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PMID: 27768875 Published · ppublish English Journal Article Research Support, U.S. Gov't, Non-P.H.S. Research Support, Non-U.S. Gov't Research Support, N.I.H., Extramural

High-Resolution Mapping of RNA-Binding Regions in the Nuclear Proteome of Embryonic Stem Cells.

Molecular cell ·Vol. 64 ·No. 2 ·2016-00-20 ·Pages 416-430

He C, Sidoli S, Warneford-Thomson R, Tatomer DC, Wilusz JE, Garcia BA, Bonasio R

Abstract

Interactions between noncoding RNAs and chromatin proteins play important roles in gene regulation, but the molecular details of most of these interactions are unknown. Using protein-RNA photocrosslinking and mass spectrometry on embryonic stem cell nuclei, we identified and mapped, at peptide resolution, the RNA-binding regions in ∼800 known and previously unknown RNA-binding proteins, many of which are transcriptional regulators and chromatin modifiers. In addition to known RNA-binding motifs, we detected several protein domains previously unknown to function in RNA recognition, as well as non-annotated and/or disordered regions, suggesting that many functional protein-RNA contacts remain unexplored. We identified RNA-binding regions in several chromatin regulators, including TET2, and validated their ability to bind RNA. Thus, proteomic identification of RNA-binding regions (RBR-ID) is a powerful tool to map protein-RNA interactions and will allow rational design of mutants to dissect their function at a mechanistic level.

MeSH Terms
Animals Binding Sites Chromatin/chemistry,metabolism,radiation effects Gene Expression HEK293 Cells Humans Mice Mouse Embryonic Stem Cells/cytology,metabolism,radiation effects Nuclear Proteins/chemistry,genetics,metabolism Nucleic Acid Conformation Peptide Mapping/methods Photochemical Processes Protein Binding Protein Interaction Domains and Motifs Protein Structure, Secondary Proteome/chemistry,genetics,metabolism Proteomics/methods RNA, Untranslated/chemistry,genetics,metabolism RNA-Binding Proteins/chemistry,genetics,metabolism Ultraviolet Rays
Chemicals
Chromatin Nuclear Proteins Proteome RNA, Untranslated RNA-Binding Proteins
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
He Chongsheng
Epigenetics Program, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Sidoli Simone
Epigenetics Program, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Warneford-Thomson Robert
Epigenetics Program, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Graduate Group in Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Tatomer Deirdre C
Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Wilusz Jeremy E
Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Garcia Benjamin A
Epigenetics Program, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Bonasio Roberto
Epigenetics Program, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address: [email protected].
References (85)
85 references, click to expand
  1. Circular RNAs: Unexpected outputs of many protein-coding genes.
    RNA Biol. 2016 Aug 29;:1-11 PMID: 27571848
  2. Structure-function studies of FMRP RGG peptide recognition of an RNA duplex-quadruplex junction.
    Nat Struct Mol Biol. 2011 Jun 05;18(7):796-804 PMID: 21642970
  3. Formation of triple-helical structures by the 3'-end sequences of MALAT1 and MENβ noncoding RNAs.
    Proc Natl Acad Sci U S A. 2012 Nov 20;109(47):19202-7 PMID: 23129630
  4. Structural basis of pyrimidine specificity in the MS2 RNA hairpin-coat-protein complex.
    RNA. 2001 Nov;7(11):1616-27 PMID: 11720290
  5. PRC2 binds active promoters and contacts nascent RNAs in embryonic stem cells.
    Nat Struct Mol Biol. 2013 Nov;20(11):1258-64 PMID: 24141703
  6. Jpx RNA activates Xist by evicting CTCF.
    Cell. 2013 Jun 20;153(7):1537-51 PMID: 23791181
  7. RNA biochemistry. Transcriptome-wide distribution and function of RNA hydroxymethylcytosine.
    Science. 2016 Jan 15;351(6270):282-5 PMID: 26816380
  8. Unique features of long non-coding RNA biogenesis and function.
    Nat Rev Genet. 2016 Jan;17(1):47-62 PMID: 26666209
  9. NF45 dimerizes with NF90, Zfr and SPNR via a conserved domain that has a nucleotidyltransferase fold.
    Nucleic Acids Res. 2012 Oct;40(18):9356-68 PMID: 22833610
  10. SAP - a putative DNA-binding motif involved in chromosomal organization.
    Trends Biochem Sci. 2000 Mar;25(3):112-4 PMID: 10694879
  11. SUMOylation promotes de novo targeting of HP1α to pericentric heterochromatin.
    Nat Genet. 2011 Mar;43(3):220-7 PMID: 21317888
  12. Insights into RNA biology from an atlas of mammalian mRNA-binding proteins.
    Cell. 2012 Jun 8;149(6):1393-406 PMID: 22658674
  13. 4-Thiouridine photosensitized RNA-protein crosslinking in mammalian cells.
    Biochem Biophys Res Commun. 1986 Dec 15;141(2):847-54 PMID: 2432896
  14. Evolution of the SNF2 family of proteins: subfamilies with distinct sequences and functions.
    Nucleic Acids Res. 1995 Jul 25;23(14):2715-23 PMID: 7651832
  15. RNA-mediated epigenetic regulation of gene expression.
    Nat Rev Genet. 2015 Feb;16(2):71-84 PMID: 25554358
  16. Long noncoding RNAs: functional surprises from the RNA world.
    Genes Dev. 2009 Jul 1;23(13):1494-504 PMID: 19571179
  17. Linking RNA biology to lncRNAs.
    Genome Res. 2015 Oct;25(10):1456-65 PMID: 26430155
  18. Widespread RNA binding by chromatin-associated proteins.
    Genome Biol. 2016 Feb 16;17 :28 PMID: 26883116
  19. Serial interactome capture of the human cell nucleus.
    Nat Commun. 2016 Apr 04;7:11212 PMID: 27040163
  20. Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5' splice site recognition.
    Elife. 2015 Jan 02;4:null PMID: 25555158
  21. Protein-RNA interactions: a structural analysis.
    Nucleic Acids Res. 2001 Feb 15;29(4):943-54 PMID: 11160927
  22. Biogenesis of multisubunit RNA polymerases.
    Trends Biochem Sci. 2012 Mar;37(3):99-105 PMID: 22260999
  23. RNA modulation of the human DNA methyltransferase 3A.
    Nucleic Acids Res. 2012 Sep 1;40(17):8550-7 PMID: 22730298
  24. RNA-binding protein L1TD1 interacts with LIN28 via RNA and is required for human embryonic stem cell self-renewal and cancer cell proliferation.
    Stem Cells. 2012 Mar;30(3):452-60 PMID: 22162396
  25. Long noncoding RNA as modular scaffold of histone modification complexes.
    Science. 2010 Aug 6;329(5992):689-93 PMID: 20616235
  26. Promiscuous RNA binding by Polycomb repressive complex 2.
    Nat Struct Mol Biol. 2013 Nov;20(11):1250-7 PMID: 24077223
  27. RNA emerging from the active site of RNA polymerase II interacts with the Rpb7 subunit.
    Nat Struct Mol Biol. 2006 Jan;13(1):49-54 PMID: 16327806
  28. Comprehensive Identification of RNA-Binding Domains in Human Cells.
    Mol Cell. 2016 Aug 18;63(4):696-710 PMID: 27453046
  29. Laser cross-linking of nucleic acids to proteins. Methodology and first applications to the phage T4 DNA replication system.
    J Biol Chem. 1986 Mar 15;261(8):3512-8 PMID: 3949776
  30. Intra-RNA cross-linking in Escherichia coli 30S ribosomal subunits: selective isolation of cross-linked products by hybridization to specific cDNA fragments.
    Nucleic Acids Res. 1988 May 25;16(10):4315-29 PMID: 2837729
  31. Systematic identification of novel protein domain families associated with nuclear functions.
    Genome Res. 2002 Jan;12(1):47-56 PMID: 11779830
  32. The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression.
    FEBS J. 2005 May;272(9):2118-31 PMID: 15853797
  33. A triple helix stabilizes the 3' ends of long noncoding RNAs that lack poly(A) tails.
    Genes Dev. 2012 Nov 1;26(21):2392-407 PMID: 23073843
  34. Coordinated methyl and RNA binding is required for heterochromatin localization of mammalian HP1alpha.
    EMBO Rep. 2002 Oct;3(10):975-81 PMID: 12231507
  35. Enhancer RNAs and regulated transcriptional programs.
    Trends Biochem Sci. 2014 Apr;39(4):170-82 PMID: 24674738
  36. Structure and mechanism of the RNA polymerase II transcription machinery.
    Nat Struct Mol Biol. 2004 May;11(5):394-403 PMID: 15114340
  37. Interaction of the RNA-binding domain of the hnRNP C proteins with RNA.
    EMBO J. 1992 Sep;11(9):3289-95 PMID: 1380452
  38. Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression.
    Proc Natl Acad Sci U S A. 2009 Jul 14;106(28):11667-72 PMID: 19571010
  39. Chromodomains are protein-RNA interaction modules.
    Nature. 2000 Sep 21;407(6802):405-9 PMID: 11014199
  40. D²P²: database of disordered protein predictions.
    Nucleic Acids Res. 2013 Jan;41(Database issue):D508-16 PMID: 23203878
  41. The RNA-binding protein repertoire of embryonic stem cells.
    Nat Struct Mol Biol. 2013 Sep;20(9):1122-30 PMID: 23912277
  42. The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3.
    Nature. 2015 May 14;521(7551):232-6 PMID: 25915022
  43. TETonic shift: biological roles of TET proteins in DNA demethylation and transcription.
    Nat Rev Mol Cell Biol. 2013 Jun;14(6):341-56 PMID: 23698584
  44. Regulation of transcription by long noncoding RNAs.
    Annu Rev Genet. 2014;48:433-55 PMID: 25251851
  45. RNA-binding proteins in Mendelian disease.
    Trends Genet. 2013 May;29(5):318-27 PMID: 23415593
  46. Ultraviolet light-induced crosslinking of mRNA to proteins.
    Nucleic Acids Res. 1979 Feb;6(2):715-32 PMID: 424311
  47. Interactions with RNA direct the Polycomb group protein SCML2 to chromatin where it represses target genes.
    Elife. 2014 Jul 01;3:e02637 PMID: 24986859
  48. Short RNAs are transcribed from repressed polycomb target genes and interact with polycomb repressive complex-2.
    Mol Cell. 2010 Jun 11;38(5):675-88 PMID: 20542000
  49. A long noncoding RNA associated with susceptibility to celiac disease.
    Science. 2016 Apr 1;352(6281):91-5 PMID: 27034373
  50. Prp43p contains a processive helicase structural architecture with a specific regulatory domain.
    EMBO J. 2010 Jul 7;29(13):2194-204 PMID: 20512115
  51. Interactions between JARID2 and noncoding RNAs regulate PRC2 recruitment to chromatin.
    Mol Cell. 2014 Jan 23;53(2):290-300 PMID: 24374312
  52. Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins.
    Nature. 2001 Mar 1;410(6824):116-20 PMID: 11242053
  53. Photo-cross-linking and high-resolution mass spectrometry for assignment of RNA-binding sites in RNA-binding proteins.
    Nat Methods. 2014 Oct;11(10):1064-70 PMID: 25173706
  54. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.
    Nat Biotechnol. 2008 Dec;26(12):1367-72 PMID: 19029910
  55. Locus-specific targeting to the X chromosome revealed by the RNA interactome of CTCF.
    Mol Cell. 2015 Jan 22;57(2):361-75 PMID: 25578877
  56. Aspartate-glutamate-alanine-histidine box motif (DEAH)/RNA helicase A helicases sense microbial DNA in human plasmacytoid dendritic cells.
    Proc Natl Acad Sci U S A. 2010 Aug 24;107(34):15181-6 PMID: 20696886
  57. Nascent RNA interaction keeps PRC2 activity poised and in check.
    Genes Dev. 2014 Sep 15;28(18):1983-8 PMID: 25170018
  58. IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content.
    Bioinformatics. 2005 Aug 15;21(16):3433-4 PMID: 15955779
  59. Nuclear factor 90 is a substrate and regulator of the eukaryotic initiation factor 2 kinase double-stranded RNA-activated protein kinase.
    J Biol Chem. 2001 Aug 31;276(35):32522-30 PMID: 11438540
  60. The RNA-binding proteomes from yeast to man harbour conserved enigmRBPs.
    Nat Commun. 2015 Dec 03;6:10127 PMID: 26632259
  61. Crystal structure of an RNA bacteriophage coat protein-operator complex.
    Nature. 1994 Oct 13;371(6498):623-6 PMID: 7523953
  62. Genome-wide identification of polycomb-associated RNAs by RIP-seq.
    Mol Cell. 2010 Dec 22;40(6):939-53 PMID: 21172659
  63. Tet-mediated formation of 5-hydroxymethylcytosine in RNA.
    J Am Chem Soc. 2014 Aug 20;136(33):11582-5 PMID: 25073028
  64. Genome regulation by long noncoding RNAs.
    Annu Rev Biochem. 2012;81:145-66 PMID: 22663078
  65. Peptide Labeling Using Isobaric Tagging Reagents for Quantitative Phosphoproteomics.
    Methods Mol Biol. 2016;1355:53-70 PMID: 26584918
  66. Chromosomes. A comprehensive Xist interactome reveals cohesin repulsion and an RNA-directed chromosome conformation.
    Science. 2015 Jul 17;349(6245):null PMID: 26089354
  67. The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics.
    Proc Natl Acad Sci U S A. 2015 Jun 9;112(23):7189-94 PMID: 26015579
  68. Systematic discovery of Xist RNA binding proteins.
    Cell. 2015 Apr 9;161(2):404-16 PMID: 25843628
  69. RBPDB: a database of RNA-binding specificities.
    Nucleic Acids Res. 2011 Jan;39(Database issue):D301-8 PMID: 21036867
  70. Phosphorylation of the PRC2 component Ezh2 is cell cycle-regulated and up-regulates its binding to ncRNA.
    Genes Dev. 2010 Dec 1;24(23 ):2615-20 PMID: 21123648
  71. How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers.
    Nat Struct Mol Biol. 2007 Nov;14(11):1025-40 PMID: 17984965
  72. The three-dimensional structures of two complexes between recombinant MS2 capsids and RNA operator fragments reveal sequence-specific protein-RNA interactions.
    J Mol Biol. 1997 Aug 1;270(5):724-38 PMID: 9245600
  73. The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts.
    Mol Cell. 2012 Jun 8;46(5):674-90 PMID: 22681889
  74. The double-stranded RNA-binding motif, a versatile macromolecular docking platform.
    FEBS J. 2005 May;272(9):2109-17 PMID: 15853796
  75. The interaction of PRC2 with RNA or chromatin is mutually antagonistic.
    Genome Res. 2016 Jul;26(7):896-907 PMID: 27197219
  76. ATRX directs binding of PRC2 to Xist RNA and Polycomb targets.
    Cell. 2014 Nov 6;159(4):869-83 PMID: 25417162
  77. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.
    Nat Protoc. 2009;4(1):44-57 PMID: 19131956
  78. Toward a consensus on the binding specificity and promiscuity of PRC2 for RNA.
    Mol Cell. 2015 Feb 5;57(3):552-8 PMID: 25601759
  79. CTCF regulates the human p53 gene through direct interaction with its natural antisense transcript, Wrap53.
    Genes Dev. 2014 Apr 1;28(7):723-34 PMID: 24696455
  80. RNA-binding proteins: modular design for efficient function.
    Nat Rev Mol Cell Biol. 2007 Jun;8(6):479-90 PMID: 17473849
  81. Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP.
    Cell. 2010 Apr 2;141(1):129-41 PMID: 20371350
  82. KH domain: one motif, two folds.
    Nucleic Acids Res. 2001 Feb 1;29(3):638-43 PMID: 11160884
  83. UniProt: a hub for protein information.
    Nucleic Acids Res. 2015 Jan;43(Database issue):D204-12 PMID: 25348405
  84. Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs.
    Cell. 2007 Jun 29;129(7):1311-23 PMID: 17604720
  85. Crystal structure of human spliceosomal U1 snRNP at 5.5 A resolution.
    Nature. 2009 Mar 26;458(7237):475-80 PMID: 19325628
Article Info
Journal
Molecular cell
Abbr.
Mol Cell
ISSN
1097-4164
DOI
10.1016/j.molcel.2016.09.034
Email
[email protected]
Published
2016-00-20
Pages
416-430
Language
English
Region
United States
NLM ID
9802571
PMCID
PMC5222606
Subset
IM
Grants
NIGMS NIH HHS · R00 GM104166 · United States
NIGMS NIH HHS · R01 GM110174 · United States
NIGMS NIH HHS · R35 GM119735 · United States
NIGMS NIH HHS · T32 GM008216 · United States
Corrections
CommentIn
PMID: 27814488
External Links
PubMed source DOI: 10.1016/j.molcel.2016.09.034 PMC Full Text View journal details
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