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

TL;DR

This study elucidates the structural requirements for DDX3X RNA helicase activity, highlighting the importance of single-stranded-double-stranded RNA regions for trimer formation and ATP hydrolysis, with implications for cancer and viral infection mechanisms.

Contribution

It reveals that single-stranded-double-stranded RNA regions are essential for DDX3X trimer formation and ATPase activity, providing new insights into its functional mechanism.

Findings

Single-stranded-double-stranded RNA regions support DDX3X binding.

Trimer formation is necessary for ATPase activity.

DNA substrates do not support ATPase activity.

Abstract

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.