Figure 5: Mutant GARS and YARS expression reduces global protein translation rates in motor and sensory neurons in vivo. | Nature Communications

Figure 5: Mutant GARS and YARS expression reduces global protein translation rates in motor and sensory neurons in vivo.

From: Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases

Figure 5

(a–d) FUNCAT labelling in motor neurons of larvae co-expressing dMetRSL262G-EGFP and two copies of GARS transgenes (OK371-GAL4). Scale bar, 10 μm. (e) Quantification of FUNCAT signal intensity revealed ∼60% reduction of translation rates in motor neurons expressing GARS_G240R and GARS_G526R. Average±s.e.m. relative to GARS_WT (100%); Mann–Whitney U-test; ***P<0.001; N=9–12. (f) Representative western blotting to detect newly synthesized proteins in CNS lysates from OK371-GAL4>UAS-dMetRSL262G-EGFP>2 × UAS-GARS_WT and OK371-GAL4>UAS-dMetRSL262G-EGFP>2 × UAS-GARS_G240R larvae. After biotin–alkyne affinity tagging, total protein concentrations were determined and samples were diluted so that each sample contained equal total protein concentrations. Part of the samples was used for NeutrAvidin affinity purification and subsequent western blotting using anti-biotin antibodies detected biotinylated proteins before (B) and after (A) purification. The full-length blot is shown in Supplementary Fig. 16a. (g) Quantification of BONCAT signal intensity after affinity purification. Averages±s.e.m. relative to GARS_WT (100%); unpaired t-test with Welch’s correction (two-tailed); **P<0.01; N=4. (h) FUNCAT revealed reduced translation rates in sensory neurons expressing any of the three GARS mutant proteins. Averages±s.e.m. relative to GARS_WT (100%); Mann–Whitney U test; *P<0.05, ***P<0.005; N=10. (i) Levels of 35S-methionine incorporation in proteins of flies expressing GARS transgenes from the adult stage onwards. 35S-methionine incorporation was determined 4 days after transgene induction and normalized to total protein content. 35S-methionine incorporation was also measured in age-matched uninduced control flies of the same genotype, and the ratio of induced to uninduced 35S-methionine incorporation is shown as percentage of 2 × GARS_WT. Ubiquitous expression of each of the mutant GARS transgenes significantly reduced 35S-methionine incorporation by ≈50%. Welch’s analysis of variance (Dunnett’s T3 post hoc test); ***P<0.005; N=10–12. (j,k) FUNCAT in motor (j) and sensory (k) neurons revealed significantly reduced translation rates induced by any of three CMT-mutant YARS proteins. mutDEL: YARS_153-156delVKQV. Averages±s.e.m. relative to YARS_WT (100%), N=22–24 (j) and 26–30 (k) Mann–Whitney U-test; *P<0.05, **P<0.01, ***P<0.001. Error bars represent s.e.m.

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