Efficient G-Quadruplex RNA Resolution Mediated by Dimeric Suv3 Helicase

G-quadruplexes (G4) are stable secondary structures that can impede DNA replication, transcription, and translation. Although the human mitochondrial helicase Suv3 (SUPV3L1) is essential for RNA unwinding in mitochondrial RNA decay pathways, its involvement in resolving G4-RNA remains poorly characterized. Here, we demonstrate that dimeric Suv3 binds G4-RNA with nanomolar affinity, independently of ATP and irrespective of a 3′ overhang. However, efficient catalytic resolution of G4-RNA by Suv3 requires both ATP hydrolysis and a minimum 3′ single-stranded overhang of approximately 12 nucleotides to serve as a loading dock. Our findings support a translocation-based mechanism whereby efficient G4 resolution is initiated by the binding of Suv3 to G4-RNA with a long 3′ overhang, and this interaction triggers ATP hydrolysis, subsequent enzyme translocation, and G4 unfolding. We further show that dimeric Suv3 is markedly more effective than its monomeric counterpart in resolving G4-RNA, particularly under physiologically relevant mitochondrial salt conditions. Structural modeling supports that dimeric Suv3 functions as an asymmetric complex, with one of the subunits together with the C-terminal tails interacting with the G4 core structure, while the other subunit tethers the long 3′ overhang to enhance enzymatic efficiency and processivity. Together, these results establish Suv3 as a mitochondrial G4-RNA resolvase and provide a mechanistic framework for understanding how Suv3 dysfunction is linked to aberrant RNA processing in mitochondria and disease.