Nanopore direct RNA sequencing maps the complexity of Arabidopsis mRNA processing and m6A modification.

Matthew T Parker,Anthony Jw Hall,Anna V Sherwood,Katarzyna Mackinnon,Nicholas J Schurch,Geoffrey J Barton,Katarzyna Knop,Gordon G Simpson,Peter D Gould

doi:10.7554/elife.49658

Matthew T Parker, Anthony Jw Hall + Show 7 more

Open Access

https://doi.org/10.7554/elife.49658

Copy DOI

Abstract

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.

Highlights

E misidentification of 3′ ends through internal priming[3], spurious antisense and splicing events produced by reverse transcriptases (RTs) template switching[4,5], and the inability to detect all base modifications in the copying process[6]
We investigated whether long-read direct RNA sequencing (DRS) with nanopores[8] could reveal the complexity of Arabidopsis mRNA processing and modifications
We incorporated synthetic External RNA Controls Consortium (ERCC) RNA Spike-In mixes into all replicates[11,12] and carried out nanopore DRS

Summary

Introduction

E misidentification of 3′ ends through internal priming[3], spurious antisense and splicing events produced by RT template switching[4,5], and the inability to detect all base modifications in the copying process[6]. Consistent with these Arabidopsis transcriptome-wide nanopore DRS data, reads mapping to the synthetic ERCC RNA Spike-Ins and in vitro transcribed RNAs lacked ~11 nt of authentic 5′ sequence (Supplementary figure 3E, F).

Results

Conclusion