• MYG1 is a Mn2+- or Mg2+-dependent 3′→5′ exonuclease with broad substrate specificity, showing highest activity on single-stranded RNA and a preference for U in ssRNA and dT in ssDNA.
• Crystal structures of MYG1 in complex with metal ions and nucleotides reveal a dimeric architecture where the active site is formed by the catalytic domain of one monomer and the substrate-binding domain of the other.
• The catalytic mechanism of MYG1 involves a two-metal ion-mediated cleavage, with key residues for metal and substrate binding identified through mutagenesis and activity assays.
• These findings provide a molecular basis for understanding MYG1's biological functions in RNA processing, mitochondrial regulation, and its potential roles in human diseases such as vitiligo and cancer.