Simultaneous identification of m6A and m5C reveals coordinated RNA modification at single-molecule resolution [mouse frontal cortex]. Simultaneous identification of m6A and m5C reveals coordinated RNA modification at single-molecule resolution [mouse frontal cortex]

The expanding field of epitranscriptomics might rival the epigenome in the diversity of biological processes impacted. However, the identification of multiple modification types in individual RNA molecules remains challenging. We present CHEUI, a new method that detects N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in individual transcript molecules in a single condition as well as differential methylation between two conditions, using nanopore signals. CHEUI processes observed and expected signals with convolutional neural networks to achieve high single-molecule accuracy and outperform other methods in detecting m6A and m5C sites and quantifying their stoichiometry. Moreover, CHEUI’s unique capability to identify different modifications in the same signal data reveals a non-random co-occurrence of m6A and m5C in transcripts in human cell lines and during mouse embryonic brain development. CHEUI unlocks the capability of studying links between multiple RNA modifications and phenotypes, enabling the discovery of new epitranscriptome functions. Furthermore, CHEUI's training and testing protocols are adaptable to other modifications, making it a versatile RNA technology. Overall design: Mouse frontal cortex tissues were extracted and sequenced using Nanopore d-RNA from embryonic day (E) 12, E15 and E18.

表观转录组学（epitranscriptomics）作为快速发展的研究领域，其影响的生物学过程多样性或可与表观基因组（epigenome）相匹敌。然而，在单个RNA分子中同时鉴定多种修饰类型仍颇具挑战。我们提出CHEUI这一全新方法，该方法可借助纳米孔信号（nanopore signals），在单一实验条件下检测单个转录本分子中的N6-甲基腺嘌呤（N6-methyladenosine，m6A）与5-甲基胞苷（5-methylcytidine，m5C），同时还可识别两种实验条件间的甲基化差异。CHEUI通过卷积神经网络（convolutional neural networks）处理观测信号与预期信号，实现高精度的单分子检测性能，在m6A与m5C位点检测及修饰化学计量比量化方面均优于其他方法。此外，CHEUI具备在同一套信号数据中识别多种修饰的独特能力，借此揭示了人类细胞系以及小鼠胚胎脑发育过程中转录本中m6A与m5C存在非随机共修饰现象。CHEUI为研究多种RNA修饰与表型之间的关联提供了可能，助力发现全新的表观转录组功能。进一步而言，CHEUI的训练与测试流程可适配其他RNA修饰类型，使其成为一款通用型RNA研究技术。总体实验设计：提取胚胎发育第12天（E12）、第15天（E15）及第18天（E18）的小鼠额叶皮层组织，采用Nanopore d-RNA测序技术进行测序。