Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Luo Q

PROVIDER: S-EPMC9512776 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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

Luo Qiang Q Mo Jiezhen J Chen Hao H Hu Zetao Z Wang Baihui B Wu Jiabing J Liang Ziyu Z Xie Wenhao W Du Kangxi K Peng Maolin M Li Yingping Y Li Tianyang T Zhang Yangyi Y Shi Xiaoyan X Shen Wen-Hui WH Shi Yang Y Dong Aiwu A Wang Hailin H Ma Jinbiao J

Nature communications 20220926 1

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 fu ...[more]

PMID: 36163360

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Project description:RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2'-O methyltransferase activities that are required for the formation of 5' type I cap (m(7)GpppAm) of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4) specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2'-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N?-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2'-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2'-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2'-O-methyladenosine. The 2'-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine) pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2'-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2'-O methylation of internal adenosine of viral RNA in vivo and host ribosomal RNAs in vitro.

Dataset Information

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

Publications

Structural insights into molecular mechanism for N<sup>6</sup>-adenosine methylation by MT-A70 family methyltransferase METTL4.

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