Fig 1: Western blot analysis of mitochondrial proteins. (A) Twenty µg of total proteins from mutant and wild type zebrafish were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with antibodies for 6 subunits of OXPHOS (four encoded by mtDNA and two encoded by nuclear genes), Kars, Lars2, Yars2, Eftu, Tfb2m as well as Tom20 as a loading control. Quantification of levels of OXPHOS subunits (B) and other mitochondrial proteins (C). Average content of Nd1, Nd6, Cytb, Co2 and Atp5a, Sdhb, Kars, Lars2, Yars2, Eftu and Tfb2m was normalized to the average content of Tom20 in mutant and wild type zebrafish. The values for the mutant zebrafish are expressed as percentages of the average values for the wild type zebrafish. The horizontal dashed lines represent the average values of four proteins encoded by mtDNA or two proteins encoded by nuclear genes for each group. The calculations were based on three independent determinations. Graph details and symbols are explained in the legend to the Figure 3.
Fig 2: Deletion of yars2 caused the eye defects in zebrafish.A, the lateral views of yars2-/-, yars2+/-, and yars2+/+ zebrafish at 5 dpf, and the eye morphologies of zebrafish were illustrated under a Leica microscope with an objective magnification of 20×. Asterisk indicated the eye pigments. B, the dorsal views of the yars2+/+, yars2+/-, and yars2-/- zebrafish at 5 dpf. C–E, quantification of the area and length of eyes as well as area of lens of yars2+/+ (n = 10), yars2+/- (n = 12), and yars2-/- (n = 14) zebrafish, as detailed elsewhere (47). The values for the mutants were expressed as percentages of the average values for the wild type. Graph details and symbols are explained in the legend to Figure 2.
Fig 3: The proposed model of YARS2 interacting with the OXPHOS supercomplex. The partial OXPHOS supercomplex was derived from (MCI2III2IV2, PDB: 5XTI) (41). The structure of Thermus thermophilus tryosyl-tRNA synthetase and tRNATyr complex (PDB ID: 1H3E) was used as an original templet for construction of the model of humanYARS2-tRNATyr complex. The structure of YARS2 was extracted from the crystal structures of human mitochondrial tyrosyl-tRNA synthetase (PDB ID: 2PID) (42). The model of human YARS2-tRNATyr complex was generated using PyMOL molecular visualization system (PyMOL Molecular Graphics System, Version 2.3.3, Schrödinger, LLC). A model of YARS2 interacting with the OXPHOS supercomplex was proposed as the YARS2 homodimer–tRNATyr complex interacts with CO2 of CIV, NDUFS1 and NDUFA9 of membrane arm of CI in the megacomplexes I2III2IV2, respectively.
Fig 4: Generation of YARS2 knockout HeLa cell lines using CRISPR/Cas9 system.A, schematic representation of CRISPR/Cas9 target site at exon 1 as used in this study. An allele, YARS2del14bp was produced by a 14 bp delete in the exon 1 and a truncated nonfunctional protein with 24 amino acids. B, western blot analysis of YARS2 in various cells. Twenty micrograms of total cellular proteins of each cell line was electrophoresed through and hybridized with antibodies specific for YARS2 (1:1000 dilution) and with TOM20 (1:2000 dilution) as a loading control. WT, wild-type cells; KO, YARS2KO; KO+YARS2, exogenous YARS2 expression in YARS2KO; KO+vector, vector transfected in YARS2KO. Three independent experiments were performed. C, in vivo aminoacylation of mitochondrial tRNA assays. Ten micrograms of total cellular RNAs purified from various cell lines under acid conditions was electrophoresed through an acid (pH 5.2) 10% polyacrylamide-7 M urea gel, electroblotted, and hybridized with DIG-labeled oligonucleotide probe specific for the tRNATyr, tRNALeu(UUR), and tRNAThr, respectively. Samples for WT cells were deacylated (DA) by heating for 10 min at 60 °C at pH 8.3, electrophoresed, and hybridized with DIG-labeled oligonucleotide probes as described above. Three independent experiments were performed.
Fig 5: Mitochondrial defects in the zebrafish retina.A and B, assessment of mitochondrial function in the retina by enzyme histochemistry (EHC) staining for COX (A) and SDH (B) in the frozen sections of retina in the yars2+/+, yars2+/-, and yars2-/- larvae at 5 dpf. Scale bar, 100 µm. C and D, mitochondrial morphology from photoreceptors (PR) (C) and RGC (D) of transmission electron microscopy. Ultrathin sections were visualized with 25,000× magnification. Scale bar, 1 µm. e, ellipsoid; m, mitochondrion; n, nucleus; os, outer segment of photoreceptors. E and F, quantification of mitochondrial numbers of photoreceptors (E) from yars2+/+ (n = 20), yars2+/- (n = 20), and yars2-/- (n = 25) and RGC (F) from yars2+/+ (n = 32), yars2+/- (n = 24), and yars2-/- (n = 25) zebrafish at 5dpf. Graph details and symbols are explained in the legend to Figure 2.
Supplier Page from Abcam for Anti-YARS2/TyRS antibody