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

Matthew T. Parker, Katarzyna Knop, Anna V. Sherwood, Nicholas J. Schurch, Katarzyna Mackinnon, Peter D. Gould, Anthony J. W. Hall, Geoffrey J. Barton (Lead / Corresponding author), Gordon G. Simpson (Lead / Corresponding author)

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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.

Original languageEnglish
Article numbere49658
Pages (from-to)1-35
Number of pages35
JournaleLife
Volume9
DOIs
Publication statusPublished - 14 Jan 2020

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Nanopores
RNA Sequence Analysis
Arabidopsis
RNA
Messenger RNA
Genome
Processing
Genes
Poly A
Transcription Initiation Site
3' Untranslated Regions
Transcriptome
Methylation
Transcription
Gene expression
Molecules

Cite this

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title = "Nanopore direct RNA sequencing maps the complexity of Arabidopsis mRNA processing and m6A modification",
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.",
author = "Parker, {Matthew T.} and Katarzyna Knop and Sherwood, {Anna V.} and Schurch, {Nicholas J.} and Katarzyna Mackinnon and Gould, {Peter D.} and Hall, {Anthony J. W.} and Barton, {Geoffrey J.} and Simpson, {Gordon G.}",
note = "{\circledC} 2020, Parker et al. This work was funded by BBSRC grants BB/J00247X/1, BB/M004155/1, BB/M010066/1, the University of Dundee Global Challenges Research Fund and the European Union’s Horizon 2020 research and innovation programme under Marie Skłodowska-Curie grant agreement No. 799300. The m6A LC-MS analysis was carried out by Abdel Atrih of the FingerPrints Proteomics Facility, University of Dundee, which is supported by a Wellcome Trust Technology Platform award (097945/B/11/Z)",
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AU - Parker, Matthew T.

AU - Knop, Katarzyna

AU - Sherwood, Anna V.

AU - Schurch, Nicholas J.

AU - Mackinnon, Katarzyna

AU - Gould, Peter D.

AU - Hall, Anthony J. W.

AU - Barton, Geoffrey J.

AU - Simpson, Gordon G.

N1 - © 2020, Parker et al. This work was funded by BBSRC grants BB/J00247X/1, BB/M004155/1, BB/M010066/1, the University of Dundee Global Challenges Research Fund and the European Union’s Horizon 2020 research and innovation programme under Marie Skłodowska-Curie grant agreement No. 799300. The m6A LC-MS analysis was carried out by Abdel Atrih of the FingerPrints Proteomics Facility, University of Dundee, which is supported by a Wellcome Trust Technology Platform award (097945/B/11/Z)

PY - 2020/1/14

Y1 - 2020/1/14

N2 - 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.

AB - 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.

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