rRNA N(6)-adenosine-methyltransferase ZCCHC4
S-adenosyl-L-methionine binding
Structure and regulation of ZCCHC4 in m<sup>6</sup>A-methylation of 28S rRNA.
N<sup>6</sup>-methyladenosine (m<sup>6</sup>A) modification provides an important epitranscriptomic mechanism that critically regulates RNA metabolism and function. However, how m<sup>6</sup>A writers attain substrate specificities remains unclear. We report the 3.1 Å-resolution crystal structure of human CCHC zinc finger-containing protein ZCCHC4, a 28S rRNA-specific m<sup>6</sup>A methyltransferase, bound to S-adenosyl-L-homocysteine. The methyltransferase (MTase) domain of ZCCHC4 is packed against N-terminal GRF-type and C2H2 zinc finger domains and a C-terminal CCHC domain, creating an integrated RNA-binding surface. Strikingly, the MTase domain adopts an autoinhibitory conformation, with a self-occluded catalytic site and a fully-closed cofactor pocket. Mutational and enzymatic analyses further substantiate the molecular basis for ZCCHC4-RNA recognition and a role of the stem-loop structure within substrate in governing the substrate specificity. Overall, this study unveils unique structural and enzymatic characteristics of ZCCHC4, distinctive from what was seen with the METTL family of m<sup>6</sup>A writers, providing the mechanistic basis for ZCCHC4 modulation of m<sup>6</sup>A RNA methylation.