Left panel: total genes with m6A peaks; right panel: genes conserved in human andArabidopsis

Left panel: total genes with m6A peaks; right panel: genes conserved in human andArabidopsis. positive correlation between m6A deposition and the mRNA abundance, suggesting a regulatory role of m6A in plant gene expression. Keywords:N6-methyladenosine (m6A), RNA methylation, plant mRNA methylome, chloroplast, gene expression == INTRODUCTION == N6-methyladenosine (m6A) is the most prevalent internal messenger RNA (mRNA) modification1-3in eukaryotes including mammals4, plants5,6,Drosophila7and yeast8, as well as viruses with a nuclear phase9. This modification is installed byN6-adenosine methyltransferase. A 70kD SAM (S-adenosylmethionine)-binding subunit methyltransferase like 3 (METTL3, also calledMT-A70) was identified as one component of a methyltransferase complex in mammalian cells10. Recent studies have characterized this complex, which consists ofMETTL3, methyltransferase like 14 (METTL14) and Wilms tumor 1 associated protein (WTAP)11-13.METTL14andMETTL3are two active methyltransferases that form a heterodimer to catalyze m6A ACH RNA methylation, 6H05 (TFA) while WTAP interacts with this complex and substantially affects the mRNA methylation inside cells but notin vitro11. Knockdowns of these methyltransferases affect mouse embryonic stem cell differentiation12. Since 2011, two m6A RNA demethylases ofFTOandALKBH5have been discovered; these demethylases are involved in mammalian development, RNA metabolism and fertility14,15. These findings reveal the first examples of reversible RNA modification and indicate regulatory functions of reversible m6A methylation on mRNA and certain non-coding RNAs that contain m6A16. Subsequent profiling of m6A distributions in mammalian transcriptomes17,18and the recent mapping of the yeast m6A methylome in the meiotic state19further confirm the dynamic nature of m6A modification. These studies revealed that m6A is enriched around the stop codon and at 3 UTRs, as well as in long internal exons and at the transcription start site (TSS)17-19. Cellular proteins have also been found to preferentially bind m6A-containing RNA17,20. The human YTH domain family 2 (YTHDF2) has recently been characterized to specifically recognize m6A-methylated mRNA and accelerate the decay of the bound mRNA20. InArabidopsis thaliana, the m6A content in mRNA varies across tissues with a high ratio of m6A/A found in flower buds6. This variation correlates with the expression levels of the plant methyltransferaseMTA(the plant homolog of humanMETTL3, encoded byAt4g10760)6. Previous studies have also shown that m6A predominantly locates at the 3 end of transcripts in a region 100-150 bp before the poly(A) tail inA. thalianamRNA21. Inactivation ofMTAprevents the progression of the developing embryo from passing the globular stage; an embryo-lethal phenotype with seed arrestment has been observed6. Reduced expression ofMTAinA. thalianaleads to decreased m6A level in mRNA and abnormal growth with reduced apical dominance, abnormal organ definition, and increased trichome branching21. These data demonstrate that m6A in mRNA plays functional roles in plant development. In order to further investigate the functions of m6A and to facilitate future studies of m6A in plants, we report here transcriptome-wide m6A profiling in two accessions ofA. thaliana, Can-0 and Hen-16. These accessions are wild-collected natural lines from the two extremes of the natural range of photosynthetically 6H05 (TFA) active radiation (PAR) in the spring22. We show that m6A is a highly conserved RNA modification in mRNA across these two accessions. Intriguingly, m6A inA. thalianais enriched not only around the stop codon and within 3 UTRs, as in yeast and mammalian systems, but also around the start codon, a property distinct from other known m6A methylomes17,18. A positive correlation between m6A deposition and mRNA levels indicates a regulatory role of m6A in plant gene expression. == RESULTS == == m6A is abundant and conserved in A. thaliana mRNA == m6A is known to be a relatively abundant internal modification inA. thalianamRNA6. We selected ten geographically diverse accessions 6H05 (TFA) ofA. thalianato grow in a common laboratory environment in order to measure the m6A/A ratio of purified mRNA (Supplementary Table 1). These wild-collected natural lines were collected from sites that vary widely in PAR values22. We observed that the ratio of m6A/A in total mRNA from these ten accessions varied within the range of 0.45-0.65% 6H05 (TFA) (Fig. 1a), although not directly related to PAR, suggesting that the m6A methylation level in mRNA is relatively stable but potentially affected by.