Home LiteratureArticle Details
PMID: 31931956 Published · epublish English Journal Article Research Support, Non-U.S. Gov't

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

eLife ·Vol. 9 ·2020-00-14

Parker MT, Knop K, Sherwood AV, Schurch NJ, Mackinnon K, Gould PD, Hall AJ, Barton GJ, Simpson GG

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.

Keywords
A. thaliana RNA chromosomes computational biology epitranscriptome gene expression nanopore polyadenylation systems biology
MeSH Terms
Adenosine/analogs & derivatives,metabolism Arabidopsis/genetics,metabolism Gene Expression Profiling Methylation Nanopores Poly A/genetics,metabolism RNA Caps RNA Processing, Post-Transcriptional RNA Splicing RNA, Messenger/chemistry,genetics,metabolism RNA, Plant/chemistry,genetics,metabolism RNA, Untranslated/chemistry,genetics Sequence Analysis, RNA
Chemicals
RNA Caps RNA, Messenger RNA, Plant RNA, Untranslated Poly A N-methyladenosine Adenosine
Authors & Affiliations
9 authors, click to expand affiliations / ORCID
Parker Matthew T ORCID
School of Life Sciences, University of Dundee, Dundee, United Kingdom.
Knop Katarzyna ORCID
School of Life Sciences, University of Dundee, Dundee, United Kingdom.
Sherwood Anna V
School of Life Sciences, University of Dundee, Dundee, United Kingdom.
Schurch Nicholas J ORCID
School of Life Sciences, University of Dundee, Dundee, United Kingdom.
Mackinnon Katarzyna
School of Life Sciences, University of Dundee, Dundee, United Kingdom.
Gould Peter D
Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.
Hall Anthony Jw
Earlham Institute, Norwich Research Park, Norwich, United Kingdom.
Barton Geoffrey J ORCID
School of Life Sciences, University of Dundee, Dundee, United Kingdom.
Simpson Gordon G ORCID
School of Life Sciences, University of Dundee, Dundee, United Kingdom. | James Hutton Institute, Invergowrie, United Kingdom.
Conflict of Interest

MP, KK, AS, NS, KM, PG, AH, GB, GS No competing interests declared

References (94)
94 references, click to expand
  1. Assessment of transcript reconstruction methods for RNA-seq.
    Nat Methods. 2013 Dec;10(12):1177-84 PMID: 24185837
  2. CPSF30 and Wdr33 directly bind to AAUAAA in mammalian mRNA 3' processing.
    Genes Dev. 2014 Nov 1;28(21):2370-80 PMID: 25301780
  3. Formation, regulation and evolution of Caenorhabditis elegans 3'UTRs.
    Nature. 2011 Jan 6;469(7328):97-101 PMID: 21085120
  4. Circadian control of messenger RNA stability. Association with a sequence-specific messenger RNA decay pathway.
    Plant Physiol. 2005 Aug;138(4):2374-85 PMID: 16055688
  5. Autoregulation of FCA pre-mRNA processing controls Arabidopsis flowering time.
    EMBO J. 2003 Jun 16;22(12):3142-52 PMID: 12805228
  6. PTGBase: an integrated database to study tandem duplicated genes in plants.
    Database (Oxford). 2015 Mar 22;2015:null PMID: 25797062
  7. Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome.
    Nat Methods. 2015 Aug;12(8):767-72 PMID: 26121403
  8. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana.
    Nature. 2000 Dec 14;408(6814):796-815 PMID: 11130711
  9. Nanopore native RNA sequencing of a human poly(A) transcriptome.
    Nat Methods. 2019 Dec;16(12):1297-1305 PMID: 31740818
  10. DART-seq: an antibody-free method for global m6A detection.
    Nat Methods. 2019 Dec;16(12):1275-1280 PMID: 31548708
  11. Ambiguous splice sites distinguish circRNA and linear splicing in the human genome.
    Bioinformatics. 2019 Apr 15;35(8):1263-1268 PMID: 30192918
  12. Highly parallel direct RNA sequencing on an array of nanopores.
    Nat Methods. 2018 Mar;15(3):201-206 PMID: 29334379
  13. Dynamic analyses of alternative polyadenylation from RNA-seq reveal a 3'-UTR landscape across seven tumour types.
    Nat Commun. 2014 Nov 20;5:5274 PMID: 25409906
  14. Biopython: freely available Python tools for computational molecular biology and bioinformatics.
    Bioinformatics. 2009 Jun 1;25(11):1422-3 PMID: 19304878
  15. Short poly(A) tails are a conserved feature of highly expressed genes.
    Nat Struct Mol Biol. 2017 Dec;24(12):1057-1063 PMID: 29106412
  16. Direct sequencing of Arabidopsis thaliana RNA reveals patterns of cleavage and polyadenylation.
    Nat Struct Mol Biol. 2012 Aug;19(8):845-52 PMID: 22820990
  17. Molecular basis for the recognition of the human AAUAAA polyadenylation signal.
    Proc Natl Acad Sci U S A. 2018 Feb 13;115(7):E1419-E1428 PMID: 29208711
  18. FIMO: scanning for occurrences of a given motif.
    Bioinformatics. 2011 Apr 1;27(7):1017-8 PMID: 21330290
  19. A majority of m6A residues are in the last exons, allowing the potential for 3' UTR regulation.
    Genes Dev. 2015 Oct 1;29(19):2037-53 PMID: 26404942
  20. N(6)-Methyladenosine RNA Modification Regulates Shoot Stem Cell Fate in Arabidopsis.
    Dev Cell. 2016 Jul 25;38(2):186-200 PMID: 27396363
  21. Deciphering the "m6A Code" via Antibody-Independent Quantitative Profiling.
    Cell. 2019 Jul 25;178(3):731-747.e16 PMID: 31257032
  22. Characterization of mRNA polyadenylation in the apicomplexa.
    PLoS One. 2018 Aug 30;13(8):e0203317 PMID: 30161237
  23. A high quality Arabidopsis transcriptome for accurate transcript-level analysis of alternative splicing.
    Nucleic Acids Res. 2017 May 19;45(9):5061-5073 PMID: 28402429
  24. What Are 3' UTRs Doing?
    Cold Spring Harb Perspect Biol. 2019 Oct 1;11(10):null PMID: 30181377
  25. Delayed fluorescence as a universal tool for the measurement of circadian rhythms in higher plants.
    Plant J. 2009 Jun;58(5):893-901 PMID: 19638147
  26. The draft genomes of five agriculturally important African orphan crops.
    Gigascience. 2019 Mar 1;8(3): PMID: 30535374
  27. Marking RNA: m6A writers, readers, and functions in Arabidopsis.
    J Mol Cell Biol. 2019 Oct 25;11(10):899-910 PMID: 31336387
  28. Reconstitution of CPSF active in polyadenylation: recognition of the polyadenylation signal by WDR33.
    Genes Dev. 2014 Nov 1;28(21):2381-93 PMID: 25301781
  29. proovread: large-scale high-accuracy PacBio correction through iterative short read consensus.
    Bioinformatics. 2014 Nov 1;30(21):3004-11 PMID: 25015988
  30. The U6 snRNA m6A Methyltransferase METTL16 Regulates SAM Synthetase Intron Retention.
    Cell. 2017 May 18;169(5):824-835.e14 PMID: 28525753
  31. Novel Stress-Inducible Antisense RNAs of Protein-Coding Loci Are Synthesized by RNA-Dependent RNA Polymerase.
    Plant Physiol. 2017 Sep;175(1):457-472 PMID: 28710133
  32. Chloroplast retrograde signal regulates flowering.
    Proc Natl Acad Sci U S A. 2016 Sep 20;113(38):10708-13 PMID: 27601637
  33. The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote.
    Nucleic Acids Res. 2013 May 1;41(10):e108 PMID: 23558742
  34. The RNA helicases AtMTR4 and HEN2 target specific subsets of nuclear transcripts for degradation by the nuclear exosome in Arabidopsis thaliana.
    PLoS Genet. 2014 Aug 21;10(8):e1004564 PMID: 25144737
  35. N6-Methyladenosine Inhibits Local Ribonucleolytic Cleavage to Stabilize mRNAs in Arabidopsis.
    Cell Rep. 2018 Oct 30;25(5):1146-1157.e3 PMID: 30380407
  36. Transcription termination and chimeric RNA formation controlled by Arabidopsis thaliana FPA.
    PLoS Genet. 2013 Oct;9(10):e1003867 PMID: 24204292
  37. Direct RNA sequencing on nanopore arrays redefines the transcriptional complexity of a viral pathogen.
    Nat Commun. 2019 Feb 14;10(1):754 PMID: 30765700
  38. DNA methylation in an intron of the IBM1 histone demethylase gene stabilizes chromatin modification patterns.
    EMBO J. 2012 Jun 29;31(13):2981-93 PMID: 22580822
  39. Comprehensive splice-site analysis using comparative genomics.
    Nucleic Acids Res. 2006;34(14):3955-67 PMID: 16914448
  40. Identification of factors required for m6 A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI.
    New Phytol. 2017 Jul;215(1):157-172 PMID: 28503769
  41. Structural basis of AAUAAA polyadenylation signal recognition by the human CPSF complex.
    Nat Struct Mol Biol. 2018 Feb;25(2):135-138 PMID: 29358758
  42. FLASH: fast length adjustment of short reads to improve genome assemblies.
    Bioinformatics. 2011 Nov 1;27(21):2957-63 PMID: 21903629
  43. Temperature-dependent regulation of flowering by antagonistic FLM variants.
    Nature. 2013 Nov 21;503(7476):414-7 PMID: 24067612
  44. m6A enhances the phase separation potential of mRNA.
    Nature. 2019 Jul;571(7765):424-428 PMID: 31292544
  45. RNA-methylation-dependent RNA processing controls the speed of the circadian clock.
    Cell. 2013 Nov 7;155(4):793-806 PMID: 24209618
  46. The m6A pathway protects the transcriptome integrity by restricting RNA chimera formation in plants.
    Life Sci Alliance. 2019 May 29;2(3): PMID: 31142640
  47. Improved annotation of 3' untranslated regions and complex loci by combination of strand-specific direct RNA sequencing, RNA-Seq and ESTs.
    PLoS One. 2014 Apr 10;9(4):e94270 PMID: 24722185
  48. Site identification in high-throughput RNA-protein interaction data.
    Bioinformatics. 2012 Dec 1;28(23):3013-20 PMID: 23024010
  49. Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences.
    F1000Res. 2015 Dec 30;4:1521 PMID: 26925227
  50. MultiQC: summarize analysis results for multiple tools and samples in a single report.
    Bioinformatics. 2016 Oct 1;32(19):3047-8 PMID: 27312411
  51. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.
    Bioinformatics. 2010 Jan 1;26(1):139-40 PMID: 19910308
  52. Apparent non-canonical trans-splicing is generated by reverse transcriptase in vitro.
    PLoS One. 2010 Aug 18;5(8):e12271 PMID: 20805885
  53. Deepbinner: Demultiplexing barcoded Oxford Nanopore reads with deep convolutional neural networks.
    PLoS Comput Biol. 2018 Nov 20;14(11):e1006583 PMID: 30458005
  54. SeqKit: A Cross-Platform and Ultrafast Toolkit for FASTA/Q File Manipulation.
    PLoS One. 2016 Oct 5;11(10):e0163962 PMID: 27706213
  55. uShuffle: a useful tool for shuffling biological sequences while preserving the k-let counts.
    BMC Bioinformatics. 2008 Apr 11;9:192 PMID: 18405375
  56. Alternative splicing and nonsense-mediated decay modulate expression of important regulatory genes in Arabidopsis.
    Nucleic Acids Res. 2012 Mar;40(6):2454-69 PMID: 22127866
  57. Direct RNA sequencing enables m6A detection in endogenous transcript isoforms at base-specific resolution.
    RNA. 2020 Jan;26(1):19-28 PMID: 31624092
  58. Unique features of the m6A methylome in Arabidopsis thaliana.
    Nat Commun. 2014 Nov 28;5:5630 PMID: 25430002
  59. Functional annotation of a full-length Arabidopsis cDNA collection.
    Science. 2002 Apr 5;296(5565):141-5 PMID: 11910074
  60. Light Controls Protein Localization through Phytochrome-Mediated Alternative Promoter Selection.
    Cell. 2017 Nov 30;171(6):1316-1325.e12 PMID: 29129375
  61. Araport11: a complete reannotation of the Arabidopsis thaliana reference genome.
    Plant J. 2017 Feb;89(4):789-804 PMID: 27862469
  62. The spen family protein FPA controls alternative cleavage and polyadenylation of RNA.
    Dev Cell. 2010 Feb 16;18(2):203-13 PMID: 20079695
  63. STAR: ultrafast universal RNA-seq aligner.
    Bioinformatics. 2013 Jan 1;29(1):15-21 PMID: 23104886
  64. External RNA Controls Consortium Beta Version Update.
    J Genomics. 2016 Jul 26;4:19-22 PMID: 27512518
  65. Integrative genomics viewer.
    Nat Biotechnol. 2011 Jan;29(1):24-6 PMID: 21221095
  66. Detecting RNA modifications in the epitranscriptome: predict and validate.
    Nat Rev Genet. 2017 May;18(5):275-291 PMID: 28216634
  67. Synthetic spike-in standards for RNA-seq experiments.
    Genome Res. 2011 Sep;21(9):1543-51 PMID: 21816910
  68. Recognition of RNA N6-methyladenosine by IGF2BP proteins enhances mRNA stability and translation.
    Nat Cell Biol. 2018 Mar;20(3):285-295 PMID: 29476152
  69. NanoPARE: parallel analysis of RNA 5' ends from low-input RNA.
    Genome Res. 2018 Dec;28(12):1931-1942 PMID: 30355603
  70. Sequencing nothing: Exploring failure modes of nanopore sensing and implications for life detection.
    Life Sci Space Res (Amst). 2018 Aug;18:80-86 PMID: 30100151
  71. Detecting differential usage of exons from RNA-seq data.
    Genome Res. 2012 Oct;22(10):2008-17 PMID: 22722343
  72. UMI-tools: modeling sequencing errors in Unique Molecular Identifiers to improve quantification accuracy.
    Genome Res. 2017 Mar;27(3):491-499 PMID: 28100584
  73. FY is an RNA 3' end-processing factor that interacts with FCA to control the Arabidopsis floral transition.
    Cell. 2003 Jun 13;113(6):777-87 PMID: 12809608
  74. Accurate detection of m6A RNA modifications in native RNA sequences.
    Nat Commun. 2019 Sep 9;10(1):4079 PMID: 31501426
  75. Proposed methods for testing and selecting the ERCC external RNA controls.
    BMC Genomics. 2005 Nov 02;6:150 PMID: 16266432
  76. Flexible expressed region analysis for RNA-seq with derfinder.
    Nucleic Acids Res. 2017 Jan 25;45(2):e9 PMID: 27694310
  77. A complete bacterial genome assembled de novo using only nanopore sequencing data.
    Nat Methods. 2015 Aug;12(8):733-5 PMID: 26076426
  78. The Sequence Alignment/Map format and SAMtools.
    Bioinformatics. 2009 Aug 15;25(16):2078-9 PMID: 19505943
  79. Earth BioGenome Project: Sequencing life for the future of life.
    Proc Natl Acad Sci U S A. 2018 Apr 24;115(17):4325-4333 PMID: 29686065
  80. Salmon provides fast and bias-aware quantification of transcript expression.
    Nat Methods. 2017 Apr;14(4):417-419 PMID: 28263959
  81. Minimap2: pairwise alignment for nucleotide sequences.
    Bioinformatics. 2018 Sep 15;34(18):3094-3100 PMID: 29750242
  82. Long-read direct RNA sequencing by 5'-Cap capturing reveals the impact of Piwi on the widespread exonization of transposable elements in locusts.
    RNA Biol. 2019 Jul;16(7):950-959 PMID: 30982421
  83. Probing N6-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA.
    RNA. 2013 Dec;19(12):1848-56 PMID: 24141618
  84. BulkVis: a graphical viewer for Oxford nanopore bulk FAST5 files.
    Bioinformatics. 2019 Jul 1;35(13):2193-2198 PMID: 30462145
  85. Dynamic RNA Modifications in Gene Expression Regulation.
    Cell. 2017 Jun 15;169(7):1187-1200 PMID: 28622506
  86. BEDTools: a flexible suite of utilities for comparing genomic features.
    Bioinformatics. 2010 Mar 15;26(6):841-2 PMID: 20110278
  87. BLAST+: architecture and applications.
    BMC Bioinformatics. 2009 Dec 15;10:421 PMID: 20003500
  88. The m(6)A Methyltransferase METTL3 Promotes Translation in Human Cancer Cells.
    Mol Cell. 2016 May 5;62(3):335-345 PMID: 27117702
  89. N6-methyladenosine-dependent regulation of messenger RNA stability.
    Nature. 2014 Jan 2;505(7481):117-20 PMID: 24284625
  90. MEME SUITE: tools for motif discovery and searching.
    Nucleic Acids Res. 2009 Jul;37(Web Server issue):W202-8 PMID: 19458158
  91. Mapping m6A at Individual-Nucleotide Resolution Using Crosslinking and Immunoprecipitation (miCLIP).
    Methods Mol Biol. 2017;1562:55-78 PMID: 28349454
  92. The genetics of plant clocks.
    Adv Genet. 2011;74:105-39 PMID: 21924976
  93. Rethinking m6A Readers, Writers, and Erasers.
    Annu Rev Cell Dev Biol. 2017 Oct 6;33:319-342 PMID: 28759256
  94. CarrierSeq: a sequence analysis workflow for low-input nanopore sequencing.
    BMC Bioinformatics. 2018 Mar 27;19(1):108 PMID: 29587645
Article Info
Journal
eLife
Abbr.
Elife
ISSN
2050-084X
DOI
10.7554/eLife.49658
Published
2020-00-14
Epub
2020-00-14
Language
English
Region
England
NLM ID
101579614
PMCID
PMC6959997
Subset
IM
Grants
Biotechnology and Biological Sciences Research Council · BB/M010066/1 · United Kingdom
Biotechnology and Biological Sciences Research Council · BB/H002286/1 · United Kingdom
Biotechnology and Biological Sciences Research Council · BB/J00247X/1 · United Kingdom
Biotechnology and Biological Sciences Research Council · BB/M004155/1 · United Kingdom
H2020 Marie Skłodowska-Curie Actions · 799300
Wellcome · 097945/B/11/Z
Databases
GEO
GSE112869
External Links
PubMed source DOI: 10.7554/eLife.49658 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2