Discontinuous transcription of ribosomal DNA in human cells
Language English Country United States Media electronic-ecollection
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
32119673
PubMed Central
PMC7051091
DOI
10.1371/journal.pone.0223030
PII: PONE-D-19-25095
Knihovny.cz E-resources
- MeSH
- Cell Nucleolus genetics MeSH
- Epithelial Cells metabolism MeSH
- Transcription, Genetic * MeSH
- HeLa Cells MeSH
- Kinetics MeSH
- Middle Aged MeSH
- Humans MeSH
- Limbus Corneae cytology MeSH
- Cadaver MeSH
- Cold Temperature MeSH
- DNA, Ribosomal genetics MeSH
- Ribosomes genetics MeSH
- RNA, Ribosomal genetics MeSH
- Aged MeSH
- Software MeSH
- Transfection MeSH
- Uridine analogs & derivatives immunology metabolism MeSH
- Uridine Triphosphate analogs & derivatives immunology metabolism MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Aged MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- 5-bromouridine triphosphate MeSH Browser
- 5-fluorouridine MeSH Browser
- DNA, Ribosomal MeSH
- RNA, Ribosomal MeSH
- Uridine MeSH
- Uridine Triphosphate MeSH
Numerous studies show that various genes in all kinds of organisms are transcribed discontinuously, i.e. in short bursts or pulses with periods of inactivity between them. But it remains unclear whether ribosomal DNA (rDNA), represented by multiple copies in every cell, is also expressed in such manner. In this work, we synchronized the pol I activity in the populations of tumour derived as well as normal human cells by cold block and release. Our experiments with 5-fluorouridine (FU) and BrUTP confirmed that the nucleolar transcription can be efficiently and reversibly arrested at +4°C. Then using special software for analysis of the microscopic images, we measured the intensity of transcription signal (incorporated FU) in the nucleoli at different time points after the release. We found that the ribosomal genes in the human cells are transcribed discontinuously with periods ranging from 45 min to 75 min. Our data indicate that the dynamics of rDNA transcription follows the undulating pattern, in which the bursts are alternated by periods of rare transcription events.
See more in PubMed
Smirnov E, Hornáček M, Vacík T, Cmarko D, Raška I. Discontinuous transcription. Nucleus. 2018;9(1):149–60. 10.1080/19491034.2017.1419112 PubMed Central PMCID: PMC5973254. PubMed DOI PMC
McKnight SL, Miller OL. Post-replicative nonribosomal transcription units in D. melanogaster embryos. Cell. 1979;17(3):551–63. 10.1016/0092-8674(79)90263-0 PubMed DOI
Bahar Halpern K, Tanami S, Landen S, Chapal M, Szlak L, Hutzler A, et al. Bursty gene expression in the intact mammalian liver. Mol Cell. 2015;58(1):147–56. 10.1016/j.molcel.2015.01.027 PubMed Central PMCID: PMC4500162. PubMed DOI PMC
Chubb JR, Trcek T, Shenoy SM, Singer RH. Transcriptional pulsing of a developmental gene. Curr Biol. 2006;16(10):1018–25. 10.1016/j.cub.2006.03.092 PubMed Central PMCID: PMC4764056. PubMed DOI PMC
Dar RD, Razooky BS, Singh A, Trimeloni TV, McCollum JM, Cox CD, et al. Transcriptional burst frequency and burst size are equally modulated across the human genome. Proc Natl Acad Sci USA. 2012;109(43):17454–9. 10.1073/pnas.1213530109 PubMed Central PMCID: PMC3491463. PubMed DOI PMC
Golding I, Paulsson J, Zawilski SM, Cox EC. Real-time kinetics of gene activity in individual bacteria. Cell. 2005;123(6):1025–36. 10.1016/j.cell.2005.09.031 PubMed DOI
Nicolas D, Phillips NE, Naef F. What shapes eukaryotic transcriptional bursting? Mol Biosyst. 2017;13(7):1280–90. 10.1039/c7mb00154a PubMed DOI
Nicolas D, Zoller B, Suter DM, Naef F. Modulation of transcriptional burst frequency by histone acetylation. Proc Natl Acad Sci USA. 2018;115(27):7153–8. 10.1073/pnas.1722330115 PubMed Central PMCID: PMC6142243. PubMed DOI PMC
Raj A, Peskin CS, Tranchina D, Vargas DY, Tyagi S. Stochastic mRNA synthesis in mammalian cells. PLoS Biol. 2006;4(10):e309 10.1371/journal.pbio.0040309 PubMed Central PMCID: PMC1563489. PubMed DOI PMC
Suter DM, Molina N, Gatfield D, Schneider K, Schibler U, Naef F. Mammalian genes are transcribed with widely different bursting kinetics. Science. 2011;332(6028):472–4. 10.1126/science.1198817 PubMed DOI
Suter DM, Molina N, Naef F, Schibler U. Origins and consequences of transcriptional discontinuity. Curr Opin Cell Biol. 2011;23(6):657–62. 10.1016/j.ceb.2011.09.004 PubMed DOI
Wang Y, Ni T, Wang W, Liu F. Gene transcription in bursting: a unified mode for realizing accuracy and stochasticity. Biol Rev Camb Philos Soc. 2018. 10.1111/brv.12452 PubMed DOI PMC
Li C, Cesbron F, Oehler M, Brunner M, Höfer T. Frequency modulation of transcriptional bursting enables sensitive and rapid gene regulation. Cell Syst. 2018;6(4):409–23.e11. 10.1016/j.cels.2018.01.012 PubMed DOI
Ochiai H, Hayashi T, Umeda M, Yoshimura M, Harada A, Shimizu Y, et al. Genome-wide analysis of transcriptional bursting-induced noise in mammalian cells. BioRxiv. 2019. 10.1101/736207 DOI
Coulon A, Ferguson ML, de Turris V, Palangat M, Chow CC, Larson DR. Kinetic competition during the transcription cycle results in stochastic RNA processing. elife. 2014;3 10.7554/eLife.03939 PubMed Central PMCID: PMC4210818. PubMed DOI PMC
Eldar A, Elowitz MB. Functional roles for noise in genetic circuits. Nature. 2010;467(7312):167–73. 10.1038/nature09326 PubMed Central PMCID: PMC4100692. PubMed DOI PMC
Raj A, van Oudenaarden A. Nature, nurture, or chance: stochastic gene expression and its consequences. Cell. 2008;135(2):216–26. 10.1016/j.cell.2008.09.050 PubMed Central PMCID: PMC3118044. PubMed DOI PMC
Chubb JR. Gene regulation: stable noise. Curr Biol. 2016;26(2):R61–R4. 10.1016/j.cub.2015.12.002 PubMed DOI
Bahar Halpern K, Itzkovitz S. Single molecule approaches for quantifying transcription and degradation rates in intact mammalian tissues. Methods. 2016;98:134–42. 10.1016/j.ymeth.2015.11.015 PubMed DOI
Femino AM, Fay FS, Fogarty K, Singer RH. Visualization of single RNA transcripts in situ. Science. 1998;280(5363):585–90. 10.1126/science.280.5363.585 PubMed DOI
Mueller F, Senecal A, Tantale K, Marie-Nelly H, Ly N, Collin O, et al. FISH-quant: automatic counting of transcripts in 3D FISH images. Nat Methods. 2013;10(4):277–8. 10.1038/nmeth.2406 PubMed DOI
Larsson AJM, Johnsson P, Hagemann-Jensen M, Hartmanis L, Faridani OR, Reinius B, et al. Genomic encoding of transcriptional burst kinetics. Nature. 2019;565(7738):251–4. 10.1038/s41586-018-0836-1 PubMed DOI PMC
Bensidoun P, Raymond P, Oeffinger M, Zenklusen D. Imaging single mRNAs to study dynamics of mRNA export in the yeast Saccharomyces cerevisiae. Methods. 2016;98:104–14. 10.1016/j.ymeth.2016.01.006 PubMed DOI
Bertrand E, Chartrand P, Schaefer M, Shenoy SM, Singer RH, Long RM. Localization of ASH1 mRNA particles in living yeast. Mol Cell. 1998;2(4):437–45. 10.1016/s1097-2765(00)80143-4 PubMed DOI
Henderson AS, Warburton D, Atwood KC. Location of ribosomal DNA in the human chromosome complement. Proc Natl Acad Sci USA. 1972;69(11):3394–8. 10.1073/pnas.69.11.3394 PubMed Central PMCID: PMC389778. PubMed DOI PMC
Long EO, Dawid IB. Repeated genes in eukaryotes. Annu Rev Biochem. 1980;49:727–64. 10.1146/annurev.bi.49.070180.003455 PubMed DOI
Moss T, Mars J-C, Tremblay MG, Sabourin-Felix M. The chromatin landscape of the ribosomal RNA genes in mouse and human. Chromosome Res. 2019;27(1–2):31–40. 10.1007/s10577-018-09603-9 PubMed DOI
Puvion-Dutilleul F, Bachellerie J-P, Puvion E. Nucleolar organization of HeLa cells as studied by in situ hybridization. Chromosoma. 1991;100(6):395–409. 10.1007/bf00337518 PubMed DOI
Raska I, Shaw PJ, Cmarko D. New insights into nucleolar architecture and activity. Int Rev Cytol. 2006;255:177–235. 10.1016/S0074-7696(06)55004-1 PubMed DOI
Sharifi S, Bierhoff H. Regulation of RNA polymerase I transcription in development, disease, and aging. Annu Rev Biochem. 2018;87:51–73. 10.1146/annurev-biochem-062917-012612 PubMed DOI
Bártová E, Horáková AH, Uhlírová R, Raska I, Galiová G, Orlova D, et al. Structure and epigenetics of nucleoli in comparison with non-nucleolar compartments. The Journal of Histochemistry and Cytochemistry. 2010;58(5):391–403. 10.1369/jhc.2009.955435 PubMed Central PMCID: PMC2857811. PubMed DOI PMC
Bersaglieri C, Santoro R. Genome Organization in and around the Nucleolus. Cells. 2019;8(6). 10.3390/cells8060579 PubMed Central PMCID: PMC6628108. 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;140(2):453–62. 10.1016/j.neuroscience.2006.02.030 PubMed DOI
Cmarko D, Smigova J, Minichova L, Popov A. Nucleolus: the ribosome factory. Histology and Histopathology. 2008;23(10):1291–8. 10.14670/HH-23.1291 PubMed DOI
Cmarko D, Verschure PJ, Rothblum LI, Hernandez-Verdun D, Amalric F, van Driel R, et al. Ultrastructural analysis of nucleolar transcription in cells microinjected with 5-bromo-UTP. Histochemistry and Cell Biology. 2000;113(3):181–7. 10.1007/s004180050437 PubMed DOI
Koberna K, Malínský J, Pliss A, Masata M, Vecerova J, Fialová M, et al. Ribosomal genes in focus: new transcripts label the dense fibrillar components and form clusters indicative of "Christmas trees" in situ. The Journal of Cell Biology. 2002;157(5):743–8. 10.1083/jcb.200202007 PubMed Central PMCID: PMC2173423. PubMed DOI PMC
Lam YW, Trinkle-Mulcahy L. New insights into nucleolar structure and function. F1000Prime Rep. 2015;7:48 10.12703/P7-48 PubMed Central PMCID: PMC4447046. PubMed DOI PMC
Scheer U, Benavente R. Functional and dynamic aspects of the mammalian nucleolus. Bioessays: News and Reviews in Molecular, Cellular and Developmental Biology. 1990;12(1):14–21. 10.1002/bies.950120104 PubMed DOI
Shaw PJ, McKeown PC. The Structure of rDNA Chromatin In: Olson MOJ, editor. The Nucleolus. New York, NY: Springer New York; 2011. p. 43–55.
Sirri V, Urcuqui-Inchima S, Roussel P, Hernandez-Verdun D. Nucleolus: the fascinating nuclear body. Histochemistry and Cell Biology. 2008;129(1):13–31. 10.1007/s00418-007-0359-6 PubMed Central PMCID: PMC2137947. PubMed DOI PMC
Raška I, Reimer G, Jarník M, Kostrouch Z, Raška K Jr. Does the synthesis of ribosomal RNA take place-within nucleolar fibrillar centers or dense fibrillar components? Biology of the cell. 1989;65(1):79–82. PubMed
Correll CC, Bartek J, Dundr M. The Nucleolus: A Multiphase Condensate Balancing Ribosome Synthesis and Translational Capacity in Health, Aging and Ribosomopathies. Cells. 2019;8(8):869. PubMed PMC
Cheutin T, O'Donohue M-F, Beorchia A, Vandelaer M, Kaplan H, Deféver B, et al. Three-dimensional organization of active rRNA genes within the nucleolus. Journal of Cell Science. 2002;115(Pt 16):3297–307. PubMed
Haaf T, Hayman DL, Schmid M. Quantitative determination of rDNA transcription units in vertebrate cells. Experimental Cell Research. 1991;193(1):78–86. 10.1016/0014-4827(91)90540-b PubMed DOI
Smirnov E, Borkovec J, Kováčik L, Svidenská S, Schröfel A, Skalníková M, et al. Separation of replication and transcription domains in nucleoli. J Struct Biol. 2014;188(3):259–66. 10.1016/j.jsb.2014.10.001 PubMed DOI
Haaf T, Ward DC. Inhibition of RNA polymerase II transcription causes chromatin decondensation, loss of nucleolar structure, and dispersion of chromosomal domains. Experimental Cell Research. 1996;224(1):163–73. 10.1006/excr.1996.0124 PubMed DOI
Tollervey D, Kiss T. Function and synthesis of small nucleolar RNAs. Curr Opin Cell Biol. 1997;9(3):337–42. 10.1016/s0955-0674(97)80005-1 PubMed DOI
Tollervey D, Lehtonen H, Jansen R, Kern H, Hurt EC. Temperature-sensitive mutations demonstrate roles for yeast fibrillarin in pre-rRNA processing, pre-rRNA methylation, and ribosome assembly. Cell. 1993;72(3):443–57. 10.1016/0092-8674(93)90120-f PubMed DOI
Iyer-Bierhoff A, Grummt I. Stop-and-Go: Dynamics of Nucleolar Transcription During the Cell Cycle. Epigenet Insights. 2019;12:2516865719849090. 10.1177/2516865719849090 PubMed Central PMCID: PMC6537492. PubMed DOI PMC
Pliss A, Kuzmin AN, Kachynski AV, Baev A, Berezney R, Prasad PN. Fluctuations and synchrony of RNA synthesis in nucleoli. Integrative Biology: Quantitative Biosciences from Nano to Macro. 2015;7(6):681–92. 10.1039/c5ib00008d PubMed DOI
Hornáček M, Kováčik L, Mazel T, Cmarko D, Bártová E, Raška I, et al. Fluctuations of pol I and fibrillarin contents of the nucleoli. Nucleus. 2017;8(4):421–32. 10.1080/19491034.2017.1306160 PubMed Central PMCID: PMC5597295. PubMed DOI PMC
Brejchova K, Trosan P, Studeny P, Skalicka P, Utheim TP, Bednar J, et al. Characterization and comparison of human limbal explant cultures grown under defined and xeno-free conditions. Experimental Eye Research. 2018;176:20–8. 10.1016/j.exer.2018.06.019 PubMed DOI
Stadnikova A, Trosan P, Skalicka P, Utheim TP, Jirsova K. Interleukin-13 maintains the stemness of conjunctival epithelial cell cultures prepared from human limbal explants. Plos One. 2019;14(2):e0211861 10.1371/journal.pone.0211861 PubMed Central PMCID: PMC6370187. PubMed DOI PMC
Dundr M, Hoffmann-Rohrer U, Hu Q, Grummt I, Rothblum LI, Phair RD, et al. A kinetic framework for a mammalian RNA polymerase in vivo. Science. 2002;298(5598):1623–6. 10.1126/science.1076164 PubMed DOI
Smirnov E, Hornáček M, Kováčik L, Mazel T, Schröfel A, Svidenská S, et al. Reproduction of the FC/DFC units in nucleoli. Nucleus. 2016;7(2):203–15. 10.1080/19491034.2016.1157674 PubMed DOI PMC
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(1):77–80. 10.1023/a:1026799818566 PubMed DOI
Golding I, Cox EC. RNA dynamics in live Escherichia coli cells. Proc Natl Acad Sci USA. 2004;101(31):11310–5. 10.1073/pnas.0404443101 PubMed Central PMCID: PMC509199. PubMed DOI PMC
Lionnet T, Czaplinski K, Darzacq X, Shav-Tal Y, Wells AL, Chao JA, et al. A transgenic mouse for in vivo detection of endogenous labeled mRNA. Nat Methods. 2011;8(2):165–70. 10.1038/nmeth.1551 PubMed Central PMCID: PMC3076588. PubMed DOI PMC