Fig. 5: NAT10 interacts with NFE2L3 and promotes its stability.

a The acetylation levels of different genes with p < 0.05 were analyzed to make a volcano map. Blue: decrease in ac4C acetylation level; red: increase in ac4C acetylation level (n = 4). b, c ac4C acetylation of genes mainly occurs in the CDS region. d There were 199 gene intersections between RNA-seq and acRIP-seq of genes with increased expression in ccRCC and genes with increased ac4C acetylation in cancer. e mRNA variation trend of 11 candidate genes after NAT10 knockdown in 786-O cells. f The abundance of ac4C on NFE2L3 mRNA transcripts in ccRCC cancer tissues was detected by acRIP-seq. g Overall analysis of ac4C in ccRCC tissues Main common sequence motifs identified in ac4C peaks. h acRIP-qPCR revealed ac4C acetylation on NFE2L3 mRNA. i RIP-qPCR showed that NAT10 could bind to NFE2L3 mRNA. Below is an anti-NAT10 that specifically pulls down the NAT10 protein. j–k QRT-PCR and western blot showed that NFE2L3 mRNA and protein level decreased after NAT10 knockdown. l The activity of NFE2L3-wild type and mutant vectors after NAT10 knockdown were detected by dual luciferase. m RNA attenuation experiment showed that the stability of NFE2L3 mRNA decreased after NAT10 knockdown. n The role of NAT10 on NFE2L3 mRNA translation efficiency. (evaluated using relative fluorescence intensity/relative RNA expression [16]). o Western blot detection of Flag levels in control group and knockdown NAT10 group verified the effect of NAT10-mediated ac4C modification on NFE2L3 protein. p The correlation of NAT10 and NFE2L3 mRNA in 20 ccRCC tissues was performed, r = 0.7309, p < 0.005.