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  • The following term was not found in PubMed: RNRNA-binding.
Nat Methods. 2014 Oct;11(10):1064-70. doi: 10.1038/nmeth.3092. Epub 2014 Aug 31.

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

Author information

1
1] Bioanalytical Mass Spectrometry Group, Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany. [2].
2
1] Center for Bioinformatics, University of Tübingen, Tübingen, Germany. [2] Department of Computer Science, University of Tübingen, Tübingen, Germany. [3].
3
1] European Molecular Biology Laboratory, Heidelberg, Germany. [2].
4
Bioanalytical Mass Spectrometry Group, Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
5
Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
6
European Molecular Biology Laboratory, Heidelberg, Germany.
7
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.
8
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.

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.

PMID:
25173706
DOI:
10.1038/nmeth.3092
[Indexed for MEDLINE]

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