Cytosine-5 RNA methylation plays an important role in several biologically and pathologically relevant processes. However, owing to methodological limitations, the transcriptome-wide distribution of this mark has remained largely unknown. We previously established RNA bisulfite sequencing as a method for the analysis of RNA cytosine-5 methylation patterns at single-base resolution. More recently, next-generation sequencing has provided opportunities to establish transcriptome-wide maps of this modification. Here, we present a computational approach that integrates tailored filtering and data-driven statistical modeling to eliminate many of the artifacts that are known to be associated with bisulfite sequencing. By using RNAs from mouse embryonic stem cells, we performed a comprehensive methylation analysis of mouse tRNAs, rRNAs, and mRNAs. Our approach identified all known methylation marks in tRNA and two previously unknown but evolutionary conserved marks in 28S rRNA. In addition, mRNAs were found to be very sparsely methylated or not methylated at all. Finally, the tRNA-specific activity of the DNMT2 methyltransferase could be resolved at single-base resolution, which provided important further validation. Our approach can be used to profile cytosine-5 RNA methylation patterns in many experimental contexts and will be important for understanding the function of cytosine-5 RNA methylation in RNA biology and in human disease.

Cytosine-5 RNA甲基化在一些生物学和病理学相关的过程中起着重要作用。然而，由于方法学上的限制，该标记在转录组范围内的分布仍是未知之数。我们之前建立了RNA亚硫酸氢盐测序，作为在单碱基分辨率下分析RNA cytosine-5甲基化模式的方法。最近，下一代测序为建立这种修饰的转录组范围内的图谱提供了机会。在这里，我们提出了一种计算方法，该方法将量身定制的过滤和数据驱动的统计建模相结合，以消除许多已知与亚硫酸氢盐测序相关的伪像。通过使用小鼠胚胎干细胞的RNA，我们对小鼠tRNA，rRNA和mRNA进行了全面的甲基化分析。我们的方法鉴定了tRNA中所有已知的甲基化标记，以及28S rRNA中两个以前未知但进化的保守标记。另外，发现mRNA非常稀疏地被甲基化或根本没有被甲基化。最后，DNMT2甲基转移酶的tRNA特异性活性可以单碱基分辨率分辨，这提供了重要的进一步验证。我们的方法可用于在许多实验情况下分析cytosine-5 RNA甲基化模式，并且对于理解cytosine-5 RNA甲基化在RNA生物学和人类疾病中的功能非常重要。DNMT2甲基转移酶的tRNA特异性活性可以单碱基分辨率分辨，这提供了重要的进一步验证。我们的方法可用于在许多实验情况下分析cytosine-5 RNA甲基化模式，并且对于理解cytosine-5 RNA甲基化在RNA生物学和人类疾病中的功能非常重要。DNMT2甲基转移酶的tRNA特异性活性可以单碱基分辨率分辨，这提供了重要的进一步验证。我们的方法可用于在许多实验情况下分析cytosine-5 RNA甲基化模式，并且对于理解cytosine-5 RNA甲基化在RNA生物学和人类疾病中的功能非常重要。