Journal
NATURE COMMUNICATIONS
Volume 12, Issue 1, Pages -Publisher
NATURE PORTFOLIO
DOI: 10.1038/s41467-021-26317-5
Keywords
-
Categories
Funding
- Ministry of Science and ICT of Korea [IBS-R008-D1]
- National Research Foundation of Korea [NRF-2021R1A2C2009336]
- Ministry of Education, Science and Technology of Korea
Ask authors/readers for more resources
The pRBS-ID method combines the advantages of photoactivatable ribonucleosides and chemical RNA cleavage to detect peptides crosslinked to ribonucleoside adducts, providing direct evidence of RNA binding and identifying RNA-binding sites with single amino acid resolution and base-specificity. This platform is widely applicable for investigating the molecular basis of post-transcriptional gene regulation.
RNA-protein interactions play critical roles in post-transcriptional gene regulation. Here the authors demonstrate pRBS-ID, an updated MS/MS-based method that combines the benefits of photoactivatable ribonucleosides and the chemical cleavage of RNA. 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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available