小柯机器人

西班牙巴塞罗那科技学院Eva Maria Novoa研究团队通过纳米孔测序，对天然RNA中的假尿苷化动力学进行了定量分析。这一研究成果于2021年5月13日在线发表在国际学术期刊《自然—生物技术》上。

纳米孔RNA测序是一种有前景的方法，可用于区分和鉴定天然RNA中不同的RNA修饰。扩展了纳米孔测序检测N⁶-甲基腺苷的能力，研究人员发现其他修饰，特别是假尿苷（Ψ）和2'-O-甲基化（Nm），也导致纳米孔中特征性的碱基识别“错误”特征数据。

着眼于Ψ修饰位点，研究人员在mRNA、非编码RNA和rRNA（包括酵母线粒体rRNA中的Pus4依赖Ψ修饰）中检测到了已知的以及以前未发现的Ψ位点。为了探索假尿苷化的动力学，研究人员用氧化、冷和热应激处理了酵母细胞，并在小核RNA、小核仁RNA和mRNA中检测到了热敏感的Ψ-修饰位点。

最后，研究人员开发了一个软件nanoRMS，该软件可以通过识别电流强度和痕量曲线改变的单分子读数来估计每个位点的修饰化学计量。这项工作表明，细胞RNA中能够检测到Nm和ΨRNA修饰，并且可以通过对天然RNA进行纳米孔测序来定量其修饰化学计量。 

附：英文原文

Title: Quantitative profiling of pseudouridylation dynamics in native RNAs with nanopore sequencing

Author: Oguzhan Begik, Morghan C. Lucas, Leszek P. Pryszcz, Jose Miguel Ramirez, Rebeca Medina, Ivan Milenkovic, Sonia Cruciani, Huanle Liu, Helaine Graziele Santos Vieira, Aldema Sas-Chen, John S. Mattick, Schraga Schwartz, Eva Maria Novoa

Issue&Volume: 2021-05-13

Abstract: 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 N6-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.

DOI: 10.1038/s41587-021-00915-6

Source: https://www.nature.com/articles/s41587-021-00915-6

Nature Biotechnology：《自然—生物技术》，创刊于1996年。隶属于施普林格·自然出版集团，最新IF：68.164
官方网址：https://www.nature.com/nbt/
投稿链接：https://mts-nbt.nature.com/cgi-bin/main.plex