Mutation linked to neurological disorders delivers double-pronged attack at cellular level

A mutation linked to neurological disorders knocks out not just one protein involved in translating the genetic code, but the entire cellular translation machinery.

That’s the discovery of a team from the University of Maryland who were investigating how editing defects in threonyl-tRNA synthetase (ThrRS) work at a cellular level using yeast as a model organism.

Editing defects cause neurodegeneration and cardioproteinopathy in mice and are associated with microcephaly in human patients. The research is published in ‘Coordination between aminoacylation and editing to protect against proteotoxicity’ which is published in Nucleic Acids Research.

Essential enzymes

“Aminoacyl-tRNA synthetases (aaRSs) universally exist in all organisms and are essential enzymes playing critical roles in protein synthesis. AaRSs defines the first step of protein synthesis by charging cognate tRNAs with amino acids to form aminoacyl-tRNAs (aa-tRNAs), which are delivered to the ribosome for nascent peptide formation,” explained Dr Hong Zhang, an Applied Microbiology International member.

“In some cases, mischarging of tRNAs by aaRSs occurs. Most organisms have evolved aaRS editing activities to deacylate the mischarged aa-tRNAs. The editing function is an essential checkpoint to ensure translation fidelity.

“An increasing number of studies show that mutations in aaRS genes result in various neurological diseases (e.g., Charcot–Marie–Tooth (CMT) disease and microcephaly), developmental delay, and cancer.

“Other factors involved in tRNA biogenesis and tRNA modifications are also linked to multiple diseases. Revealing the physiological effects of aaRS mutations is thus critical to understand the development of neurodegenerative diseases and searching for effective drugs to treat these diseases.”

Yeast model

The team decided to investigate the cellular impact of cytoplasmic ThrRS editing deficiency in eukaryotes using haploid budding yeast BY4741 as a model.

“We used CRISPR/Cas9 genome editing to introduce the ths1 mutation to abolish editing of ThrRS protein in Saccharomyces cerevisiae and checked the physiology impact,” Dr Zhang said.

They created several point mutation strains of yeast threonyl-tRNA synthetase (ThrRS) for editing defects, and ThrRS knockdown strains for aminoacylation impairment.

“We found that yeast threonyl-tRNA synthetase (ThrRS) editing defect mutation C268A causes serine misincorporation into yeast proteome at Threonine (Thr) codon and leads to severe growth defect under heat stress,” Dr Zhang said.

Heat stress

“However, ThrRS editing defect mutation C268S does not result in growth defect under heat stress. Finally, we found that ThrRS editing defect mutation C268A protein itself is not stable and can be degraded under heat stress, which caused the aminoacylation defect. Besides, we found the aminoacylation impaired strain alone also grow normally. In total, editing defect alone is not sufficient to cause heat sensitivity and coordination between charging (aminoacylation) and proof reading (editing) can protect cells against proteotoxicity.”

The team found that the ThrRS editing defective strain C268A can grow well under normal culture conditions but has a severe growth defect under heat stress conditions.

“We optimized bacteria β-lactamase gene and constructed the reporters to detect serine mistranslation rate in yeast. It turned out serine mistranslation rate in yeast ThrRS editng defective strain C268A, much higher than the wildtype yeast.

“Further, to understand how the ThrRS editing-defective mutation causes heat sensitivity, we performed three independent evolution experiments of the ThrRS-C268A strain at 37 °C. After 15 rounds (~100 generations), all three replicates (E15A, E15B, and E15C) had evolved to restore growth at 37 °C.

Additional mutation

“Whole-genome sequencing shows that E15B contains a ths1-L264F mutation in addition to the ths1-C268A mutation and maintained a single copy of chromosome IX. The E15A and E15C strains contain the duplicated chromosome IX, but also each acquired an additional mutation at the P173 position of one copy of THS1 (ths1-P173L and ths1-P173R, respectively). In vitro aminoacylation assay determining Thr aminoacylation by ThrRS variants also showed that evolved ThrRS proteins have much higher levels of charged Thr-tRNAThr under heat stress (at 37 °C).

“We measured the overall protein translation rate by using a methionine analog homopropargylglycine (HPG) and found that HPG incorporation in the editing defect C268A strain decreased over 50%, which means overall protein translation decreased more than 50% in editing defect C268A strain under 37 °C heat stress. However, all 3 evolved strains protein synthesis recovered to normal level under 37 °C heat stress.

“Editing deficiency of many aaRSs are well tolerated under normal growth conditions. Serine mistranslation leads to protein misfolding, and heat stress exacerbates this misfolding and degradation.

Degrading quickly

“Our western blot showed that editing defect C268A protein is not stable and degrades quickly under heat stress. As expected, using acidic northern blot, we showed that the ths1-C268A cells have a significantly lower percentage of aa-tRNAThr at 37 °C. In total, the higher ThrRS expression, the increased correct chargeing and recovered total protein synthesis may contribute to the evolved strains’ normal growth under heat stress.”

To clarify the contribution of aminoacylation and editing defects to heat sensitivity, the researchers constructed additional aminoacylation impaired mutants (lower ThrRS expression level) and editing defect mutants, by using the powerful CRISPR/Cas9 genome editing tool.

“We saw the aminoacylation defect alone (lower level of ThrRS protein) or the editing defect alone does not contribute to the heat sensitivity, but combined aminoacylation and editing defects caused heat sensitivity,” Dr Zhang said.

“In addition to being used by the ribosome during canonical protein synthesis, Thr-tRNAThr and Ala-tRNAAla synthesized by ThrRS and AlaRS are also used in the ribosome-associated quality control (RQC) pathway. RQC senses stalled ribosomes and adds C-terminal Ala and Thr (CAT) tails to stalled polypeptides.

“Formation of the CAT tail facilitates degradation of truncated polypeptides, thus protecting cells against proteotoxicity. We show that the ths1-C268A mutation destabilizes ThrRS and leads to a lower tRNAThr aminoacylation level, which in turn impairs both global protein synthesis and the RQC pathway. We further show that decreasing ThrRS aminoacylation or editing efficacy alone is insufficient to cause proteotoxicity.“

Surprising find

What was surprising was that the evolved strains could grow normally under heat stress, but their ThrRS protein in the cells were still editing defective, but the team later discovered that their ThrRS protein in the cell increased significantly.

“The editing defective C268A strain is sensitive to heat stress, but the editing defective C268S strain does not. We discovered that ThrRS protein in the editing defect C268S strain does not change, however, ThrRS protein in editing defect C268A strain decreased significantly under stress. The northern blot also showed the editing defect strain C268A has aminoacylation defect. Both aminoacylation and editing defect contributes to the proteotoxicity,” Dr Zhang said.

“ISR usually is activated by amino acids starvation. In our previous study, the integrated stress response (ISR), also called the general amino acid control (GAAC) pathway in yeast, was activated in the yeast Alanyl-tRNA synthetase (AlaRS) editing defective strain (Zhang et al., 2021). Induction of gcn2/p-eIF2α/gcn4 (GAAC) pathway can quickly downregulate the overall protein biosynthesis while allowing translation of specific genes to support adapting to all kinds of adverse environments. The integrated stress response (ISR) is an adaptive mechanism in response to multiple stresses. However, chronic activation of the ISR contributes to the development of multiple diseases (e.g., neuropathies).

Next steps

“For in the editing defective strain C268A, showed by the northern blot assay, there are uncharged tRNAThr, which may activate the ISR. In the next step, it inspired us to detect whether ISR was activated in the ThrRS editing deficiency strain. We can check the cellular GCN4 expression by using gcn4 expression reporters, and phosphorylation level of eIF2α by using p-eIF2α antibody.

“Also, we detected the serine mistranslation into Threonine rate is 0.2% in this paper. We expect there will be much more serine mistranslation if we overexpress a tRNASer with its anticodon mutated to base pair and decode the Thr codon. And check the phenotype and physiology effect.”

The PI, Dr. Jiqiang (Lanny) Ling in the University of Maryland, led this study. Dr Zhang is now an assistant research scientist in Lanny’s lab.