PUS1 (pseudouridine synthase 1) is an RNA-modifying enzyme that catalyzes the formation of pseudouridine in both tRNAs and mRNAs. In mitochondria, PUS1 modifies specific positions in tRNAs (27/28 in the anticodon stem and positions 34 and 36 in intron-containing tRNAs), where its activity is essential for maintaining mitochondrial tRNA levels and supporting proper mitochondrial protein synthesis 1. PUS1 also pseudouridylates mRNAs at consensus sequences (5'-UGUAG-3') and regulates pre-mRNA splicing by modifying sites near alternatively spliced regions and splice sites 2. The enzyme functions co-transcriptionally, directly affecting splicing efficiency and mRNA 3'-end processing 2. PUS1 deficiency causes mitochondrial myopathy, lactic acidosis, and sideroblastic anemia 1 (MLASA), characterized by impaired erythropoiesis due to compromised mitochondrial function and protein synthesis 1. In cancer contexts, PUS1 acts as an oncogene, promoting tumor progression in hepatocellular carcinoma by enhancing translation of oncogenic mRNAs through pseudouridylation 3, and facilitating metastasis in prostate cancer through non-enzymatic stabilization of EIF3b protein 4. Clinically, PUS1 serves as a prognostic biomarker and potential therapeutic target in multiple cancer types 35.