Fig. 4
Interaction with mitochondrial mRNA and translation machinery shows GATD3A is necessary for preventing AGE formation. A CRISPR-Cas9-mediated insertion of a FLAG allele in the C-terminus of GATD3A using a donor 151mer oligonucleotide. PCR genotyping shows insertion of 24-bp fragment in the knock-in mice. Immunoblot analysis detects FLAG epitope in knock-in mice only. GAPDH is used as a loading control. B Co-elution of EFTu and ATP5A with GATD3A by FLAG immunoprecipitation from Gatd3aFLAG/FLAG mouse heart mitochondria. C Coomassie gel showing consistent co-elution of the bacterial elongation factor EFTu with recombinant human GATD3A:6X-His from E. coli by fast protein liquid chromatography. D Model showing GATD3A interactors (arrowheads) at the interface of mitochondrial mRNA maturation and stabilization, and mitochondrial protein translation (representative of n = 3 mice used in immunoprecipitation). E CRISPR-mediated removal of a part of Gatd3a coding region between exons 1 and 3, leading to total loss of the mature GATD3A protein. PCR genotyping shows a large deletion of ~850 bp in the knockout allele. gRNA—guideRNA, E—exon, F—forward primer, R—reverse primer, UTR—untranslated region. F Transmission electron microscopy (TEM) indicates significant loss of electron dense mitochondrial structure (arrowheads) compared to electron density of muscle fibers in left ventricle cardiac tissue of 1-year-old Gatd3a−/− mice (p = ***<0.001, two-tailed Student’s t test, n = 3 per genotype, N = 4 analyzed sections per genotype, scale bars = ±SD, TEM scale bar = 500 nm). G Aged (1 year old) GATD3A knockout mice do not have differential protein expression of deglycase DJ-1, or oxidative phosphorylation components. GAPDH is used as a loading control. H Augmented AGE immunoreactivity of mitochondrial ribosomal 16S and 12S RNA species in one-year-old Gatd3a−/− mouse heart ventricle. The arrowhead denotes the increased AGE reactivity of the 12S RNA in Gatd3a−/− mouse. Representative blot of n = 3 mice per genotype. I Enhanced 1,2-dicarbonyl and AGE immunoreactivity in heart tissue of one-year-old Gatd3a−/− mice as detected by slot blot assay. GAPDH is used as a loading control. (n = 3 per genotype, p = *<0.05, **<0.01, two-tailed Student’s t test, +SEM)