oa Hydrogen peroxide induces stress granule formation independent of eukaryotic initiation factor 2α phosphorylation

Background and Objectives: In cells exposed to environmental stress, inhibition of translation initiation conserves energy for the repair of cellular damage. Untranslated mRNAs that accumulate in these cells move to discrete cytoplasmic foci known as stress granules (SGs). The assembly of SGs helps cells to survive under adverse environmental conditions. SGs are formed as a consequence of eIF2α phosphorylation that inhibits translation initiation. However, this is not the sole mechanism by which SGs are assembled. Some compounds inhibit translation and induce SG formation via the interaction with eIF4A. During oxidative stress conditions, reactive oxygen species (ROS) levels increase dramatically causing a significant alteration in cell metabolism including protein synthesis. The most stable form of the ROS is hydrogen peroxide (H2O2), which was found to inhibit protein synthesis in different types of cells. The objective of our study is to analyze the mechanism by which H2O2-induced oxidative stress inhibits translation initiation and induces SG assembly in mammalian cells. Methods: Cells were treated with different concentrations of H2O2 and then fixed, stained with SG markers, and visualized using fluorescence microscopy to quantify SGs. To test the effect of oxidative stress treatment on eIF4F complex formation we pulled down the eIF4F complex from lysates of SA or H2O2- treated U2OS cells using m7GTP-Sepharose. Results: H2O2 inhibits translation and induces the assembly of SGs. The assembly of H2O2-induced SGs is independent of the phosphorylation of eIF2α, a major trigger of SG assembly, but requires remodeling of the cap-binding eIF4F complex. Moreover, H2O2-induced SGs are compositionally distinct from canonical SGs, and targeted knockdown of eIF4E, a protein required for canonical translation initiation, inhibits H2O2-induced SG assembly. Conclusions: In conclusion, our data suggest that mammalian cells can assemble different types of SGs utilizing different mechanisms. These different routes of assembly are stress-specific and dictate recruitment of selective SG constituents. In analogy to amino acid starvation-induced SGs that selectively sequester mRNAs bearing 5'-terminal oligopyrimidine tracts, we propose that different classes of SGs selectively recruit specific mRNAs from translating ribosomes. In turn, this selective mRNA re-localization causes stress-specific changes in protein translation allowing adaptation to stress conditions.