In Arabidopsis thaliana, the METTL3 homolog, mRNA adenosine methylase (MTA) introduces N6-methyladenosine (m6A) into various coding and noncoding RNAs of the plant transcriptome. Here, we show that an MTA-deficient mutant (mta) has decreased levels of microRNAs (miRNAs) but accumulates primary miRNA transcripts (pri-miRNAs). Moreover, pri-miRNAs are methylated by MTA, and RNA structure probing analysis reveals a decrease in secondary structure within stem–loop regions of these transcripts in mta mutant plants. We demonstrate interaction between MTA and both RNA Polymerase II and TOUGH (TGH), a plant protein needed for early steps of miRNA biogenesis. Both MTA and TGH are necessary for efficient colocalization of the Microprocessor components Dicer-like 1 (DCL1) and Hyponastic Leaves 1 (HYL1) with RNA Polymerase II. We propose that secondary structure of miRNA precursors induced by their MTA-dependent m6A methylation status, together with direct interactions between MTA and TGH, influence the recruitment of Microprocessor to plant pri-miRNAs. Therefore, the lack of MTA in mta mutant plants disturbs pri-miRNA processing and leads to the decrease in miRNA accumulation. Furthermore, our findings reveal that reduced miR393b levels likely contributes to the impaired auxin response phenotypes of mta mutant plants.

m 6 A, one of the most abundant mRNA modifications, has been associated with various metabolic processes in plants. Here we show that m 6 A also plays a role in miRNA biogenesis in Arabidopsis thaliana. Significant reductions in plant m 6 A/MTA levels results in lower accumulation of miRNAs whereas pri-miRNA levels tend to be higher in such plants. m 6 A-IP Seq and MTA-GFP RIP were used to show that many pri-miRNAs are m 6 A methylated and are bound by MTA, further demonstrating that pri-miRNAs can also be substrates for m 6 A methylation by MTA. We report that MTA interacts with RNA Pol II, supporting the assumption that m 6 A methylation is a co-transcriptional process, and also identify TGH, a known miRNA biogenesis related protein, as a novel protein that interacts with MTA. Finally, reduced levels of 2 miR393b may partially explain the strong auxin insensitivity seen in Arabidopsis plants with reduced m 6 A levels.Introduction: N 6 -methyladenosine (m 6 A), one of the most abundant mRNA modifications in eukaryotic cells can regulate eukaryote gene expression at multiple post-and cotranscriptional levels. m 6 A methylation in animal mRNAs is associated with several biological processes, ranging from cancer 1 , viral infections 2,3 to cell development 4,5 with the underpinning mechanisms including m 6 A regulated pre-mRNA splicing patterns, mRNA export, mRNA stability and changes in translational efficiency 6 . A group of proteins that collectively form the RNA methylation "writer" complex have been characterized and are well conserved between plants and animals. The mammalian m 6 A methyltranserase complex consists of Methyltransferase Like 3 (METTL3) 7 , Methyltransferase Like 14 (METTL14) 8 , Wilms' Tumour1-Associating Protein (WTAP) 9 , VIRMA (KIAA1429) 10 , RNA-binding motif protein 15 (RBM15) 11 and Zinc Finger CCCH-Type Containing 13 (ZC3H13) 12,13 . While METTL3 has been identified as the catalytic protein in this complex 7 , auxiliary proteins provide specificity and/or help with proper localization of the complex 6 . The m 6 A mark can be recognized by various "readers", the best characterized of which belong to the YT521-B homology (YTH) domain family [14][15][16][17] . The modification can also be removed from transcripts by "erasers", which in humans include fat mass and obesity-associated protein (FTO) 18 and α-ketoglutaratedependent dioxygenase alkB homolog 5 (ALKBH5) 19 .In Arabidopsis thaliana, the presence of m 6 A was first reported in 2008 and was shown to be dependent upon the activity of mRNA adenosine methylase (MTA) [homolog of human METTL3], the catalytic component of Arabidopsis m 6 A methyltransferase complex 20 . FKBP12 interacting protein 37 kDa (FIP37, homolog of WTAP) was the first identified methyltransferase

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