Global analysis of RNA-binding protein dynamics by comparative and enhanced RNA interactome capture.

Nature Protocols
Joel I Perez-PerriAlfredo Castello

Abstract

Interactions between RNA-binding proteins (RBPs) and RNAs are critical to cell biology. However, methods to comprehensively and quantitatively assess these interactions within cells were lacking. RNA interactome capture (RIC) uses in vivo UV crosslinking, oligo(dT) capture, and proteomics to identify RNA-binding proteomes. Recent advances have empowered RIC to quantify RBP responses to biological cues such as metabolic imbalance or virus infection. Enhanced RIC exploits the stronger binding of locked nucleic acid (LNA)-containing oligo(dT) probes to poly(A) tails to maximize RNA capture selectivity and efficiency, profoundly improving signal-to-noise ratios. The subsequent analytical use of SILAC and TMT proteomic approaches, together with high-sensitivity sample preparation and tailored statistical data analysis, substantially improves RIC's quantitative accuracy and reproducibility. This optimized approach is an extension of the original RIC protocol. It takes 3 d plus 2 weeks for proteomics and data analysis and will enable the study of RBP dynamics under different physiological and pathological conditions.

References

Feb 1, 1979·Nucleic Acids Research·J R Greenberg
Jul 11, 2006·Journal of Proteome Research·Jeroen KrijgsveldAlbert J R Heck
Apr 5, 2007·Nature Protocols·Shao-En Ong, Matthias Mann
Jul 4, 2008·Trends in Genetics : TIG·Kiven E LukongStéphane Richard
Apr 21, 2009·Nature Methods·Jacek R WiśniewskiMatthias Mann
Jul 6, 2010·Nature Structural & Molecular Biology·Julian KönigJernej Ule
Jan 25, 2011·Journal of Proteome Research·Jürgen CoxMatthias Mann
Jun 5, 2012·Cell·Alfredo CastelloMatthias W Hentze
Jun 12, 2012·Molecular Cell·Alexander G BaltzMarkus Landthaler
Oct 23, 2012·Journal of Proteomics·A F Maarten AltelaarShabaz Mohammed
Feb 16, 2013·Nature Protocols·Alfredo CastelloMatthias W Hentze
Feb 19, 2013·Trends in Genetics : TIG·Alfredo CastelloThomas Preiss
Aug 6, 2013·Nature Structural & Molecular Biology·S Chul KwonV Narry Kim
Nov 5, 2013·Epigenetics : Official Journal of the DNA Methylation Society·Alan L Jiao, Frank J Slack
Mar 26, 2014·RNA·Claudia StreinAlfredo Castello
Nov 2, 2014·Molecular Systems Biology·Christopher S HughesJeroen Krijgsveld
Jan 22, 2015·Nucleic Acids Research·Matthew E RitchieGordon K Smyth
Oct 27, 2015·Seminars in Cell & Developmental Biology·Pooja Kumari, Karuna Sampath
Nov 2, 2015·Trends in Endocrinology and Metabolism : TEM·Alfredo CastelloThomas Preiss
Nov 12, 2015·Analytical Chemistry·Baby Rorielyn Tacla Dimayacyac-EsletaYu-Ju Chen
Nov 26, 2015·Nature Structural & Molecular Biology·Ana M Matia-GonzálezAndré P Gerber
Dec 4, 2015·Nature Communications·Benedikt M BeckmannMatthias W Hentze
Apr 7, 2016·Cell Communication and Signaling : CCS·Aino I JärvelinAlfredo Castello
May 20, 2016·Genome Research·Hans-Hermann WesselsMarkus Landthaler
Jun 10, 2016·Molecular & Cellular Proteomics : MCP·Anke LiepeltAntje Ostareck-Lederer
Jul 6, 2016·Nature Communications·Vasiliy O SysoevAnne Ephrussi
Jul 7, 2016·Genome Biology·Evelien M BunnikKarine G Le Roch
Jul 28, 2016·Molecular Cell·Alfredo CastelloMatthias W Hentze
Oct 13, 2016·The Plant Cell·Marlene ReichelAnthony A Millar
Nov 4, 2016·Nature Protocols·Stefka TyanovaJuergen Cox
Jan 28, 2017·Cell·Martina U MuckenthalerBruno Galy
Apr 7, 2017·Genome Research·Vladimir DespicKarla M Neugebauer
May 10, 2017·RNA·Birgit RogellMatthias W Hentze
Sep 28, 2017·Journal of Proteome Research·Malte SielaffUte Distler
Nov 21, 2017·Wiley Interdisciplinary Reviews. RNA·Abul ArifPaul L Fox
Dec 8, 2017·Current Opinion in Genetics & Development·Samantha MooreAlfredo Castello
Jan 18, 2018·Nature Reviews. Molecular Cell Biology·Matthias W HentzeThomas Preiss
Feb 13, 2018·Nature Methods·Xichen BaoMiguel A Esteban

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Citations

Mar 9, 2021·Current Genetics·Ofri Levi, Yoav S Arava
Jun 1, 2021·Frontiers in Molecular Biosciences·Carlos H Vieira-Vieira, Matthias Selbach
Nov 5, 2021·Nature Reviews. Nephrology·Lisa SeufertRoman-Ulrich Müller

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Methods Mentioned

BETA
eCLIP
Protein Assay
PCR
irradiating
electrophoresis
acetylation
RNAseq
CLIP
RIP

Software Mentioned

OOPS
Andromeda
DAVID
limma
R packages
PAR
TMT
MaxQuant
R
eRIC

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