Figure 6: Impaired protein translation is independent of tRNA^Gly aminoacylation and may causally contribute to CMT-like phenotypes.

(a) In vitro aminoacylation assay on total protein extracts from larvae that ubiquitously express GARS (actin5C-GAL4^weak). dGars overexpression was used as a positive control. Experiments were performed in triplicate and error bars represent s.e.m. (b,c) Steady-state in vivo aminoacylation levels of the two cytoplasmic tRNA^Gly variants in Drosophila (b: tRNA₁^Gly; c: tRNA₂^Gly) were determined by acid urea PAGE and northern blotting. The ratio of aminoacylated versus non-aminoacylated tRNA^Ala serves as internal standard. ac: tRNA isolated under acidic conditions; OH: deacylation by base treatment. The full-length blots are shown in Supplementary Fig. 16b,c. (d) FUNCAT revealed that dGars co-overexpression does not rescue GARS_G240R induced protein synthesis defects in larval motor neurons; Mann–Whitney U-test; **P<0.01 versus control; NS, not significant; N=9–10. (e) Expression of constitutively active d4E-BP reduced translation rates in larval motor neurons as determined by FUNCAT; Mann–Whitney U-test; *P<0.05 versus control; N=22 (control), 13 (d4EBP-LL), 8 (d4EBP-TA). (f) Expression of constitutively active d4E-BP in motor neurons results in muscle denervation in third instar larvae; χ²-Test; ***P<0.005 versus control; N=19. (g) Expression of constitutively active d4E-BP reduced protein translation rates in larval sensory neurons as determined by FUNCAT. N=16–20; ***P<0.001 versus control. (h) Expression of constitutively active d4E-BP in class IV multidendritic sensory neurons significantly reduces the percentage of dendritic coverage; Mann–Whitney U-test; ***P<0.001 versus control; N=15. Error bars represent s.e.m.