A radiolabeling-free, qPCR-based method for locus-specific pseudouridine detection

Pseudouridine (Ψ) is the most abundant post-transcriptional RNA modification. Various methods have been developed to achieve locus-specific Ψ detection; however, the existing methods often involve radiolabeling of RNA, require advanced experimental skills and can be time-consuming. Herein we report a radiolabeling-free，qPCR-based method to detect locus-specific Ψs in rRNA and mRNA. This method is based on Ψ chemical adduct (Ψ-CMC) induced mutation/deletion during reverse transcription (RT), leading to qPCR products of different melting temperatures. Utilizing high-throughput sequencing, we demonstrate that such misincorporation is a general feature of Ψ-CMC adduct during our improved RT conditions. We validated this method on known Ψ sites in rRNA and showed that the melting curves correlate with the modification level. Moreover, we successfully detected Ψs in mRNA and lncRNA of different abundance, and identified Ψ synthase that targets mRNA. Our facile method takes only 1.5 days to complete, and with slight adjustment it can be applied to detect other epitranscriptomic marks in the transcriptome. Here we report a radiolabeling-free, qPCR based method to rapidly detect Ψ in a locus-specific manner. The method is based on Ψ labelling adduct induced misincorporation during reverse transcription (RT), generating qPCR products of different melting temperatures. To demonstrate that read-through induced misincorporation is a general feature of Ψ sites under our improved RT condition, we utilized high-throughput sequencing to comprehensively examine the pattern of misincorporation for all Ψ sites in rRNA using HEK293T cells. Besides, to address the detection resolution of the method, three qPCR amplicons examined with our locus-specific approach were subjected to high-throughput sequencing (for MALAT1, EEF1A1 and 18S rRNA).