Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain.
Garvan Institute of Medical Research, Darlinghurst, NSW, Australia.
Nat Biotechnol. 2021 Oct;39(10):1278-1291. doi: 10.1038/s41587-021-00915-6. Epub 2021 May 13.
Nanopore RNA sequencing shows promise as a method for discriminating and identifying different RNA modifications in native RNA. Expanding on the ability of nanopore sequencing to detect N-methyladenosine, we show that other modifications, in particular pseudouridine (Ψ) and 2'-O-methylation (Nm), also result in characteristic base-calling 'error' signatures in the nanopore data. Focusing on Ψ modification sites, we detected known and uncovered previously unreported Ψ sites in mRNAs, non-coding RNAs and rRNAs, including a Pus4-dependent Ψ modification in yeast mitochondrial rRNA. To explore the dynamics of pseudouridylation, we treated yeast cells with oxidative, cold and heat stresses and detected heat-sensitive Ψ-modified sites in small nuclear RNAs, small nucleolar RNAs and mRNAs. Finally, we developed a software, nanoRMS, that estimates per-site modification stoichiometries by identifying single-molecule reads with altered current intensity and trace profiles. This work demonstrates that Nm and Ψ RNA modifications can be detected in cellular RNAs and that their modification stoichiometry can be quantified by nanopore sequencing of native RNA.
纳米孔 RNA 测序有望成为一种区分和鉴定天然 RNA 中不同 RNA 修饰的方法。在纳米孔测序检测 N6-甲基腺苷的能力的基础上，我们表明其他修饰，特别是假尿嘧啶 (Ψ) 和 2'-O-甲基化 (Nm)，也会导致纳米孔数据中特征性的碱基调用“错误”特征。我们专注于 Ψ 修饰位点，在 mRNA、非编码 RNA 和 rRNA 中检测到已知和以前未报道的 Ψ 位点，包括酵母线粒体 rRNA 中 Pus4 依赖性的 Ψ 修饰。为了探索假尿嘧啶化的动态，我们用氧化、冷和热应激处理酵母细胞，并在小核 RNA、小核仁 RNA 和 mRNA 中检测到对热敏感的 Ψ 修饰位点。最后，我们开发了一种软件 nanoRMS，通过识别电流强度和轨迹图谱改变的单分子读取来估计每个位点的修饰化学计量。这项工作表明，Nm 和 Ψ RNA 修饰可以在细胞 RNA 中检测到，并且可以通过对天然 RNA 进行纳米孔测序来定量它们的修饰化学计量。
