mRNA adenosine methylase (MTA) deposits m⁶A on pri-miRNAs to modulate miRNA biogenesis in Arabidopsis thaliana

Abstract

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

Occurrence and Functions of m6A and Other Covalent Modifications in Plant mRNA

“…It is a question of outstanding importance to define if and how the labeling of these many housekeeping transcripts by m 6 A contributes to plant growth and development. Finally, a recent report deposited in bioRxiv shows that primary microRNA transcripts in Arabidopsis also contain m 6 A and proposes that the modification regulates microRNA biogenesis (Bhat et al, 2019), as previously proposed in animals (Alarcón et al, 2015;Berulava et al, 2015).…”

Functional Implications of Active N6-Methyladenosine in Plants

“…More than 150 RNA modifications have been identified as post-transcriptional regulatory markers in a variety of RNA species, including messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small non-coding RNA (snRNA), and long non-coding RNA (lncRNA), RNA methylation is one of the post-transcriptional modifications of RNA, and N 6 -methyladenosine (m 6 A) is the most common type of RNA methylation modification, accounting for more than 80% of RNA methylation modifications in organism. Current study suggests that the m 6 A modification plays an important role in RNA fate, such as RNA splicing (Liu et al, 2015(Liu et al, , 2017Haussmann et al, 2016;Lence et al, 2016;Xiao et al, 2016;Pendleton et al, 2017), RNA stability (Wang et al, 2014;Du et al, 2016;Mishima and Tomari, 2016;Huang et al, 2018), RNA export (Roundtree et al, 2017;Edens et al, 2019), 3 untranslated region (UTR) processing (Ke et al, 2015;Bartosovic et al, 2017;Wei et al, 2018;Yue et al, 2018), translation (Zhou et al, 2015;Choi et al, 2016;Li et al, 2017;Shi et al, 2017), and miRNA processing (Alarcón et al, 2015a,b;Bhat et al, 2019). Although the presence of m 6 A was detected in mammals (Desrosiers et al, 1974;Wei et al, 1975;Schibler et al, 1977) and plants (Kennedy and Lane, 1979;Nichols, 1979) in the 1970s, it had not received much attention because it was considered to be "static" due to the method of detecting m 6 A sites.…”

Plant microRNAs: Biogenesis, Homeostasis, and Degradation

“…Second, several regulatory proteins affecting miRNA transcription (e.g., CDC5, NOT2, and ELP2) have been shown to interact with processing machinery proteins (Wang et al, 2013; Zhang et al, 2013; Fang et al, 2015a). Notably, a recent study showed that mRNA adenosine methylase (MTA), a homologous protein of animal METTL3, deposits m 6 A onto pri-miRNAs and may impact miRNA biogenesis via its dual interaction with Pol II and Tough (TGH), a known miRNA processing regulator (Ren et al, 2012b; Bhat et al, 2019). Another intriguing finding is that many proteins affecting MIRNA gene transcription, pri-miRNA stability, and/or processing have reported or proposed functions in RNA splicing (Yu et al, 2017b).…”