DEAD-box helicases, DDX3 and eukaryotic initiation factor 4A (eIF4A) are critical players in translation initiation, each containing intrinsically disordered regions (IDRs) that guide molecular interactions, respond to cellular cues, and promote biomolecular condensation. Through multi-parameter confocal spectroscopy, we uncovered that the sex chromosome-encoded helicases DDX3X and DDX3Y spontaneously form nanometer-scale RNA-protein clusters (RPCs) with several RNA and numerous protein subunits at nanomolar bulk concentrations, well below the micromolar threshold required for liquid-liquid phase separation. The helicase is active inside the clusters. In contrast, eIF4A, which harbors much smaller IDRs, does not form RPCs unless assisted by additional protein cofactors. Fluorescence correlation spectroscopy showed faster diffusion for eIF4A bound to labeled duplex RNA, forming small complexes with just two subunits, and showing minimal helicase activity. Addition of the cofactors eIF4B, which is largely unstructured, and the scaffold protein eIF4G, led to the formation of RPCs (∼2 MDa) accompanied by enhanced helicase activity, suggesting a link between RPC formation and full strand separation. To assess the role of eIF4B’s RNA recognition motif (RRM), we disrupted eIF4B-RNA interaction by introducing a point mutation, F139A within its RRM domain. This mutation significantly decreased both helicase activity and cluster size, suggesting that a functional RRM domain is crucial not only for RNA-protein interactions that drive cluster formation but also for maintaining helicase activity. Further, we examined eIF4H, a homolog of eIF4B that shares a similar RRM but possesses smaller and less complex IDRs. Substituting eIF4H for eIF4B led to slower helicase activity and smaller cluster size. Are nano-RPCs involved in translation initiation? Our results underscore the pivotal role of IDRs in driving nano-RPC formation and modulating helicase activity, revealing the dynamic behavior of these subunits within clusters.
Shweta et al. (Sun,) studied this question.