Photoactivatable ribonucleosides mark base-specific RNA-binding sites.

Area of Science:

• Molecular Biology
• Biochemistry
• Genomics

Background:

• RNA-protein interactions are crucial for gene regulation but identifying specific binding sites remains challenging.
• Existing methods struggle to pinpoint RNA-binding sites (RBSs) or provide direct evidence of RNA binding.
• Current techniques lack single amino acid resolution and base specificity.

Purpose of the Study:

• To develop a robust method for identifying RNA-binding sites (RBSs) with single amino acid resolution and base specificity.
• To provide direct evidence of RNA-protein interactions using photoactivatable ribonucleosides (PAR) and chemical RNA cleavage.
• To characterize base-specific RNA-protein interactions, particularly for uridine and guanosine.

Main Methods:

• Developed pRBS-ID, integrating UVA-based photoactivatable ribonucleoside (PAR) crosslinking with chemical RNA cleavage.
• pRBS-ID detects peptides crosslinked to PAR adducts, enabling identification of RNA-binding peptides.
• Utilized a search pipeline to analyze pRBS-ID and existing UVC-based RBS-ID datasets.

Main Results:

• pRBS-ID robustly identifies RNA-binding peptides and pinpoints RNA-binding sites (RBSs) at single amino acid resolution.
• The method achieves base specificity, distinguishing between uridine (U) and guanosine (G) contacts.
• Profiling of uridine-contacting RBSs and discovery of guanosine-contacting RBSs were achieved.
• Analysis of combined datasets identified 3,077 RBSs across 532 human proteins.

Conclusions:

• pRBS-ID is a powerful platform for investigating RNA-protein interactions and the molecular basis of posttranscriptional regulation.
• The method offers direct evidence of RNA binding and high-resolution mapping of RNA-binding sites.
• pRBS-ID significantly advances the study of base-specific RNA-protein interactions and their functional implications.