Uncoupling Stress Granule Assembly and Translation Initiation Inhibition

Cytoplasmic stress granules( SGs) are specialized regulatory sitesof mRNA translation that form under different stress conditions known to inhibit translation initiation. The formation of SGoccurs via two pathways; the eukaryotic initiation factor(eIF) 2α phosphorylation-dependent pathway mediated by stress and the eIF2α phosphorylation-independent pathway mediated by inactivation of the translation initiation factors eIF4Aand eIF4G. In this study, we investigated the effects of targeting different translation initiation factorsand steps in SGformation in HeLa cells. By depleting eIF2α, we demonstrate that reduced levels of the eIF2.GTP.Met-tRNAiMet ternary translation initiationcomplexes is sufficient to induce SGs. Likewise, reduced levels of eIF4B, eIF4H, or polyA-binding protein, also trigger SGformation. In contrast, depletion of the cap-binding protein eIF4Eor preventing its assembly into eIF4F results in modest SGformation. Intriguingly, interfering with the last step of translation initiationby blocking the recruitment of 60S ribosome either with 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamideis or through depletion of the large ribosomal subunits protein L28 does not induce SGassembly. Our study identifies translation initiationsteps and factors involved in SGformation as well as those that can be targeted without induction of SGs.