State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China.
Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China.
Nat Chem Biol. 2023 Oct;19(10):1185-1195. doi: 10.1038/s41589-023-01304-7. Epub 2023 Mar 30.
Pseudouridine (Ψ) is an abundant post-transcriptional RNA modification in ncRNA and mRNA. However, stoichiometric measurement of individual Ψ sites in human transcriptome remains unaddressed. Here we develop 'PRAISE', via selective chemical labeling of Ψ by bisulfite to induce nucleotide deletion signature during reverse transcription, to realize quantitative assessment of the Ψ landscape in the human transcriptome. Unlike traditional bisulfite treatment, our approach is based on quaternary base mapping and revealed an ~10% median modification level for 2,209 confident Ψ sites in HEK293T cells. By perturbing pseudouridine synthases, we obtained differential mRNA targets of PUS1, PUS7, TRUB1 and DKC1, with TRUB1 targets showing the highest modification stoichiometry. In addition, we quantified known and new Ψ sites in mitochondrial mRNA catalyzed by PUS1. Collectively, we provide a sensitive and convenient method to measure transcriptome-wide Ψ; we envision this quantitative approach would facilitate emerging efforts to elucidate the function and mechanism of mRNA pseudouridylation.
假尿嘧啶核苷 (Ψ) 是 ncRNA 和 mRNA 中转录后 RNA 修饰的一种丰富成分。然而，人类转录组中单个 Ψ 位点的化学计量测量仍未得到解决。在这里，我们通过亚硫酸氢盐选择性化学标记 Ψ 来开发 'PRAISE'，在逆转录过程中诱导核苷酸缺失特征，以实现人类转录组中 Ψ 景观的定量评估。与传统的亚硫酸氢盐处理不同，我们的方法基于季铵碱基映射，在 HEK293T 细胞中揭示了 2209 个可信 Ψ 位点的中位数修饰水平约为 10%。通过干扰假尿嘧啶核苷合成酶，我们获得了 PUS1、PUS7、TRUB1 和 DKC1 的差异 mRNA 靶标，其中 TRUB1 靶标显示出最高的修饰化学计量。此外，我们还定量了 PUS1 催化的线粒体 mRNA 中的已知和新 Ψ 位点。总之，我们提供了一种灵敏、便捷的方法来测量全转录组 Ψ；我们设想这种定量方法将有助于阐明 mRNA 假尿嘧啶化的功能和机制。
