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Tuning tRNA synthetase inhibition reveals parabolicinduction of stress granules limited in size and RNA contentMAX BAYMILLER, 1,2,3 NOAH S. HELTON, 1,2 BENJAMIN DODD, 1,2 and STEPHANIE L. MOON 1,21 Department of Human Genetics, University of Michigan, Ann Arbor, Michigan 48109, USA2 Center for RNA Biomedicine, University of Michigan, Ann Arbor, Michigan 48109, USA3 Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06459, USAABSTRACTTranslation elongation defects cause ribosome stalling and activate the integrated stress response (ISR). During the ISR,translationinitiationsuppressionandribosomerunoffdrivemRNAcondensationintostressgranules.However,theeffectsof partial translation elongation inhibition on stress granules are poorly defined. We demonstrate that intermediate levelsof tRNA synthetase inhibitors activate the ISR and cause assembly of stress granules in a parabolic dose–response pattern.These stress granules arelimited in size and number due to ribosome association with mRNAs. Assemblyofstress granulesby intermediate levels of the prolyl-tRNA synthetase inhibitor halofuginone requires the canonical stress granule scaffold-ing proteins G3BP1/2 and GCN2-mediated ISR activation. We performed a candidate-based comparative analysis of thecomposition of stress granules induced by intermediate levelsof halofuginone orcanonical stressors arsenite or thapsigar-gin. The stress granules induced by halofuginone, arsenite, or thapsigargin harbor polyadenylated RNA and the canonicalstress granule proteins PABPC1, G3BP1, and UBAP2L. We observe stress- and transcript-specific differences in the local-ization of candidate RNA molecules to stress granules. These results demonstrate that partial translation elongation inhi-bition permits stress granule assembly through the balance of ISR activation and mRNA association with ribosomes, withimplicationsforthestressresponseassociatedwithaminoacidortRNAdeficiency,therapeutictRNAsynthetaseinhibition,or diseases associated with tRNA synthetase mutations.Keywords: stressgranules;tRNAsynthetase;halofuginone;translation;translationelongation;integratedstressresponseINTRODUCTIONThe integrated stress response (ISR) is a conserved signal-ing pathway activated in response to physiological chal-lenges including nutrient deprivation, toxic metalloidexposure, RNA damage, and ER stress. During the ISR,translation initiation is suppressed when stress-sensing ki-nases GCN2 (general control nonderepressible 2), HRI(heme regulated inhibitor), PKR (protein kinase R), orPERK (PKR-like endoplasmic reticulum kinase) phosphory-late the translation initiation factor eIF2α (Pakos-Zebruckaet al. 2016; Costa-Mattioli and Walter 2020). Inhibition oftranslation initiation by P-eIF2α promotes stress-inducedgene expression (Young and Wek 2016) and drives the as-semblyofbiomolecularcondensatescalledstressgranules(Kedersha et al. 2005). Stress granules are implicated incellular stress resilience and form through multivalentRNA–RNA, RNA–protein, and protein–protein interac-tions, typically driven by G3BP1-mediated phase separa-tion (Kedersha et al. 2016; Guillén-Boixet et al. 2020;Sanders et al. 2020; Yang et al. 2020). Stress granules areenriched in translationally repressed mRNAs and RNAbinding proteins such as G3BP1 (G3BP stress granule as-sembly factor 1), PABPC1 (poly(A) binding protein), andUBAP2L (ubiquitin associated protein 2 like) (Kedershaet al. 2005; Khong et al. 2017; Moon et al. 2019). Stressgranule formation depends on both translation initiationsuppression and continued translation elongation, andthe association of mRNAs with one or more ribosomes in-hibits their assembly into stress granules (Kedersha et al.2000, 2005; Mollet et al. 2008; Khong and Parker 2018;Moon et al. 2019, 2020; Helton et al. 2025). Thus, the re-lease ofmRNAsfromribosomes iscritical for stress granuleassembly during the ISR.Translation elongation stresses (e.g., UV stress, aminoacid deprivation, RNA damage, or loss of tRNA synthetaseCorresponding author: smslmoon@umich.eduHandling editor: Eric PhizickyArticle is online at http://www.rnajournal.org/cgi/doi/10.1261/rna.080883.125. Freely available online through the RNA Open Accessoption.© 2026 Baymiller et al. This article, published in RNA, is available un-der a Creative Commons License (Attribution-NonCommercial 4.0International), as described at http://creativecommons.org/licenses/by-nc/4.0/.ARTICLE870 RNA (2026) 32:870–884; Published by Cold Spring Harbor Laboratory Press for the RNA Society Cold Spring Harbor Laboratory Press on July 2, 2026 - Published by rnajournal.cshlp.org Downloaded from