Understanding genome organization and gene regulation requires insight into RNA transcription, processing and modification. We adapted nanopore direct RNA sequencing to examine RNA from a wild-type accession of the model plant Arabidopsis thaliana and a mutant defective in mRNA methylation (m⁶A). Here we show that m⁶A can be mapped in full-length mRNAs transcriptome-wide and reveal the combinatorial diversity of cap-associated transcription start sites, splicing events, poly(A) site choice and poly(A) tail length. Loss of m⁶A from 3’ untranslated regions is associated with decreased relative transcript abundance and defective RNA 3′ end formation. A functional consequence of disrupted m⁶A is a lengthening of the circadian period. We conclude that nanopore direct RNA sequencing can reveal the complexity of mRNA processing and modification in full-length single molecule reads. These findings can refine Arabidopsis genome annotation. Further, applying this approach to less well-studied species could transform our understanding of what their genomes encode.

中文翻译：

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纳米孔直接RNA测序可绘制拟南芥mRNA加工和m6A修饰的复杂性

了解基因组的组织和基因调控需要深入了解RNA转录，加工和修饰。我们适应了纳米孔直接RNA测序，以检查来自模型植物拟南芥的野生型登录基因和mRNA甲基化缺陷的突变体（m ⁶ A）的RNA 。在这里，我们显示m ⁶ A可以在转录组范围内的全长mRNA中进行定位，并揭示了与帽相关的转录起始位点，剪接事件，poly（A）位点选择和poly（A）尾长的组合多样性。来自3'非翻译区的m ⁶ A的丧失与相对转录丰度的降低和RNA 3'末端形成的缺陷有关。m ⁶中断的功能后果A是昼夜节律周期的延长。我们得出的结论是，纳米孔直接RNA测序可以揭示全长单分子读取中mRNA加工和修饰的复杂性。这些发现可以完善拟南芥基因组注释。此外，将这种方法应用于研究较少的物种可能会改变我们对其基因组编码的理解。