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Localization of Nopp140 within mammalian cells during interphase and mitosis

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Abstract

We investigated distribution of the nucleolar phosphoprotein Nopp140 within mammalian cells, using immunofluorescence confocal microscopy and immunoelectron microscopy. During interphase, three-dimensional image reconstructions of confocal sections revealed that nucleolar labelling appeared as several tiny spheres organized in necklaces. Moreover, after an immunogold labelling procedure, gold particles were detected not only over the dense fibrillar component but also over the fibrillar centres of nucleoli in untreated and actinomycin D-treated cells. Labelling was also consistently present in Cajal bodies. After pulse-chase experiments with BrUTP, colocalization was more prominent after a 10- to 15-min chase than after a 5-min chase. During mitosis, confocal analysis indicated that Nopp140 organization was lost. The protein dispersed between and around the chromosomes in prophase. From prometaphase to telophase, it was also detected in numerous cytoplasmic nucleolus-derived foci. During telophase, it reappeared in the reforming nucleoli of daughter nuclei. This strongly suggests that Nopp140 could be a component implicated in the early steps of pre-rRNA processing.

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References

  • Andrade LE, Chan EK, Raska I, Peebles CL, Roos G, Tan EM (1991) Human autoantibody to a novel protein of the nuclear coiled body: immunological characterization and cDNA cloning of p80-coilin. J Exp Med 173:1407–1419

    Article  PubMed  CAS  Google Scholar 

  • Baran V, Brochard V, Renard JP, Flechon JE (2001) Nopp 140 involvement in nucleologenesis of mouse preimplantation embryos. Mol Reprod Dev 59:277–284

    Article  PubMed  CAS  Google Scholar 

  • Bendayan M (1984) Protein A-gold electron microscopic immunocytochemistry: methods, applications and limitations. J Electron Microsc Tech 1:243–270

    Article  CAS  Google Scholar 

  • Cadwell C, Yoon HJ, Zebarjadian Y, Carbon J (1997) The yeast nucleolar protein Cbf5p is involved in rRNA biosynthesis and interacts genetically with the RNA polymerase I transcription factor RRN3. Mol Cell Biol 17:6175–6183

    PubMed  CAS  Google Scholar 

  • Cairns C, McStay B (1995) Identification and cDNA cloning of a Xenopus nucleolar phosphoprotein, xNopp180, that is the homolog of the rat nucleolar protein Nopp140. J Cell Sci 108:3339–3347

    PubMed  CAS  Google Scholar 

  • Carmo-Fonseca M, Pepperkok R, Carvalho MT, Lamond AI (1992) Transcription-dependent colocalization of the U1, U2, U4/U6, and U5 snRNPs in coiled bodies. J Cell Biol 117:1–14

    Article  PubMed  CAS  Google Scholar 

  • Chan EK, Imai H, Hamel JC, Tan EM (1991) Human autoantibody to RNA polymerase I transcription factor hUBF. Molecular identity of nucleolus organizer region autoantigen NOR-90 and ribosomal RNA transcription upstream binding factor. J Exp Med 174:1239–1244

    Article  PubMed  CAS  Google Scholar 

  • Chen HK, Pai CY, Huang JY, Yeh NH (1999) Human Nopp140, which interacts with RNA polymerase I: implications for rRNA gene transcription and nucleolar structural organization. Mol Cell Biol 19:8536–8546

    PubMed  CAS  Google Scholar 

  • Cheutin T, O’Donohue MF, Beorchia A, Vandelaer M, Kaplan H, Deféver B, Ploton D, Thiry M (2002) Three-dimensional organization of active rRNA genes within the nucleolus. J Cell Sci 115:3297–3307

    PubMed  CAS  Google Scholar 

  • Christensen ME, Moloo J, Swischuk JL, Schelling ME (1986) Characterization of the nucleolar protein, B-36, using monoclonal antibodies. Exp Cell Res 166:77–93

    Article  PubMed  CAS  Google Scholar 

  • Chu S, Archer RH, Zengel JM, Lindahl L (1994) The RNA of RNase MRP is required for normal processing of ribosomal RNA. Proc Natl Acad Sci USA 91:659–663

    Article  PubMed  CAS  Google Scholar 

  • Derenzini M, Pasquinelli G, O’Donohue MF, Ploton D, Thiry M (2006) Structural and functional organization of ribosomal genes within the mammalian cell nucleolus. J Histochem Cytochem 54:131–145

    Article  PubMed  CAS  Google Scholar 

  • DiMario P (2004) Cell and molecular biology of nucleolar assembly and disassembly. Int Rev Cytol 239:99–178

    Article  PubMed  CAS  Google Scholar 

  • Dundr M, Olson MO (1998) Partially processed pre-rRNA is preserved in association with processing components in nucleolus-derived foci during mitosis. Mol Biol Cell 9:2407–2422

    PubMed  CAS  Google Scholar 

  • Dundr M, Meier UT, Lewis N, Rekosh D, Hammarskjold ML, Olson MO (1997) A class of nonribosomal nucleolar components is located in chromosome periphery and in nucleolus-derived foci during anaphase and telophase. Chromosoma 105:407–417

    Article  PubMed  CAS  Google Scholar 

  • Dundr M, Misteli T, Olson MO (2000) The dynamics of postmitotic reassembly of the nucleolus. J Cell Biol 150:433–446

    Article  PubMed  CAS  Google Scholar 

  • Fomproix N, Hernandez-Verdun D (1999) Effects of anti-PM-Scl 100 (Rrp6p exonuclease) antibodies on prenucleolar body dynamics at the end of mitosis. Exp Cell Res 251:452–464

    Article  PubMed  CAS  Google Scholar 

  • Guldner HH, Szostecki C, Vosberg HP, Lakomek HJ, Penner E, Bautz FA (1986) Scl 70 autoantibodies from scleroderma patients recognize a 95 kDa protein identified as DNA topoisomerase I. Chromosoma 94:132–138

    Article  PubMed  CAS  Google Scholar 

  • Heliot L, Kaplan H, Lucas L, Klein C, Beorchia A, Doco-Fenzy M, Menager M, Thiry M, O’Donohue MF, Ploton D (1997) Electron tomography of metaphase nucleolar organizer regions: evidence for a twisted-loop organization. Mol Biol Cell 8:2199–2216

    PubMed  CAS  Google Scholar 

  • Hernandez-Verdun D, Gautier T (1994) The chromosome periphery during mitosis. Bioessays 16:179–185

    Article  PubMed  CAS  Google Scholar 

  • Hughes JM (1996) Functional base-pairing interaction between highly conserved elements of U3 small nucleolar RNA and the small ribosomal subunit RNA. J Mol Biol 259:645–654

    Article  PubMed  CAS  Google Scholar 

  • Isaac C, Yang Y, Meier UT (1998) Nopp140 functions as a molecular link between the nucleolus and the coiled bodies. J Cell Biol 142:319–329

    Article  PubMed  CAS  Google Scholar 

  • Jacobson MR, Cao LG, Wang YL, Pederson T (1995) Dynamic localization of RNase MRP RNA in the nucleolus observed by fluorescent RNA cytochemistry in living cells. J Cell Biol 131:1649–1658

    Article  PubMed  CAS  Google Scholar 

  • Jimenez-Garcia LF, Segura-Valdez ML, Ochs R, Rothblum LI, Hanwan R, Spector DL (1994) Nucleologenesis: U3 snRNA-containing prenucleolar bodies move to sites of active pre-rRNA transcription after mitosis. Mol Biol Cell 5:955–966

    PubMed  CAS  Google Scholar 

  • Jordan P, Mannervik M, Tora L, Carmo-Fonseca M (1996) In vivo evidence that TATA-binding protein/SL1 colocalizes with UBF and RNA polymerase I when rRNA synthesis is either active or inactive. J Cell Biol 133:225–234

    Article  PubMed  CAS  Google Scholar 

  • Kass S, Tyc K, Steitz JA, Sollner-Webb B (1990) The U3 small nucleolar ribonucleoprotein functions in the first step of preribosomal RNA processing. Cell 60:897–908

    Article  PubMed  CAS  Google Scholar 

  • Kelly S, Singleton W, Wickstead B, Ersfeld K, Gull K (2006) Characterization and differential nuclear localization of Nopp140 and a novel Nopp140-like protein in trypanosomes. Eukaryotic Cell 5:876–879

    Article  PubMed  CAS  Google Scholar 

  • Klein C, Cheutin T, O’Donohue MF, Rothblum L, Kaplan H, Beorchia A, Lucas L, Heliot L, Ploton D (1998) The three-dimensional study of chromosomes and upstream binding factor-immunolabeled nucleolar organizer regions demonstrates their nonrandom spatial arrangement during mitosis. Mol Biol Cell 9:3147–3159

    PubMed  CAS  Google Scholar 

  • Lafontaine DL, Tollervey D (1998) Birth of the snoRNPs: the evolution of the modification-guide snoRNAs. Trends Biochem Sci 23:383–388

    Article  PubMed  CAS  Google Scholar 

  • Lafontaine DL, Bousquet-Antonelli C, Henry Y, Caizergues-Ferrer M, Tollervey D (1998) The box H + ACA snoRNAs carry Cbf5p, the putative rRNA pseudouridine synthase. Genes Dev 12:527–537

    Article  PubMed  CAS  Google Scholar 

  • Li D, Meier UT, Dobrowolska G, Krebs EG (1997) Specific interaction between casein kinase 2 and the nucleolar protein Nopp140. J Biol Chem 272:3773–3779

    Article  PubMed  CAS  Google Scholar 

  • Matera AG, Tycowski KT, Steitz JA, Ward DC (1994) Organization of small nucleolar ribonucleoproteins (snoRNPs) by fluorescence in situ hybridization and immunocytochemistry. Mol Biol Cell 5:1289–1299

    PubMed  CAS  Google Scholar 

  • Meier UT (1996) Comparison of the rat nucleolar protein nopp140 with its yeast homolog SRP40. Differential phosphorylation in vertebrates and yeast. J Biol Chem 271:19376–19384

    PubMed  CAS  Google Scholar 

  • Meier UT (2005) The many facets of H/ACA ribonucleoproteins. Chromosoma 114:1–14

    Article  PubMed  CAS  Google Scholar 

  • Meier UT, Blobel G (1990) A nuclear localization signal binding protein in the nucleolus. J Cell Biol 111:2235–2245

    Article  PubMed  CAS  Google Scholar 

  • Meier UT, Blobel G (1992) Nopp140 shuttles on tracks between nucleolus and cytoplasm. Cell 70:127–138

    Article  PubMed  CAS  Google Scholar 

  • Meier UT, Blobel G (1994) NAP57, a mammalian nucleolar protein with a putative homolog in yeast and bacteria. J Cell Biol 127:1505–1514

    Article  PubMed  CAS  Google Scholar 

  • Miau LH, Chang CJ, Tsai WH, Lee SC (1997) Identification and characterization of a nucleolar phosphoprotein, Nopp140, as a transcription factor. Mol Cell Biol 17:230–239

    PubMed  CAS  Google Scholar 

  • Pai CY, Chen HK, Sheu HL, Yeh NH (1995) Cell-cycle-dependent alterations of a highly phosphorylated nucleolar protein p130 are associated with nucleologenesis. J Cell Sci 108:1911–1920

    PubMed  CAS  Google Scholar 

  • Peculis BA, Steitz JA (1993) Disruption of U8 nucleolar snRNA inhibits 5.8S and 28S rRNA processing in the Xenopus oocyte. Cell 73:1233–1245

    Article  PubMed  CAS  Google Scholar 

  • Puvion-Dutilleul F, Mazan S, Nicoloso M, Christensen ME, Bachellerie JP (1991) Localization of U3 RNA molecules in nucleoli of HeLa and mouse 3T3 cells by high resolution in situ hybridization. Eur J Cell Biol 56:178–186

    PubMed  CAS  Google Scholar 

  • Reimer G, Raska I, Scheer U, Tan EM (1988) Immunolocalization of 7–2-ribonucleoprotein in the granular component of the nucleolus. Exp Cell Res 176:117–128

    Article  PubMed  CAS  Google Scholar 

  • Roth J, Bendayan M, Carlemalm E, Villiger W, Garavito M (1981) Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue. J Histochem Cytochem 29:663–671

    PubMed  CAS  Google Scholar 

  • Roussel P, André C, Comai L, Hernandez-Verdun D (1996) The rDNA transcription machinery is assembled during mitosis in active NORs and absent in inactive NORs. J Cell Biol 133:235–246

    Article  PubMed  CAS  Google Scholar 

  • Savino R, Gerbi SA (1990) In vivo disruption of Xenopus U3 snRNA affects ribosomal RNA processing. EMBO J 9:2299–2308

    PubMed  CAS  Google Scholar 

  • Savino TM, Bastos R, Jansen E, Hernandez-Verdun D (1999) The nucleolar antigen Nop52, the human homologue of the yeast ribosomal RNA processing RRP1, is recruited at late stages of nucleologenesis. J Cell Sci 112:1889–1900

    PubMed  CAS  Google Scholar 

  • Savino TM, Gebrane-Younes J, De Mey J, Sibarita JB, Hernandez-Verdun D (2001) Nucleolar assembly of the rRNA processing machinery in living cells. J Cell Biol 153:1097–1110

    Article  PubMed  CAS  Google Scholar 

  • Scheer U, Rose K (1984) Localization of RNA polymerase I in interphase cells and mitotic chromosomes by light and electron microscopic immunocytochemistry. Proc Natl Acad Sci USA 81:1431–1435

    Article  PubMed  CAS  Google Scholar 

  • Scheer U, Thiry M, Goessens G (1993) Structure, function and assembly of the nucleolus. Trends Cell Biol 3:236–241

    Article  PubMed  CAS  Google Scholar 

  • Schmidt-Zachmann MS, Hugle B, Scheer U, Franke WW (1984) Identification and localization of a novel nucleolar protein of high molecular weight by a monoclonal antibody. Exp Cell Res 153:327–346

    Article  PubMed  CAS  Google Scholar 

  • Schmitt M, Clayton D (1993) Nuclear RNase MRP is required for correct processing of pre-5.8S in Saccharomyces cerevisiae. Mol Cell Biol 13:7935–7941

    PubMed  CAS  Google Scholar 

  • Sirri V, Roussel P, Hernandez-Verdun D (1999) The mitotically phosphorylated form of the transcription termination factor TTF-1 is associated with the repressed rDNA transcription machinery. J Cell Sci 112:3259–3268

    PubMed  CAS  Google Scholar 

  • Thiry M, Goessens G (1996) The nucleolus during the cell cycle. RG Landes Company/Chapman & Hall, New York

    Google Scholar 

  • Thiry M, Cheutin T, O’Donohue MF, Kaplan H, Ploton D (2000) Dynamics and three-dimensional localization of ribosomal RNA within the nucleolus. RNA 6:1750–1761

    Article  PubMed  CAS  Google Scholar 

  • Thiry M, Lamaye F, Thelen N, Chatron-Colliet A, Lalun N, Bobichon H, Ploton D (2008) A protocol for studying the kinetics of RNA within cultured cells: application to ribosomal RNA. Nat Protoc 3:1997–2004

    Article  PubMed  CAS  Google Scholar 

  • Tsai Y-T, Lin C-I, Chen H-K, Lee K-M, Hsu C-Y, Yang S-J, Yeh N-H (2008) Chromatin tethering effects of hNopp140 are involved in the spatial organization of the nucleolus and the rRNA gene transcription. J Biomed Sci 15:471–486

    Article  PubMed  CAS  Google Scholar 

  • Vandelaer M, Thiry M (1998) The phosphoprotein pp135 is an essential constituent of the fibrillar components of nucleoli and of coiled bodies. Histochem Cell Biol 110:169–177

    Article  PubMed  CAS  Google Scholar 

  • Verheggen C, Almouzni G, Hernandez-Verdun D (2000) The ribosomal RNA processing machinery is recruited to the nucleolar domain before RNA polymerase I during Xenopus laevis development. J Cell Biol 149:293–306

    Article  PubMed  CAS  Google Scholar 

  • Verheggen C, Le Panse S, Almouzni G, Hernandez-Verdun D (2001a) Maintenance of nucleolar machineries and pre-rRNAs in remnant nucleolus of erythrocyte nuclei and remodeling in Xenopus egg extracts. Exp Cell Res 269:23–34

    Article  PubMed  CAS  Google Scholar 

  • Verheggen C, Mouaikel J, Thiry M, Blanchard JM, Tollervey D, Bordonne R, Lafontaine DL, Bertrand E (2001b) Box C/D small nucleolar RNA trafficking involves small nucleolar RNP proteins, nucleolar factors and a novel nuclear domain. EMBO J 20:5480–5490

    Article  PubMed  CAS  Google Scholar 

  • Waggener JM, DiMario PJ (2002) Two splice variants of Nopp140 in Drosophila melanogaster. Mol Biol Cell 13:362–381

    Article  PubMed  CAS  Google Scholar 

  • Weibel E (1969) Stereological principles for morphometry in electron microscopic cytology. Int Rev Cytol 26:235–302

    Article  PubMed  CAS  Google Scholar 

  • Weisenberger D, Scheer U (1995) A possible mechanism for the inhibition of ribosomal RNA gene transcription during mitosis. J Cell Biol 129:561–575

    Article  PubMed  CAS  Google Scholar 

  • Yang Y, Isaac C, Wang C, Dragon F, Pogacic V, Meier UT (2000) Conserved composition of mammalian box H/ACA and box C/D small nucleolar ribonucleoprotein particles and their interaction with the common factor Nopp140. Mol Biol Cell 11:567–577

    PubMed  Google Scholar 

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Acknowledgments

The authors wish to thank Drs F. Amalric, R. Deltour and F. Puvion-Dutilleul for their generous gift of antibodies. They also acknowledge the skillful technical provided by F. Skivée and D. Bourguignon. This work received financial support from the “Fonds de la Recherche Scientifique Médicale” (grant no. 3. 3.4540.06) to M. Thiry, from the “Ligue Régionale de la Marne”, from the “Fondation pour la Recherche Médicale” (grant no. 40001837–01) to D. Ploton, and from the National Institute of Health (HL079566) to U.T. Meier, F. Lamaye and N. Thelen are Ph.D. grant holders of the F.N.R.S.

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  1. Thierry Cheutin

    Present address: Institut de Génétique Humaine CNRS, 141 rue de la Cardonille, 34396, Montpellier Cedex 5, France

  2. Marie-Françoise O’Donohue

    Present address: Laboratoire de Biologie Moléculaire Eucaryote CNRS, Université de Toulouse, 118 route de Narbonne, 31062, Toulouse Cedex 9, France

Authors and Affiliations

  1. Laboratoire de Biologie Cellulaire et Tissulaire, Université de Liège, 20 rue de Pitteurs, 4020, Liege, Belgium

    Marc Thiry, Françoise Lamaye & Nicolas Thelen

  2. UMRCNRS 6237, Université de Reims Champagne-Ardenne, 51 rue Cognacq-Jay, 51095, Reims Cedex, France

    Thierry Cheutin, Marie-Françoise O’Donohue & Dominique Ploton

  3. Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York, NY, 10461, USA

    U. Thomas Meier

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  1. Marc Thiry
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Thiry, M., Cheutin, T., Lamaye, F. et al. Localization of Nopp140 within mammalian cells during interphase and mitosis. Histochem Cell Biol 132, 129–140 (2009). https://doi.org/10.1007/s00418-009-0599-8

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