Exonuclease-assisted identification of pseudouridine sites in single-stranded RNA

Pseudouridine is considered as the most abundant modified nucleotides in RNA, playing an indispensable role in various life processes. The identification of pseudouridine sites provides the basis for the study of their function. Here, we proposed a new method for the identification of pseudouridine sites with a higher signal-to-noise ratio, combining CMC-specific labeling of pseudouridine sites and exonuclease-assisted strategy. We called this method exonuclease-assisted identification of pseudouridine sites (EAIPS). By using exonuclease XRN1 to digest the RNA strands that were not labeled by CMC, we could greatly reduce the background signal of unlabeled strands, while the pseudouridine sites that were specifically labeled by CMC could terminate exonuclease digestion at one nucleotide 5’ to the pseudouridine sites due to the steric hindrance of CMC. Moreover, we used EAIPS to distinguish pseudouridine sites in single-stranded and double-stranded regions of RNA. Our results indicated that CMC could only specifically label pseudouridine sites in single-stranded regions, but not in double-stranded regions, and thus EAIPS enabled selectively identification of pseudouridine sites in the single-stranded RNA.