The eIF4B RNA recognition motif promotes higher-order organization of the translation initiation machinery during stress granule assembly.

Cells respond to environmental stress by rapidly remodeling translation and assembling stress granules (SGs), which are dynamic ribonucleoprotein condensates that contain untranslated mRNAs, translation initiation factors, and 40S ribosomal subunits. Although the translation initiation factor eIF4B has been implicated in SG biology, the contribution of its highly conserved RNA recognition motif (RRM) to SG assembly has remained unclear. Here, we developed a quantitative live-cell imaging framework that resolves distinct kinetic phases of SG assembly at single-cell resolution and combines these measurements with single-cell analysis of protein synthesis. Using this approach, we show that disruption of the eIF4B RRM delays SG nucleation, slows SG assembly, and reduces the number of SGs formed, while having little effect on mature SG size. Biochemical analyses revealed that the RRM mutant retained high-affinity binding to both RNA and the 40S ribosomal subunit and exhibited only a modest reduction in eIF4A helicase stimulation activity but displayed altered RNA engagement, consistent with impaired RNA-dependent organization of the translation initiation machinery. Coupling SG kinetics with single-cell measurements of protein synthesis further revealed that delayed SG nucleation is associated with reduced translational repression during oxidative stress. Together, our findings identify the conserved eIF4B RRM as a regulator of productive higher-order organization of the translation initiation machinery and establish a quantitative framework for investigating how SG assembly and translational remodeling are coordinated during cellular stress.