Quantitative Mass Spectrometry and PAR-CLIP to Identify RNA-protein Interactions

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2012 Aug 14

PMID 22885304

Citations 31

Authors

Marion Scheibe

Falk Butter

Markus Hafner

Thomas Tuschl

Matthias Mann

Affiliations

Soon will be listed here.

Abstract

Systematic analysis of the RNA-protein interactome requires robust and scalable methods. We here show the combination of two completely orthogonal, generic techniques to identify RNA-protein interactions: PAR-CLIP reveals a collection of RNAs bound to a protein whereas SILAC-based RNA pull-downs identify a group of proteins bound to an RNA. We investigated binding sites for five different proteins (IGF2BP1-3, QKI and PUM2) exhibiting different binding patterns. We report near perfect agreement between the two approaches. Nevertheless, they are non-redundant, and ideally complement each other to map the RNA-protein interaction network.

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References

Nagaraj N, Wisniewski J, Geiger T, Cox J, Kircher M, Kelso J . Deep proteome and transcriptome mapping of a human cancer cell line. Mol Syst Biol. 2011; 7:548. PMC: 3261714. DOI: 10.1038/msb.2011.81. View

Licatalosi D, Mele A, Fak J, Ule J, Kayikci M, Chi S . HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature. 2008; 456(7221):464-9. PMC: 2597294. DOI: 10.1038/nature07488. View

Ule J, Jensen K, Ruggiu M, Mele A, Ule A, Darnell R . CLIP identifies Nova-regulated RNA networks in the brain. Science. 2003; 302(5648):1212-5. DOI: 10.1126/science.1090095. View

Takizawa P, DeRisi J, Wilhelm J, Vale R . Plasma membrane compartmentalization in yeast by messenger RNA transport and a septin diffusion barrier. Science. 2000; 290(5490):341-4. DOI: 10.1126/science.290.5490.341. View

Olsen J, Macek B, Lange O, Makarov A, Horning S, Mann M . Higher-energy C-trap dissociation for peptide modification analysis. Nat Methods. 2007; 4(9):709-12. DOI: 10.1038/nmeth1060. View

Hafner M, Landthaler M, Burger L, Khorshid M, Hausser J, Berninger P . Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell. 2010; 141(1):129-41. PMC: 2861495. DOI: 10.1016/j.cell.2010.03.009. View

Ward A, Bidet K, Yinglin A, Ler S, Hogue K, Blackstock W . Quantitative mass spectrometry of DENV-2 RNA-interacting proteins reveals that the DEAD-box RNA helicase DDX6 binds the DB1 and DB2 3' UTR structures. RNA Biol. 2011; 8(6):1173-86. PMC: 3256426. DOI: 10.4161/rna.8.6.17836. View

Tsai B, Wang X, Huang L, Waterman M . Quantitative profiling of in vivo-assembled RNA-protein complexes using a novel integrated proteomic approach. Mol Cell Proteomics. 2011; 10(4):M110.007385. PMC: 3069349. DOI: 10.1074/mcp.M110.007385. View

Bell A, Deutsch E, Au C, Kearney R, Beavis R, Sechi S . A HUPO test sample study reveals common problems in mass spectrometry-based proteomics. Nat Methods. 2009; 6(6):423-30. PMC: 2785450. DOI: 10.1038/nmeth.1333. View

10.

Weidensdorfer D, Stohr N, Baude A, Lederer M, Kohn M, Schierhorn A . Control of c-myc mRNA stability by IGF2BP1-associated cytoplasmic RNPs. RNA. 2008; 15(1):104-15. PMC: 2612774. DOI: 10.1261/rna.1175909. View

11.

Srisawat C, Engelke D . Streptavidin aptamers: affinity tags for the study of RNAs and ribonucleoproteins. RNA. 2001; 7(4):632-41. PMC: 1370116. DOI: 10.1017/s135583820100245x. View

12.

Dignam J, Lebovitz R, Roeder R . Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983; 11(5):1475-89. PMC: 325809. DOI: 10.1093/nar/11.5.1475. View

13.

Kishore S, Jaskiewicz L, Burger L, Hausser J, Khorshid M, Zavolan M . A quantitative analysis of CLIP methods for identifying binding sites of RNA-binding proteins. Nat Methods. 2011; 8(7):559-64. DOI: 10.1038/nmeth.1608. View

14.

Butter F, Scheibe M, Morl M, Mann M . Unbiased RNA-protein interaction screen by quantitative proteomics. Proc Natl Acad Sci U S A. 2009; 106(26):10626-31. PMC: 2697111. DOI: 10.1073/pnas.0812099106. View

15.

Cox J, Mann M . MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol. 2008; 26(12):1367-72. DOI: 10.1038/nbt.1511. View