NAT10 and N⁴-acetylcytidine restrain R-loop levels and related inflammatory responses

N⁴-acetylcytidine (ac⁴C) is deposited on diverse RNAs by N-acetyltransferase 10 (NAT10), a protein with high biological relevance for aging and cancer. We performed a comprehensive survey of ac⁴C using metabolic labeling, sodium cyanoborohydride chemical treatment coupled to next-generation sequencing (NGS), and ac⁴C antibody-based cell and molecular biology techniques. Our analysis shows that NAT10-dependent ac⁴C-acetylation is robust in rRNA and specific tRNAs but low/spurious in mRNA. It also revealed an inflammatory signature and mutagenesis at transcriptionally active sites in NAT10-KO cells. This finding led us to explore the role of NAT10 in R-loops, which were recently linked to APOBEC3B-mediated mutagenesis. Our analysis showed that R-loops are ac⁴C-acetylated in a NAT10-dependent manner. Furthermore, NAT10 restrains the levels of R-loops at a subset of differentially expressed genes in a catalytic activity-dependent manner. Together with cellular biology data showing ac⁴C-modified RNA in endosomal structures, we propose that increased levels of ac⁴C-unmodified RNAs, likely derived from R-loops, in endosomal structures induce inflammatory responses.