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Method Article
Mapping protein-RNA interactions with single residue resolution by CLIR-MS/MS
Ruedi Aebersold
Allain group
Corresponding Author
Frédéric H.-T. Allain
Allain group
Corresponding Author
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Protein-RNA complexes are key regulators involved in all steps of RNA metabolism and are thus crucial for cellular function. The precise knowledge of the RNA/protein interaction sites in such complexes provides essential information for understanding ribonucleoprotein (RNP) regulated processes in health and disease. We established an efficient technique that combines segmental isotope labeling of RNA with photo-crosslinking and tandem mass spectrometry. It resolves protein-RNA interactions in RNPs assembled in vitro, at single amino acid and single nucleotide level resolution in the same analysis. Information on these zero-length crosslinks can be used to guide further functional assays and as intermolecular distance restraints, thus supporting atomic scale modeling of protein-RNA complexes in an integrative structural biology approach. Optimally, the mapping of protein-RNA interactions can be achieved for a given complex within three weeks. This protocol accompanies Dorn et al, Nature Methods, published online 27 March 2017 (doi:10.1038/nmeth.4235).
Keywords
protein RNA interactions, RNPs, LC-MS/MS, UV-crosslinking
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Associated Publications
Structural modeling of protein–RNA complexes using crosslinking of segmentally isotope-labeled RNA and MS/MS
by Georg Dorn, Alexander Leitner, Julien Boudet, +5
Nature Methods (30 March, 2017)
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This is a preprint. It has not completed peer review.
Method Article
Mapping protein-RNA interactions with single residue resolution by CLIR-MS/MS
Ruedi Aebersold
Allain group
Corresponding Author
Frédéric H.-T. Allain
Allain group
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 30 Mar, 2017
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Protein-RNA complexes are key regulators involved in all steps of RNA metabolism and are thus crucial for cellular function. The precise knowledge of the RNA/protein interaction sites in such complexes provides essential information for understanding ribonucleoprotein (RNP) regulated processes in health and disease. We established an efficient technique that combines segmental isotope labeling of RNA with photo-crosslinking and tandem mass spectrometry. It resolves protein-RNA interactions in RNPs assembled in vitro, at single amino acid and single nucleotide level resolution in the same analysis. Information on these zero-length crosslinks can be used to guide further functional assays and as intermolecular distance restraints, thus supporting atomic scale modeling of protein-RNA complexes in an integrative structural biology approach. Optimally, the mapping of protein-RNA interactions can be achieved for a given complex within three weeks. This protocol accompanies Dorn et al, Nature Methods, published online 27 March 2017 (doi:10.1038/nmeth.4235).
Figure 1
Figure 2
Associated Publications
Structural modeling of protein–RNA complexes using crosslinking of segmentally isotope-labeled RNA and MS/MS
by Georg Dorn, Alexander Leitner, Julien Boudet, +5
Nature Methods (30 March, 2017)