Chemical RNA digestion enables robust RNA-binding site mapping at single amino acid resolution.

Nature Structural & Molecular Biology
Jong Woo BaeJong-Seo Kim

Abstract

RNA-binding sites (RBSs) can be identified by liquid chromatography and tandem mass spectrometry analyses of the protein-RNA conjugates created by crosslinking, but RBS mapping remains highly challenging due to the complexity of the formed RNA adducts. Here, we introduce RBS-ID, a method that uses hydrofluoride to fully cleave RNA into mono-nucleosides, thereby minimizing the search space to drastically enhance coverage and to reach single amino acid resolution. Moreover, the simple mono-nucleoside adducts offer a confident and quantitative measure of direct RNA-protein interaction. Using RBS-ID, we profiled ~2,000 human RBSs and probed Streptococcus pyogenes Cas9 to discover residues important for genome editing.

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Citations

Nov 26, 2020·Nature Reviews. Genetics·Fátima GebauerMatthias W Hentze
Jun 1, 2021·Frontiers in Molecular Biosciences·Carlos H Vieira-Vieira, Matthias Selbach
Oct 17, 2021·Nature Communications·Jong Woo BaeJong-Seo Kim

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Datasets Mentioned

BETA
016254

Methods Mentioned

BETA
acetylation
transfection
flow
flow cytometry
immunoprecipitation
affinity purification
PCR
in

Software Mentioned

RNP xl )
ImageJ
RawConverter
GF
DAVID
PANDA
PeptideIons
mzid
ID
MSFragger

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