Detection and Quantification of Pseudouridine in RNA

Pseudouridylation is the most abundant of all RNA modifications. Pseudouridylation is dynamic and widespread among many different types of RNAs in living organisms, thus drawing a lot of recent interest from the RNA and epigenetics communities. To successfully carry out an investigation into RNA pseudouridylation, it is desirable to have a convenient and effective method capable of detection and quantification of pseudouridylation. Here, we present two such methods: one relies on pseudouridine (Ψ)-specific CMCT modification followed by reverse transcription/primer-extension (semiquantitative), and the other is based on site-specific cleavage and radiolabeling followed by nuclease digestion and TLC (quantitative). Although only semiquantitative, the CMCT and reverse transcription-based method is capable of detecting multiple Ψs (present in the same RNA molecule) in one reaction. In contrast, the second method, based on site-specific cleavage/labeling, nuclease digestion, and TLC, is quantitative, but can be used to analyze only one site at a time. These two methods can be used independently or in combination.