Yanas A, Shweta H, Owens MC, Liu KF, Goldman YE. DDX3X and DDX3Y constitutively form nano-sized RNA-protein clusters that foster enzymatic activity. bioRxiv [Preprint]. 2023 Nov 29:2023.11.29.569239. doi: 10.1101/2023.11.29.569239. PMID: 38077005; PMCID: PMC10705435.


DEAD-box helicases, which are crucial for many aspects of RNA metabolism, often contain intrinsically disordered regions (IDRs), whose functions remain unclear. Using multiparameter confocal microscopy, we reveal that sex chromosome-encoded homologous RNA helicases, DDX3X and DDX3Y, form nano-sized RNA-protein clusters (RPCs) that foster their catalytic activities in vitro and in cells. The IDRs are critical for the formation of these RPCs. A thorough analysis of the catalytic cycle of DDX3X and DDX3Y by ensemble biochemistry and single molecule photon bursts in the confocal microscope showed that RNA release is a major step that differentiates the unwinding activities of DDX3X and DDX3Y. Our findings provide new insights that the nano-sized helicase RPCs may be the normal state of these helicases under non-stressed conditions that promote their RNA unwinding and act as nucleation points for liquid-liquid phase separation under stress. This mechanism may apply broadly among other members of the DEAD-box helicase family.