Action mechanism of DDX3X: An RNA helicase implicated in cancer propagation and viral infection

DDX3X is a human DEAD-box RNA helicase implicated in many cancers and in viral progression. In addition to the RecA-like catalytic core, DDX3X contains N- and C-terminal domains. Here, we investigate the substrate and protein requirements to support the ATPase activity of a DDX3X construct lacking 80 residues from its C-terminal domain. Our data confirmed previous results that for an RNA molecule to support the ATPase activity of DDX3X it must contain a single-stranded-double-stranded region. We investigated protein and RNA structural reasons for this requirement. First, the RNA substrates consisting only of a double-helix were unable to support DDX3X binding. A single-stranded RNA substrate supported DDX3X binding, while an RNA substrate consisting of a single-stranded-double-stranded region not only supported the binding of DDX3X to RNA, but also promoted DDX3X trimer formation. Thus, the single-stranded-double-stranded RNA region is needed for DDX3X trimer formation, and trimer formation is required for ATPase activity. Interestingly, the dependence of ATP hydrolysis on the protein concentration suggests that the DDX3X trimer hydrolyzes only two molecules of ATP. Lastly, a DNA substrate that contains single-stranded-double-stranded regions does not support the ATPase activity of DDX3X.