RNA helicases DDX3X and DDX3Y form nanometer-scale RNA-protein clusters that support catalytic activity.

DEAD-box helicases, 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 nanometer-scale 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. The N-terminal IDRs of DDX3X and DDX3Y are both the drivers of RPC formation and the major differentiators of their enzymatic activities. Our findings provide new insights that the nanoscale helicase RPCs may be the normal state of these helicases under non-stressed conditions that promote their RNA unwinding and might act as nucleation points for stress granule formation. This mechanism may apply broadly among other members of the DEAD-box helicase family.