Fig 1: In vitro studies of the human mt-AlaRS p.Arg580Trp mutant. (A) ATP-PPi exchange determination of human mt-AlaRS (•) and the p.Arg580Trp mutant (▪). A reaction at the absence of Ala was included for a control (▴). (B) Aminoacylation activity of human mt-AlaRS (•) and the p.Arg580Trp mutant (▪). (C) A representative TLC showing the mischarging of mt-tRNAAla by human mt-AlaRS and the p.Arg580Trp mutant. Nuclease S1-generated Ser-[32P]AMP (reflecting Ser-[32P]tRNAAla) and [32P]AMP (reflecting free [32P]tRNAAla) were separated by TLC. (D) Graph of the mischarging activity of human mt-AlaRS (•) and the p.Arg580Trp mutant (▪). In all graphs, the data represent the mean values with error bars indicating standard deviation (SD). (E) Steady-state protein level of overexpressed human mt-AlaRS and the p.Arg580Trp mutant in HEK293T cells. Genes encoding C-terminal FLAG-tagged human mt-AlaRS and p.Arg580Trp mutant were overexpressed in HEK293T cells and the proteins were detected by FLAG antibodies. GAPDH was detected as a loading control.
Fig 2: Genetic analysis of identified AARS2 variants. (A) Family pedigrees showing Sanger sequencing confirmation of the c.1008dupT (p.Asp337*) and c.1738C>T (p.Arg580Trp) AARS2 variants for Patient 1 and segregation of the c.1738C>T (p.Arg580Trp) variant for Patient 2. (B) Long-range PCR confirmation of a maternally inherited, heterozygous intragenic 4.1 kb deletion on the short arm of chromosome 6p21.1 encompassing exons 5–7 of AARS2. A non-specific product at ~2 kb does not affect segregation analysis. The wild-type allele (5 kb) and the deleted allele (1 kb) are denoted by a solid red arrow.
Fig 3: Western blot, northern blot and aminoacylation analysis in AARS2 patient fibroblast lysates. (A) Steady-state mt-AlaRS and OXPHOS subunit protein levels in Patient 1 (P1) and two control fibroblast lysates. Also shown are two reported AARS2 patients in the literature who were homozygous or compound heterozygous for the p.Arg592Trp founder mutation; Patient x (homozygous p.Arg592Trp) corresponds to Patient 11, and Patient y (p.Arg592Trp and c.2882C>T (p.Ala961Val)) corresponds to Patient 7 in (24). Antibodies against mt-AlaRS, NDUFB8 (CI), SDHA (CII), UQCRC2 (CIII), MT-COI (CIV) and ATP5B (CV) were used, with β-actin as a loading control. (B) Graph of relative mt-AlaRS protein levels (n = 3) in controls, Patient 1 (P1), Patient x (x) and Patient y (y) fibroblasts. All data were normalized to SDHA and represented as mean ± standard error of the mean. Significant difference between controls and patient fibroblasts is indicated by asterisks above the columns (*, P-value < 0.05; **, P-value < 0.01 by two-tailed paired students t-test). (C) Northern blot analysis of mt-tRNAAla levels in patient fibroblasts. (D) Aminoacylation assay showing aminoacylated (‘charged’) and deacylated (‘uncharged’) mt-tRNAAla in patient fibroblasts. Mitochondrial tRNAArg was used as a loading control. Lower bands in the dAc lanes denote fully deacylated control tRNA species. Patient z was homozygous for the p.Arg592Trp founder mutation and has been previously reported, corresponding to Patient 1 in (19).
Fig 4: In silico modelling of the human mt-AlaRS Arg580 residue and p.Arg580Trp variant. (A) Multiple sequence alignment of the mt-AlaRS Arg580 residue across species. (B) In silico modelling of the p.Arg580Trp variant on mt-AlaRS protein stability and intramolecular bonds. Editing site is highlighted with transparent green sphere. It contains zinc-binding residues His632, His636, His753 and Cys749.
Fig 5: Western blot and northern blot analysis in AARS2 patient skeletal and cardiac muscle homogenates. (A) Steady-state mt-AlaRS and OXPHOS subunit protein levels in Patient 2 skeletal and cardiac muscle homogenates. Antibodies against mt-AlaRS, NDUFB8 (CI), SDHA (CII), UQCRC2 (CIII), MT-COI (CIV) and ATP5B (CV) were used, with SDHA as a loading control. (B) Graphs of relative mt-AlaRS protein levels in control and Patient 2 (P2) skeletal (n = 3) and cardiac muscle (n = 2) homogenate. All data were normalized to SDHA (skeletal muscle) or VDAC (cardiac muscle) and represented as mean ± standard error of the mean. Significant difference between controls and patient homogenates is indicated by asterisks above the columns (*, P-value < 0.05; **, P-value < 0.01 by two-tailed paired students t-test). (C) Northern blot analysis of mt-tRNAAla levels in Patient 2 and Patient z who was homozygous for the p.Arg592Trp founder mutation, corresponding to Patient 1 in (19). Mitochondrial tRNASer(AGY) was used as a loading control.
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