The RNA helicase domain of NAT10 promotes the biogenesis of hypomodified ribosomes to enhance cancer cell proliferation.

N-acetyltransferase 10 (NAT10) is a multifunctional enzyme that harbors RNA acetyltransferase and RNA helicase domains and has emerged as a therapeutic vulnerability in solid and hematological malignancies. By coupling Proteolysis Targeting Chimera-mediated degradation of NAT10 with a deep mutational scanning assay, followed by validations in biochemical assays, human cell lines, and female mouse xenografts, we find that the RNA helicase domain of NAT10 enhances cancer cell proliferation and tumor growth. This proliferative function of NAT10 is independent of RNA acetylation but requires its RNA-binding activity. The RNA helicase domain of NAT10 is required for 18S rRNA binding, promoting biogenesis of the 40S ribosomal subunit, while simultaneously interfering with the deposition of the conserved 18S rRNA modification m¹acp³Ψ. Loss of m¹acp³Ψ in 18S rRNA enhances cancer cell proliferation, revealing that NAT10 promotes the biogenesis of hypomodified ribosomes to facilitate tumor growth. These findings uncover a mechanism by which NAT10 promotes cancer cell proliferation and establish its RNA helicase domain as a potential therapeutic target.