tRNA synthetase activity is required for stress granule and P-body assembly

Why I picked this preprint

I chose to highlight this preprint because it provides important insights into the mechanistic relationship between ribosomes, tRNA charging, and the formation of RNA-protein granules during cellular stress. The authors use a comprehensive array of techniques—including immunofluorescence microscopy, polysome profiling, and biochemical assays—to demonstrate how inhibition of tRNA synthetase activity prevents stress granule assembly despite activation of the integrated stress response. This work reveals a previously unrecognized requirement for tRNA charging in the formation of two critical types of RNA-protein condensates and challenges current models of stress response pathways.

Background

The integrated stress response (ISR) is a conserved signaling pathway in which phosphorylation of the translation initiation factor eIF2α leads to translation inhibition and reprogramming of gene expression^1,2. During the ISR, non-translating mRNAs can condense with RNA-binding proteins to form membraneless organelles called stress granules (SGs) and processing bodies (P-bodies or PBs)^3,4. These ribonucleoprotein (RNP) granules are believed to concentrate specific RNAs and proteins, potentially modulating the stress response and cellular resilience. Inhibitors of tRNA charging, like halofuginone which specifically blocks prolyl-tRNA synthetase activity, activate the ISR through GCN2, a kinase that can sense uncharged tRNAs and/or ribosome collisions^5,6. However, it remains unclear how tRNA charging inhibition affects the assembly of stress-induced RNP granules and whether quality control pathways like ribosome-associated quality control (RQC) can rescue stalled ribosomes to enable RNP granule formation.

Main goal

In this preprint, Baymiller and colleagues examined how inhibiting tRNA synthetase activity impacts the integrated stress response, translation, and RNP granule formation. They focused on whether persistent ribosome stalling caused by tRNA charging defects can be resolved by cellular quality control pathways and how this influences stress adaptation.

Key Findings

tRNA synthetase inhibition activates the ISR but blocks stress granule assembly

The researchers first confirmed that halofuginone treatment suppressed protein synthesis by approximately 84%, similar to the translational suppression observed with arsenite and thapsigargin (Fig. 1B). Surprisingly, despite robust ISR activation, halofuginone-treated cells did not form SG  (Fig. 1C). Baymiller and colleagues found that halofuginone not only blocked SG assembly (Fig. 1E), but also disassembled existing P-bodies and prevented their formation during stress.

Figure 1. tRNA charging inhibition triggers ISR but not stress granule formation.  (C) U-2 OS cells expressing GFP-G3BP1 were treated with conditions that inhibit tRNA charging. (D) Cells were further analyzed using immunofluorescence and FISH to detect SG-associated proteins UBAP2L and PABPC1, as well as poly(A) RNA. This was done in two independent experiments. (E) Cells were treated with halofuginone (HF), arsenite, or thapsigargin, and SG formation was quantified. Image made available under a CC-BY-NC-ND 4.0 International license.

tRNA synthetase inhibition causes persistent ribosome stalling

To understand why RNP granules fail to form despite ISR activation, the researchers performed polysome profiling to examine ribosome association with mRNAs. While thapsigargin treatment caused polysomes to collapse (indicating mRNAs released from translation), halofuginone-treated cells maintained robust polysome profiles (Fig. 2A), suggesting that ribosomes remained stalled on mRNAs. Co-treatment with puromycin, which causes premature ribosome release from mRNAs, rescued polysome collapse in halofuginone-treated cells and restored SG assembly (Figure 2B). This suggests that ribosome stalling due to the lack of charged tRNAs prevents mRNAs from being released to form RNP granules. The authors also demonstrated that these stalled ribosomes are surprisingly persistent, as halofuginone treatment prevented SG formation for up to 16 hours, with continued high levels of phosphorylated eIF2α and preserved polysomes throughout this period.

Ribosome collisions contribute to ISR activation during tRNA synthetase inhibition

The researchers showed a low level of ribosome collisions during halofuginone treatment (Fig. 3A). They also observed increased P-eIF2α levels during halofuginone treatment, suggesting that ribosome collisions do contribute to ISR activation, which the RQC pathway normally limits by clearing collided ribosomes (Fig. 3C).

Figure 3. Ribosome Collisions Underlie ISR Activation by tRNA Synthetase Inhibition. (A) U-2 OS cells treated with emetine or varying doses of halofuginone (HF) and quantified in three replicates by measuring Ub-eS10:eS10 ratios. (B) ISR activation (via p-eIF2α) was assessed after treating cells with HF, arsenite, or thapsigargin, with or without puromycin. (C) Comparison of wild-type (WT) and ZNF598 knockout (KO) cells in the absence of ZNF598. (D) P-eIF2α reduction in ZNF598 KO cells treated with HF. Image made available under a CC-BY-NC-ND 4.0 International license.

 

In a key experiment, the researchers showed that puromycin treatment rescued  formation in halofuginone-treated cells (Fig. 4B of the preprint) Similarly, preprint Figure 4D shows that puromycin increased the percentage of cells with P-bodies during halofuginone treatment.

Even more strikingly, Figure 4C and 4E of the preprint show that puromycin also rescued SG and P-body formation in cells treated with halofuginone plus arsenite or thapsigargin, confirming that mRNA sequestration is the primary mechanism by which halofuginone prevents granule formation.

The long-term persistence of this effect is remarkable. As shown in preprint Figure 5A, halofuginone-treated cells did not form stress granules for up to 16 hours. This was accompanied by sustained P-eIF2α levels (preprint Fig. 5B) and preserved polysomes (preprint Fig. 5C), indicating that ribosome stalls are extremely stable and resist clearance by endogenous mechanisms.

Main Takeaways

• This study demonstrates that tRNA synthetase inhibitors activate the ISR but prevent SG and P-body assembly, uncoupling these processes.
• The authors show that ribosome stalls caused by tRNA charging inhibition persist for extended periods, trapping mRNAs in polysomes and preventing RNP granule formation.
• This work reveals that ribosome collisions contribute to ISR activation during tRNA synthetase inhibition, but the RQC pathway is insufficient to resolve all stalled ribosomes.

Questions/Future Directions

• What determines whether stalled ribosomes lead to collisions that activate quality control versus persistent isolated stalls?
• How does the uncoupling of RNP granule assembly from the ISR affect cellular resilience and recovery after stress?
• Do other translation elongation stresses exhibit similar uncoupling of RNP granule assembly from the ISR?

References

1. Costa-Mattioli M, Walter P. The integrated stress response: From mechanism to disease. Science. 2020;368(6489):eaat5314. doi:10.1126/science.aat5314
2. Pakos‐Zebrucka K, Koryga I, Mnich K, Ljujic M, Samali A, Gorman AM. The integrated stress response. EMBO Rep. 2016;17(10):1374-1395. doi:10.15252/embr.201642195
3. Sanders DW, Kedersha N, Lee DSW, et al. Competing Protein-RNA Interaction Networks Control Multiphase Intracellular Organization. Cell. 2020;181(2):306-324.e28. doi:10.1016/j.cell.2020.03.050
4. Baymiller M, Moon SL. Stress Granules as Causes and Consequences of Translation Suppression. Antioxid Redox Signal. 2023;39(4-6):390-409. doi:10.1089/ars.2022.0164
5. Keller TL, Zocco D, Sundrud MS, et al. Halofuginone and other febrifugine derivatives inhibit prolyl-tRNA synthetase. Nat Chem Biol. 2012;8(3):311-317. doi:10.1038/nchembio.790
6. Harding HP, Ordonez A, Allen F, et al. The ribosomal P-stalk couples amino acid starvation to GCN2 activation in mammalian cells. Sonenberg N, Manley JL, Sattlegger E, eds. eLife. 2019;8:e50149. doi:10.7554/eLife.50149

Tags: p-bodies, ribonucleoproteins, ribosomes, stress granules, stress response, translation

doi: https://doi.org/10.1242/prelights.40477

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