RPUSD1 (RNA pseudouridine synthase domain containing 1) is a stand-alone pseudouridine synthase that catalyzes pseudouridylation of specific tRNA substrates. As an RluA family enzyme, RPUSD1 specifically installs the canonical Ψ30 modification in tRNA-Ile and Ψ72 in tRNA-Arg isoacceptors 1. RPUSD1 functions alongside other PUS enzymes (PUS1, PUS7) to modify the tRNA anticodon stem-loop, exhibiting unexpected interplay where nearby Ψ sites can influence pseudouridylation catalyzed by other PUSs 1. Comprehensive mapping confirms RPUSD1 targets specific cytosolic tRNAs, with modifications introduced at distinct pre-tRNA processing stages 2. Beyond its canonical RNA modification role, RPUSD1 demonstrates oncogenic activity in non-small cell lung cancer (NSCLC). Its RluA catalytic domain enhances eIF4E mRNA stability, which increases NBS1 expression; elevated NBS1 competitively inhibits PI3K p85-p110 binding, activating the PI3K/AKT signaling pathway and promoting cell proliferation, migration, and invasion 3. RPUSD1 expression is elevated in NSCLC tissue and associates with malignant phenotypes 3. Additionally, RPUSD1 was identified among 12 tumor-discriminating RNA modification enzymes overexpressed in hepatocellular carcinoma tumor-infiltrating cells 4 and was associated with DNA methylation signatures linked to maternal education during pregnancy 5. These findings suggest RPUSD1 functions in both fundamental RNA metabolism and cancer-associated signaling.