NAT10 governs development through the Xrp1-mediated stress response rather than its RNA acetylation activity.

N4-acetylcytidine (ac4C) is installed by N-acetyltransferase 10 (NAT10) and represents the only known acetylation mark on messenger RNA. Since NAT10 also acetylates transfer RNA, ribosomal RNA (rRNA), and proteins, its in vivo molecular mechanisms remain elusive. Here, we demonstrate that knockdown of Drosophila NAT10 induces an eye-to-antenna transformation, c-Jun amino-terminal kinase activation, and cell apoptosis. NAT10 facilitates ac4C modification in rRNA, and its loss impairs rRNA processing and ribosomal assembly. Depletion of NAT10 activates the integrated stress response, ultimately leading to reduced global protein synthesis. Crucially, Xrp1 plays a key role in the stress response and its ablation rescues most NAT10 loss-of-function defects and transcriptomic alterations. We also performed acetylated RNA immunoprecipitation and sequencing (acRIP-seq) on control and NAT10 knockdown flies. Furthermore, a catalytically deficient form of NAT10, which is unable to mediate ac4C acetylation, completely rescues the lethality of NAT10 mutants. Collectively, these findings establish that NAT10's primary developmental function stems from its roles in ribosome biogenesis and Xrp1 activation, which are independent of its RNA acetylation activity.