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

Author:

Parker Matthew T1ORCID,Knop Katarzyna1ORCID,Sherwood Anna V1,Schurch Nicholas J1ORCID,Mackinnon Katarzyna1,Gould Peter D2,Hall Anthony JW3,Barton Geoffrey J1ORCID,Simpson Gordon G14ORCID

Affiliation:

1. School of Life Sciences, University of Dundee, Dundee, United Kingdom

2. Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom

3. Earlham Institute, Norwich Research Park, Norwich, United Kingdom

4. James Hutton Institute, Invergowrie, United Kingdom

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.

Funder

Biotechnology and Biological Sciences Research Council

H2020 Marie Skłodowska-Curie Actions

University of Dundee

Wellcome

Publisher

eLife Sciences Publications, Ltd

Subject

General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Medicine,General Neuroscience

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