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Katharina Kramer, Timo Sachsenberg, Benedict M Beckmann, Saadia Qamar, Kum-Loong Boon, Matthias W Hentze, Oliver Kohlbacher, and Henning Urlaub (2014)

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

Nat. Methods, 11(10):1064-70.

RNA–protein complexes play pivotal roles in many central biological processes. While methods based on next-generation 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 site and the cross-linked RNA oligonucleotide moiety 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, RNPxl, is available as part of the OpenMS project.
25173706
10.1038/nmeth.3092