. Prog. Nucleic Acids Res. Mol. Biol. 1994; 49: 197-239Crossref PubMed Scopus (180) Google Scholar). FIB is also found in Cajal bodies, subnuclear organelles that contain distinct components involved in RNA transcription and editing such as mRNA splicing and small nuclear (nucleolar) ribonucleoprotein (sn(o)RNP) biogenesis (3.Snaar S. Wiesmeijer K. Jochemsen A G. Tanke H.J. Dirks R.W. J. Cell Biol. 2000; 151: 653-662Crossref PubMed Scopus (111) Google Scholar, 4.Spector D.L. J. Cell Sci. 2001; 114: 2891-2893Crossref PubMed Google Scholar). FIB is a component of a ribonucleoprotein (RNP) complex that contains U3, U8, and U13 small nucleolar RNAs that exhibit consensus sequence elements denoted box C (5′-UGAUGA-3′) and box D (5′-CUGA-3′) (5.Smith C.M. Steitz J.A. Cell. 1997; 89: 669-672Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). The FIB RNP associates with Nop56p, Nop5p/58p, and a 15.5-kDa protein (a counterpart of yeast Snu13p) to form box C/D snoRNP complexes that function in site-specific 2′-O-methylation of pre-rRNA (6.Kiss-Laszlo Z. Henry Y. Bachellerie J.P. Caizergues-Ferrer M. Kiss T. Cell. 1996; 85: 1077-1088Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar, 7.Tycowski K.T. Smith C.M. Shu M.D. Steitz J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14480-14485Crossref PubMed Scopus (143) Google Scholar, 8.Tyc K. Steitz J.A. EMBO J. 1989; 8: 3113-3119Crossref PubMed Scopus (305) Google Scholar, 9.Baserga S.J. Yang X.D. Steitz J.A. EMBO J. 1991; 10: 2645-2651Crossref PubMed Scopus (131) Google Scholar). FIB is the methyltransferase that catalyzes this 2′-O-methylation (10.Omer A.D. Ziesche S. Ebhardt H. Dennis P.P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5289-5294Crossref PubMed Scopus (158) Google Scholar). FIB, or Nop1p in the yeast Saccharomyces cerevisiae, is highly conserved in eukaryotes with respect to sequence, structure, and function (11.Schimmang T. Tollervey D. Kern H. Frank R. Hurt E.C. EMBO J. 1989; 8: 4015-4024Crossref PubMed Scopus (236) Google Scholar, 12.Henriquez R. Blobel G. Aris J.P. J. Biol. Chem. 1990; 265: 2209-2215Abstract Full Text PDF PubMed Google Scholar, 13.Lapeyre B. Mariottini P. Mathieu C. Ferrer P. Amaldi F. Amalric F. Caizergues-Ferrer M. Mol. Cell Biol. 1990; 10: 430-434Crossref PubMed Scopus (106) Google Scholar, 14.Aris J.P. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 931-935Crossref PubMed Scopus (156) Google Scholar, 15.Jansen R.P. Hurt E.C. Kern H. Lehtonen H. Carmo-Fonseca M. Lapeyre B. Tollervey D. J. Cell Biol. 1991; 113: 715-729Crossref PubMed Scopus (132) Google Scholar, 16.Turley S.J. Tan E.M. Pollard K.M. Biochim. Biophys. Acta. 1993; 1216: 119-122Crossref PubMed Scopus (41) Google Scholar, 17.David E. McNeil J.B. Basile V. Pearlman R.E. Mol. Biol. Cell. 1997; 8: 1051-1061Crossref PubMed Scopus (21) Google Scholar). Deletion of the Nop1 gene in yeast results in inhibition of 2′-O-ribose methylation and pre-rRNA processing at sites A0 to A2, indicating that Nop1p is directly involved in both pre-rRNA methylation and processing and ultimately in ribosome assembly (18.Tollervey D. Lehtonen H. Jansen R. Kern H. Hurt E.C. Cell. 1993; 72: 443-457Abstract Full Text PDF PubMed Scopus (407) Google Scholar). Although human FIB is the functional homolog of yeast Nop1p, it only partially complements a yeast nop1-defective mutant (15.Jansen R.P. Hurt E.C. Kern H. Lehtonen H. Carmo-Fonseca M. Lapeyre B. Tollervey D. J. Cell Biol. 1991; 113: 715-729Crossref PubMed Scopus (132) Google Scholar). Human FIB is a nucleolar autoantigen for the non-hereditary immune disease scleroderma (14.Aris J.P. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 931-935Crossref PubMed Scopus (156) Google Scholar). FIB co-localizes with the survival motor neuron (SMN) gene product in both nucleoli and Cajal bodies/gems of primary neurons (19.Jones K.W. Gorzynski K. Hales C.M. Fischer U. Badbanchi F. Terns R.M. Terns M.P. J. Biol. Chem. 2001; 276: 38645-38651Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 20.Wehner K.A. Ayala L. Kim Y. Young P.J. Hosler B.A. Lorson C.L. Baserga S.J. Francis J.W. Brain Res. 2002; 945: 160-173Crossref PubMed Scopus (26) Google Scholar). SMN is linked to one of the most common inheritable causes of childhood mortality, spinal muscular atrophy (19.Jones K.W. Gorzynski K. Hales C.M. Fischer U. Badbanchi F. Terns R.M. Terns M.P. J. Biol. Chem. 2001; 276: 38645-38651Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). In fact, a direct interaction between FIB and SMN has been demonstrated, although no functional basis for this interaction has been established, including any involvement of FIB in the pathogenesis of spinal muscular atrophy. Another protein, the nuclear DEAD box protein p68, an RNA-dependent ATPase and RNA helicase, co-localizes with FIB specifically in nascent nucleoli during telophase (21.Nicol S.M. Causevic M. Prescott A.R. Fuller-Pace F.V. Exp. Cell Res. 2000; 257: 272-280Crossref PubMed Scopus (58) Google Scholar). As with SMN, no physiological role of its interaction with FIB has been established. Human FIB (∼36 kDa) comprises 321 amino acids and three structural domains (14.Aris J.P. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 931-935Crossref PubMed Scopus (156) Google Scholar) and is 66% identical to yeast Nop1p. The N-terminal 80 residues comprise a glycine- and arginine-rich (GAR) domain (14.Aris J.P. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 931-935Crossref PubMed Scopus (156) Google Scholar) that is also present in Nop1p and Xenopus FIB (Fig. 1) but not in Tetrahymena FIB (17.David E. McNeil J.B. Basile V. Pearlman R.E. Mol. Biol. Cell. 1997; 8: 1051-1061Crossref PubMed Scopus (21) Google Scholar) or Methanococcus jannaschii FIB (22.Wang H. Boisvert D. Kim K.K. Kim R. Kim S.H. EMBO J. 2000; 19: 317-323Crossref PubMed Scopus (143) Google Scholar). The GAR domain is methylated at arginine residues, although the arginine methyltransferase responsible for in vivo methylation has not been identified (23.Lischwe M.A. Cook R.G. Ahn Y.S. Yeoman L.C. Busch H. Biochemistry. 1985; 24: 6025-6028Crossref PubMed Scopus (111) Google Scholar, 24.Lischwe M.A. Ochs R.L. Reddy R. Cook R.G. Yeoman L.C. Tan E.M. Reichlin M. Busch H. J. Biol. Chem. 1985; 260: 14304-14310Abstract Full Text PDF PubMed Google Scholar). The GAR domain is responsible for the interaction of FIB with both SMN protein and the DEAD box RNA helicase p68 (19.Jones K.W. Gorzynski K. Hales C.M. Fischer U. Badbanchi F. Terns R.M. Terns M.P. J. Biol. Chem. 2001; 276: 38645-38651Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 21.Nicol S.M. Causevic M. Prescott A.R. Fuller-Pace F.V. Exp. Cell Res. 2000; 257: 272-280Crossref PubMed Scopus (58) Google Scholar). A centrally located 90-residue sequence resembles an RNA-binding domain (RBD) present in various snRNPs. This RBD together with the C-terminal α-helix domain and the intervening spacer (spacer region 2) constitutes a methyltransferase-like domain that contains an S-adenosyl methionine-binding motif (14.Aris J.P. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 931-935Crossref PubMed Scopus (156) Google Scholar, 22.Wang H. Boisvert D. Kim K.K. Kim R. Kim S.H. EMBO J. 2000; 19: 317-323Crossref PubMed Scopus (143) Google Scholar). Replacement of two residues in the Nop1p S-adenosyl methionine-binding motif results in temperature sensitivity and a drastic reduction in nascent rRNA transcript methylation under restrictive conditions (18.Tollervey D. Lehtonen H. Jansen R. Kern H. Hurt E.C. Cell. 1993; 72: 443-457Abstract Full Text PDF PubMed Scopus (407) Google Scholar). Thus, the methyltransferase-like domain is responsible for FIB methyltransferase activity. The C-terminal α-helix domain is composed of ∼30 residues, and although this domain probably targets FIB to Cajal bodies, spacer region 2 appears to target FIB to the fibrillar regions. However, the targeting of FIB in both instances occurs only in the presence of the RBD (3.Snaar S. Wiesmeijer K. Jochemsen A G. Tanke H.J. Dirks R.W. J. Cell Biol. 2000; 151: 653-662Crossref PubMed Scopus (111) Google Scholar). Although it is well established that FIB plays a role in ribosome biogenesis within the nucleolus, its role in Cajal bodies is not understood. Our recent studies have used proteomic methodology to characterize a series of preribosomal ribonucleoprotein (pre-rRNP) complexes formed in mammalian cells. We have thus far isolated and analyzed the pre-rRNP complexes associated with human nucleolin (25.Yanagida M. Shimamoto A. Nishikawa K. Furuichi Y. Isobe T. Takahashi N. Proteomics. 2001; 1: 1390-1404Crossref PubMed Scopus (61) Google Scholar), parvulin (26.Fujiyama S. Yanagida M. Hayano T. Miura Y. Uchida T. Fujimori F. Isobe T. Takahashi N. J. Biol. Chem. 2002; 277: 23773-23780Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), and Nop56p (27.Hayano T. Yanagida M. Yamauchi Y. Shinkawa T. Isobe T. Takahashi N. J. Biol. Chem. 2003; 278: 34309-34319Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). These studies demonstrate the applicability of proteomic analysis to the study of human ribosome biogenesis and have identified a number of mammalian counterparts of yeast trans-acting factors involved in this process. Furthermore, several candidate mammalian trans-acting factors were identified that were not previously identified in yeast. Here, we present a proteomic analysis of FIB-associated protein complexes. In addition to the association of FIB with pre-rRNP complexes, we found that this protein interacts with a sub-complex containing a minimal set of proteins including SF2A-p32, PRMT5, tubulin α3, tubulin β1, and PRMT1. The FIB GAR domain and the spacer region 1 interact directly with SF2A-p32, whereas the methyltransferase-like domain interacts with PRMT5. These results provide new clues to the precise functions of FIB not only in ribosome biogenesis but also in sn(o)RNP biogenesis and mRNA processing in Cajal bodies. Materials—Human kidney cell line 293EBNA, Opti-MEM, and LipofectAMINE were purchased from Invitrogen (Grand Island, NY). Dulbecco's modified Eagle's medium, anti-FLAG M2 affinity gel, FLAG peptide, IGEPAL CA630, RNase A, and α-cyano-4-hydroxycinnamic acid were from Sigma-Aldrich Chemical (Steinheim, Germany). Alkaline phosphatase-conjugated anti-mouse IgG was from Amersham Biosciences (Uppsala, Sweden). Alexa Fluor 488-conjugated rabbit anti-mouse IgG antibody was from Molecular Probes, Inc. (Eugene, OR). Trypsin (sequence grade) was from Promega (Madison, WI) and Achromobacter lyticus protease I (Lys-C) was from WAKO Pure Chemicals (Osaka, Japan). ZipTipC18 was from Millipore (Billerica, MA). LATaq DNA polymerase was from Takara (Shiga, Japan). Protease inhibitor mixture Complete Mini was from Roche Diagnostics (Mannheim, Germany). Collagen I-coated Biocoat 8-well culture slides were from BD Biosciences (Franklin Lakes, NJ). All other reagents were from WAKO Pure Chemicals. Vectors for Epitope-tagged FIB and FIB Truncation Mutants and Expression in 293EBNA Cells—The FIB expression plasmid was constructed using a PCR-amplified DNA fragment comprising the FIB open reading frame with the FLAG tag at its N terminus (Fig. 1). This fragment was introduced between the NheI and the BamHI sites of the mammalian expression vector, pcDNA3.1 (+) (Invitrogen). The PCR primer set was 5′-ATATATCTAGAGCCACCATGGACTACAAGGACGACGACGACAAGAAGCCAGGATTCAGTCCCCGT-3′ and 5′-TATAGGATCCTCAGTTCTTCACCTTGGGGGG-3′, and human placenta cDNA (OriGene Technologies, Inc., Rockville, MD) was used as the template. The DNA fragment encoding the FLAG tag along with the nucleolar localization signal (NLS) of HIV Rex (TRRRPRRSQRKR) (28.Hofer L. Weichselbraun I. Quick S. Farrington G.K. Bohnlein E. Hauber J. J. Virol. 1991; 65: 3379-3383Crossref PubMed Google Scholar) and the SV40 nuclear localization signal (NS) (PKKKRKV) (29.Goldfarb D.S. Gariepy J. Schoolnik G. Kornberg R.D. Nature. 1986; 322: 641-644Crossref PubMed Scopus (328) Google Scholar) was synthesized by PCR using the oligonucleotide sets 5′-TATAGCTAGCGCCACCATGGACTACAAGGACGACGACGACAAGACCCGTCGGAGGCCCCG-3′ and 5′-TCTTTTTCTTTGGGATCGGCGGGGCCTCCGACGGGT-3′, and 5′-GATCCCAAAGAAAAAGAGCCAGCCCAAAAAAGAAGAGAAA-3′ and 5′-ATATAGGATCCTACCTTTCTCTTCTTTTTTGG-3′. The amplified fragment was subcloned between the NheI and the BamHI sites of pcDNA3.1(+), and the resulting plasmid was designated pcDNA3.1-NLS. All the expression plasmids of the FIB deletion mutants were constructed by introducing PCR-amplified fragments between the BamHI and XhoI sites downstream of the FLAG tag/NLS/NS in pcDNA3.1-NLS. Primer sets used for the amplification of FIB deletion mutants were as follows; 5′-ATATAGGATCCAAGCCAGGATTCAGTCCCCGT-3′ and 5′-TATATCTCGAGTCATTTTCCTCCCCGACCACGACC-3′ for FIB I (residues 2–77), 5′-ATATAGGATCCAGAGGAAACCAGTCGGGGAAG-3′ and 5′-TATATCTCGAGTCATCGGTACTCAAATTTGTCATC-3′ for FIB II (residues 78–135), 5′-ATATAGGATCCGCCTGGAACCCCTTCCGCTCC-3′ and 5′-TATATCTCGAGTCAGTTCTTCACCTTGGGGGG-3′ for FIB III (residues 136–321), 5′-ATATAGGATCCAAGCCAGGATTCAGTCCCCGT-3′ and 5′-TATATCTCGAGTCATCGGTACTCAAATTTGTCATC-3′ for FIB IV (residues 2–135), 5′-ATATAGGATCCAGAGGAAACCAGTCGGGGAAG-3′ and 5′-TATATCTCGAGTCAGTTCTTCACCTTGGGGGG-3′ for FIB V (residues 78–321). Human 293EBNA cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, streptomycin (0.1 μg/ml), and penicillin G (100 units/ml) at 37 °C in an incubator under 5% CO2. Subconfluent cells in 90-mm dishes were transfected with 10 μg of expression plasmid DNA using LipofectAMINE, and the transfected cells were grown for 48 h at 37 °C. Isolation of FIB- and Its Truncated Mutant-associated Complexes—At 48 h post-transfection, 293EBNA cells were harvested and washed with PBS and lysed in lysis buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.5% IGEPAL CA630) containing a protease inhibitor mixture on ice for 30 min. The soluble fraction was obtained by centrifugation at 15,000 rpm for 30 min at 4 °C and was incubated with 20 μl of anti-FLAG M2-agarose beads for 4 h at 4 °C for immunoprecipitation of FIB-associated complexes or overnight at 4 °C for deletion mutant-associated complexes. After washing the agarose beads five times with lysis buffer and once with 50 mm Tris-HCl, pH 8.0, 150 mm NaCl, the complexes bound to the agarose beads were eluted with 20 μl of 50 mm Tris-HCl, pH 8.0, 150 mm NaCl containing 500 μg/ml FLAG peptide. The eluted complexes were analyzed by SDS-PAGE. Ribonuclease Treatment of the FIB- and Truncation Mutant-associated Complexes—The immunoprecipitated FIB and truncation mutant-associated complexes described above were incubated with 50 mm Tris-HCl, pH 8.0, 150 mm NaCl containing 1 μg/ml RNase A for 10 min at 37 °C, washed twice with lysis buffer, and then once with 50 mm Tris-HCl, pH 8.0, 150 mm NaCl, and eluted with buffer containing the FLAG peptide as described above. Immunocytochemistry—293EBNA cells were grown on Collagen I-coated 8-well culture slides and transfected with expression plasmids using LipofectAMINE. Prior to fixation, cells were washed with PBS followed by incubation with 3.7% formaldehyde in PBS. After several washes with PBS-T (PBS containing 0.05% (w/v) Tween 20), the cells were incubated with PBS containing 0.1% (w/v) Triton X-100 for 5 min at room temperature. The cells were then blocked by 3% (w/v) nonfat dried milk in PBS and were incubated with anti-FLAG for 1 h at room temperature. The cells were rinsed in PBS-T and then incubated with Alexa Fluor 488-conjugated anti-mouse IgG for 1 h at room temperature, followed by three washes with PBS-T. The resulting cells were examined with a confocal laser-scanning microscope TCS (Leica Microsystems AG, Wetzlar, Germany). Protein Identification by the Peptide Mass Fingerprinting Method— Protein-containing SDS-PAGE gel fragments were subjected to in-gel digestion with trypsin as previously described (25.Yanagida M. Shimamoto A. Nishikawa K. Furuichi Y. Isobe T. Takahashi N. Proteomics. 2001; 1: 1390-1404Crossref PubMed Scopus (61) Google Scholar). The resulting peptides were recovered and analyzed for peptide mass fingerprints using a PE Biosystems MALDI-TOF MS (Voyager DE-STR) as described previously (25.Yanagida M. Shimamoto A. Nishikawa K. Furuichi Y. Isobe T. Takahashi N. Proteomics. 2001; 1: 1390-1404Crossref PubMed Scopus (61) Google Scholar). Peptide masses were searched with 50 ppm mass accuracy using the data base fitting program MS-Fit (available at prospector.ucsf.edu), and protein identification was performed according to the criteria described previously (25.Yanagida M. Shimamoto A. Nishikawa K. Furuichi Y. Isobe T. Takahashi N. Proteomics. 2001; 1: 1390-1404Crossref PubMed Scopus (61) Google Scholar). Protein Identification by LC-MS/MS Analysis—The FIB- and truncation mutant-associated complexes were digested with Lys-C, and the resulting peptides were analyzed using a nanoscale LC-MS/MS system as described (30.Natsume T. Yamauchi Y. Nakayama H. Shinkawa T. Yanagida M. Takahashi N. Isobe T. Anal. Chem. 2002; 74: 4725-4733Crossref PubMed Scopus (180) Google Scholar). The peptide mixture was applied to a Mightysil-RP-18 (3-μm particle, Kanto Chemical, Osaka, Japan) frit-less column (45 mm × 0.150 mm i.d.) and separated using a 0–40% gradient of aceto-nitrile containing 0.1% formic acid over 80 min at a flow rate of 50 or 25 nl/min. Eluted peptides were sprayed directly into a quadrupole time-of-flight hybrid mass spectrometer (Q-Tof 2, Micromass Wythenshawe, UK). MS/MS spectra were acquired by data-dependent collision-induced dissociation, and MS/MS data were analyzed using the MASCOT software (Matrix Science, London, UK) for peptide assignment. The criteria were in accordance with the manufacturer's definitions. If necessary, match acceptance of automated batch processes was confirmed by manual inspection of each set of raw MS/MS spectra in which the major product ions were matched with theoretically predicted product ions from the data base-matched peptides. Proteins in the mock eluate from anti-FLAG antibody with FLAG-peptide were analyzed by the same LC-MS/MS method as used for the fibrillarin-associated complexes and then subtracted from the proteins identified in the total fibrillarin-associated complexes. Thus, those proteins identified in the mock eluate were not included in the fibrillarin-associated proteins unless the quantitative increase was confirmed. Ultracentrifugation of FIB-associated RNP Complexes—The anti-FLAG immunoprecipitate obtained from FLAG-tagged FIB gene-transfected 293EBNA cells after elution with the FLAG peptide was analyzed on a 12–50% sucrose gradient in 50 mm Tris, pH 7.5, 25 mm KCl, 5mm MgCl2. The gradients were centrifuged in an SW65 rotor at 50,000 rpm (180,000 × g) for 3 h at 4 °C. A total of 18 fractions of 300 μl each were collected. The migration of the 40 S/60 S/80 S ribosomal complexes was determined by comparison to the ultraviolet absorption profile of cytosolic ribosomes fractionated by ultracentrifugation under identical experimental conditions. Isolation of FLAG-tagged FIB-associated Protein Complexes—Because endogenous FIB is found primarily in the nucleolus of mammalian cells, the subcellular localization of the FLAG-tagged protein in transfected 293EBNA cells was confirmed by immunofluorescence microscopy using an antibody to FLAG (Supplementary Fig. 1). Although weak staining was observed in the cytoplasm and nucleoplasm of the FLAG·FIB-transfected cells, the nucleolus exhibited intense staining thus confirming the correct localization of FLAG-tagged FIB. Complexes associated with FIB were isolated from FLAG·FIB-transfected cells via immunoprecipitation using the FLAG antibody. A typical silver-stained SDS-PAGE gel of the immunoprecipitated fraction showed that FIB-associated complexes contained many proteins spanning a wide range of molecular weight (Fig. 2). In contrast, only four protein bands were apparent in a mock immunoprecipitate prepared from untransfected control cells (Fig. 2). In addition, when cells were transfected with unrelated FLAG-tagged proteins, an entirely different pattern of protein bands was obtained (data not shown). These results indicated that most of the factors immunoprecipitated from FLAG·FIB-transfected cells represented de facto FIB-associated proteins. RNA Integrity in FIB-associated Complexes—RNA integrity is requisite for protein association in pre-rRNP complexes associated with human nucleolin, parvulin, and Nop56p, all of which may be involved in ribosome biogenesis as reported in our previous studies (25.Yanagida M. Shimamoto A. Nishikawa K. Furuichi Y. Isobe T. Takahashi N. Proteomics. 2001; 1: 1390-1404Crossref PubMed Scopus (61) Google Scholar, 26.Fujiyama S. Yanagida M. Hayano T. Miura Y. Uchida T. Fujimori F. Isobe T. Takahashi N. J. Biol. Chem. 2002; 277: 23773-23780Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 27.Hayano T. Yanagida M. Yamauchi Y. Shinkawa T. Isobe T. Takahashi N. J. Biol. Chem. 2003; 278: 34309-34319Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Therefore, the requirement for RNA integrity was also analyzed for FLAG·FIB-associated complexes. RNase treatment prompted the dissociation of the majority of the protein components of FLAG·FIB-associated complexes. However, at least four major protein-staining bands as well as several minor bands remained associated with FLAG·FIB after RNase treatment (Fig. 2). The four major protein bands appeared to exhibit approximate equivalent stoichiometry with respect to staining intensity and were representative of the more abundant proteins present in the non-RNase-treated complexes (Fig. 2, compare lanes 1 and 2). In addition, the proteins released by RNase treatment could not be distinguished from those present in the RNase-untreated complexes except for the four major protein bands and several minor protein bands that remained associated with FLAG·FIB after RNase treatment, as judged by SDS-PAGE analysis (data not shown). These results suggested that at least two distinct groups of proteins are present in FIB-associated complexes, one whose association is dependent on RNA integrity (the FIB-associated RNPs) and another that associates directly with FIB (independent of RNA integrity). However, it was uncertain as to whether these two groups of proteins were associated with each other or present independently. Identification of Protein Components in FIB-associated RNPs—Given the involvement of FIB in ribosome biogenesis, we expected isolated FIB-associated RNP complexes to contain trans-acting factors involved in this process, as well as ribosomal proteins. We therefore identified the protein components of the FIB-associated RNPs. We previously described a highly sensitive "direct nano-flow LC-MS/MS" system to identify proteins in limited amounts of multiprotein complexes (30.Natsume T. Yamauchi Y. Nakayama H. Shinkawa T. Yanagida M. Takahashi N. Isobe T. Anal. Chem. 2002; 74: 4725-4733Crossref PubMed Scopus (180) Google Scholar). Immunoisolated FIB-associated complexes were digested with Lys-C and analyzed directly using the nano-LC-MS/MS system. In addition to the criteria for match acceptance described under "Experimental Procedures," more stringent criteria were adopted to conclusively identify proteins. Namely, at least two different peptides had to be identified in a single nano-LC-MS/MS analysis, and/or at least one peptide had to be identified at least twice (with highly significant data base matching scores) among four separate analyses. A total of 1426 peptides were identified via sequence data base searches using the collision-induced dissociation spectra obtained from four nano-LC-MS/MS runs of a Lys-C digest of the FIB-associated complexes. These peptide data identified 170 proteins (excluding the bait protein and the proteins present in mock) that met our identification criteria. Although we do not exclude that some of the proteins identified may be nonspecifically associated proteins, we believe most of the protein components identified in the fibrillarin-associated complexes are specifically associated with fibrillarin. Of the 170 proteins, 73 were ribosomal proteins (43 from the large subunit and 30 from the small subunit; Supplementary Table SI) and 97 were non-ribosomal proteins (Supplementary Tables SII and SIII). Of the non-ribosomal proteins, 24 were assigned as probable trans-acting factors involved in ribosome biogenesis based on their homology to yeast proteins known or expected to be involved in ribosome biogenesis (Table I and Supplementary Table SII).Table IPutative fibrillarin-associated trans-acting factors involved in ribosome biogenesis In addition to the putative trans-acting factors that were assigned, the FIB-associated complexes contained 68 more non-ribosomal proteins, including a number of RNA-binding proteins, splicing factors, DNA-topoisomerase, Myb-binding protein, components of DNA-dependent protein kinase, components of the signal recognition particle, nuclear matrix proteins as well as many hypothetical/unknown proteins (Supplementary Table SIII). Of these, at least 44 proteins reportedly localize to the nucleolus or the nucleus (31.Andersen J.S. Lyon C.E. Fox A.H. Leung A.K.L. Lam Y.W. Steen H. Mann M. Lamond A.I. Curr. Biol. 2002; 12: 1-11Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar, 32.Scheri A. Coute Y. Deon C. Calle A. Kindbeiter K. Sanchez J.-C. Greco A. Hochstrasser D. Diaz J.-J. Mol. Biol. Cell. 2002; 13: 4100-4109Crossref PubMed Google Scholar) (Supplementary Table SIII). Identification of Proteins Associated with FIB without RNA Integrity—The proteins that remained associated with FIB after the RNase treatment were identified by MALDI-TOF

Referência(s)