Photoactivatable ribonucleosides mark base-specific RNA-binding sites.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 34654832.
- Also identified by DOI 10.1038/s41467-021-26317-5 and PMC identifier 8519950.
- Licence recorded as CC BY.
- The licence permits redistribution, so the abstract is shown in full and the full text is available from the publisher.
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.
Medical subject headings
- Binding Sites
- RNA
- RNA-Binding Proteins
- Ribonucleosides