show Abstracthide AbstractPseudouridine (?) is the most abundant RNA modification, yet little is known about its content, dynamics and function in mRNA and ncRNA. Here, we perform quantitative MS analysis and develop CAP-seq for transcriptome-wide ? profiling. The unexpected high ? content (?/U ratio: ~ 0.2% to 0.6%) indicates that pseudouridylation in mammalian mRNA is much more prevalent and comprehensive than previously believed. In concordance, CAP-seq identified 2,084 ? sites within 1,929 human transcripts. We prove four previously unknown ? sites in rRNA and EEF1A1 mRNA. Genetic and biochemical analysis uncover PUS1 as a major human mRNA ? synthase. In response to stimuli, ? level and sites are dynamically modulated in stimulus-specific manners. Comparisons between human and mouse pseudouridylation reveal conserved and unique sites across tissue and species. We observe stop codon pseudouridylation and readthrough events simultaneously for HSPB1 mRNA, indicating a role in nonsense suppression. Together, these approaches allow in-depth analysis of transcriptome-wide pseudouridylation events and our comprehensive study provides a resource for functional studies of ?-mediated epigenetic regulation. Overall design: Here we report a transcriptome-wide profiling method that utilizes a chemically synthesized N3-CMC, which pre-enriches the ?-containing RNAs and blocks the reverse transcription.Mapping the ? sites in human transcriptome was performed using HEK293T and PUS1 dependent ? sites were identified by comparing PUS1 knock out cells with wildtype cells. Stress inducible or suppressed ? sites were identified by comparing stress treated cells with untreated cells. And mouse brain and liver were used to map ? sites in mouse transcriptome.