title: 4-thiouridine inhibits rRNA synthesis and causes a nucleolar stress response.
abstract: High concentrations (> 100 µM) of the ribonucleoside analog 4-thiouridine (4sU) is widely used in methods for RNA analysis like photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) and nascent messenger (m)RNA labeling (4sU-tagging). Here, we show that 4sU-tagging at low concentrations ≤ 10 µM can be used to measure production and processing of ribosomal (r)RNA. However, elevated concentrations of 4sU (> 50 µM), which are usually used for mRNA labeling experiments, inhibit production and processing of 47S rRNA. The inhibition of rRNA synthesis is accompanied by nucleoplasmic translocation of nucleolar nucleophosmin (NPM1), induction of the tumor suppressor p53, and inhibition of proliferation. We conclude that metabolic labeling of RNA by 4sU triggers a nucleolar stress response, which might influence the interpretation of results. Therefore, functional ribosome biogenesis, nucleolar integrity, and cell cycle should be addressed in 4sU labeling experiments.
authors.affiliation: Department of Molecular Epigenetics; Helmholtz Center Munich; Center for Integrated Protein Science Munich (CIPSM); Munich, Germany.
authors.forename: Kaspar
authors.initials: K
authors.lastname: Burger
authors.affiliation: Department of Molecular Epigenetics; Helmholtz Center Munich; Center for Integrated Protein Science Munich (CIPSM); Munich, Germany.
authors.forename: Bastian
authors.initials: B
authors.lastname: Mühl
authors.affiliation: Department of Molecular Epigenetics; Helmholtz Center Munich; Center for Integrated Protein Science Munich (CIPSM); Munich, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Kellner
authors.affiliation: Department of Molecular Epigenetics; Helmholtz Center Munich; Center for Integrated Protein Science Munich (CIPSM); Munich, Germany.
authors.forename: Michaela
authors.initials: M
authors.lastname: Rohrmoser
authors.affiliation: Department of Molecular Epigenetics; Helmholtz Center Munich; Center for Integrated Protein Science Munich (CIPSM); Munich, Germany.
authors.forename: Anita
authors.initials: A
authors.lastname: Gruber-Eber
authors.affiliation: Institute for Informatics, Teaching and Research Unit Bioinformatics; Ludwig-Maximilians-University Munich; Munich, Germany.
authors.forename: Lukas
authors.initials: L
authors.lastname: Windhager
authors.affiliation: Institute for Informatics, Teaching and Research Unit Bioinformatics; Ludwig-Maximilians-University Munich; Munich, Germany.
authors.forename: Caroline C
authors.initials: CC
authors.lastname: Friedel
authors.affiliation: Department of Medicine; Addenbrooke's Hospital; University of Cambridge; Cambridge, UK.
authors.forename: Lars
authors.initials: L
authors.lastname: Dölken
authors.affiliation: Department of Molecular Epigenetics; Helmholtz Center Munich; Center for Integrated Protein Science Munich (CIPSM); Munich, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Eick
mesh_terms: D000818:Animals; D002453:Cell Cycle; D002466:Cell Nucleolus / Q000502:physiology; D051379:Mice; D000090243:Nucleophosmin; D012323:RNA Processing, Post-Transcriptional / Q000187:drug effects*; D012335:RNA, Ribosomal / Q000235:genetics*; D012270:Ribosomes / Q000187:drug effects; D013194:Staining and Labeling / Q000379:methods*; D013312:Stress, Physiological; D013891:Thiouridine / Q000009:adverse effects* / Q000494:pharmacology
chemical_list: C000715570:Npm1 protein, mouse; D012335:RNA, Ribosomal; D000090243:Nucleophosmin; D013891:Thiouridine
keywords: 4-thiouridine; RNA labeling; nucleolar stress; nucleophosmin; p53; rRNA processing; ribosomal RNA
references.citation: Mol Cell. 2011 Nov 18;44(4):660-6
references.pmid: 22099312
references.citation: Proc Natl Acad Sci U S A. 2007 Apr 10;104(15):6164-9
references.pmid: 17405863
references.citation: Mol Cell. 2010 Oct 22;40(2):216-27
references.pmid: 20965417
references.citation: Cell. 1993 Jun 18;73(6):1233-45
references.pmid: 8513505
references.citation: Mol Cell. 2003 Nov;12(5):1151-64
references.pmid: 14636574
references.citation: Cancer Lett. 1996 Feb 27;100(1-2):191-7
references.pmid: 8620441
references.citation: J Cell Biol. 2011 Sep 5;194(5):689-703
references.pmid: 21893597
references.citation: Curr Protoc Cell Biol. 2008 Jun;Chapter 22:Unit 22.11
references.pmid: 18551418
references.citation: Proc Natl Acad Sci U S A. 1967 Nov;58(5):1984-90
references.pmid: 5237493
references.citation: Genome Res. 2012 Oct;22(10):2031-42
references.pmid: 22539649
references.citation: Nature. 2011 May 19;473(7347):337-42
references.pmid: 21593866
references.citation: J Cell Biol. 2005 Aug 1;170(3):367-78
references.pmid: 16043514
references.citation: Nucleic Acids Res. 2007;35(2):550-8
references.pmid: 17169984
references.citation: J Biol Chem. 2013 Jul 19;288(29):21173-21183
references.pmid: 23744076
references.citation: Cancer Cell. 2004 May;5(5):465-75
references.pmid: 15144954
references.citation: Wiley Interdiscip Rev RNA. 2010 Nov-Dec;1(3):415-31
references.pmid: 21956940
references.citation: Mol Cell Biol. 1997 Jul;17(7):3702-13
references.pmid: 9199304
references.citation: Nat Biotechnol. 2005 Feb;23(2):232-7
references.pmid: 15685165
references.citation: Mol Biosyst. 2009 Nov;5(11):1271-8
references.pmid: 19823741
references.citation: J Biol Chem. 2010 Apr 16;285(16):12416-25
references.pmid: 20159984
references.citation: Curr Opin Cell Biol. 2002 Jun;14(3):313-8
references.pmid: 12067653
references.citation: J Biol Chem. 2004 Oct 22;279(43):44475-82
references.pmid: 15308643
references.citation: Cancer Cell. 2003 Jun;3(6):577-87
references.pmid: 12842086
references.citation: Nucleic Acids Res. 2009 Sep;37(17):e115
references.pmid: 19561200
references.citation: Cell Mol Life Sci. 2008 Aug;65(15):2334-59
references.pmid: 18408888
references.citation: Cell. 2010 Apr 2;141(1):129-41
references.pmid: 20371350
references.citation: Mol Cell Biol. 2004 Sep;24(17):7654-68
references.pmid: 15314173
references.citation: RNA. 2008 Sep;14(9):1959-72
references.pmid: 18658122
references.citation: Cancer Res. 1994 Feb 1;54(3):632-6
references.pmid: 8306322
references.citation: Mol Cell Biol. 2010 Jun;30(12):2947-56
references.pmid: 20404093
references.citation: Neoplasia. 2000 May-Jun;2(3):208-25
references.pmid: 10935507
references.citation: Nat Biotechnol. 2011 May;29(5):436-42
references.pmid: 21516085
references.citation: EMBO J. 1999 Mar 15;18(6):1660-72
references.pmid: 10075936
references.citation: Exp Cell Res. 2001 Oct 15;270(1):66-77
references.pmid: 11597128
references.citation: Nat Rev Genet. 2012 Jan 18;13(2):77-83
references.pmid: 22251872
references.citation: EMBO J. 2004 Jun 16;23(12):2402-12
references.pmid: 15152193