Abstract
RNA ligation is critical for RNA processing pathways including pre-tRNA splicing. RTCB is the only essential RNA ligase in humans and the catalytic subunit of the tRNA ligase complex (tRNA-LC). Its active site is sensitive to oxidation and strictly depends on oxidoreductase PYROXD1, the only tRNA-LC subunit with multiple known pathogenic protein-coding variants. The role of RNA ligation in PYROXD1 myopathy remained contentious: loss of PYROXD1 primarily affects mitochondria, and ligation-linked readouts are inconsistent in existing assays. Here, we reveal that PYROXD1 is physically, genetically and functionally committed to the tRNA-LC. Selectively impairing RTCB activation reproduces hallmarks of PYROXD1 dysfunction, including mitochondrial deregulation. Using degron-based depletion of endogenous PYROXD1, we show that all six single amino acid variants reported in patients fail to sustain RNA ligation in proportion to their clinical severity. The variants segregate into three mechanistic classes: they lower protein stability, disrupt the NADPH-mediated conformational switch, or impair RTCB engagement by the C-terminal tail, a sequence unique to PYROXD1 and directly affected in the most severe variant D500N. Variant-level measurements in the degron model predict outcomes in homozygous and compound heterozygous patient fibroblasts. Together, these data establish impaired RNA ligation as the driver of PYROXD1 pathogenicity.