title: 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. 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.
authors.affiliation: 1] Bioanalytical Mass Spectrometry Group, Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany. [2].
authors.forename: Katharina
authors.initials: K
authors.lastname: Kramer
authors.affiliation: 1] Center for Bioinformatics, University of Tübingen, Tübingen, Germany. [2] Department of Computer Science, University of Tübingen, Tübingen, Germany. [3].
authors.forename: Timo
authors.identifier: 0000000228336070
authors.initials: T
authors.lastname: Sachsenberg
authors.affiliation: 1] European Molecular Biology Laboratory, Heidelberg, Germany. [2].
authors.forename: Benedikt M
authors.initials: BM
authors.lastname: Beckmann
authors.affiliation: Bioanalytical Mass Spectrometry Group, Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
authors.forename: Saadia
authors.initials: S
authors.lastname: Qamar
authors.affiliation: Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
authors.forename: Kum-Loong
authors.initials: KL
authors.lastname: Boon
authors.affiliation: European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Matthias W
authors.initials: MW
authors.lastname: Hentze
authors.affiliation: 1] Center for Bioinformatics, University of Tübingen, Tübingen, Germany. [2] Department of Computer Science, University of Tübingen, Tübingen, Germany. [3] Quantitative Biology Center, University of Tübingen, Tübingen, Germany. [4] Faculty of Medicine, University of Tübingen, Tübingen, Germany.
authors.forename: Oliver
authors.identifier: 0000000317394598
authors.initials: O
authors.lastname: Kohlbacher
authors.affiliation: 1] Bioanalytical Mass Spectrometry Group, Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany. [2] Bioanalytics Research Group, Department of Clinical Chemistry, University Medical Center, Göttingen, Germany.
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D001331:Automation; D001665:Binding Sites; D003198:Computer Simulation; D003432:Cross-Linking Reagents / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D020543:Proteome; D040901:Proteomics / Q000379:methods; D012313:RNA / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000737:chemistry*; D012984:Software
chemical_list: D000596:Amino Acids; D003432:Cross-Linking Reagents; D005656:Fungal Proteins; D009841:Oligonucleotides; D010455:Peptides; D020543:Proteome; D016601:RNA-Binding Proteins; D012313:RNA