Absolute quantitative and base-resolution sequencing reveals comprehensive landscape of pseudouridine across the human transcriptome

• Published in: Nature methods Vol. 21; no. 11; pp. 2024 - 2033
• Main Authors: Xu, Haiqi, Kong, Linzhen, Cheng, Jingfei, Al Moussawi, Khatoun, Chen, Xiufei, Iqbal, Aleema, Wing, Peter A. C., Harris, James M., Tsukuda, Senko, Embarc-Buh, Azman, Wei, Guifeng, Castello, Alfredo, Kriaucionis, Skirmantas, McKeating, Jane A., Lu, Xin, Song, Chun-Xiao
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.11.2024; Nature Publishing Group
• Subjects: 631/1647/514/1949; 631/92/500; Adenosine; Adenosine - analogs & derivatives; Adenosine - chemistry; Adenosine - genetics; Adenosine - metabolism; Bioinformatics; Biological Microscopy; Biological Techniques; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Epstein-Barr virus; Gene sequencing; Herpesvirus 4, Human - genetics; Humans; Intramolecular Transferases; Life Sciences; Mutation; Nucleoli; Nucleotide sequence; Proteomics; Pseudouridine - genetics; Pseudouridine - metabolism; Pseudouridylation; RNA viruses; RNA, Ribosomal - genetics; RNA, Small Nuclear - chemistry; RNA, Small Nuclear - genetics; RNA, Small Nuclear - metabolism; RNA, Small Nucleolar - genetics; RNA, Small Nucleolar - metabolism; RNA, Transfer - chemistry; RNA, Transfer - genetics; RNA, Viral - genetics; rRNA; Sequence Analysis, RNA - methods; snoRNA; snRNA; Transcriptome; Transcriptomes; Transfer RNA; tRNA; Uridine
• ISSN: 1548-7091; 1548-7105; 1548-7105
• DOI: 10.1038/s41592-024-02439-8

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 1 -methyladenosine. Depletion of pseudouridine synthases TRUB1, PUS7 and PUS1 elucidated their targets and sequence motifs. We further identified a highly abundant Ψ 114 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. This study introduces a chemical method, BACS, that generates Ψ-to-C mutation signatures, allowing for sequencing and quantification of Ψ at single-base resolution.