Ludwig Institute for Cancer Research, Nuffield Department of Medicine, University of Oxford, Oxford, UK.
Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK.
Nat Methods. 2024 Nov;21(11):2024-2033. doi: 10.1038/s41592-024-02439-8. Epub 2024 Sep 30.
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-methyladenosine. Depletion of pseudouridine synthases TRUB1, PUS7 and PUS1 elucidated their targets and sequence motifs. We further identified a highly abundant Ψ 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.
假尿嘧啶核苷 (Ψ) 是细胞 RNA 中最丰富的修饰之一。然而，由于缺乏高度敏感和准确的检测方法，其功能仍然难以捉摸。在这里，我们引入了 2-溴丙烯酰胺辅助环化测序 (BACS)，它可以实现 Ψ 到 C 的转变，从而实现单碱基分辨率下 Ψ 的定量分析。BACS 允许精确识别 Ψ 位置，特别是在修饰密度高的 Ψ 区域和连续的尿嘧啶序列中。BACS 检测了人类 rRNA 和剪接体小核 RNA 中的所有已知 Ψ 位点，并生成了人类小核仁 RNA 和 tRNA 的定量 Ψ 图谱。此外，BACS 还可以同时检测腺苷到肌苷的编辑位点和 N6-甲基腺苷。假尿嘧啶核苷合成酶 TRUB1、PUS7 和 PUS1 的耗竭阐明了它们的靶标和序列基序。我们进一步鉴定了 Epstein-Barr 病毒编码的小 RNA EBER2 中的一个高度丰富的 Ψ 位点。令人惊讶的是，将 BACS 应用于一组 RNA 病毒表明它们的病毒转录本或基因组中不存在 Ψ，这揭示了不同病毒家族中假尿嘧啶化的差异。
