Photoactivatable ribonucleosides mark base-specific RNA-binding sites

Jong Woo Bae, Sangtae Kim, V. Narry Kim () and Jong-Seo Kim ()
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Jong Woo Bae: Institute for Basic Science
Sangtae Kim: Seer Inc.
V. Narry Kim: Institute for Basic Science
Jong-Seo Kim: Institute for Basic Science

Nature Communications, 2021, vol. 12, issue 1, 1-10

Abstract: Abstract RNA-protein interaction can be captured by crosslinking and enrichment followed by tandem mass spectrometry, but it remains challenging to pinpoint RNA-binding sites (RBSs) or provide direct evidence for RNA-binding. To overcome these limitations, we here developed pRBS-ID, by incorporating the benefits of UVA-based photoactivatable ribonucleoside (PAR; 4-thiouridine and 6-thioguanosine) crosslinking and chemical RNA cleavage. pRBS-ID robustly detects peptides crosslinked to PAR adducts, offering direct RNA-binding evidence and identifying RBSs at single amino acid-resolution with base-specificity (U or G). Using pRBS-ID, we could profile uridine-contacting RBSs globally and discover guanosine-contacting RBSs, which allowed us to characterize the base-specific interactions. We also applied the search pipeline to analyze the datasets from UVC-based RBS-ID experiments, altogether offering a comprehensive list of human RBSs with high coverage (3,077 RBSs in 532 proteins in total). pRBS-ID is a widely applicable platform to investigate the molecular basis of posttranscriptional regulation.

Date: 2021
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DOI: 10.1038/s41467-021-26317-5

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