Home LiteratureArticle Details
PMID: 28503769 Published · ppublish English Journal Article

Identification of factors required for m6 A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI.

The New phytologist ·Vol. 215 ·No. 1 ·2017-00-00 ·Pages 157-172

Růžička K, Zhang M, Campilho A, Bodi Z, Kashif M, Saleh M, Eeckhout D, El-Showk S, Li H, Zhong S, De Jaeger G, Mongan NP, Hejátko J, Helariutta Y, Fray RG

Abstract

N6-adenosine methylation (m6 A) of mRNA is an essential process in most eukaryotes, but its role and the status of factors accompanying this modification are still poorly understood. Using combined methods of genetics, proteomics and RNA biochemistry, we identified a core set of mRNA m6 A writer proteins in Arabidopsis thaliana. The components required for m6 A in Arabidopsis included MTA, MTB, FIP37, VIRILIZER and the E3 ubiquitin ligase HAKAI. Downregulation of these proteins led to reduced relative m6 A levels and shared pleiotropic phenotypes, which included aberrant vascular formation in the root, indicating that correct m6 A methylation plays a role in developmental decisions during pattern formation. The conservation of these proteins amongst eukaryotes and the demonstration of a role in writing m6 A for the E3 ubiquitin ligase HAKAI is likely to be of considerable relevance beyond the plant sciences.

Keywords
Arabidopsis HAKAI N6-adenosine methylation (m6A) VIRILIZER mRNA methylation protoxylem
MeSH Terms
Adenosine/metabolism Arabidopsis/metabolism Arabidopsis Proteins/genetics,metabolism,physiology Conserved Sequence Methylation Methyltransferases/genetics,metabolism,physiology Plants, Genetically Modified/metabolism RNA, Messenger/metabolism Sequence Alignment Ubiquitin-Protein Ligases/genetics,metabolism,physiology
Chemicals
Arabidopsis Proteins RNA, Messenger 6-methyladenine mRNA methyltransferase Methyltransferases Ubiquitin-Protein Ligases Adenosine
Authors & Affiliations
15 authors, click to expand affiliations / ORCID
Růžička Kamil
Functional Genomics and Proteomics of Plants, Central European Institute of Technology and National Centre for Biomolecular Research, Masaryk University, 62500, Brno, Czech Republic. | Institute of Biotechnology, University of Helsinki, 00014, Helsinki, Finland.
Zhang Mi
Plant Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK.
Campilho Ana
Institute of Biotechnology, University of Helsinki, 00014, Helsinki, Finland. | Research Center in Biodiversity and Genetic Resources, University of Porto, 4485-661 Vairão, Portugal.
Bodi Zsuzsanna
Plant Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK.
Kashif Muhammad
Institute of Biotechnology, University of Helsinki, 00014, Helsinki, Finland.
Saleh Mária
Functional Genomics and Proteomics of Plants, Central European Institute of Technology and National Centre for Biomolecular Research, Masaryk University, 62500, Brno, Czech Republic.
Eeckhout Dominique
Department of Plant Systems Biology, VIB, 9052, Gent, Belgium. | Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052, Gent, Belgium.
El-Showk Sedeer
Institute of Biotechnology, University of Helsinki, 00014, Helsinki, Finland.
Li Hongying
Plant Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK. | Key Laboratory of Crop Gene Resources and Germplasm Enhancement on Loess Plateau, Ministry of Agriculture, Taiyuan, Shanxi, 030031, China.
Zhong Silin
Plant Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK. | The State Key Laboratory of Agrobiotechnology, The School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China.
De Jaeger Geert
Department of Plant Systems Biology, VIB, 9052, Gent, Belgium. | Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052, Gent, Belgium.
Mongan Nigel P
School of Veterinary Medicine and Sciences, University of Nottingham, Sutton Bonington, Loughborough, LE12 5RD, UK.
Hejátko Jan
Functional Genomics and Proteomics of Plants, Central European Institute of Technology and National Centre for Biomolecular Research, Masaryk University, 62500, Brno, Czech Republic.
Helariutta Ykä
Institute of Biotechnology, University of Helsinki, 00014, Helsinki, Finland. | Sainsbury Laboratory, University of Cambridge, Cambridge, CB2 1LR, UK.
Fray Rupert G
Plant Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK.
References (89)
89 references, click to expand
  1. N(6)-methyladenosine Modulates Messenger RNA Translation Efficiency.
    Cell. 2015 Jun 4;161(6):1388-99 PMID: 26046440
  2. Arabidopsis PIS1 encodes the ABCG37 transporter of auxinic compounds including the auxin precursor indole-3-butyric acid.
    Proc Natl Acad Sci U S A. 2010 Jun 8;107(23 ):10749-53 PMID: 20498067
  3. AtPIN4 mediates sink-driven auxin gradients and root patterning in Arabidopsis.
    Cell. 2002 Mar 8;108(5):661-73 PMID: 11893337
  4. Transcriptome-wide N⁶-methyladenosine profiling of rice callus and leaf reveals the presence of tissue-specific competitors involved in selective mRNA modification.
    RNA Biol. 2014;11(9):1180-8 PMID: 25483034
  5. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega.
    Mol Syst Biol. 2011 Oct 11;7:539 PMID: 21988835
  6. Yeast m6A Methylated mRNAs Are Enriched on Translating Ribosomes during Meiosis, and under Rapamycin Treatment.
    PLoS One. 2015 Jul 17;10(7):e0132090 PMID: 26186436
  7. Jalview Version 2--a multiple sequence alignment editor and analysis workbench.
    Bioinformatics. 2009 May 1;25(9):1189-91 PMID: 19151095
  8. The Pfam protein families database.
    Nucleic Acids Res. 2012 Jan;40(Database issue):D290-301 PMID: 22127870
  9. rMATS: robust and flexible detection of differential alternative splicing from replicate RNA-Seq data.
    Proc Natl Acad Sci U S A. 2014 Dec 23;111(51):E5593-601 PMID: 25480548
  10. agriGO: a GO analysis toolkit for the agricultural community.
    Nucleic Acids Res. 2010 Jul;38(Web Server issue):W64-70 PMID: 20435677
  11. Adenosine Methylation in Arabidopsis mRNA is Associated with the 3' End and Reduced Levels Cause Developmental Defects.
    Front Plant Sci. 2012 Mar 23;3:48 PMID: 22639649
  12. Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons.
    Cell. 2012 Jun 22;149(7):1635-46 PMID: 22608085
  13. Mapping of N6-methyladenosine residues in bovine prolactin mRNA.
    Proc Natl Acad Sci U S A. 1984 Sep;81(18):5667-71 PMID: 6592581
  14. Purification and cDNA cloning of the AdoMet-binding subunit of the human mRNA (N6-adenosine)-methyltransferase.
    RNA. 1997 Nov;3(11):1233-47 PMID: 9409616
  15. The immunophilin-interacting protein AtFIP37 from Arabidopsis is essential for plant development and is involved in trichome endoreduplication.
    Plant Physiol. 2004 Apr;134(4):1283-92 PMID: 15047892
  16. Co-localisation studies of Arabidopsis SR splicing factors reveal different types of speckles in plant cell nuclei.
    Exp Cell Res. 2008 Oct 15;314(17 ):3175-86 PMID: 18674533
  17. A comprehensive two-hybrid analysis to explore the yeast protein interactome.
    Proc Natl Acad Sci U S A. 2001 Apr 10;98(8):4569-74 PMID: 11283351
  18. Structure prediction and phylogenetic analysis of a functionally diverse family of proteins homologous to the MT-A70 subunit of the human mRNA:m(6)A methyltransferase.
    J Mol Evol. 2002 Oct;55(4):431-44 PMID: 12355263
  19. ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility.
    Mol Cell. 2013 Jan 10;49(1):18-29 PMID: 23177736
  20. Localization and dynamics of nuclear speckles in plants.
    Plant Physiol. 2012 Jan;158(1):67-77 PMID: 22045923
  21. SHOREmap: simultaneous mapping and mutation identification by deep sequencing.
    Nat Methods. 2009 Aug;6(8):550-1 PMID: 19644454
  22. N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO.
    Nat Chem Biol. 2011 Oct 16;7(12 ):885-7 PMID: 22002720
  23. N6-methyladenosine-dependent regulation of messenger RNA stability.
    Nature. 2014 Jan 2;505(7481):117-20 PMID: 24284625
  24. Cytokinin signaling and its inhibitor AHP6 regulate cell fate during vascular development.
    Science. 2006 Jan 6;311(5757):94-8 PMID: 16400151
  25. Insights into nuclear organization in plants as revealed by the dynamic distribution of Arabidopsis SR splicing factors.
    Plant Cell. 2006 Nov;18(11):3218-34 PMID: 17114353
  26. Novel roles of hakai in cell proliferation and oncogenesis.
    Mol Biol Cell. 2009 Aug;20(15):3533-42 PMID: 19535458
  27. Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set.
    Plant J. 2009 Jul;59(1):169-78 PMID: 19309456
  28. Induction of sporulation in Saccharomyces cerevisiae leads to the formation of N6-methyladenosine in mRNA: a potential mechanism for the activity of the IME4 gene.
    Nucleic Acids Res. 2002 Oct 15;30(20):4509-18 PMID: 12384598
  29. Differential expression analysis for sequence count data.
    Genome Biol. 2010;11(10):R106 PMID: 20979621
  30. Tissue-specific expression and dynamic organization of SR splicing factors in Arabidopsis.
    Mol Biol Cell. 2004 Jun;15(6):2664-73 PMID: 15034145
  31. Improving yeast two-hybrid screening systems.
    Brief Funct Genomic Proteomic. 2007 Dec;6(4):302-12 PMID: 18218650
  32. Transcriptome-wide high-throughput deep m(6)A-seq reveals unique differential m(6)A methylation patterns between three organs in Arabidopsis thaliana.
    Genome Biol. 2015 Dec 14;16:272 PMID: 26667818
  33. RNA N6-methyladenosine methylation in post-transcriptional gene expression regulation.
    Genes Dev. 2015 Jul 1;29(13):1343-55 PMID: 26159994
  34. Identification of Wilms' tumor 1-associating protein complex and its role in alternative splicing and the cell cycle.
    J Biol Chem. 2013 Nov 15;288(46):33292-302 PMID: 24100041
  35. STAR: ultrafast universal RNA-seq aligner.
    Bioinformatics. 2013 Jan 1;29(1):15-21 PMID: 23104886
  36. Morphometric analysis of root shape.
    New Phytol. 2005 Feb;165(2):641-51 PMID: 15720674
  37. HNRNPA2B1 Is a Mediator of m(6)A-Dependent Nuclear RNA Processing Events.
    Cell. 2015 Sep 10;162(6):1299-308 PMID: 26321680
  38. Identification of genes required for embryo development in Arabidopsis.
    Plant Physiol. 2004 Jul;135(3):1206-20 PMID: 15266054
  39. A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation.
    Nat Chem Biol. 2014 Feb;10(2):93-5 PMID: 24316715
  40. The gene virilizer is required for female-specific splicing controlled by Sxl, the master gene for sexual development in Drosophila.
    Development. 1995 Dec;121(12):4017-26 PMID: 8575302
  41. RNA methylation by the MIS complex regulates a cell fate decision in yeast.
    PLoS Genet. 2012;8(6):e1002732 PMID: 22685417
  42. MultiSite Gateway-Compatible Cell Type-Specific Gene-Inducible System for Plants.
    Plant Physiol. 2016 Feb;170(2):627-41 PMID: 26644504
  43. N(6)-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions.
    Nature. 2015 Feb 26;518(7540):560-4 PMID: 25719671
  44. Biochemical function of female-lethal (2)D/Wilms' tumor suppressor-1-associated proteins in alternative pre-mRNA splicing.
    J Biol Chem. 2003 Jan 31;278(5):3040-7 PMID: 12444081
  45. A mutually inhibitory interaction between auxin and cytokinin specifies vascular pattern in roots.
    Curr Biol. 2011 Jun 7;21(11):917-26 PMID: 21620702
  46. MODOMICS: a database of RNA modification pathways--2013 update.
    Nucleic Acids Res. 2013 Jan;41(Database issue):D262-7 PMID: 23118484
  47. Recombinational cloning with plant gateway vectors.
    Plant Physiol. 2007 Dec;145(4):1144-54 PMID: 18056864
  48. NIH Image to ImageJ: 25 years of image analysis.
    Nat Methods. 2012 Jul;9(7):671-5 PMID: 22930834
  49. N6-methyladenosine modification destabilizes developmental regulators in embryonic stem cells.
    Nat Cell Biol. 2014 Feb;16(2):191-8 PMID: 24394384
  50. The Arabidopsis epitranscriptome.
    Curr Opin Plant Biol. 2015 Oct;27:17-21 PMID: 26048078
  51. Essential requirement for RING finger E3 ubiquitin ligase Hakai in early embryonic development of Drosophila.
    Genes Cells. 2009 Sep;14 (9):1067-77 PMID: 19682089
  52. GABI-Kat SimpleSearch: new features of the Arabidopsis thaliana T-DNA mutant database.
    Nucleic Acids Res. 2012 Jan;40(Database issue):D1211-5 PMID: 22080561
  53. m6A modulates neuronal functions and sex determination in Drosophila.
    Nature. 2016 Dec 8;540(7632):242-247 PMID: 27919077
  54. SMART 7: recent updates to the protein domain annotation resource.
    Nucleic Acids Res. 2012 Jan;40(Database issue):D302-5 PMID: 22053084
  55. Dynamic m(6)A mRNA methylation directs translational control of heat shock response.
    Nature. 2015 Oct 22;526(7574):591-4 PMID: 26458103
  56. N(6)-Methyladenosine RNA Modification Regulates Shoot Stem Cell Fate in Arabidopsis.
    Dev Cell. 2016 Jul 25;38(2):186-200 PMID: 27396363
  57. An in vitro system for accurate methylation of internal adenosine residues in messenger RNA.
    Science. 1988 Nov 25;242(4882):1159-62 PMID: 3187541
  58. A tandem affinity purification-based technology platform to study the cell cycle interactome in Arabidopsis thaliana.
    Mol Cell Proteomics. 2007 Jul;6(7):1226-38 PMID: 17426018
  59. Targeted interactomics reveals a complex core cell cycle machinery in Arabidopsis thaliana.
    Mol Syst Biol. 2010 Aug 10;6:397 PMID: 20706207
  60. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq.
    Nature. 2012 Apr 29;485(7397):201-6 PMID: 22575960
  61. Structure and binding specificity of the receiver domain of sensor histidine kinase CKI1 from Arabidopsis thaliana.
    Plant J. 2011 Sep;67(5):827-39 PMID: 21569135
  62. N6-methyladenosine marks primary microRNAs for processing.
    Nature. 2015 Mar 26;519(7544):482-5 PMID: 25799998
  63. Plant development. Arabidopsis NAC45/86 direct sieve element morphogenesis culminating in enucleation.
    Science. 2014 Aug 22;345(6199):933-7 PMID: 25081480
  64. Regulatory mechanisms for specification and patterning of plant vascular tissues.
    Annu Rev Cell Dev Biol. 2010;26:605-37 PMID: 20590454
  65. Role of transcription factor Kar4 in regulating downstream events in the Saccharomyces cerevisiae pheromone response pathway.
    Mol Cell Biol. 2007 Feb;27(3):818-29 PMID: 17101777
  66. Hakai, a c-Cbl-like protein, ubiquitinates and induces endocytosis of the E-cadherin complex.
    Nat Cell Biol. 2002 Mar;4(3):222-31 PMID: 11836526
  67. Nuclear m(6)A Reader YTHDC1 Regulates mRNA Splicing.
    Mol Cell. 2016 Feb 18;61(4):507-19 PMID: 26876937
  68. Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5' sites.
    Cell Rep. 2014 Jul 10;8(1):284-96 PMID: 24981863
  69. Boosting tandem affinity purification of plant protein complexes.
    Trends Plant Sci. 2008 Oct;13(10):517-20 PMID: 18771946
  70. Drosophila Inducer of MEiosis 4 (IME4) is required for Notch signaling during oogenesis.
    Proc Natl Acad Sci U S A. 2011 Sep 6;108(36):14855-60 PMID: 21873203
  71. 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
  72. Polar delivery in plants; commonalities and differences to animal epithelial cells.
    Open Biol. 2014 Apr 16;4:140017 PMID: 24740985
  73. The dynamic epitranscriptome: N6-methyladenosine and gene expression control.
    Nat Rev Mol Cell Biol. 2014 May;15(5):313-26 PMID: 24713629
  74. Molecular identification of virilizer, a gene required for the expression of the sex-determining gene Sex-lethal in Drosophila melanogaster.
    Genetics. 2001 Feb;157(2):679-88 PMID: 11156988
  75. Characterization and partial purification of mRNA N6-adenosine methyltransferase from HeLa cell nuclei. Internal mRNA methylation requires a multisubunit complex.
    J Biol Chem. 1994 Jul 1;269(26):17697-704 PMID: 8021282
  76. Stem cells. m6A mRNA methylation facilitates resolution of naïve pluripotency toward differentiation.
    Science. 2015 Feb 27;347(6225):1002-6 PMID: 25569111
  77. A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome.
    Plant Mol Biol. 1992 Dec;20(6):1203-7 PMID: 1463857
  78. m6A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination.
    Nature. 2016 Dec 8;540(7632):301-304 PMID: 27919081
  79. Unique features of the m6A methylome in Arabidopsis thaliana.
    Nat Commun. 2014 Nov 28;5:5630 PMID: 25430002
  80. Nuclear speckles.
    Cold Spring Harb Perspect Biol. 2011 Feb 01;3(2):null PMID: 20926517
  81. RNA-methylation-dependent RNA processing controls the speed of the circadian clock.
    Cell. 2013 Nov 7;155(4):793-806 PMID: 24209618
  82. Isolation of transcription factor complexes from Arabidopsis cell suspension cultures by tandem affinity purification.
    Methods Mol Biol. 2011;754:195-218 PMID: 21720954
  83. MTA is an Arabidopsis messenger RNA adenosine methylase and interacts with a homolog of a sex-specific splicing factor.
    Plant Cell. 2008 May;20(5):1278-88 PMID: 18505803
  84. Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase.
    Cell Res. 2014 Feb;24(2):177-89 PMID: 24407421
  85. Biological influence of Hakai in cancer: a 10-year review.
    Cancer Metastasis Rev. 2012 Jun;31(1-2):375-86 PMID: 22349934
  86. Identification of fusion genes in breast cancer by paired-end RNA-sequencing.
    Genome Biol. 2011;12(1):R6 PMID: 21247443
  87. PLETHORA gradient formation mechanism separates auxin responses.
    Nature. 2014 Nov 6;515(7525):125-9 PMID: 25156253
  88. Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease.
    Nat Biotechnol. 2013 Aug;31(8):691-3 PMID: 23929340
  89. The Arabidopsis SeedGenes Project.
    Nucleic Acids Res. 2003 Jan 1;31(1):90-3 PMID: 12519955
Article Info
Journal
The New phytologist
Abbr.
New Phytol
ISSN
1469-8137
DOI
10.1111/nph.14586
Published
2017-00-00
Epub
2017-00-15
Pages
157-172
Language
English
Region
England
NLM ID
9882884
PMCID
PMC5488176
Subset
IM
Grants
European Research Council · 323052 · International
Biotechnology and Biological Sciences Research Council · BB/C513369/1 · United Kingdom
External Links
PubMed source DOI: 10.1111/nph.14586 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