Chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome

• Published in: Nature chemical biology Vol. 11; no. 8; pp. 592 - 597
• Main Authors: Li, Xiaoyu, Zhu, Ping, Ma, Shiqing, Song, Jinghui, Bai, Jinyi, Sun, Fangfang, Yi, Chengqi
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.08.2015; Nature Publishing Group
• Subjects: 101/58; 13/1; 13/100; 13/109; 13/89; 38/22; 38/23; 38/77; 38/90; 38/91; 42/70; 45/29; 631/1647/296; 631/337/1645/2570; 631/553; 631/92/500; 82/80; Analysis; Animals; Biochemical Engineering; Biochemistry; Bioorganic Chemistry; Cell Biology; Chemistry; Chemistry/Food Science; Epigenesis, Genetic; Humans; Hydro-Lyases - chemistry; Hydro-Lyases - genetics; Hydro-Lyases - metabolism; Mammals; Mice; Organ Specificity; Peptide Elongation Factor 1 - chemistry; Peptide Elongation Factor 1 - genetics; Peptide Elongation Factor 1 - metabolism; Pseudouridine - chemistry; Pseudouridine - genetics; Pseudouridine - metabolism; Ribonucleic acid; RNA; RNA Processing, Post-Transcriptional; RNA, Messenger - chemistry; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA, Ribosomal - chemistry; RNA, Ribosomal - genetics; RNA, Ribosomal - metabolism; Staining and Labeling - methods; Stress, Physiological; Studies; Transcriptome
• ISSN: 1552-4450; 1552-4469; 1552-4469
• DOI: 10.1038/nchembio.1836

Pseudouridine (ψ) is a C-linked uracil modification originally discovered in tRNA. MS analysis and CeU-Seq, a method that permits chemical tagging, pulldown and sequencing of ψ residues, reveal that these modifications are more abundant in the mammalian transcriptome than previously thought. 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 N 3 -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.