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Solution structure of the RNA recognition domain of METTL3-METTL14 N6-methyladenosine methyltransferase.

ABSTRACT: N6-methyladenosine (m6A), a ubiquitous RNA modification, is installed by METTL3-METTL14 complex. The structure of the heterodimeric complex between the methyltransferase domains (MTDs) of METTL3 and METTL14 has been previously determined. However, the MTDs alone possess no enzymatic activity. Here we present the solution structure for the zinc finger domain (ZFD) of METTL3, the inclusion of which fulfills the methyltransferase activity of METTL3-METTL14. We show that the ZFD specifically binds to an RNA containing 5'-GGACU-3' consensus sequence, but does not to one without. The ZFD thus serves as the target recognition domain, a structural feature previously shown for DNA methyltransferases, and cooperates with the MTDs of METTL3-METTL14 for catalysis. However, the interaction between the ZFD and the specific RNA is extremely weak, with the binding affinity at several hundred micromolar under physiological conditions. The ZFD contains two CCCH-type zinc fingers connected by an anti-parallel ?-sheet. Mutational analysis and NMR titrations have mapped the functional interface to a contiguous surface. As a division of labor, the RNA-binding interface comprises basic residues from zinc finger 1 and hydrophobic residues from ?-sheet and zinc finger 2. Further we show that the linker between the ZFD and MTD of METTL3 is flexible but partially folded, which may permit the cooperation between the two domains during catalysis. Together, the structural characterization of METTL3 ZFD paves the way to elucidate the atomic details of the entire process of RNA m6A modification.

SUBMITTER: Huang J

PROVIDER: S-EPMC6418081 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Solution structure of the RNA recognition domain of METTL3-METTL14 N6-methyladenosine methyltransferase.

Huang Jinbo J Dong Xu X Gong Zhou Z Qin Ling-Yun LY Yang Shuai S Zhu Yue-Ling YL Wang Xiang X Zhang Delin D Zou Tingting T Yin Ping P Tang Chun C

Protein & cell 20180314 4

N6-methyladenosine (m6A), a ubiquitous RNA modification, is installed by METTL3-METTL14 complex. The structure of the heterodimeric complex between the methyltransferase domains (MTDs) of METTL3 and METTL14 has been previously determined. However, the MTDs alone possess no enzymatic activity. Here we present the solution structure for the zinc finger domain (ZFD) of METTL3, the inclusion of which fulfills the methyltransferase activity of METTL3-METTL14. We show that the ZF ...[more]

PMID: 29542011

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Project description:Rheumatoid arthritis (RA) is largely caused by the inflammatory response triggered by macrophage polarization. Through epigenetic reprogramming, the inflammatory state of macrophages can be modified. Macrophage polarization is associated with the RNA epigenetic alteration N6-methyladenosine (m6A) RNA methylation. However, the specific function and underlying mechanisms of m6A methylation in the role of macrophage polarization in RA remain to be elucidated. The mRNA expression levels of m6A methylase genes and signaling pathway components associated with RA macrophages were determined in the present study using reverse-transcription quantitative PCR. Methyltransferase 14 (METTL14) protein expression levels were determined using western blot analysis, and the levels of specific cellular secretion factors were determined using ELISA and flow cytometry. The results of the present study demonstrated that elevated METTL14 expression was associated with joint tenderness, and METTL14 expression was positively correlated with both C-reactive protein and rheumatoid factor expression levels. Moreover, METTL14 exhibited potential in the prediction of visual analogue scale. Pro-inflammatory cytokines (TNF-α) and M1 macrophage markers (CD68+CD86+) were also positively associated with METTL14 expression. The results of the Kyoto Encyclopedia of Genes and Genomes analysis revealed that METTL14 was strongly associated with the MAPK signaling pathway. Notably, JNK and ERK2 exhibited a positive correlation with the M1 macrophage marker, CD68+CD86+, which was positively associated with the pro-inflammatory factor, TNF-α. JNK and ERK2 expression levels were markedly increased in the METTL14 high-expression group, compared with in the low-expression group; however, p38 and ERK1 expression levels were not significantly different between these groups. Collectively, the results of the present study demonstrated that METTL14 expression was significantly increased in the peripheral blood and synovial tissue of patients with RA, highlighting the potential association with both immunoinflammatory markers and clinical symptoms. In addition, it was suggested that METTL14 may exacerbate the downstream inflammatory response, through mediating macrophage polarization via the MAPK pathway.

Dataset Information

Solution structure of the RNA recognition domain of METTL3-METTL14 N6-methyladenosine methyltransferase.

Publications

Solution structure of the RNA recognition domain of METTL3-METTL14 N<sup>6</sup>-methyladenosine methyltransferase.

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