Sep 1, 2014

Photo-cross-linking and high-resolution mass spectrometry for assignment of RNA-binding sites in RNA-binding proteins

Nature Methods
Katharina KramerHenning Urlaub

Abstract

RNA-protein complexes play pivotal roles in many central biological processes. Although methods based on high-throughput sequencing have advanced our ability to identify the specific RNAs bound by a particular protein, there is a need for precise and systematic ways to identify RNA interaction sites on proteins. We have developed an experimental and computational workflow combining photo-induced cross-linking, high-resolution mass spectrometry and automated analysis of the resulting mass spectra for the identification of cross-linked peptides, cross-linking sites and the cross-linked RNA oligonucleotide moieties of such RNA-binding proteins. The workflow can be applied to any RNA-protein complex of interest or to whole proteomes. We applied the approach to human and yeast mRNA-protein complexes in vitro and in vivo, demonstrating its powerful utility by identifying 257 cross-linking sites on 124 distinct RNA-binding proteins. The open-source software pipeline developed for this purpose, RNP(xl), is available as part of the OpenMS project.

Mentioned in this Paper

Computer Software
RNA Aptamers
In Silico
In Vivo
Projections and Predictions
Complex (molecular entity)
Yeasts
Computer Programs and Programming
Mass Spectrometry
Proteomics

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