Quantitative sequencing using BID-seq uncovers abundant pseudouridines in mammalian mRNA at base resolution
Qing Dai, Lisheng Zhang, Hui‐Lung Sun, Kinga Pajdzik, Lei Yang, Chang Ye, Cheng‐Wei Ju, Shun Liu, Yuru Wang, Zhong Zheng, Linda Zhang, Bryan T. Harada, Xiaoyang Dou, Iryna Irkliyenko, Xinran Feng, Wen Zhang, Tao Pan, Chuan He
Abstract
Functional characterization of pseudouridine (Ψ) in mammalian mRNA has been hampered by the lack of a quantitative method that maps Ψ 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 Ψ 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 Ψ in mammalian mRNA and assign different 'writer' proteins to individual Ψ deposition. Our results reveal a transcript stabilization role of Ψ sites installed by TRUB1 in human cancer cells. We also detect the presence of Ψ within stop codons of mammalian mRNA and confirm the role of Ψ in promoting stop codon readthrough in vivo. BID-seq will enable future investigations of the roles of Ψ in diverse biological processes.