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

Abstract

RNA–protein complexes play pivotal roles in many central biological processes. While methods based on next-generation sequencing have profoundly advanced our ability to identify the specific RNAs bound by a particular protein, there is a dire need for precise and systematic ways to identify RNA interaction sites on proteins. We have developed an integrated 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 and exact amino acids with their cross-linked RNA oligonucleotide moiety of such RNA-binding proteins. The generic workflow can be applied to any RNA–protein complex of interest. Application to human and yeast mRNA–protein complexes in vitro and in vivo demonstrates the powerful utility of the approach by identification of 257 cross-linking sites on 124 distinct RNA-binding proteins. The software pipeline developed for this purpose is available as open-source software as part of the OpenMS project.