Using single-molecule fluorescence techniques to understand the enzymatic cycle of sex-biased RNA helicases DDX3X and DDX3Y.

Abstract

Sex differences are pervasive in human health and disease with the most striking differences lying in the sex chromosomes, which encode a group of sex-specific protein homologs. DDX3X and DDX3Y are a pair of sexually dimorphic non-processive ATP-dependent RNA helicases. We previously showed that DDX3Y forms larger, less dynamic phase separated condensates compared to DDX3X, possibly due to the lower ATPase activity of DDX3Y and different dynamic interactions with RNA. We used single-molecule FRET of immobilized, fluorescently labeled RNA duplexes and reported that DDX3X and DDX3Y show different binding dynamics and partial unwinding activities (Shen and Yanas et al., Molecular Cell 2022). To further investigate the enzymatic activities of DDX3, we used multiparameter confocal spectroscopy to obtain unwinding, time-resolved anisotropy, diffusion coefficients, and structural changes by FRET of freely diffusing RNA duplexes upon addition of DDX3, with and without ATP. Duplex RNA with a 5’ overhang was labeled with a Cy3 and Alexa647 FRET pair at the chain termini. The proportion of duplex RNA was quantified and used as a readout for helicase activity, and DDX3X shows higher helicase activity than DDX3Y. With ns time-resolved fluorescence anisotropy, we found that the shorter, non-overhanging strand RNA is released with helicase activity, whereas protein remains bound to the longer RNA strand. Diffusion constants from fluorescence correlation spectroscopy revealed formation of enzymatically active protein clusters at low DDX concentrations. Removal of N- and C-terminal intrinsically disordered regions suppressed formation of clusters and reduced enzymatic activity. We hypothesize that these clusters act as “helicase hubs,” possibly tiny nuclei of liquid granules, which may be translational centers in the cell, leading to efficient translation initiation.