Fig 2: Wild-type GlyRS binds the SIRT2 to maintain the acetylated a-tubulin. (a–c) The recombinant SIRT2(1µmol) and recombinant GlyRSWT (a), GlyRSG526R (b), or GlyRSE71G (c) were incubated with NSC-34 lysate at the indicated ratios(concentration measured as a monomer). The reaction products were detected by Western blotting for acetylated tubulin, a-tubulin, and His-tag. Data are presented as mean ± SD, n = 3 biological replicates per group, from three independent experiments. (d) Representative immunoblotting of 3 independent experiments shows that both knockdowns of GlyRS and overexpression of SIRT2 result in decreased tubulin acetylation. HEK293 cells were transfected with GlyRS siRNA or control siRNA 24 h later, and SIRT2-Myc was overexpressed in cells transfected with control siRNA. After an additional 48 h, cells were harvested and acetylation of tubulin was detected by Western blot. (e) Western blot analysis detecting the level of a-tubulin acetylation in NSC-34 cells transfected with wild-type GlyRS and GlyRS mutants

Fig 3: Mutant GARS and YARS expression reduces global protein translation rates in motor and sensory neurons in vivo.(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.

Fig 4: HDAC6 co-immunoprecipitates with GlyRS. (A) N2a cells were transiently transfected with either pCMV-DDK-tagged human cDNA encoding GlyRS or pCMV-dsRed-tagged human cDNA encoding HDAC6. Co-immunoprecipitation was performed using agarose beads conjugated to an antibody directed against the DDK peptide. Transfected N2a cells were pre-treated with 1 µM tubastatin A (TubA) or vehicle (DMSO). Western blot analysis was performed to check for equal protein expression and co-immunoprecipitation of HDAC6. (B) N2a cells were transiently transfected with either pCMV-DDK-tagged human cDNA encoding wild-type or C175R mutant GlyRS or pCMV-dsRed-tagged human cDNA encoding HDAC6. Co-immunoprecipitation was performed using agarose beads conjugated to an antibody directed against the DDK peptide. Transfected N2a cells were pretreated with 1 µM tubastatin A or vehicle (DMSO). Western blot analysis was performed to check for equal protein expression and co-immunoprecipitation of HDAC6.

Fig 5: Tubastatin A treatment increases the acetylation of a-tubulin in sciatic nerve and DRG homogenates from GarsC201R/+ mice. (A and D) The levels of acetylated of a-tubulin and Gars expression in sciatic nerve homogenates was determined by western blot. Densitometry was used to assess the ratio of acetylation a-tubulin to total a-tubulin levels in sample from vehicle and tubastatin A-treated mice. All values were normalized to samples from vehicle treated mice: vehicle 1.00 ± 0.18, n = 3 mice versus tubastatin A 1.35 ± 0.29, n = 3 mice; unpaired t-test t = 7.636, P = 0.0016. (B and E) The acetylation status of a-tubulin and Gars expression in DRGs from vehicle- and tubastatin A-treated mice was determined using western blot. The ratio between the acetylation of a-tubulin and total a-tubulin levels was quantified by densitometry. All values were normalized to samples from vehicle-treated mice: vehicle 1.00 ± 0.30, n = 3 mice versus tubastatin A 2.13 ± 0.15, n = 3 mice; unpaired t-test t = 5.799, P = 0.0044. (C and F) Spinal cord samples were collected from vehicle- and tubastatin A-treated mice to assess the acetylation status of a-tubulin. Densitometry was used to quantify the ratio between acetylated and total a-tubulin levels. All values were normalized to samples from vehicle-treated mice: vehicle 1.00 ± 0.18, n = 3 mice versus tubastatin A 1.35 ± 0.29, n = 3 mice; unpaired t-test t = 1.8, P = 0.1463.