Fig. 5: NAT10 regulates MYC protein expression.

A The highly enriched motif within ac4C peaks was analyzed using acRIPseq. B The proportion of ac4C peak distribution in the TSS, 5’UTR, start codon, stop codon, and 3’UTR regions across the entire set of mRNA transcripts. C Density distribution of ac4C peaks across mRNA transcripts. D Seven candidate genes, including Phf2, Myc, Wwc2, Kmt2a, Gigyf1, Timeless, and Nufip2, were identified using acRIP-seq and label-free quantitative proteomics. E The expression levels of MYC in sgNAT10 TC1 and MCA205 tumor cells were analyzed by Western blotting. F The peaks of myc in WT and sgNAT10 TC1 cells from acRIP-seq data were visualized using the IGV software. G Schematic representation of the positions of ac4C motifs in Myc mRNA (upper panel). The ac4C sites in the 3’UTR of Myc mRNA were mutated to eliminate ac4C sites as much as possible. The lower panel shows the schematic representation of the mutated 3’UTR of the pEZX-MT06 vector for studying the roles of ac4C in Myc mRNA stability. H Effect of NAT10 on pEZX-MT06-Myc reporter. TC1 tumor cells were cultured in 24-well plates and transfected with Lipofectamine 3000 reagent according to the manufacturer’s instructions. Specifically, 100 ng/well of pEZX-MT06-Myc and either 0, 150, or 300 ng/well of VP64-NAT10 or empty vector were co-transfected. Additionally, Renilla luciferase plasmids (30 ng/well) were co-transfected as a normalization control for transcription efficiency. Luciferase activity was measured 24 h after transfection. The results are presented as relative luciferase activity (luciferase activity normalized to Renilla activity); ***P < 0.001; ***P < 0.001. I Anti-NAT10 antibody-based RIP-PCR analysis of Myc mRNA in TC1 cells. J The mRNA levels of MYC were detected in sgNAT10 TC1 cells after treatment with Act-D. The statistical method used was two-way ANOVA; **P = 0.0023. Unless specified otherwise, the data are presented as means ± SEM (error bar). Source data are provided as a Source Data file.