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 highthroughput 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 crosslinking, 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.RNA molecules bind to proteins to form ribonucleoprotein complexes (RNPs). These are indispensable for the synthesis, stability, transport and activity of mRNAs 1 and noncoding RNAs 2,3 . RNA-binding proteins (RBPs) assume numerous functions in RNPs. RBPs can modulate or stabilize RNA structures, thereby making RNA catalytically active, for example, during pre-mRNA splicing 4 . RNA can also guide a catalytically active RBP to its destination; examples of this are microRNA-or long noncoding RNA-mediated translational control and epigenetic modulation 5,6 . RBPs are also involved in splicing and can recruit or repel other proteins, induce hydrolysis of RNA or protect RNA from degradation.

Analysis of protein–RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry

“…The database search, performed to identify the cross-linked peptide region with its cross-linked nucleotides, is as important as the entire purification procedure, since it differs from the searches typically performed for modified peptides. However, in this article only the principle of the modified database search is described, and we refer to a more detailed description of the database search of raw MS data in a recent publication [25]. The step-by-step description of the workflow includes sample preparation, UV-induced protein-RNA cross-linking, endoproteinase and nuclease digestion of proteins and RNAs, enrichment of peptide-RNA oligonucleotide cross-links, liquid chromatography (LC)-coupled electrospray ionization (ESI) tandem mass spectrometry (MS/MS) analysis and database search of raw MS data.…”

“…Note that DTT acts as a protein-RNA cross-linker under UV irradiation, as it reacts with the uridine base and with cysteine and generates a spacer between cysteine and uridines. This reaction is strictly UV-dependent ( [25]; U.Z. and H.U., unpublished data).…”

“…ex vivo protein-RNA complexes that are isolated from cells and which contain a larger RNA moiety, irradiation times of max. 2 min are recommended [25,34]. Irradiation at longer wavelength, e.g.…”

“…We have now developed a straightforward approach that utilizes UV-induced cross-linking and mass spectrometry, not only to identify proteins that cross-link to RNA but also to identify unambiguously the cross-linked amino-acid and the cross-linked nucleotide(s) [25]. The approach is easily applicable to single (e.g., recombinant) proteins that interact with RNA but whose structure cannot be determined in complex with RNA.…”

OpenMS: a flexible open-source software platform for mass spectrometry data analysis

“…Case studies Some recent examples where researchers have used OpenMS to build complex analysis tools include improvements to a label-free quantification pipeline [1] , the development of a metabolomics workflow [33] , the integration of RNA cross-linking workflows [34] and the addition of a workflow for targeted data analysis using SWATH-MS [35] . In addition, OpenMS has been extended to include a probabilistic scoring engine [36] , SILAC analysis [37] , isobaric quantification, random-access XML file parsing [38] and an MS simulation framework [39] .…”