Pseudouridine is the most abundant post-transcriptional RNA modification.Although pseudouridine is well established in non-coding RNAs, little is known about its prevalence and function in mRNA.To determine the pseudouridine content in mRNA, we performed quantitative mass spectrometry analysis in mammalian mRNAs using stringent mRNA purification procedures and found that pseudouridine is much more prevalent than previously believed.The ψ/U ratio ranges from 0.2% to 0.6%, which is comparable to the ratio of m6A in mRNA, the most abundant internal mRNA modification.To study the pseudouridine in the transcriptome, we developed N3-CMC-enriched pseudouridine sequencing (CeU-Seq), which is enabled via a chemically-synthesized CMC derivative that pre-enriches the ψ-containing RNA through biotin pulldown.Indeed, CeU-Seq allowed us to identify 2084 ψ sites in the transcriptome of HEK293T cells, and also 1741 and 1543 ψ sites in mouse brain and liver, respectively.Combining CeU-Seq and quantitative MS analysis,we showed that several pseudouridine synthases could act on mRNA and identified 77 PUS 1-dependent ψ sites.Furthermore, via CeU-Seq we identified several hundreds of stress-inducible ψ sites under heat shock or hydrogen peroxide conditions, respectively.Interestingly, the inducible ψ sites displayed strong stimulus-specific patterns and were essentially non-overlapping, indicating that mRNA pseudouridylation is dynamically regulated.Work is ongoing to map the ψ profiles of embryonic stem cells and model animals in different developmental stages.Functional roles of pseudouridine in different biological processes are also under investigation.