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An autoinhibitory mechanism in regulating m6A‐methylation of 28S rRNA

N6‐methyladenosine (m6A) modification represents an evolutionarily conserved epitranscriptomic mechanism that critically regulates RNA metabolism and function. m6A is widely present in mRNA and long noncoding RNA, as well as in ribosomal RNA (rRNA) and small nuclear RNA, mediated by an array of distinct writer enzymes. However, how m6A writers attain substrate specificities remains unclear. CCHC zinc finger‐containing protein ZCCHC4 is a newly identified RNA methyltransferase that specifically mediates the m6A modification on the site 4220 of 28S rRNA, which in turns impacts translation, cell proliferation and tumor growth. To provide mechanistic insights into the ZCCHC4‐mediated m6A RNA methylation, we performed structural characterization of the enzyme‐cofactor complex of human ZCCHC4. Our work reveals that the regulatory domains and the methyltransferase (MTase) domain pack into an integrated fold, thereby creating a multi‐domain RNA‐binding platform. Importantly, the MTase domain adopts an autoinhibitory conformation, in which the active site is occluded from RNA binding by an inter‐domain loop segment. Mutational and enzymatic analyses further indicate that this loop segment directly engages substrate recognition, providing an autoinhihitory mechanism by which the activity of ZCCHC4 is coupled with specific substrate binding. In addition, analysis of the substrate sequence and structure suggests a role of the stem‐loop structure within substrate in governing the substrate specificity. Together, our study uncovers an autoinhibitory mechanism that ensures specific interplay between ZCCHC4 and the RNA substrate, reminiscent of what was seen with the DNA methylation writers.