RPUSD4 (RNA pseudouridine synthase D4) is a mitochondrial enzyme that catalyzes pseudouridylation, the conversion of uridine to pseudouridine in RNA substrates 1. Its primary function involves modifying position 1397 in 16S mitochondrial ribosomal RNA (16S mt-rRNA), a modification essential for 16S mt-rRNA stability and assembly into functional mitochondrial ribosomes 1. RPUSD4 also pseudouridylates position 39 in mitochondrial tRNA(Phe) 2. As part of a conserved protein-RNA module with RCC1L, NGRN, RPUSD3, TRUB2, and FASTKD2, RPUSD4 controls 16S mt-rRNA abundance and is required for intra-mitochondrial translation and oxidative phosphorylation 3. Beyond mitochondrial functions, RPUSD4 regulates nuclear pre-mRNA splicing and 3'-end processing by mediating pseudouridylation near splice sites and alternatively spliced regions, thereby controlling widespread changes in alternative pre-mRNA processing 4. RPUSD4 depletion causes severe mitochondrial translation defects and oxidative phosphorylation assembly failures 1. Dysregulated RPUSD4 expression correlates with immune infiltration in osteoarthritis 5, and the gene serves as a hub in disease networks affecting mitochondrial protein synthesis 6. These findings establish RPUSD4 as essential for both mitochondrial biogenesis and nuclear gene expression regulation.