N 6 -Methyladenosine is a ubiquitous modification identified in the mRNA of numerous eukaryotes, where it is present within both coding and noncoding regions. However, this base modification does not alter the coding capacity, and its biological significance remains unclear. We show that Arabidopsis thaliana mRNA contains N 6 -methyladenosine at levels similar to those previously reported for animal cells. We further show that inactivation of the Arabidopsis ortholog of the yeast and human mRNA adenosine methylase (MTA) results in failure of the developing embryo to progress past the globular stage. We also demonstrate that the arrested seeds are deficient in mRNAs containing N 6 -methyladenosine. Expression of MTA is strongly associated with dividing tissues, particularly reproductive organs, shoot meristems, and emerging lateral roots. Finally, we show that MTA interacts in vitro and in vivo with At FIP37, a homolog of the Drosophila protein FEMALE LETHAL2D and of human WILMS' TUMOUR1-ASSOCIATING PROTEIN. The results reported here provide direct evidence for an essential function for N 6 -methyladenosine in a multicellular eukaryote, and the interaction with At FIP37 suggests possible RNA processing events that might be regulated or altered by this base modification.

(2016) m6A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination. Nature, 540 (7632). pp. 301-304. ISSN 1476-4687 Access from the University of Nottingham repository: http://eprints.nottingham.ac.uk/39448/1/Nature_m6A_2016.pdf Copyright and reuse:The Nottingham ePrints service makes this work by researchers of the University of Nottingham available open access under the following conditions. This article is made available under the University of Nottingham End User licence and may be reused according to the conditions of the licence. For more details see: http://eprints.nottingham.ac.uk/end_user_agreement.pdf A note on versions:The version presented here may differ from the published version or from the version of record. If you wish to cite this item you are advised to consult the publisher's version. Please see the repository url above for details on accessing the published version and note that access may require a subscription. sex bias towards maleness. This is because m6A is required for female-specific AS of Sxl, 37 which determines female physiognomy, but also translationally represses male-specific 38 lethal2 (msl-2) to prevent dosage compensation normally occurring in males. We further 39show that the m6A reader protein YT521-B decodes m6A in the sex-specifically spliced 40 intron of Sxl, as its absence phenocopies dIME4 mutants. Loss of m6A also affects AS of 41 additional genes, predominantly in the 5'UTR, and has global impacts on the expression of 42 metabolic genes. Requirement of m6A and its reader YT521-B for female-specific Sxl AS 43 reveal that this hitherto enigmatic mRNA modification constitutes an ancient and specific 44 mechanism to adjust levels of gene expression. 45In mature mRNA the m6A modification is most prevalently found around the stop codon as well 46 as in 5'UTRs and in long exons in mammals, plants and yeast 2,3,6,7,15 . Since methylosome 47 components predominantly localize to the nucleus it has been speculated that m6A localized in 48Haussmann et al.3 pre-mRNA introns could have a role in AS regulation in addition to such a role when present in 49 long exons [9][10][11][12]16 . This prompted us to investigate whether m6A is required for Sxl AS, which 50 determines female sex and prevents dosage compensation in females 13 . We generated a null 51 allele of the Drosophila METTL3 methyltransferase homologue dIME4 by imprecise excision of 52 a P-element inserted in the promoter region. The excision ∆22-3 deletes most of the protein-53 coding region including the catalytic domain and is thus referred to as dIME4 null (Fig 1a). These 54 flies are viable and fertile, but flightless, and this phenotype can be rescued by a genomic 55 construct restoring dIME4 (Fig 1a and b). dIME4 shows increased expression in the brain, and 56 like in mammals and plants 17, localizes to the nucleus (Fig 1c,d). 57Following RNAse T1 digestion and 32 P end-labeling of RNA fragments we detected m6A after G 58 in polyA mRNA of adult flies at relatively low l...

Summary N6‐adenosine methylation (m6A) 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 m6A writer proteins in Arabidopsis thaliana.The components required for m6A in Arabidopsis included MTA, MTB, FIP37, VIRILIZER and the E3 ubiquitin ligase HAKAI. Downregulation of these proteins led to reduced relative m6A levels and shared pleiotropic phenotypes, which included aberrant vascular formation in the root, indicating that correct m6A 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 m6A for the E3 ubiquitin ligase HAKAI is likely to be of considerable relevance beyond the plant sciences.

We previously showed that the N6-methyladenosine (m6A) mRNA methylase is essential during Arabidopsis thaliana embryonic development. We also demonstrated that this modification is present at varying levels in all mature tissues. However, the requirement for the m6A in the mature plant was not tested. Here we show that a 90% reduction in m6A levels during later growth stages gives rise to plants with altered growth patterns and reduced apical dominance. The flowers of these plants commonly show defects in their floral organ number, size, and identity. The global analysis of gene expression from reduced m6A plants show that a significant number of down-regulated genes are involved in transport, or targeted transport, and most of the up-regulated genes are involved in stress and stimulus response processes. An analysis of m6A distribution in fragmented mRNA suggests that the m6A is predominantly positioned toward the 3′ end of transcripts in a region 100–150 bp before the poly(A) tail. In addition to the analysis of the phenotypic changes in the low methylation Arabidopsis plants we will review the latest advances in the field of mRNA internal methylation

N6-Methyladenosine (m6A) is a modified base present in the mRNA of all higher eukaryotes and in Saccharomyces cerevisiae, where there is an increase in m6A levels during sporulation. The methyltransferase, Ime4, is responsible for this modification and has a role in the initiation of meiosis. However, neither the function, nor the extent of distribution of this nucleotide modification is established. We demonstrate that in S. cerevisiae, substantial levels of internal adenosine methylation are present in the GpA context in mRNA from sporulating cells, which is consistent with the preferred methylation consensus of higher eukaryotes. Based upon our quantification data, every second transcript could contain one m6A during meiosis. As methylation is distributed across all mRNA size ranges, it is likely that m6A is not limited to a small population of messages. We developed a new antibody based method for identifying m6A containing messages, and using this method the transcripts of three key, early regulators of meiosis, IME1, IME2 and IME4 itself, were identified as being methylated. The position of m6A in IME2 was narrowed down to a region in the 3′-end. Methylation of these and other targets suggests mechanisms by which IME4 could control developmental choices leading to meiosis.