Abstract
N6-methyladenosine (m6A) modification provides an important epitranscriptomic mechanism that critically regulates RNA metabolism and function. However, how m6A 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 m6A 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 m6A writers, providing the mechanistic basis for ZCCHC4 modulation of m6A RNA methylation.
Highlights
N6-methyladenosine (m6A) modification provides an important epitranscriptomic mechanism that critically regulates RNA metabolism and function
MRNA and long noncoding RNA (lncRNA) are primarily methylated by the METTL3–METTL14 heterodimeric complex[11,12,13,14,15], which recognizes a DRACH (D: A, G, U; R: G, A; H: A, C, U) consensus sequence near 3′ UTR16–18, whereas a subset of mRNA and small nuclear RNA (snRNA) sites is distinctly methylated by RNA methyltransferase METTL1619, which recognizes a UACAGAGAA motif embedded in a stem-loop structure[20,21]
The N-terminal regions of all the other ZCCHC4 molecules are not involved in domain swapping, in line with the fact that ZCCHC4 exists as a monomer in solution, as demonstrated by our size-exclusion chromatography analysis (Supplementary Fig. 2c)
Summary
N6-methyladenosine (m6A) modification provides an important epitranscriptomic mechanism that critically regulates RNA metabolism and function. One of the most prevalent RNA modifications is N6-methyladenosine (m6A), which is widely present in mRNA and long noncoding RNA (lncRNA)[2], as well as in the ribosomal RNA (rRNA)[3,4] and small nuclear RNA (snRNA)[5,6] Specific methylation of these RNA molecules functions to modulate RNA structure and protein–RNA interactions, which in turn influences RNA metabolism, cell signaling, cell survival, and differentiation[7,8]. Recent studies have further demonstrated that the m6A modification on the site 4220 of 28S rRNA and site 1832 of 18S rRNA is, respectively, mediated by CCHC zinc finger-containing protein ZCCHC4 and the methyltransferase METTL59,23 These identified RNA methyltransferases bear no sequence homology, except for a remotely related methyltransferase (MTase) domain (Supplementary Fig. 1), raising a question of the molecular basis underlying their distinct substrate specificities. This work reveals the previously unappreciated molecular mechanisms that underlie activity autoregulation and substrate binding of ZCCHC4 in the modulation of cellular m6A RNA methylation
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