RPUSD2 is a pseudouridine synthase that catalyzes pseudouridylation of RNA molecules, a post-transcriptional modification abundant in human cells. As a member of the RluA family of stand-alone pseudouridine synthases, RPUSD2 exhibits substrate specificity for mitochondrial tRNAs, specifically pseudouridylating Ψ31 of mt-tRNALeu(CUN), Ψ32 of mt-tRNAPro, and mt-tRNACys 1. RPUSD2 functions alongside other PUS enzymes in tRNA anticodon stem-loop regions, where it participates in an unexpected interplay with enzymes like PUS1 and PUS7 that can influence each other's pseudouridylation activity 1. The enzyme contributes to comprehensive pseudouridine modification patterns across human tRNAs at distinct pre-tRNA processing stages 2. Clinically, RPUSD2 is significantly overexpressed in basal-subtype breast cancer, where its elevated levels are linked to poor survival outcomes 3. Additionally, RPUSD2 was identified as a target of the tumor-suppressive microRNA hsa-let-7i in colorectal cancer 4, suggesting dysregulation in cancer contexts. These findings position RPUSD2 as both a critical epitranscriptomic regulator and a potential cancer biomarker.