Exonuclease-assisted enrichment and base resolution analysis of pseudouridine in single-stranded RNA

Pseudouridine (Ψ) is one of the most abundant RNA modifications, playing crucial roles in various biological processes. Identifying Ψ sites is vital for understanding their functions. In this study, we proposed a novel method for identifying Ψ sites with an improved signal-to-noise ratio. This method, called RNA exonuclease-assisted identification of pseudouridine sites (RIPS), combines specific CMC-labeling of Ψ sites with an exonuclease-assisted digestion strategy for the detection of Ψ sites. Utilizing exonuclease XRN1 to digest RNA strands not labeled by CMC, RIPS significantly reduces the background signal from unlabeled strands and enhances the positive signal of Ψ sites labeled by CMC, which terminates exonuclease digestion. As a result, we can enrich Ψ sites and identify them at single-base resolution. Considering the unique functions of single-stranded RNA (ssRNA), we employed RIPS to distinguish Ψ sites in single-stranded and double-stranded regions of RNA. Our results indicated that CMC could specifically label Ψ sites in ssRNA under natural conditions, enabling RIPS to selectively identify Ψ sites in ssRNA, which may facilitate the study on the functions of Ψ sites.