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

Abstract

N-methyladenosine (mA), 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 β-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 β-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 mA modification.

The RNA Epitranscriptome of DNA Viruses

“…METTL3 exists in a heterodimer with METTL14, and depletion of either subunit significantly impairs methyltransferase activity in vitro (16,17). This observation agrees with structural data showing that METTL14 has a catalytically inactive methyltransferase domain that enhances RNA binding of the METTL3-METTL14 dimer (18)(19)(20)(21). Photoactivatable ribonucleosideenhanced crosslinking and immunoprecipitation (PAR-CLIP) and in vitro methylation studies show that these proteins bind to and methylate mRNA at RRACH motifs (17).…”

Roles of METTL3 in cancer: mechanisms and therapeutic targeting

“…Huang et al characterized the ZFD solution structure using nuclear magnetic resonance (NMR), showing that the domain contains two tandem CCCH-type zinc fingers (ZnF1 and ZnF2) connected by an anti-parallel β-sheet (Fig. 1 c), which is responsible for target recognition, specifically for binding to single-stranded RNAs containing 5′-GGACU-3′ consensus sequence [ 55 ]. The structure of the methyltransferase domain of METTL3, named MT-A70, has been determined using X-ray crystallography in a complex with the corresponding domain of METTL14 by three independent groups [ 52 – 54 ].…”

Structural Insights into N 6-Methyladenosine (m6A) Modification in the Transcriptome

“…Bioinformatics analyses reveal that two CCCH-type zinc finger domains (ZFDs) are present preceding the MTD in the N-terminus of METTL3 [62] , and biochemistry assay proved that the ZFDs are necessary for the methylation activity [31] , [64] . Recently we have determined the solution structure of ZFD [126] . The ZFD serves as the target recognition domain and specifically targets to GGACU-containing RNAs [126] .…”

“…Recently we have determined the solution structure of ZFD [126] . The ZFD serves as the target recognition domain and specifically targets to GGACU-containing RNAs [126] . Besides, the secondary structure of mRNA might affect the writer’s catalyzing activity [52] in vivo .…”