Quantitative sequencing using BID-seq uncovers abundant pseudouridines in mammalian mRNA at base resolution

Pseudouridine sites in mRNA are detected at base resolution and functionally investigated. Functional characterization of pseudouridine (psi) in mammalian mRNA has been hampered by the lack of a quantitative method that maps psi in the whole transcriptome. We report bisulfite-induced deletion sequencing (BID-seq), which uses a bisulfite-mediated reaction to convert pseudouridine stoichiometrically into deletion upon reverse transcription without cytosine deamination. BID-seq enables detection of abundant psi sites with stoichiometry information in several human cell lines and 12 different mouse tissues using 10-20 ng input RNA. We uncover consensus sequences for psi in mammalian mRNA and assign different 'writer' proteins to individual psi deposition. Our results reveal a transcript stabilization role of psi sites installed by TRUB1 in human cancer cells. We also detect the presence of psi within stop codons of mammalian mRNA and confirm the role of psi in promoting stop codon readthrough in vivo. BID-seq will enable future investigations of the roles of psi in diverse biological processes.