Department of Chemistry, The University of Chicago, Chicago, Illinois 60637, United States.
Howard Hughes Medical Institute, The University of Chicago, Chicago, Illinois 60637, United States.
ACS Chem Biol. 2024 Aug 16;19(8):1813-1819. doi: 10.1021/acschembio.4c00387. Epub 2024 Jul 16.
Pseudouridine (Ψ) is a widespread RNA modification found in various RNA species, including rRNA, tRNA, snRNA, mRNA, and long noncoding RNA (lncRNA). Understanding the function of Ψ in these RNA types requires a robust method for the detection and quantification of the Ψ level at single-nucleotide resolution. A previously used method utilizes Ψ labeling by N-cyclohexyl-'-β-(4-methylmorpholinium)ethylcarbodiimide (CMC). The quantification of Ψ is based on the stop ratio after reverse transcription. However, the use of CMC followed by strong alkaline treatment causes severe RNA degradation, often requiring a large amount of RNA. The removal of CMC and recovery of RNA by ethanol precipitation are also time-consuming. Here, we introduce a isulfite ncorporation ered ligation-based method (BIHIND), which can detect and quantify Ψ sites on rRNA, mRNA, and noncoding RNA. BIHIND can be coupled with quantitative PCR (BIHIND-qPCR) for quantitative detection of Ψ fraction at individual modification sites, as well as with next-generation sequencing (BIHIND-seq) for high-throughput sequencing of Ψ without requiring reverse transcription. We validated the robustness of BIHIND with the elucidation of Ψ dynamics following pseudouridine synthase depletion.
假尿嘧啶核苷（Ψ）是一种广泛存在于各种 RNA 物种中的 RNA 修饰物，包括 rRNA、tRNA、snRNA、mRNA 和长非编码 RNA（lncRNA）。要了解 Ψ 在这些 RNA 类型中的功能，需要一种能够在单核苷酸分辨率下检测和定量 Ψ 水平的强大方法。以前使用的方法利用 N-环己基-'-β-（4-甲基吗啉基）乙基碳二亚胺（CMC）对 Ψ 进行标记。Ψ 的定量是基于反转录后的停止比。然而，CMC 随后进行强碱性处理会导致严重的 RNA 降解，通常需要大量的 RNA。CMC 的去除和 RNA 通过乙醇沉淀回收也很耗时。在这里，我们引入了一种亚硫酸氢盐掺入连接法（BIHIND），它可以检测和定量 rRNA、mRNA 和非编码 RNA 上的 Ψ 位点。BIHIND 可以与定量 PCR（BIHIND-qPCR）结合使用，用于在单个修饰位点定量检测 Ψ 分数，也可以与下一代测序（BIHIND-seq）结合使用，用于在不需要反转录的情况下高通量测序 Ψ。我们通过阐明假尿嘧啶核苷合酶耗竭后 Ψ 的动态变化来验证 BIHIND 的稳健性。
