Absolute quantitative and base-resolution sequencing reveals comprehensive landscape of pseudouridine across the human transcriptome
Pseudouridine (Ψ) is one of the most abundant modifications in cellular RNA. However, its function remains elusive, mainly due to the lack of highly sensitive and accurate detection methods. Here, we introduced 2-bromoacrylamide-assisted cyclization sequencing (BACS), which enables Ψ-to-C transitions, for quantitative profiling of Ψ at single-base resolution. BACS allowed the precise identification of Ψ positions, especially in densely modified Ψ regions and consecutive uridine sequences. BACS detected all known Ψ sites in human rRNA and spliceosomal small nuclear RNAs and generated the quantitative Ψ map of human small nucleolar RNA and tRNA. Furthermore, BACS simultaneously detected adenosine-to-inosine editing sites and N<sup>1</sup>-methyladenosine. Depletion of pseudouridine synthases TRUB1, PUS7 and PUS1 elucidated their targets and sequence motifs. We further identified a highly abundant Ψ<sub>114</sub> site in Epstein-Barr virus-encoded small RNA EBER2. Surprisingly, applying BACS to a panel of RNA viruses demonstrated the absence of Ψ in their viral transcripts or genomes, shedding light on differences in pseudouridylation across virus families.
Authors
- Peter A. C. Wing (ORCID: https://orcid.org/0000-0002-2354-3281)
- Guifeng Wei (ORCID: https://orcid.org/0000-0002-5698-426X)
- Aleema Iqbal (ORCID: https://orcid.org/0009-0009-7352-3033)
- Jane A. McKeating (ORCID: https://orcid.org/0000-0002-7229-5886)
- Senko Tsukuda (ORCID: https://orcid.org/0000-0002-7289-3018)
- Haiqi Xu (ORCID: https://orcid.org/0009-0006-4486-5075)
- Jingfei Cheng (ORCID: https://orcid.org/0000-0001-9615-2719)
- Xiufei Chen (ORCID: https://orcid.org/0000-0001-6412-4385)
- Skirmantas Kriaučionis (ORCID: https://orcid.org/0000-0002-2273-5994)
- Chun‐Xiao Song (ORCID: https://orcid.org/0000-0002-7781-6521)
- James Michael Harris (ORCID: https://orcid.org/0000-0003-1880-3496)
- Alfredo Castelló (ORCID: https://orcid.org/0000-0002-1499-4662)
- Azman Embarc‐Buh (ORCID: https://orcid.org/0000-0002-1980-942X)
- Linzhen Kong
- Khatoun Al Moussawi (ORCID: https://orcid.org/0000-0003-1790-7330)
- Xin Lü (ORCID: https://orcid.org/0000-0002-6587-1152)
Institutions
- Chinese Academy of Medical Sciences & Peking Union Medical College (CN)
- University of Oxford (GB)
- Ludwig Cancer Research (GB)
- MRC University of Glasgow Centre for Virus Research (GB)
Publication Details
- Journal
- Nature Methods
- Published
- 2024-09-30
- DOI
- https://doi.org/10.1038/s41592-024-02439-8
- Citations
- 42
- Primary Topic
- RNA modifications and cancer
- Type
- article
- Field-Weighted Citation Impact
- 9.82
Funders
- Fundación Ramón Areces
- Ludwig Institute for Cancer Research
- National Institute for Health and Care Research
- University of Oxford
- University of Glasgow
- Universität Heidelberg
- China Scholarship Council
- Chinese Academy of Medical Sciences
- Medical Research Council