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

  1. Max Baymiller
  2. Noah S. Helton
  3. Benjamin Dodd
  4. Stephanie L. Moon

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

In response to stress, translation initiation is suppressed and ribosome runoff via translation elongation drives mRNA assembly into ribonucleoprotein (RNP) granules including stress granules and P-bodies. Defects in translation elongation activate the integrated stress response. If and how stalled ribosomes are removed from mRNAs during translation elongation stress to drive RNP granule assembly is not clear. We demonstrate the integrated stress response is induced upon tRNA synthetase inhibition in part via ribosome collision sensing. However, saturating levels of tRNA synthetase inhibitors do not induce stress granules or P-bodies and prevent RNP granule assembly upon exogenous stress. The loss of tRNA synthetase activity causes persistent ribosome stalls that can be released with puromycin but are not rescued by ribosome-associated quality control pathways. Therefore, tRNA synthetase activity is required for ribosomes to run off mRNAs during stress to scaffold cytoplasmic RNP granules. Our findings suggest ribosome stalls can persist in human cells and uniquely uncouple ribonucleoprotein condensate assembly from the integrated stress response.

Article activity feed

  1. Life Science Editors Foundation

    Review coordinated by Life Science Editors Foundation Reviewed by: Dr. Angela Andersen, Life Science Editors Foundation & Life Science Editors. Potential Conflicts of Interest: None.

    PUNCHLINE: tRNA Synthetase Inhibition Paradoxically Blocks Stress Granule Formation by Causing Unresolved Ribosome Stalling Despite Activation of the Integrated Stress Response.

    BACKGROUND: Stress granules (SGs) and P-bodies (PBs) are dynamic ribonucleoprotein (RNP) condensates that form in response to cellular stress and contribute to post-transcriptional gene regulation. SGs are implicated in various physiological and pathological contexts, including neurodegenerative diseases, viral infections, and cancer. Mutations in proteins involved in SG dynamics have been implicated in Charcot-Marie-Tooth disease (CMT2), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). The assembly of SGs and PBs is thought to depend on sequestering translationally repressed mRNAs following translational initiation inhibition. The integrated stress response (ISR) mediates translational control under stress via phosphorylation of eIF2α, leading to global translation suppression and the formation of SGs. Ribosome stalling and stress-induced ISR activation should facilitate SG formation, but SGs require ribosome-free mRNAs, and trapped ribosomes block SG assembly. Hypothesis: Persistent stalled ribosomes due to tRNA synthetase inhibition prevent SG and PB assembly. • How does tRNA synthetase inhibition affect the formation of stress-induced RNP granules, including SGs and PBs? • Does the uncoupling of ISR activation from RNP granule assembly indicate a fundamental regulatory mechanism of stress responses?

    SUMMARY: This study demonstrates that tRNA synthetase activity is crucial for stress granule (SG) and P-body (PB) formation, challenging the idea that integrated stress response (ISR) activation alone is sufficient. The authors show that tRNA synthetase inhibition prevents SG/PB assembly by inducing persistent ribosome stalling, which traps mRNAs and disrupts RNP granule formation. By uncoupling ISR activation from stress granule assembly, these findings refine our understanding of how translation and RNP granule dynamics respond to stress, with significant implications for neurodegenerative diseases and therapeutic strategies.

    Key Results

    • • tRNA Synthetase Inhibition Activates ISR but Prevents SG and PB Assembly
    • o While ISR activation via eIF2α phosphorylation typically leads to SG formation, the inhibition of tRNA synthetase paradoxically prevents it.
    • o tRNA synthetase inhibitors (e.g., halofuginone, borrelidin) strongly suppress translation but fail to promote SG/PB formation.
    • • Persistent Ribosome Stalls Trap mRNA in Polysomes
    • o Polysome profiling shows that mRNAs remain trapped in ribosomes instead of being released for SG/PB formation.
    • o Unlike typical translation inhibition (which allows ribosome runoff and mRNA sequestration into SGs), tRNA synthetase inhibition causes prolonged ribosome stalling.
    • • Ribosome Rescue Pathways Are Insufficient to Resolve Stalls
    • o The ribosome-associated quality control (RQC) pathway, mediated by ZNF598, does not effectively clear stalled ribosomes.
    • o These unresolved stalls prevent normal RNP granule assembly.
    • • Puromycin Restores SG/PB Assembly by Releasing Stalled Ribosomes
    • o Adding puromycin, which forces premature translation termination, rescues SG formation, confirming that unresolved ribosome stalls are the key factor blocking RNP granule assembly.

    STRENGTHS

    • Refines the Link Between ISR and SGs. ISR activation alone is not sufficient for SG formation if ribosome stalling persists. This challenges the conventional view that ISR and SG assembly are always tightly linked.
    • Reveals a New Role for tRNA Synthetases in Stress Adaptation. Beyond their canonical role in aminoacylation, tRNA synthetases regulate cellular stress responses by influencing ribosome dynamics.
    • Potential Disease Connections. Mutations in tRNA synthetases are linked to Charcot-Marie-Tooth disease (CMT2) and neurodegeneration, conditions associated with defective SG dynamics. This study suggests that stalled ribosomes, rather than ISR dysfunction alone, might contribute to these diseases.
    • Therapeutic Considerations: tRNA synthetase inhibitors are explored for cancer, fibrosis, and autoimmune diseases, but their impact on stress granule formation could have unintended consequences. Targeting ribosome rescue pathways may mitigate these effects.
    • Use of Multiple Stressors and Experimental Approaches. The study employs various stress conditions (e.g., sodium arsenite, thapsigargin) to show that tRNA synthetase inhibition uniquely prevents SG/PB formation while still activating the ISR. The authors use puromycin to rescue stalled ribosomes, demonstrating that ribosome stalling, rather than translation repression per se, is the key inhibitory factor.

    CONCEPTUAL LIMITATIONS

    • Generalizability to Other Translational Stress Conditions. While the study demonstrates that halofuginone and borrelidin, inhibitors of prolyl- and threonyl-tRNA synthetases, prevent SG/PB assembly, it remains unclear if this effect extends to all aminoacyl-tRNA synthetase inhibitors. Do other ribosome-stalling conditions, such as premature termination caused by nonsense mutations, similarly prevent RNP granule formation.?
    • Role of Ribosome-Associated Quality Control (RQC) Pathway. The study shows that the ZNF598-mediated RQC pathway is insufficient to resolve ribosome stalls caused by tRNA synthetase inhibition. Do other ribosome rescue mechanisms, such as Pelota/HBS1 or GTPBP1/2, play a role in resolving these stalled ribosomes?

    TECHNICAL LIMITATIONS

    • Use of a Limited Number of Cell Lines. The experiments rely heavily on U-2 OS cells, which may not fully recapitulate the stress responses of other cell types, particularly neurons or immune cells, where stress granules have key functional roles. Future studies could extend these findings to primary neurons or disease models.
    • Lack of Direct Evidence for mRNA Sequestration Failure. While the study infers that mRNA is retained in polysomes due to ribosome stalling, it does not directly measure which mRNAs fail to enter SGs/PBs. RNA imaging or transcriptomic analysis of polysome-bound mRNAs would provide stronger support for the hypothesis that stalled ribosomes trap mRNAs and prevent RNP granule formation.
  2. Version published to 10.1101/2025.03.10.642431v1 on bioRxiv