Structural And Functional Study Of Zinc Finger Domain Of RNA M~6-Methyladenosine Methyltransferase METTL3

More than 100 chemical modifications have been identified in RNA,and RNA N~6-methyladenosine(m~6A)is the most abundant in mRNA and lncRNA.RNA m~6A has been found in a conserved motif containing the RRACH(where A is methylated;R=purine,and H=A,C or U)and is enriched in the 3'UTR.RNA m~6A is evolutionally conservative in various species,like virus,bacteria,yeast,plants,C.elegans,Drosophila and mammals.RNA m~6A modification is involved in many functions almostly in all aspects of biological process.The methylation is regulated by three types of proteins,namely writers("encoder"),erasers("decoder"),and readers.Writers is a large complex containing several components.The core enzymes METTL3 and METTL14 interact as a heterodimer to perform the catalysis,and the others are involed in the regulation.Erasers include FTO and ALKBH5,which are responsible for the demethylation of m~6A.Readers mainly consist of YTHDC1-DC2 and YTHDF1-DF3that recognize the modification and determine the fate of m~6A-containing RNA.Here we are working on the mechanistic study of m~6A modification.Prior to this paper,we have determined the crystal structure of the methyltransferase domain(MTD)of METTL3-METTL14.But the activity assay shows the complex is catalytically inactive.Bioinformatics analysis indicate that there are two CCCH type zinc finger domains(ZFD)preceding MTD in METTL3.Biochemical test shows the inclusion of ZFD completely fulfills the activity of METTL3-METTL14 MTD complex as full-length,indicating that ZFD is indispensable in holo METTL3-METTL14 complex.However,it remains unknown of how the ZFD regulates the full activity.In this paper,we determined the solution structure of human METTL3 ZFD by combining some biophysical and biochemical methods,like Nuclear Magnetic Resonance(NMR),small-angle x-ray scattering(SAXS)and Isothermal Titration Calorimetry(ITC).We found that ZFD serves as a target recognition domain(TRD),cooperates with MTD of METTL3 for the regulation of full activity.Mutational analysis and NMR titrations mapped the interface between ZFD and substrate RNA,suggesting some positively charged and hydrophobic residues are involved in the interaction between RNA and ZFD.During the catalysis process,we proposed that ZFD transfers the“captured”RNA to catalytic pocket of MTD,then rapidly releases it and returns to its original position,continuing to recruit next target.The weak interaction between two of them facilitates the release process and increases the catalytic efficiency,explaining the highly dynamics of METTL3-METTL14 holo-complex during catalysis.Together,our research paved the way to elucidate the molecular mechanism of RNA m~6A methylation.