Chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome

Xiaoyu Li; Ping Zhu; Shiqing Ma; Jinghui Song; Jinyi Bai; Fangfang Sun; Chengqi Yi

doi:10.1038/nchembio.1836

Chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome

Nat Chem Biol. 2015 Aug;11(8):592-7. doi: 10.1038/nchembio.1836. Epub 2015 Jun 15.

Authors

Xiaoyu Li¹, Ping Zhu², Shiqing Ma³, Jinghui Song¹, Jinyi Bai³, Fangfang Sun¹, Chengqi Yi⁴

Affiliations

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 Biodynamic Optical Imaging Center, School of Life Sciences, Peking University, Beijing, China.
³ 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.
⁴ 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.

PMID: 26075521
DOI: 10.1038/nchembio.1836

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Epigenesis, Genetic*
Humans
Hydro-Lyases / chemistry
Hydro-Lyases / genetics
Hydro-Lyases / metabolism
Mice
Organ Specificity
Peptide Elongation Factor 1 / chemistry
Peptide Elongation Factor 1 / genetics
Peptide Elongation Factor 1 / metabolism
Pseudouridine / chemistry
Pseudouridine / genetics
Pseudouridine / metabolism*
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
Transcriptome*

Substances

EEF1A1 protein, human
Peptide Elongation Factor 1
RNA, Messenger
RNA, Ribosomal
Pseudouridine
Hydro-Lyases
pseudouridylate synthetase

Associated data

GEO/GSE63655
PubChem-Substance/251916864
PubChem-Substance/251916865
PubChem-Substance/251916866
PubChem-Substance/251916867
PubChem-Substance/251916868