Structural insights into molecular mechanism for N⁶-adenosine methylation by MT-A70 family methyltransferase METTL4

METTL4 belongs to a subclade of MT-A70 family members of methyltransferase (MTase) proteins shown to mediate N⁶-adenosine methylation for both RNA and DNA in diverse eukaryotes. Here, we report that Arabidopsis METTL4 functions as U2 snRNA MTase for N⁶-2'-O-dimethyladenosine (m⁶Am) in vivo that regulates flowering time, and specifically catalyzes N⁶-methylation of 2'-O-methyladenosine (Am) within a single-stranded RNA in vitro. The apo structures of full-length Arabidopsis METTL4 bound to S-adenosyl-L-methionine (SAM) and the complex structure with an Am-containing RNA substrate, combined with mutagenesis and in vitro enzymatic assays, uncover a preformed L-shaped, positively-charged cavity surrounded by four loops for substrate binding and a catalytic center composed of conserved residues for specific Am nucleotide recognition and N⁶-methylation activity. Structural comparison of METTL4 with the mRNA m6A enzyme METTL3/METTL14 heterodimer and modeling analysis suggest a catalytic mechanism for N⁶-adenosine methylation by METTL4, which may be shared among MT-A70 family members.