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. 2021 Sep 27;49(17):10007-10017.
doi: 10.1093/nar/gkab730.

Charcot-Marie-Tooth mutation in glycyl-tRNA synthetase stalls ribosomes in a pre-accommodation state and activates integrated stress response

Samantha Mendonsa  1   2 , Nicolai von Kuegelgen  1   2 , Lucija Bujanic  1 , Marina Chekulaeva  1
Affiliations

Charcot-Marie-Tooth mutation in glycyl-tRNA synthetase stalls ribosomes in a pre-accommodation state and activates integrated stress response

Samantha Mendonsa et al. Nucleic Acids Res. .

Abstract

Toxic gain-of-function mutations in aminoacyl-tRNA synthetases cause a degeneration of peripheral motor and sensory axons, known as Charcot-Marie-Tooth (CMT) disease. While these mutations do not disrupt overall aminoacylation activity, they interfere with translation via an unknown mechanism. Here, we dissect the mechanism of function of CMT mutant glycyl-tRNA synthetase (CMT-GARS), using high-resolution ribosome profiling and reporter assays. We find that CMT-GARS mutants deplete the pool of glycyl-tRNAGly available for translation and inhibit the first stage of elongation, the accommodation of glycyl-tRNA into the ribosomal A-site, which causes ribosomes to pause at glycine codons. Moreover, ribosome pausing activates a secondary repression mechanism at the level of translation initiation, by inducing the phosphorylation of the alpha subunit of eIF2 and the integrated stress response. Thus, CMT-GARS mutant triggers translational repression via two interconnected mechanisms, affecting both elongation and initiation of translation.

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Figures

Figure 1.
Figure 1.
Overexpression of CMT-GARS mutants, E71G, G240R and ΔETAQ, but not WT GARS, represses protein production in cultured cells. (A) Schematic representation of constructs used in transfection experiments: RL and FL are reporter constructs encoding Renilla and firefly luciferase, correspondingly. Positions of analyzed CMT-GARS mutations in the context of GARS domain structure are shown. (B) Repression of RL and FL mRNAs by GARS mutants. Human HEK293T cells were co-transfected with plasmids encoding RL, FL, and myc-tagged GARS, either WT or indicated mutant. As a negative control, empty vector was used instead of GARS-encoding plasmid. RL and FL activities are presented as a percentage of luciferase activity produced in the presence of empty vector. Values represent means ± SD from three experiments. (C) Expression levels of myc-fusion proteins were estimated by western blotting with antibodies directed against myc-tag. Beta-actin was used as a loading control. (D) Puromycylation assay confirms the role of CMT-GARS in global translational repression. HEK293 cells were transfected with plasmids encoding WT GARS, indicated GARS mutants or an empty vector. After puromycin treatment, cells were lyzed and lysates were analyzed by western blotting with anti-puromycin antibody. PAAG stained with coomassie is provided to visualize equal total protein loading between the samples.
Figure 2.
Figure 2.
Ribosome profiling detects pausing of ribosome with Gly codons in open A-site in G240R-GARS-expressing cells. (A) Scheme showing stages of elongation and the length of ribosome protected fragments (RPFs) generated by each ribosomal state. aa-tRNA: aminoacyl-tRNA, aaRS: aa-tRNA synthetase. (B) Length distributions of CDS-mapped ribosome footprints in libraries prepared HEK293T cells expressing either WT or G240R-GARS (in triplicates). (C) Metagene aggregate plots displaying distance of 21-nt ribosome footprints from annotated start codon. (D) Scatter plots comparing frequencies of 64 codons in the ribosomal A-site between cells expressing WT (X) and G240R-GARS (Y), for 21 nt (left) and 29 nt (right) RPFs. Ribosome frequencies represent the means of triplicates. Glycine codons are labelled and marked in red. Insets show differences between codon frequencies in G240R and WT samples as bar plots.
Figure 3.
Figure 3.
CMT-GARS mutants do not impair overall glycylation activity, but have increased capacity to bind tRNAGly. (A) Northern blotting shows that aminoacylation levels of tRNAGly remain substantially unaffected in the presence of CMT-GARS mutants. HEK293T cells were transfected with plasmids encoding WT, E71G, G240R, ΔETAQ GARS or an empty vector, total RNA was isolated and aminoacylation levels of tRNAGly were evaluated by acid-urea PAGE and northern blotting. Acidic: lanes showing RNA analysed under acidic pH, that preserves ester bonds linking amino acids to tRNAs. Basic: lanes showing RNA analysed under basic pH, that leads to tRNA deacylation. (B) Immunoprecipitation and northern blotting show that CMT-GARS mutants have increased capacity to retain bound tRNAGly. HEK293T cells were transfected with plasmids encoding WT, E71G, G240R or ΔETAQ GARS-myc, with the amounts of plasmid adjusted to achieve equal protein expression. GARS-myc was immunoprecipitated with anti-myc antibody and inputs and immunoprecipitates were analyzed by PAGE and northern blotting to evaluate the levels of GARS-myc-bound tRNAGly. To control for equal efficiency of RNA recovery, immunoprecipitates were supplemented with RNA spike-in before RNA extraction. Duplicates of immunoprecipitates are shown. Western blotting for inputs and immunoprecipitated GARS-myc is shown as a loading control.
Figure 4.
Figure 4.
Overexpression of CMT-GARS mutants, E71G, G240R and ΔETAQ, induces integrated stress response (ISR) via phosphorylation of eIF2a. (A) Expression of CMT-GARS mutants induces phosphorylation of eIF2a. HEK293T cells were transfected with plasmids encoding WT, E71G, G240R, ΔETAQ GARS or an empty vector. For a positive control of eIF2a phosphorylation, cells were treated with thapsigargin. Cell lysates were analyzed by western blotting using antibodies against phosphorylated eIF2a (P-eIF2a), eIF2a (loading control), and myc (GARS-myc), as indicated on the left. (B) Schematic representation of reporter constructs used in transfection experiments: RL and FL are the same as in Figure 2A. ATF4-RL carries 5′UTR of ATF4 gene. (C) E71G, G240R and ΔETAQ GARS mutants activate expression of ATF4 reporters. Human HEK293T cells were co-transfected with plasmids encoding one of the Renilla luciferase reporters (RL, ATF4-RL), FL and myc-tagged GARS, either WT or indicated mutant. As additional controls, empty vector and NSP1-encoding plasmids were used instead of GARS plasmid. RL activity was normalized to that of FL and presented as a percentage of luciferase activity produced in the presence of empty vector for each Renilla reporter. Values represent means ± SD from three experiments. (D) Puromycylation assay shows that CMT-GARS-mediated translational repression is preserved upon inhibition of eIF2a phosphorylation. HEK293T cells were transfected with plasmids encoding WT, E71G, G240R or ΔETAQ GARS and inhibitor of eIF2a phosphorylation phosphorylation GCN2-IN-1 was added were indicated. After puromycin treatment, cells were lyzed and lysates were analyzed by western blotting with antibodies against phosphorylated eIF2a (P-eIF2a), eIF2a (loading control), myc (GARS-myc) and puromycin, as indicated on the left. PAAG stained with coomassie is provided to show total protein loading.
Figure 5.
Figure 5.
Model illustrating the mechanism of CMT-mutant GARS function in translational regulation. CMT-mutant GARS protein inhibits accommodation of glycyl-tRNA into the ribosomal A-site, possibly via decreasing the pool of available charged glycyl-tRNA, and leads to ribosome stalling on glycine codons. Ribosome stalling results in phosphorylation of eIF2a and activation of integrated stress response. In particular, phosphorylation of eIF2a leads to reduction in the levels of ternary complex eIF2:GTP:Met-tRNAi, which downregulates global translation initiation and upregulates expression of selected transcripts with uORFs, such as ATF4. When levels of ternary complex are low, ribosomes bypass uORF, which allows them to initiate translation on the main ATF4 ORF. ATF4 is a transcription factor that induces stress response genes.
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