Wordprocessing

System-wide identification of RNA-binding proteins by interactome capture

Abstract:

Owing to their preeminent biological functions, the repertoire of expressed RNA-binding proteins (RBPs) and their activity states are highly informative about cellular systems. We have developed a novel and unbiased technique, called interactome capture, for identifying the active RBPs of cultured cells. By making use of in vivo UV cross-linking of RBPs to polyadenylated RNAs, covalently bound proteins are captured with oligo(dT) magnetic beads. After stringent washes, the mRNA interactome is determined by quantitative mass spectrometry (MS). The protocol takes 3 working days for analysis of single proteins by western blotting, and about 2 weeks for the determination of complete cellular mRNA interactomes by MS. The most important advantage of interactome capture over other in vitro and in silico approaches is that only RBPs bound to RNA in a physiological environment are identified. When applied to HeLa cells, interactome capture revealed hundreds of novel RBPs. Interactome capture can also be broadly used to compare different biological states, including metabolic stress, cell cycle, differentiation, development or the response to drugs.

23411631

Projects: B1.3: Linking modulation of iron metabolism with the impact of macrophag...

Nat Protoc
Nat Protoc 8(3): 491-500
14th Feb 2013

Alfredo Castello, Rastislav Horos, Claudia Strein, Bernd Fischer, Katrin Eichelbaum, Lars M Steinmetz, Jeroen Krijgsveld, Matthias W Hentze

help Authors

[Rastislav Horos] [Matthias Hentze]

help Attributions

None

help Scales


Not Specified
Views: 1525
  • Created: 14th Jan 2014 at 14:41

Related items

Ajax-loader-large

Log in / Register

Need an account?
Sign up

Forgotten password?

Front Page

Virtual Liver Network

(v.0.22.0)

Related Projects and friends


Imprint Taverna workflow workbench myExperiment JWS Online ISATAB myGrid Sabio-RK BioPortal Semantic SBML

Powered by:

Ror-logo-32

Icons:
Silk icons 1.3
Crystal Clear icons