The essential structural components of the nucleoli, Fibrillar Centers (FC) and Dense Fibrillar Components (DFC), together compose FC/DFC units, loci of rDNA transcription and early RNA processing. In the present study we followed cell cycle related changes of these units in 2 human sarcoma derived cell lines with stable expression of RFP-PCNA (the sliding clamp protein) and GFP-RPA43 (a subunit of RNA polymerase I, pol I) or GFP-fibrillarin. Correlative light and electron microscopy analysis showed that the pol I and fibrillarin positive nucleolar beads correspond to individual FC/DFC units. In vivo observations showed that at early S phase, when transcriptionally active ribosomal genes were replicated, the number of the units in each cell increased by 60-80%. During that period the units transiently lost pol I, but not fibrillarin. Then, until the end of interphase, number of the units did not change, and their duplication was completed only after the cell division, by mid G1 phase. This peculiar mode of reproduction suggests that a considerable subset of ribosomal genes remain transcriptionally silent from mid S phase to mitosis, but become again active in the postmitotic daughter cells.

b Institute of Biophysics of the CAS Brno Czech Republic

Charles University Prague 1st Faculty of Medicine Institute of Cellular Biology and Pathology Prague Czech Republic

Zobrazit více v PubMed

Henderson AS, Warburton D, Atwood KC. Location of ribosomal DNA in the human chromosome complement. Proc Natl Acad Sci USA 1972; 69:3394-8; PMID:4508329; http://dx.doi.org/10.1073/pnas.69.11.3394 PubMed DOI PMC

Long EO, Dawid IB. Repeated genes in eukaryotes. Annu Rev Biochem 1980; 49:727-64; PMID:6996571; http://dx.doi.org/10.1146/annurev.bi.49.070180.003455 PubMed DOI

Puvion-Dutilleul F, Bachellerie JP, Puvion E. Nucleolar organization of HeLa cells as studied by in situ hybridization. Chromosoma 1991; 100:395-409; PMID:1893795; http://dx.doi.org/10.1007/BF00337518 PubMed DOI

Raska I. Oldies but goldies: searching for Christmas trees within the nucleolar architecture. Trends Cell Biol 2003; 13:517-25; PMID:14507479; http://dx.doi.org/10.1016/j.tcb.2003.08.003 PubMed DOI

Raska I, Shaw PJ, Cmarko D. New insights into nucleolar architecture and activity. Int Rev Cytol 2006a; 255:177-235; http://dx.doi.org/10.1016/S0074-7696(06)55004-1 PubMed DOI

Raska I, Shaw PJ, Cmarko D. Structure and function of the nucleolus in the spotlight. Curr Opin Cell Biol 2006b; 18: 325-34; http://dx.doi.org/10.1016/j.ceb.2006.04.008 PubMed DOI

Cmarko D, Smigova J, Minichova L, Popov A. Nucleolus: the ribosome factory. Histol Histopathol 2008; 23(10): 1291-8; PMID:18712681 PubMed

Sirri V, Urcuqui-Inchima S, Roussel P, Hernandez-Verdun D. Nucleolus: the fascinating nuclear body. Histochem Cell Biol 2008; 129:13-31; PMID:18046571; http://dx.doi.org/10.1007/s00418-007-0359-6 PubMed DOI PMC

Bártová E, Horáková AH, Uhlírová R, Raska I, Galiová G, Orlova D, Kozubek S. Structure and epigenetics of nucleoli in comparison with non-nucleolar compartments. J Histochem Cytochem 2010; 58(5):391-403; http://dx.doi.org/10.1369/jhc.2009.955435 PubMed DOI PMC

Warner JR. The economics of ribosome biosynthesis in yeast. Trends Biochem Sci 1999; 24:437-40; PMID:10542411; http://dx.doi.org/10.1016/S0968-0004(99)01460-7 PubMed DOI

Grummt I, Pikaard CS. Epigenetic silencing of RNA polymerase I transcription. Nat Rev Mol Cell Biol 2003; 4:641-9; PMID:12923526; http://dx.doi.org/10.1038/nrm1171 PubMed DOI

Santoro R. The silence of the ribosomal RNA genes. Cell Mol Life Sci 2005; 62(18):2067-79; PMID:16041568; http://dx.doi.org/10.1007/s00018-005-5110-7 PubMed DOI PMC

Santoro R. Analysis of chromatin composition of repetitive sequences: the ChIP-Chop assay. Methods Mol Biol 2014; 1094:319-28; PMID:24162999; http://dx.doi.org/10.1007/978-1-62703-706-8_25 PubMed DOI

Berger C, Horlebein A, Gogel E, Grummt F. Temporal order of replication of mouse ribosomal RNA genes during the cell cycle. Chromosoma 1997; 106:479-84; PMID:9426279; http://dx.doi.org/10.1007/s004120050269 PubMed DOI

Li J, Santoro R, Koberna K, Grummt I. The chromatin remodeling complex NoRC controls replication timing of rRNA genes. EMBO J 2004; 24(1):120-7; PMID:15577942; http://dx.doi.org/10.1038/sj.emboj.7600492 PubMed DOI PMC

Raška I, Rychter Z, Smetana K. Fibrillar centers and condensed nucleolar chromatin in resting and stimulated human lymphocytes. Zeitschrift fur mikroskopischanatomische Forschung 1983a; 97(1):15-32 PubMed

Raška I, Armbruster BL, Frey JR, Smetana K. Analysis of ring-shaped nucleoli in serially sectioned human lymphocytes. Cell Tissue Res 1983b; 234(3):707-11 PubMed

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; PMID:2539876; http://dx.doi.org/10.1016/0248-4900(89)90013-0 PubMed DOI

Scheer U, Benavente R. Functional and dynamic aspects of the mammalian nucleolus. Bioessays 1990; 12(1):14-21; PMID:2181998; http://dx.doi.org/10.1002/bies.950120104 PubMed DOI

Dundr M, Raska I. Nonisotopic ultrastructural mapping of transcription sites within the nucleolus. Exp Cell Res 1993; 208(1):275-81; PMID:8359221; http://dx.doi.org/10.1006/excr.1993.1247 PubMed DOI

Raška I, Dundr M, Koberna K, Melcak I, Risueno MM, Torok I. Does the synthesis of ribosomal RNA take place within nucleolar fibrillar centres or dense fibrillar components? A critical appraisal. J Struct Biol 1995; 114:1-22; http://dx.doi.org/10.1006/jsbi.1995.1001 PubMed DOI

Melčak I, Risueno MC, Raška I. Ultrastructural non-isotopic mapping of nucleolar transcription sites in onion protoplasts. J Struct Biol 1996; 116(2): 253-63; PMID:8812981; http://dx.doi.org/10.1006/jsbi.1996.0040 PubMed DOI

Cmarko D, Verschure PJ, Rothblum LI, Hernandez-Verdun D, Amalric F, van Driel R, Fakan S. Ultrastructural analysis of nucleolar transcription in cells microinjected with 5-bromo-UTP. Histochem Cell Biol 2000; 113:181-7; PMID:10817672; http://dx.doi.org/10.1007/s004180050437 PubMed DOI

Koberna K, Malínský J, Pliss A, Masata M, Vecerova J, Fialová M, Bednár J, Raska I. Ribosomal genes in focus: new transcripts label the dense fibrillar components and form clusters indicative of “Christmas trees” in situ. J Cell Biol 2002; 157:743-8; PMID:12034768; http://dx.doi.org/10.1083/jcb.200202007 PubMed DOI PMC

Casafont I, Navascués J, Pena E, Lafarga M, Berciano MT. Nuclear organization and dynamics of transcription sites in rat sensory ganglia neurons detected by incorporation of 5′- fluorouridine into nascent RNA. Neuroscience 2006; 14:453-62; http://dx.doi.org/10.1016/j.neuroscience.2006.02.030 PubMed DOI

Shaw PJ, McKeown PC. The structure of rDNA chromatin. In The Nucleus. Protein reviews 2011; 15:43-55

Ochs RL, Lischwe MA, Spohn WH, Busch H. Fibrillarin: a new protein of the nucleolus identified by autoimmune sera. Biol Cell 1985; 54(2):123-33; PMID:2933102; http://dx.doi.org/10.1111/j.1768-322X.1985.tb00387.x PubMed DOI

Dragon F, Gallagher JE, Compagnone-Post PA, Mitchell BM, Porwancher KA, Wehner KA, Wormsley S, Settlage RE, Shabanowitz J, Osheim Y, Beyer AL, Hunt DF, Baserga SJ. A large nucleolar U3 ribonucleoprotein required for 18S ribosomal RNA biogenesis. Nature 2002; 417:967-70; PMID:12068309; http://dx.doi.org/10.1038/nature00769 PubMed DOI PMC

Haaf T, Hayman DL, Schmid M. Quantitative determination of rDNA transcription units in vertebrate cells. Exp Cell Res 1991; 193(1): 78-86; PMID:1995304; http://dx.doi.org/10.1016/0014-4827(91)90540-B PubMed DOI

Haaf T, Ward DC. Inhibition of RNA polymerase II transcription causes chromatin decondensation, loss of nucleolar structure, and dispersion of chromosomal domains. Exp Cell Res 1996; 224(1):163-73; PMID:8612682; http://dx.doi.org/10.1006/excr.1996.0124 PubMed DOI

Cheutin T, O'Donohue MF, Beorchia A, Vandelair M, Kaplan H, Deféver B, Ploton D, Thiry M. Three-dimentional organization of active rRNA genes within nucleolus. J Cell Sci 2002; 115(Pt16): 3297-307; PMID:12140261 PubMed

Denissov S, Lessard F, Mayer C, Stefanovsky V, van Driel M, Grummt I, Moss T, Stunnenberg HG. A model for the topology of active ribosomal RNA genes. EMBO Rep 2011; 12(3):231-7; PMID:21331097; http://dx.doi.org/10.1038/embor.2011.8 PubMed DOI PMC

Miller OL Jr, Beatty BR. Visualization of nucleolar genes. Science 1969; 164:955-7; PMID:5813982; http://dx.doi.org/10.1126/science.164.3882.955 PubMed DOI

Albert B, Léger-Silvestre I, Normand C, Ostermaier MK, Pérez-Fernández J, Panov KI, Zomerdijk JC, Schultz P, Gadal O. RNA polymerase I-specific subunits promote polymerase clustering to enhance the rRNA gene transcription cycle. J Cell Biol 2011; 192:277-93; PMID:21263028; http://dx.doi.org/10.1083/jcb.201006040 PubMed DOI PMC

Trendelenberg M, Puvion-Duteul F. Electron microscopy in molecular biology, a practical approach (eds. Sommerville J. and Scheer U.), 1987: 101-146. Oxford, IRL Press.

Mougey EB, O'Reilly M, Osheim Y, Miller OL Jr, Beyer A, Sollner-Webb B. The terminal balls characteristic of eukaryotic rRNA transcription units in chromatin spreads are rRNA processing complexes. Genes Dev 1993; 7(8):1609-19; PMID:8339936; http://dx.doi.org/10.1101/gad.7.8.1609 PubMed DOI

Németh A, Längst G. Chromatin organization of active ribosomal RNA genes. Epigenetics 2008; 3(5):243-5; http://dx.doi.org/10.4161/epi.3.5.6913 PubMed DOI

Shiue CN, Berkson RG, Wright AP. c-Myc induces changes in higher order rDNA structure on stimulation of quiescent cells. Oncogene 2009; 28(16):1833-42; PMID:19270725; http://dx.doi.org/10.1038/onc.2009.21 PubMed DOI

Shiue CN, Nematollahi-Mahani A, Wright AP. Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture. Nucleic Acids Res 2014; 42(9):5505-17; PMID:24609384; http://dx.doi.org/10.1093/nar/gku183 PubMed DOI PMC

Arabi A, Wu S, Ridderstråle K, Bierhoff H, Shiue C, Fatyol K, Fahlén S, Hydbring P, Söderberg O, Grummt I, et al.. c-Myc associates with ribosomal DNA and activates RNA polymerase I transcription. Nat Cell Biol 2005; 7:303-10; PMID:15723053; http://dx.doi.org/10.1038/ncb1225 PubMed DOI

Gomez-Roman N, Felton-Edkins ZA, Kenneth NS, Goodfellow SJ, Athineos D, Zhang J, Ramsbottom BA, Innes F, Kantidakis T, Kerr ER, et al.. Activation by c-Myc of transcription by RNA polymerases I, II and III. Biochem Soc Symp 2006; (73):141-54; PMID:16626295; http://dx.doi.org/10.1042/bss0730141 PubMed DOI

Grandori C, Gomez-Roman N, Felton-Edkins ZA, Ngouenet C, Galloway DA, Eisenman RN, White RJ. c-Myc binds to human ribosomal DNA and stimulates transcription of rRNA genes by RNA polymerase I. Nat Cell Biol 2005; 7:311-318; PMID:15723054; http://dx.doi.org/10.1038/ncb1224 PubMed DOI

Grewal SS, Li L, Orian A, Eisenman RN, Edgar BA. Myc-dependent regulation of ribosomal RNA synthesis during Drosophila development. Nat Cell Biol 2005; 7:295-302; PMID:15723055; http://dx.doi.org/10.1038/ncb1223 PubMed DOI

Lykke-Andersen S, Mapendano CK, Jensen TH. An ending is a new beginning: transcription termination supports re-initiation. Cell Cycle 2011; 10:863-5; PMID:21325895; http://dx.doi.org/10.4161/cc.10.6.14931 PubMed DOI

Xie W, Ling T, Zhou Y, Feng W, Zhu Q, Stunnenberg HG, Grummt I, Tao W. The chromatin remodeling complex NuRD establishes the poised state of rRNA genes characterized by bivalent histone modifications and altered nucleosome positions. Proc Natl Acad Sci USA 2012; 109(21):8161-6; PMID:22570494; http://dx.doi.org/10.1073/pnas.1201262109 PubMed DOI PMC

Diermeier SD, Németh A, Rehli M, Grummt I, Längst G. Chromatin-specific regulation of mammalian rDNA transcription by clustered TTF-I binding sites. PLoS Genet 2013; 9(9):e1003786; PMID:24068958; http://dx.doi.org/10.1371/journal.pgen.1003786 PubMed DOI PMC

Németh A, Längst G. Genome organization in and around the nucleolus. Trends Genet 2011; 27(4):149-56; http://dx.doi.org/10.1016/j.tig.2011.01.002 PubMed DOI

Li Z, Hann SR. Nucleophosmin is essential for c-Myc nucleolar localization and c-Myc-mediated rDNA transcription. Oncogene 2013; 32(15):1988-94; PMID:22665062; http://dx.doi.org/10.1038/onc.2012.227 PubMed DOI PMC

Dundr M, McNally JG, Cohen J, Misteli T. Quantitation of GFP-fusion proteins in single living cells. J Struct Biol 2002; 140(1-3):92-9; PMID:12490157; http://dx.doi.org/10.1016/S1047-8477(02)00521-X PubMed DOI

Schermelleh L, Solovei I, Zink D, Cremer T. Two-color fluorescence labeling of early and mid-to-late replicating chromatin in living cells. Chromosome Res 2001; 9:77-80; PMID:11272795; http://dx.doi.org/10.1023/A:1026799818566 PubMed DOI

Smirnov E, Borkovec J, Kováčik L, Svidenská S, Schröfel A, Skalníková M, Švindrych Z, Křížek P, Ovesný M, Hagen GM, et al.. Separation of replication and transcription domains in nucleoli. J Struct Biol 2014; 188(3):259-66; PMID:25450594; http://dx.doi.org/10.1016/j.jsb.2014.10.001 PubMed DOI

Bezrookove V, Smits R, Moeslein G, Fodde R, Tanke HJ, Raap AK, Darroudi F. Premature chromo-some condensation revisited: a novel chemical approach permits efficient cytogenetic analysis of cancers. Gen Chrom Cancer 2003; 38:177-186; http://dx.doi.org/10.1002/gcc.10268 PubMed DOI

Tosuji H, Fusetani N, Seki Y. Calyculin A causes the activation of histone H1 kinase and condensation of chromosomes in unfertilized sea urchin eggs independently of the maturation-promoting factor. Comp Biochem Physiol C. Toxicol Pharmacol 2003; 135:415-24 PubMed

Bui HT, Yamaoka E, Miyano T. Involvement of histone H3 (Ser10) phosphorylation in chromosome condensation without Cdc2 kinase and mitogen-activated protein kinase activation in pig oocytes. Biol Reprod 2004; 70:1843-51; PMID:14960481; http://dx.doi.org/10.1095/biolreprod.103.026070 PubMed DOI

Smirnov E, Kalmárová M, Koberna K, Zemanová Z, Malínský J, Masata M, Cvacková Z, Michalová K, Raska I. NORs and their transcription competence during the cell cycle. Folia Biol (Praha) 2006; 52(3):59-70; PMID:17089916 PubMed PMC

Sirri V, Roussel P, Hernandez-Verdun D. The mitotically phosphorylated form of the transcription termination factor TTF-1 is associated with the repressed rDNA transcription machinery. J Cell Sci 1999; 112:3259-68; PMID:10504331 PubMed

Conconi A, Widmer RM, Koller T, Sogo JM. Two different chromatin structures coexist in ribosomal RNA genes throughout the cell cycle. Cell 1989; 57:753-61; PMID:2720786; http://dx.doi.org/10.1016/0092-8674(89)90790-3 PubMed DOI

Kalmárová M, Kovácik L, Popov A, Testillano SP, Smirnov E. Asymmetrical distribution of the transcriptionally competent NORs in mitosis. J Struct Biol 2008; 163(1):40-4; http://dx.doi.org/10.1016/j.jsb.2008.04.002 PubMed DOI PMC

Discontinuous transcription of ribosomal DNA in human cells

PCNA is recruited to irradiated chromatin in late S-phase and is most pronounced in G2 phase of the cell cycle

Fluctuations of pol I and fibrillarin contents of the nucleoli