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

N4-acetylcytidine (ac4C) is an evolutionarily conserved RNA modification that is deposited on diverse RNAs by N-acetyltransferase 10 (NAT10), a protein with high biological significance for aging and cancer. Here, 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 NGS analysis confirms robust ac4C modification of rRNA and specific tRNA species in a NAT10-dependent manner, but suggests low or spurious ac4C acetylation in mRNA. Analysis of RNA-seq data also revealed an induction of inflammatory responses as well as mutagenesis at transcriptionally active sites in NAT10-KO cells. This finding led us to further explore the role of NAT10 in R-loops, which have recently been shown to induce APOBEC3B-mediated mutagenesis. Our analysis revealed that R-loops are modified with ac4C in a NAT10-dependent manner. Furthermore, NAT10 restrains the levels of R-loops at a subset of differentially expressed genes in a manner dependent on its catalytic activity. Together with cellular biology data showing ac4C containing RNA in endosomal structures, we propose that increased levels of ac4C-unmodified RNAs, likely derived from R-loops, in endosomal structures induce inflammatory responses.