title: Quantitative proteomic analysis reveals concurrent RNA-protein interactions and identifies new RNA-binding proteins in Saccharomyces cerevisiae.
abstract: A growing body of evidence supports the existence of an extensive network of RNA-binding proteins (RBPs) whose combinatorial binding affects the post-transcriptional fate of every mRNA in the cell-yet we still do not have a complete understanding of which proteins bind to mRNA, which of these bind concurrently, and when and where in the cell they bind. We describe here a method to identify the proteins that bind to RNA concurrently with an RBP of interest, using quantitative mass spectrometry combined with RNase treatment of affinity-purified RNA-protein complexes. We applied this method to the known RBPs Pab1, Nab2, and Puf3. Our method significantly enriched for known RBPs and is a clear improvement upon previous approaches in yeast. Our data reveal that some reported protein-protein interactions may instead reflect simultaneous binding to shared RNA targets. We also discovered more than 100 candidate RBPs, and we independently confirmed that 77% (23/30) bind directly to RNA. The previously recognized functions of the confirmed novel RBPs were remarkably diverse, and we mapped the RNA-binding region of one of these proteins, the transcriptional coactivator Mbf1, to a region distinct from its DNA-binding domain. Our results also provided new insights into the roles of Nab2 and Puf3 in post-transcriptional regulation by identifying other RBPs that bind simultaneously to the same mRNAs. While existing methods can identify sets of RBPs that interact with common RNA targets, our approach can determine which of those interactions are concurrent-a crucial distinction for understanding post-transcriptional regulation.
authors: Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305, USA.
authors: Daniel M
authors: DM
authors: Klass
authors: Marion
authors: M
authors: Scheibe
authors: Falk
authors: F
authors: Butter
authors: Gregory J
authors: GJ
authors: Hogan
authors: Matthias
authors: M
authors: Mann
authors: Patrick O
authors: PO
authors: Brown
mesh_terms: D016000:Cluster Analysis; D008954:Models, Biological; D029543:Nucleocytoplasmic Transport Proteins / Q000378:metabolism; D011485:Protein Binding; D040901:Proteomics / Q000379:methods*; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism
chemical_list: C080773:NAB2 protein, S cerevisiae; D029543:Nucleocytoplasmic Transport Proteins; C510155:PUF3 protein, S cerevisiae; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators
references: EMBO J. 2002 Apr 2;21(7):1800-10
references: 11927564
references: J Mol Biol. 2010 Jun 18;399(4):562-75
references: 20435044
references: J Cell Biol. 2008 Oct 20;183(2):223-39
references: 18936248
references: Genes Dev. 2005 Jan 1;19(1):90-103
references: 15630021
references: Proc Natl Acad Sci U S A. 1997 Oct 14;94(21):11456-60
references: 9326631
references: Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):5860-5
references: 11972065
references: RNA. 2011 May;17(5):835-42
references: 21460238
references: Methods Mol Biol. 2012;896:297-318
references: 22821533
references: Bioinformatics. 2011 Feb 1;27(3):431-2
references: 21149340
references: Genetics. 2010 Jun;185(2):513-22
references: 20382833
references: Nat Genet. 2003 Feb;33(2):155-61
references: 12524544
references: Nat Methods. 2004 Nov;1(2):119-26
references: 15782174
references: Biochem Soc Trans. 2010 Dec;38(6):1602-7
references: 21118134
references: J Cell Biol. 2011 Feb 21;192(4):583-98
references: 21339329
references: Nat Rev Mol Cell Biol. 2004 Oct;5(10):827-35
references: 15459663
references: PLoS Biol. 2008 Oct 28;6(10):e255
references: 18959479
references: PLoS One. 2008 Jun 04;3(6):e2293
references: 18523582
references: Cell. 2011 Dec 23;147(7):1473-83
references: 22196725
references: Nat Rev Mol Cell Biol. 2011 Jun;12(6):377-84
references: 21602906
references: Genome Biol. 2011;12(1):R3
references: 21232131
references: J Biol Chem. 2004 Mar 12;279(11):10756-64
references: 14679214
references: Cell. 2010 Nov 12;143(4):552-63
references: 21074047
references: Nature. 2008 Oct 30;455(7217):1251-4
references: 18820680
references: RNA. 2005 Apr;11(4):517-31
references: 15769879
references: J Biol Chem. 2008 Oct 3;283(40):27130-43
references: 18682389
references: Nature. 2003 Oct 16;425(6959):737-41
references: 14562106
references: Bioinformatics. 2007 Jan 15;23(2):257-8
references: 17098774
references: Science. 1988 May 13;240(4854):924-8
references: 2452485
references: Nat Rev Mol Cell Biol. 2006 Dec;7(12):952-8
references: 17139335
references: RNA. 2012 Mar;18(3):449-61
references: 22271760
references: Trends Cell Biol. 2011 Feb;21(2):104-12
references: 21115348
references: Proc Natl Acad Sci U S A. 2009 Jun 16;106(24):9613-8
references: 19482942
references: Nucleic Acids Res. 1980 Dec 11;8(23):5685-701
references: 7465424
references: Proc Natl Acad Sci U S A. 2009 Jun 30;106(26):10626-31
references: 19541640
references: Nat Biotechnol. 2008 Dec;26(12):1367-72
references: 19029910
references: Proc Natl Acad Sci U S A. 1988 Apr;85(7):2171-5
references: 3127826
references: Proc Natl Acad Sci U S A. 1987 Oct;84(19):6730-4
references: 3477805
references: Nature. 2006 Mar 30;440(7084):637-43
references: 16554755
references: Mol Cell. 2009 Nov 25;36(4):583-92
references: 19941819
references: Nucleic Acids Res. 2012 Jan;40(Database issue):D700-5
references: 22110037
references: Curr Biol. 2009 Dec 29;19(24):2114-20
references: 19962308
references: Science. 1987 Dec 11;238(4833):1570-3
references: 3685996
references: Mol Cell. 2012 Jun 8;46(5):674-90
references: 22681889
references: PLoS One. 2010 Nov 11;5(11):e15499
references: 21124907
references: Mol Cell Proteomics. 2007 Mar;6(3):439-50
references: 17200106
references: Nucleic Acids Res. 2012 Oct;40(19):9897-902
references: 22885304
references: Nature. 2006 Mar 30;440(7084):631-6
references: 16429126
references: Cell Logist. 2011 Jan;1(1):37-40
references: 21686103
references: RNA. 2005 Apr;11(4):383-93
references: 15703440
references: J Biol Chem. 2002 Mar 8;277(10):7752-60
references: 11779864
references: Cold Spring Harb Symp Quant Biol. 2007;72:157-65
references: 18419273
references: Genome Res. 2009 Feb;19(2):284-93
references: 19015324
references: Nat Rev Genet. 2007 Jul;8(7):533-43
references: 17572691
references: J Biol Chem. 1999 Oct 8;274(41):29031-7
references: 10506153
references: Methods. 2005 Dec;37(4):376-86
references: 16314267
references: Nature. 1997 Jan 23;385(6614):357-61
references: 9002523
references: Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3496-500
references: 7682707
references: Proc Natl Acad Sci U S A. 1989 Aug;86(15):5768-72
references: 2474819
references: Nucleic Acids Res. 1979 Feb;6(2):715-32
references: 424311
references: Mol Cell. 2007 Dec 14;28(5):850-9
references: 18082609
references: EMBO J. 2010 Jun 16;29(12):2026-36
references: 20453830
references: PLoS One. 2010 Sep 10;5(9):
references: 20844764
references: Cell. 2012 Jun 8;149(6):1393-406
references: 22658674
references: PLoS Biol. 2004 Mar;2(3):E79
references: 15024427
references: J Cell Biol. 2010 May 17;189(4):739-54
references: 20479470
references: Mol Cell. 2007 Jun 22;26(6):795-809
references: 17588515
references: FEBS Lett. 2008 Jun 18;582(14):1987-96
references: 18394429