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.

Abstract SERRATE/ARS2 is a conserved RNA effector protein involved in transcription, processing and export of different types of RNAs. In Arabidopsis, the best-studied function of SERRATE (SE) is to promote miRNA processing. Here, we report that SE interacts with the nuclear exosome targeting (NEXT) complex, comprising the RNA helicase HEN2, the RNA binding protein RBM7 and one of the two zinc-knuckle proteins ZCCHC8A/ZCCHC8B. The identification of common targets of SE and HEN2 by RNA-seq supports the idea that SE cooperates with NEXT for RNA surveillance by the nuclear exosome. Among the RNA targets accumulating in absence of SE or NEXT are miRNA precursors. Loss of NEXT components results in the accumulation of pri-miRNAs without affecting levels of miRNAs, indicating that NEXT is, unlike SE, not required for miRNA processing. As compared to se-2, se-2 hen2-2 double mutants showed increased accumulation of pri-miRNAs, but partially restored levels of mature miRNAs and attenuated developmental defects. We propose that the slow degradation of pri-miRNAs caused by loss of HEN2 compensates for the poor miRNA processing efficiency in se-2 mutants, and that SE regulates miRNA biogenesis through its double contribution in promoting miRNA processing but also pri-miRNA degradation through the recruitment of the NEXT complex.

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

N6-methyladenosine (m6A) is known to occur in plant and animal messenger RNAs (mRNAs) since the 1970s. However, the scope and function of this modification remained un-explored till very recently. Since the beginning of this decade, owing to major technological breakthroughs, the interest in m6A has peaked again. Similar to animal mRNAs, plant mRNAs are also m6A methylated, within a specific sequence motif which is conserved across these kingdoms. m6A has been found to be pivotal for plant development and necessary for processes ranging from seed germination to floral development. A wide range of proteins involved in methylation of adenosine have been identified alongside proteins that remove or identify m6A. This review aims to put together the current knowledge regarding m6A in Arabidopsis thaliana.

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Since their discovery, microRNAs have led to a huge shift in our understanding of the regulation of key biological processes. The discovery of epigenetic modifications that affect microRNA expression has added another layer of complexity to the already tightly controlled regulatory machinery. Modifications like uridylation, adenylation and RNA editing have been shown to have variable effects on miRNA biogenesis and action. Methylation of the N6 adenosine has been studied extensively in mRNA. Presence of the N6-methyl-adenosine (mA) mark and its critical importance in miRNA biogenesis in animals adds to our understanding of the regulatory mechanisms, while its effect on miRNA biogenesis in plants is yet to be understood.

SERRATE/ARS2 is a conserved RNA effector protein involved in transcription, processing and export of different types of RNAs. In Arabidopsis, the best-studied function of SERRATE (SE) is to promote miRNA processing. Here, we report that SE interacts with the Nuclear Exosome Targeting (NEXT) complex, comprising the RNA helicase HEN2, the RNA binding protein RBM7 and one of the two zincknuckle proteins ZCCHC8A/ZCCHC8B. The identification of common targets of SE and HEN2 by RNAseq supports the idea that SE cooperates with NEXT for RNA surveillance by the nuclear exosome.Among the RNA targets accumulating in absence of SE or NEXT are miRNA precursors. Loss of NEXT components results in the accumulation of pri-miRNAs without affecting levels of miRNAs, indicating that NEXT is, unlike SE, not required for miRNA processing. As compared to se-2, se-2 hen2-2 double mutants showed increased accumulation of pri-miRNAs, but partially restored levels of mature miRNAs and attenuated developmental defects. We propose that the slow degradation of pri-miRNAs caused by loss of HEN2 compensates for the poor miRNA processing efficiency in se-2 mutants, and that SE regulates miRNA biogenesis through its double contribution in promoting miRNA processing but also pri-miRNA degradation through the recruitment of the NEXT complex. IntroductionIn plants, many miRNAs are encoded by independent RNA polymerase II transcription units. The primary transcripts contain a 5' cap structure as well as a poly(A) tail at the 3' end (1), and sometimes introns. The primary or spliced pri-miRNAs adopt stem loop structures which are processed by the nuclear endonuclease Dicer like 1 (DCL1) (2) in two sequential reactions. The first step creates shorter miRNA precursors called pre-miRNAs. The second step produces miRNA/miRNA* duplexes of mostly 21 nt with 2 nt overhangs at both 3' ends. DCL1 associates with the double stranded RNA binding 2 protein HYPONASTIC LEAVES 1 (HYL1) and the zinc finger domain-containing RNA effector protein SERRATE (3-5). Both HYL1 and SE interact with DCL1 as well as with each other (6-8). DCL1, SE and HYL1 form the core of the plant Microprocessor complex and co-localize within the nucleus in special structures known as dicing bodies (D-bodies) (7). In absence of HYL1 or upon reduced activity of SE, dicing by DCL1 is not completely abolished but less efficient and less accurate (3-5). The importance of SE for the biogenesis of miRNAs is illustrated by the phenotype of se mutants. Complete loss of SE expression as in the null mutant se-4 is embryonic lethal, while the se-1 mutation, which results in the expression of a truncated SE lacking 20 amino acids at its C-terminus, severely affects developmental timing, phylotaxy, meristem function and patterning of leaves and flowers (4,9). The se-2 mutation, in which the SE protein is truncated by 40 amino acids from the C-terminus, additionally displays the hyponastic leave shape that is characteristic for many miRNA biogenesis mutants including hyl1-2, hst-1 and ago1-25 (10-13).In ad...

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