title: Chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome.
abstract: Pseudouridine (Ψ) 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 Ψ is much more prevalent (Ψ/U ratio ∼0.2-0.6%) in mammalian mRNA than previously believed. We developed N3-CMC-enriched pseudouridine sequencing (CeU-Seq), a selective chemical labeling and pulldown method, to identify 2,084 Ψ sites within 1,929 human transcripts, of which four (in ribosomal RNA and EEF1A1 mRNA) are biochemically verified. We show that hPUS1, a known Ψ 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 Ψ-mediated epigenetic regulation.
authors.affiliation: State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Li
authors.affiliation: Biodynamic Optical Imaging Center, School of Life Sciences, Peking University, Beijing, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Zhu
authors.affiliation: 1] State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China. [2] Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China.
authors.forename: Shiqing
authors.initials: S
authors.lastname: Ma
authors.affiliation: State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China.
authors.forename: Jinghui
authors.initials: J
authors.lastname: Song
authors.affiliation: 1] State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China. [2] Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China.
authors.forename: Jinyi
authors.initials: J
authors.lastname: Bai
authors.affiliation: State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China.
authors.forename: Fangfang
authors.initials: F
authors.lastname: Sun
authors.affiliation: 1] State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China. [2] Synthetic and Functional Biomolecules Center, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing, China.
authors.forename: Chengqi
authors.initials: C
authors.lastname: Yi
mesh_terms: D000818:Animals; D044127:Epigenesis, Genetic; D006801:Humans; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009928:Organ Specificity; D020648:Peptide Elongation Factor 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011560:Pseudouridine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods; D013312:Stress, Physiological; D059467:Transcriptome
chemical_list: C094814:EEF1A1 protein, human; D020648:Peptide Elongation Factor 1; D012333:RNA, Messenger; D012335:RNA, Ribosomal; D011560:Pseudouridine; D006836:Hydro-Lyases; C022479:pseudouridylate synthetase