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Chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome

Nature Chemical Biology volume 11, pages 592–597 (2015)Cite this article

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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.

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Figure 1: Quantitative analysis of pseudouridylation in mammalian mRNA.
Figure 2: CeU-Seq pre-enriches Ψ-containing RNA and identifies known and new pseudouridylation sites in rRNA.
Figure 3: CeU-Seq identifies thousands of pseudouridylation sites in human mRNA and ncRNA.
Figure 4: Inducible pseudouridylation displays stimulus-specific patterns.
Figure 5: Human and mouse pseudouridylation exhibits both shared and unique sites across tissues and species.

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Acknowledgements

The authors would like to thank R. Meng and S. Huang for technical assistance and N. Liu, X. Wang and X. Liu for discussion. This work was supported by the National Basic Research Foundation of China (no. 2014CB964900) and the National Natural Science Foundation of China (nos. 21472009 and 31270838).

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Author notes

  1. Xiaoyu Li, Ping Zhu and Shiqing Ma: These authors contributed equally to this work.

Authors and 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

    Xiaoyu Li, Shiqing Ma, Jinghui Song, Jinyi Bai, Fangfang Sun & Chengqi Yi

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

    Ping Zhu

  3. Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China

    Shiqing Ma & Jinyi Bai

  4. Department of Chemical Biology, Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking University, Beijing, China

    Chengqi Yi

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Contributions

X.L., S.M. and C.Y. conceived the project, designed the experiments and wrote the manuscript. X.L. and S.M. performed the experiments. P.Z. designed and performed the bioinformatics analysis; J.S. contributed in making figures, J.B. synthesized the N3-CMC derivative, and F.S. contributed in Ψ quantification. All authors commented on and approved the paper.

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Correspondence to Chengqi Yi.

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Supplementary Results, Supplementary Tables 1–6, Supplementary Figures 1–11 and Supplementary Note. (PDF 2795 kb)

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Li, X., Zhu, P., Ma, S. et al. Chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome. Nat Chem Biol 11, 592–597 (2015). https://doi.org/10.1038/nchembio.1836

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