Structure and regulation of ZCCHC4 in m6A-methylation of 28S rRNA

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.

Interaction between N6-methyladenosine (m6A) modification and noncoding RNAs in cancer

“…In patients with HCC, the expression of ZCCHC4 and m 6 A on 28S rRNA in cancer tissues was significantly increased as compared to that in the surrounding healthy tissues, whereas ZCCHC4 knockout in a xenograft mouse model eliminates m 6 A modification on 28S rRNA and then decreases global translation activity, which contributes to the inhibition of HCC cell proliferation and reduction in liver tumor size; thus, highlighting the essential role of m 6 A rRNA modification in mRNA translation and tumor progression [26]. Consistent with this finding, van Tran et al confirmed ZCCHC4 as a 28S rRNA m 6 A4220 methyltransferase that acts only on rRNAs [82], and further studies on the structure and regulation of ZCCHC4 in m 6 A methylation of 28S rRNA showed that the specific binding and methylation of ZCCHC4 to substrates depended on the stem loop structure of 28S rRNA [83]. In addition, the findings that ZCCHC4 is located in nucleoli, where the ribosomes are assembled, and ZCCHC4 interacts with proteins involved in RNA metabolism provide a possible mechanism by which ZCCHC4 influences RNA translation by regulating ribosomal assembly and biogenesis [84].…”

Roles of METTL3 in cancer: mechanisms and therapeutic targeting

“…In addition to the MTC, other m 6 A writers have also been identified in recent years, including METTL16, METTL5, and ZCCHC4 ( Fig. 1a and Table 1), which are responsible for the deposition of m 6 A into structured RNAs, such as U6 snRNA, 28S rRNA, and 18S rRNA, and in some cases, the introns of mRNA [15][16][17][18][19][20][21][22]. The MTC core component METTL3-METTL14 heterodimer catalyzes most of m 6 A methylations in mRNA, with METTL3 being the only catalytic subunit that uses Sadenosylmethionine (SAM) as the methyl donor [52][53][54].…”

The emerging roles of m6A modification in liver carcinogenesis

“…International Publisher demethylases (FTO and ALKBH5) [29,30], and recognized by RNA binding proteins, including YTHDF1/2/3, YTHDC1/2, IGF2BP1/2/3, hnRNPs [14,16,[31][32][33][34][35][36][37][38][39]. m 6 A modification of U6 snRNA and structural RNA, 18 S rRNA and 28 S rRNA, the 5'cap m 6 A m , and U2 snRNA internal m 6 A m are methylated by METTL16 [10,11,40], METTL5 [41], ZCCHC4 [9,42] and PCIF1 [43,44], respectively. Recently, METTL4 was shown to be a U2 snRNA internal m 6 A m methyltransferase [45].…”