RPUSD3 (RNA pseudouridine synthase D3) is a mitochondrial enzyme that catalyzes pseudouridylation, the conversion of uridine to pseudouridine in specific RNA substrates. It functions primarily in mitochondrial mRNA modification, acting at position 390 in COXI mt-mRNA and positions 697-699 in COXIII mt-mRNA 1. RPUSD3 is a core component of a functional protein-RNA module containing RCC1L, NGRN, RPUSD4, TRUB2, FASTKD2, and 16S mt-rRNA that regulates mitochondrial 16S rRNA abundance and supports mitochondrial ribosome biogenesis 23. Depletion of RPUSD3 causes specific mitochondrial protein synthesis defects and impairs oxidative phosphorylation assembly without affecting mitochondrial mRNA levels 1. However, recent evidence suggests RPUSD3 lacks enzymatic activity on cytosolic tRNAs in human cells, indicating its function is primarily restricted to mitochondrial targets 45. Clinically, elevated RPUSD3 expression correlates with poor prognosis and unfavorable therapeutic responses in prostate cancer 6. RPUSD3's essential role in mitochondrial translation and oxidative phosphorylation establishes it as critical for cellular energy metabolism and viability.