title: Systematic discovery of Xist RNA binding proteins.
abstract: Noncoding RNAs (ncRNAs) function with associated proteins to effect complex structural and regulatory outcomes. To reveal the composition and dynamics of specific noncoding RNA-protein complexes (RNPs) in vivo, we developed comprehensive identification of RNA binding proteins by mass spectrometry (ChIRP-MS). ChIRP-MS analysis of four ncRNAs captures key protein interactors, including a U1-specific link to the 3' RNA processing machinery. Xist, an essential lncRNA for X chromosome inactivation (XCI), interacts with 81 proteins from chromatin modification, nuclear matrix, and RNA remodeling pathways. The Xist RNA-protein particle assembles in two steps coupled with the transition from pluripotency to differentiation. Specific interactors include HnrnpK, which participates in Xist-mediated gene silencing and histone modifications but not Xist localization, and Drosophila Split ends homolog Spen, which interacts via the A-repeat domain of Xist and is required for gene silencing. Thus, Xist lncRNA engages with proteins in a modular and developmentally controlled manner to coordinate chromatin spreading and silencing.
authors.affiliation: Howard Hughes Medical Institute and Program in Epithelial Biology, Stanford University School of Medicine, Stanford, CA 94305, USA; Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore.
authors.forename: Ci
authors.initials: C
authors.lastname: Chu
authors.affiliation: Howard Hughes Medical Institute and Program in Epithelial Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
authors.forename: Qiangfeng Cliff
authors.initials: QC
authors.lastname: Zhang
authors.affiliation: Institut Curie, CNRS UMR3215, INSERM U934, 26 rue d'Ulm, Paris 75248, France.
authors.forename: Simão Teixeira
authors.initials: ST
authors.lastname: da Rocha
authors.affiliation: Howard Hughes Medical Institute and Program in Epithelial Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
authors.forename: Ryan A
authors.initials: RA
authors.lastname: Flynn
authors.affiliation: Howard Hughes Medical Institute and Program in Epithelial Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
authors.forename: Maheetha
authors.initials: M
authors.lastname: Bharadwaj
authors.affiliation: Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA.
authors.forename: J Mauro
authors.initials: JM
authors.lastname: Calabrese
authors.affiliation: Department of Genetics and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA.
authors.forename: Terry
authors.initials: T
authors.lastname: Magnuson
authors.affiliation: Institut Curie, CNRS UMR3215, INSERM U934, 26 rue d'Ulm, Paris 75248, France.
authors.forename: Edith
authors.initials: E
authors.lastname: Heard
authors.affiliation: Howard Hughes Medical Institute and Program in Epithelial Biology, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address: howchang@stanford.edu.
authors.forename: Howard Y
authors.initials: HY
authors.lastname: Chang
mesh_terms: D000818:Animals; D002843:Chromatin / Q000378:metabolism; D005260:Female; D020868:Gene Silencing; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D062085:RNA, Long Noncoding / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000032:analysis
chemical_list: D002843:Chromatin; D062085:RNA, Long Noncoding; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; C077885:XIST non-coding RNA
references.citation: Nature. 2013 Jul 18;499(7458):360-3
references.pmid: 23792564
references.citation: J Vis Exp. 2012;(61). pii: 3912. doi: 10.3791/3912
references.pmid: 22472705
references.citation: Nature. 2013 Dec 19;504(7480):465-9
references.pmid: 24162848
references.citation: Mol Cell. 2014 Jan 23;53(2):301-16
references.pmid: 24462204
references.citation: Nat Struct Mol Biol. 2014 Jun;21(6):569-71
references.pmid: 24837194
references.citation: Mol Cell. 2000 Apr;5(4):695-705
references.pmid: 10882105
references.citation: Nature. 2001 Jan 25;409(6819):539-42
references.pmid: 11206553
references.citation: Cell Stem Cell. 2012 Apr 6;10(4):425-39
references.pmid: 22482507
references.citation: Cell Stem Cell. 2012 May 4;10(5):583-94
references.pmid: 22560079
references.citation: Mol Cell Proteomics. 2012 Oct;11(10):1036-47
references.pmid: 22822199
references.citation: Cell. 2012 Jul 6;150(1):53-64
references.pmid: 22770214
references.citation: Annu Rev Biochem. 2012;81:145-66
references.pmid: 22663078
references.citation: Nature. 2012 Oct 25;490(7421):556-60
references.pmid: 23023127
references.citation: Cell. 2012 Nov 21;151(5):951-63
references.pmid: 23178118
references.citation: Mol Cell. 2013 Feb 21;49(4):601-4
references.pmid: 23438856
references.citation: Genes Dev. 2001 May 1;15(9):1140-51
references.pmid: 11331609
references.citation: Nat Genet. 2002 Feb;30(2):167-74
references.pmid: 11780141
references.citation: Nature. 2002 Sep 12;419(6903):182-5
references.pmid: 12226669
references.citation: Immunity. 2003 Feb;18(2):301-12
references.pmid: 12594956
references.citation: Science. 2003 Apr 4;300(5616):131-5
references.pmid: 12649488
references.citation: Nature. 1975 Aug 21;256(5519):640-2
references.pmid: 1152998
references.citation: Cell. 2014 Jun 5;157(6):1445-59
references.pmid: 24856970
references.citation: Nucleic Acids Res. 2014 Jun;42(10):6742-52
references.pmid: 24748666
references.citation: Cell Rep. 2014 Jun 12;7(5):1456-70
references.pmid: 24857660
references.citation: Mol Cell. 2014 Sep 4;55(5):791-802
references.pmid: 25155612
references.citation: RNA. 2015 Jan;21(1):135-43
references.pmid: 25411354
references.citation: Nat Struct Mol Biol. 2015 Jan;22(1):29-35
references.pmid: 25565030
references.citation: Genes Dev. 2006 Aug 15;20(16):2223-37
references.pmid: 16912274
references.citation: Chromosoma. 1982;85(2):275-86
references.pmid: 6180866
references.citation: J Embryol Exp Morphol. 1982 Oct;71:11-24
references.pmid: 6185603
references.citation: Nature. 1994 Mar 10;368(6467):154-6
references.pmid: 8139659
references.citation: Development. 1994 Dec;120(12):3529-36
references.pmid: 7529677
references.citation: J Cell Biol. 1996 Feb;132(3):259-75
references.pmid: 8636206
references.citation: Dev Biol. 1996 Dec 15;180(2):618-30
references.pmid: 8954732
references.citation: Nat Genet. 1999 Aug;22(4):323-4
references.pmid: 10431231
references.citation: Genes Dev. 1999 Aug 1;13(15):1924-35
references.pmid: 10444591
references.citation: Dev Cell. 2004 Nov;7(5):663-76
references.pmid: 15525528
references.citation: J Biol Chem. 2004 Dec 17;279(51):52812-5
references.pmid: 15509584
references.citation: Mol Cell. 2005 Nov 23;20(4):601-11
references.pmid: 16307923
references.citation: PLoS Genet. 2006 May;2(5):e66
references.pmid: 16680199
references.citation: Nucleic Acids Res. 2008 Jan;36(Database issue):D646-50
references.pmid: 17965090
references.citation: Methods Mol Biol. 2008;463:297-308
references.pmid: 18951174
references.citation: Science. 2008 Oct 31;322(5902):750-6
references.pmid: 18974356
references.citation: Annu Rev Genet. 2008;42:733-72
references.pmid: 18729722
references.citation: Development. 2009 Apr;136(7):1063-9
references.pmid: 19224983
references.citation: Nature. 2009 Mar 26;458(7237):475-80
references.pmid: 19325628
references.citation: Cell Stem Cell. 2009 Jun 5;4(6):493-8
references.pmid: 19497276
references.citation: Dev Cell. 2010 Sep 14;19(3):469-76
references.pmid: 20833368
references.citation: Nature. 2010 Dec 2;468(7324):664-8
references.pmid: 20881964
references.citation: Development. 2011 Dec;138(23):5049-55
references.pmid: 22069183
references.citation: Mol Cell. 2011 Nov 18;44(4):667-78
references.pmid: 21963238
references.citation: Cell. 2011 Dec 23;147(7):1498-510
references.pmid: 22196727
references.citation: Nature. 2012 Feb 9;482(7384):221-5
references.pmid: 22297846
references.citation: Cell. 2012 Feb 17;148(4):664-78
references.pmid: 22325148
references.citation: Science. 2013 Aug 16;341(6147):1237973
references.pmid: 23828888