Nanopore-based native RNA sequencing of human transcriptomes reveals the complexity of mRNA modifications and crosstalk between RNA regulatory features

The identification and functional characterization of chemical modifications on an mRNA molecule, in particular N6-methyladenosine (m6A) modification, significantly broadened our understanding of RNA function and regulation. While interactions between RNA modifications and other RNA features have been proposed, direct evidence showing correlation is limited. Here, using Oxford Nanopore long-read direct RNA sequencing (dRNA-seq), we simultaneously interrogate the transcriptome and epitranscriptome of a human leukemia cell line to investigate the correlation between m6A modifications, mRNA abundance, polyA tail length and alternative splicing. We demonstrated that high quality dRNA-seq is critically important for unbiased and large-scale correlative analyses of these RNA features. The genome wide landscape of RNA methylation was captured with single nucleotide and individual isoform resolution. The length of polyA tails were ascribed to individual transcripts and genes, allowing for unprecedent measurement of polyA tail length distribution. Global analysis indicated negative association between polyA tail length and mRNA abundance while uncovering pathway-specific responses upon depletion of m6A forming enzyme METTL3. Overall, our study presented a rich dRNA-seq data resource which has been validated and can be further exploited to inquire into the complexity of RNA modifications and potential interplays between RNA regulatory elements.