The intrinsically disordered N-terminal region of eIF5B nucleates RNA-driven phase separation

Abstract

Recently, we found that eukaryotic Initiation Factor 5B (eIF5B) forms cytoplasmic puncta under conditions of cellular stress and that the puncta formation drives an increase in alternative translation initiation. The formation of these puncta depends on the intrinsically disordered N-terminal region of eIF5B (eIF5B-NTR). To address the driving force behind this observation, we reconstituted a purified, binary, in vitro system to study the phase separation potential of the eIF5B-NTR. Utilizing this system, we show that eIF5B-NTR forms RNA-driven condensates at physiological concentrations. Furthermore, we defined the properties of the eIF5B condensates and the amino acid sequence requirements of this polypeptide to form complex liquid-liquid condensates. Truncation mapping identified a central arginine-rich core (residues ~200-400) that drives condensation but forms nearly static droplets in the absence of the rest of the polypeptide. Finally, we tested this model in cells to determine if the in vitro observations apply to the complex cellular environment.