Fig 2: Translation and assembly of OXPHOS subunits are disrupted in Fars2-deficient samples. A–C Western blotting for OXPHOS complexes and the mitochondrial marker TOMM20 in control and cKO mice embryo cortex at indicated embryonic ages (mean ± SD; two-tailed unpaired t-Test, ns: non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n = 4). D–F Western blotting for ND1, CO2 and CYTB in control and cKO mice embryo cortex at the indicated embryonic ages (mean ± SD; two-tailed unpaired t-Test, ns: non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n = 4). G–H Western blotting for OXPHOS complexes and the mitochondrial marker TOMM20 in control and Fars2 knocked-down neurons at the indicated days of culture (mean ± SD; two-tailed unpaired t-Test, ns: non-significant; *P < 0.05; n = 3). I–J Western blotting for ND1 in control and Fars2 knocked-down neurons at 16 days of infection (mean ± SD; two-tailed unpaired t-Test, *P < 0.05; n = 3). K–L Quantification of TOMM20 in A, B and G (mean ± SD; two-tailed unpaired t-Test, ns: non-significant; n = 3–4). M RT-PCR analysis of OXPHOS subunits transcription levels in control and cKO mice embryo cortex at E 17.5 (mean ± SD; Two-tailed unpaired t-Test, ns: non-significant; *P < 0.05; **P < 0.01; n = 3–4). N qRT-PCR analysis of OXPHOS subunits transcription levels in control and Fars2 knocked-down neurons at 16 days after infection (mean ± SD; Two-tailed unpaired t-Test, ns: non-significant; *P < 0.05; **P < 0.01; n = 3)

Fig 3: Effects of Fars2 deficiency on mitochondria. A–B ATP and ROS levels in the cortex of the cKO mouse embryo compared with control at E 14.5 (mean ± SD; Two-tailed unpaired t-Test, **P < 0.01; ****P < 0.0001; n = 6 for ATP; n = 3 for ROS). C–D TP and ROS levels in control and Fars2 knocked-down neurons at the indicated days of culture (mean ± SD; two-tailed unpaired t-Test, ns: non-significant; **P < 0.01; ****P < 0.0001; n = 4–5 for ATP; n = 27 for ROS). E Relative mitochondrial membrane potential in the cortex of the cKO mouse embryo compared with control at E 14.5 (mean ± SD; two-tailed unpaired t-Test, ***P < 0.001; n = 3). F Relative NADtotal level in the cortex of the cKO mouse embryo compared with control at E 17.5 (mean ± SD; two-tailed unpaired t-Test, ns: non-significant; **P < 0.01; n = 4). G NAD+ and NADH levels of mitochondria pool in control and cKO mice embryo cortex at E 17.5 (mean ± SD; two-tailed unpaired t-Test, ns: non-significant; **P < 0.01; n = 4). H–INADtotal levels and NAD+/NADH ratio in control and Fars2 knocked-down neurons at the indicated days of culture (mean ± SD; two-tailed unpaired t-Test, **P < 0.01; ***P < 0.001; ****P < 0.0001; n = 5–6). J Transmission electron micrograph of mitochondria in cortex from control and cKO mice embryos at the indicated embryonic ages. Scale bars: 0.5 µm for E 14.5 and E 17.5 samples and 5 µm for P 0.5 samples. K Percentage of mitochondria with abnormal cristae in CTRL and cKO cortex at indicated embryonic ages (mean ± SD; two-tailed unpaired t-Test, *P < 0.05; n = 3). L Transmission electron micrograph of mitochondria in control and Fars2 knocked-down neurons at the indicated days of culture. Linearized mitochondrial cristae and the geometrical shapes are indicated by red lines and asterisks. Scale bars: 0.5 µm. M Percentage of mitochondria with abnormal cristae in control and Fars2 knocked-down neurons at the indicated days of culture (mean ± SD; two-tailed unpaired t-Test, **P < 0.01; ***P < 0.001; n = 3)

Fig 4: RNA-sequencing analysis of Fars2 knockdown effect in mouse neurons in vitro. A Pearson correlation value of gene expression between samples based on RNA-seq. Each row and column of the figure both represent samples, the number and the square color below represent the correlation value between pair of samples. The more two samples are similar with each other, the closer the correlation value is to 1. B Venn diagram showing the distribution of unique and common DEGs among comparisons at different culture days. C qRT-PCR-based validation of Dctn3 expression in primary cultured neurons at different time points. (mean ± SD; two-tailed unpaired t-Test, **P < 0.01; ****P < 0.0001; n = 3). D qRT-PCR-based validation of Dctn3 expression in mouse cortex at different embryonic day. (mean ± SD; two-tailed unpaired t-Test, *P < 0.05; **P < 0.01; n = 3-4). E DEGs numbers in shFars2 vs shCtrl group at 1, 3, 5, 7, 10 days post-infection (n = 3 from different littermates for each group). Adjust P < 0.05 and |logFC|= 2 were set as the cut-off criteria. F Volcano plot of the DEGs in shFars2 vs shCtrl group at 3, 5,7,10 days after infection. Red dots represent up-regulated genes, green dots represent down-regulated genes, black dots represent genes with no significantly changes. G GO term enrichment analysis of DEGs between the Fars2-knockdown group versus the control group at 3, 5, 7, 10 days post-infection. The P-value has undergone FDR Correction (FDR = 0.05). Gradation of dot color represents statistical significance and the size represents the number of implicated genes. H GSEA of primary cultured neuron transcriptomes revealed that genes involved in axon ensheathment and NF-?B signaling pathway are respectively upregulated at 7th and 10th day after infection in Fars2 knocked-down group

Fig 5: Distribution and conservation analysis of FARS2 pathogenic mutations. A Distribution of 30 missense mutations, 2 nonsense mutations, 6 inframe or outframe deletions and 1 splice-site mutation on human mtPheRS. B A graphical illustration of mtPheRS sequence conservation (black line) based on the ConSurf conservation score. Mutation sites (dots) are indicated