Tuning tRNA synthetase inhibition reveals parabolic induction of stress granules limited in size and RNA content

Abstract

Translation elongation defects cause ribosome stalling and activate the integrated stress response (ISR). During the ISR, translation initiation suppression and ribosome runoff drive mRNA condensation into stress granules. However, the effects of partial translation elongation inhibition on stress granules are poorly defined. We demonstrate that intermediate levels of tRNA synthetase inhibitors activate the ISR and cause assembly of stress granules in a parabolic dose–response pattern. These stress granules are limited in size and number due to ribosome association with mRNAs. Assembly of stress granules by intermediate levels of the prolyl-tRNA synthetase inhibitor halofuginone requires the canonical stress granule scaffolding proteins G3BP1/2 and GCN2-mediated ISR activation. We performed a candidate-based comparative analysis of the composition of stress granules induced by intermediate levels of halofuginone or canonical stressors arsenite or thapsigargin. The stress granules induced by halofuginone, arsenite, or thapsigargin harbor polyadenylated RNA and the canonical stress granule proteins PABPC1, G3BP1, and UBAP2L. We observe stress- and transcript-specific differences in the localization of candidate RNA molecules to stress granules. These results demonstrate that partial translation elongation inhibition permits stress granule assembly through the balance of ISR activation and mRNA association with ribosomes, with implications for the stress response associated with amino acid or tRNA deficiency, therapeutic tRNA synthetase inhibition, or diseases associated with tRNA synthetase mutations.