A broad diversity of modifications decorate RNA molecules. Originally conceived as static components, evidence is accumulating that some RNA modifications may be dynamic, contributing to cellular responses to external signals and environmental circumstances. A major difficulty in studying these modifications, however, is the need of tailored protocols to map each modification type individually. Here, we present a new approach that uses direct RNA nanopore sequencing to identify and quantify RNA modifications present in native RNA molecules. First, we show that each RNA modification type results in a distinct and characteristic base-calling 'error' signature, which we validate using a battery of genetic strains lacking either pseudouridine (Y) or 2'-O-methylation (Nm) modifications. We then demonstrate the value of these signatures for de novo prediction of Y modifications transcriptome-wide, confirming known Y-modified sites as well as uncovering novel Y sites in mRNAs, ncRNAs and rRNAs, including a previously unreported Pus4-dependent Y modification in yeast mitochondrial rRNA, which we validate using orthogonal methods. To explore the dynamics of pseudouridylation across environmental stresses, we treat the cells with oxidative, cold and heat stresses, finding that yeast ribosomal rRNA modifications do not change upon environmental exposures, contrary to the general belief. By contrast, our method reveals many novel heat-sensitive Y-modified sites in snRNAs, snoRNAs and mRNAs, in addition to recovering previously reported sites. Finally, we develop a novel software, nanoRMS, which we show can estimate per-site modification stoichiometries from individual RNA molecules by identifying the reads with altered current intensity and trace profiles, and quantify the RNA modification stoichiometry changes between two conditions. Our work demonstrates that Y RNA modifications can be predicted de novo and in a quantitative manner using native RNA nanopore sequencing.

中文翻译：

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使用纳米孔测序对天然RNA修饰及其动力学进行定量分析

各种各样的修饰修饰了RNA分子。最初被认为是静态成分，越来越多的证据表明某些RNA修饰可能是动态的，从而导致细胞对外部信号和环境的反应。但是，研究这些修改的主要困难是需要定制协议来分别映射每个修改类型。在这里，我们提出了一种使用直接RNA纳米孔测序来鉴定和量化天然RNA分子中存在的RNA修饰的新方法。首先，我们表明每种RNA修饰类型均会导致独特且特征性的碱基识别“错误”特征，我们使用一系列缺乏伪尿苷（Y）或2'-O-甲基化（Nm）修饰的遗传菌株进行验证。然后，我们证明这些签名的价值对于从头到尾预测整个转录组的Y修饰，证实了已知的Y修饰位点以及在mRNA，ncRNA和rRNA中发现新颖的Y位点，包括酵母中以前未报道的Pus4依赖性Y修饰线粒体rRNA，我们使用正交方法进行了验证。为了探索在环境压力下假尿嘧啶化的动力学，我们用氧化，冷和热压力处理细胞，发现酵母核糖体rRNA修饰在环境暴露下不会改变，这与普遍的看法相反。相比之下，我们的方法揭示了snRNA，snoRNA和mRNA中除了恢复先前报道的位点外，还有许多新颖的热敏Y修饰位点。最后，我们开发了一个新颖的软件nanoRMS，我们展示出可以通过识别具有改变的电流强度和痕量图谱的读数，从单个RNA分子估计每个位点的修饰化学计量，并定量两种条件之间的RNA修饰化学计量变化。我们的工作表明，可以使用天然RNA纳米孔测序从头开始并以定量方式预测Y RNA修饰。