Nanopore Direct RNA Sequencing Maps the Arabidopsis m6A Epitranscriptome

Understanding genome organization and gene regulation requires insight into RNA transcription, processing and modification. We adapted Oxford 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 terminal exons 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 refine Arabidopsis genome annotation and, if applied to less well-studied species, this approach has the potential to transform our understanding of what their genomes encode.