Fig. 3. ZNF598 aborts stalled translation of mitochondrial outer membrane-associated C-I30 mRNA and rescues mitochondrial and neuromuscular defects of PINK1 mutant flies.
a Immunostaining of ZNF598 and TOM20 or GFP in U2OS cells under normal, CCCP treatment, and MTSGFP-nonstop OE conditions. b Immunoblots of ZNF598 in isolated mitochondria from normal and CCCP treatment cells, followed by proteinase K + Triton X-100 treatment. VDAC as out membrane, CHCHD3 as inner mitochondrial membrane, and ACTB as cytosolic protein markers. c Localization of HA-tagged early RQC factors ZNF598 and Rack1 to mitochondria of adult DA neurons. DA neuron mitochondria are marked with TH-Gal4-driven mito-GFP expression. d Western blot analysis showing effect of ZNF598 and Rack1 OE on CAT-tailed C-I30-u formation in PINK1B9 fly muscle. e, f Effect of ZNF598 OE (by UAS-ZNF598-HA or ZNF598-EP) or RNAi on abnormal wing posture in PINK1B9 flies. g Effect of ZNF598 OE on mitochondrial morphology in PINK1B9 fly muscle. Red arrowheads mark aggregated mitochondria. Mito-GFP labels mitochondria. Bar graph shows quantification of damaged mitochondria. Effect of ZNF598 OE on DA neuron number in the PPL1 cluster of PINK1B9 adult brains (h). Bar graph shows data quantification (i). Data are representative of at least three biologically independent experiments (mean ± SD), n = 4 and 5 in (e), n = 3 in (f, g, i). Representative blot or image is from at least three independent experiments with similar results. p values in (e) and (g) were calculated by unpaired two-tailed t-test. p-values in (f) were calculated by one-way ANOVA (Tukey’s test). p values in (i) were calculated by two-way ANOVA (Sidak’s test). Source data are provided as a Source Data file.