Chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome

Pseudouridine (psi) is the most abundant post-transcriptional RNA modification, yet little is known about its prevalence, mechanism and function in mRNA. Here, we performed quantitative MS analysis and show that psi is much more prevalent (psi/U ratio similar to 0.2-0.6%) in mammalian mRNA than previously believed. We developed N-3-CMC-enriched pseudouridine sequencing (CeU-Seq), a selective chemical labeling and pulldown method, to identify 2,084 psi sites within 1,929 human transcripts, of which four (in ribosomal RNA and EEF1A1 mRNA) are biochemically verified. We show that hPUS1, a known psi synthase, acts on human mRNA; under stress, CeU-Seq demonstrates inducible and stress-specific mRNA pseudouridylation. Applying CeU-Seq to the mouse transcriptome revealed conserved and tissue-specific pseudouridylation. Collectively, our approaches allow comprehensive analysis of transcriptome-wide pseudouridylation and provide tools for functional studies of psi-mediated epigenetic regulation.