Human mitochondrial leucyl tRNA synthetase can suppress non cognate pathogenic mt-tRNA mutations

Abstract

Disorders of the mitochondrial genome cause a wide spectrum of disease, these present mainly as neurological and/or muscle related pathologies. Due to the intractability of the human mitochondrial genome there are currently no effective treatments for these disorders. The majority of the pathogenic mutations lie in the genes encoding mitochondrial tRNAs. Consequently, the biochemical deficiency is due to mitochondrial protein synthesis defects, which manifest as aberrant cellular respiration and ATP synthesis. It has previously been reported that overexpression of mitochondrial aminoacyl tRNA synthetases has been effective, in cell lines, at partially suppressing the defects resulting from mutations in their cognate mt-tRNAs. We now show that leucyl tRNA synthetase is able to partially rescue defects caused by mutations in non-cognate mt-tRNAs. Further, a C terminal peptide alone can enter mitochondria and interact with the same spectrum of mt-tRNAs as the entire synthetase, in intact cells. These data support the possibility that a small peptide could correct at least the biochemical defect associated with many mt-tRNA mutations, inferring a novel therapy for these disorders.

Figure 1

In each case aaRS overexpression was induced by 3 days tetracycline (Tet) treatment, indicated by + or − symbols. All data and images represent a minimum of 3 independent experiments.

1. Cell growth under glycolytic or respiratory conditions with or without overexpression of aaRS. Equal numbers of 143B.206 Rho⁺, T1, T1V1 and T1L1 cells were seeded in medium containing either glucose or galactose as the sole sugar source. The extent of growth and viability of each cell line with or without aaRS overexpression was determined at 72 h using the neutral red assay. Statistically significant comparisons for uninduced and induced cells are indicated with * ( P = 0.016), ** ( P = 0.0086) n = 3.

2. Overexpression of aaRS. Mitochondrial proteins were isolated from uninduced and aaRS overexpressors. Mitochondrial lysates from T1V1 (100 μg) and T1L1 (50 μg) were subjected to western blot analysis using antibodies against VARS2L or FLAG to confirm overexpression. Mitochondrial ribosomal protein DAP3 was used to confirm equal loading.

3. Analysis of steady state levels of OXPHOS proteins following aaRS induction. Cell lysates (15 μg) from 143B.206 Rho⁺ and T1V1 and T1L1 cells with or without induction were subjected to western blot analysis. Membranes were probed with antibodies directed against OXPHOS proteins and porin as a loading control.

4. Blue Native-PAGE analysis of respiratory chain complex levels. Mitochondria (25 μg) from uninduced and induced T1V1 and T1L1 cells were solubilised for BN-PAGE. Subsequent western blot analysis used antibodies against complex I (NDUFA9), complex IV (COX4), complex III (CORE2) and as a non-mitochondrially encoded control, complex II (SDHA).

5. In gel enzyme activity assay of respiratory chain complexes. BN-PAGE was performed on solubilised mitochondria (50 μg) from uninduced and induced T1V1 and T1L1 cells. Enzyme activities for complexes I, IV and II were examined.

Figure 2

In each case aaRS overexpression was induced by 3 days tetracycline (Tet) treatment (indicated by + or −), except for XF24 analysis. All data and images represent a minimum of 3 independent experiments. Statistically significant P values are as indicated.

1. Oxygen consumption by XF24 analyzer. Representative traces of oxygen consumption rates performed under basal conditions, following the addition of oligomycin (1 μg/ml), uncoupler FCCP (1.5 μM then 3 μM) and antimycin A (2.5 μM) are presented for 143B.206 Rho⁺, T1, T1V1 and T1L1 cell lines with or without 2 days aaRS overexpression.

2. Respiratory chain activity improves on cognate aaRS or LARS2 overexpression. Mitochondria were isolated from 143B.206 Rho⁺ cells and T1V1 and T1L1 transfectants with and without aaRS induction. Enzyme activities of complexes I, IV and II were measured spectrophotometrically and are presented as ratios against citrate synthase.

3. Steady state levels of mt-tRNA^val increase following VARS2 or LARS2 overexpression. Equal amounts of RNA (4 μg) isolated from 143B.206 Rho⁺ cells and T1V1 and T1L1 transfectants with and without aaRS induction were analysed by high resolution northern blotting. Probes were specific for mitochondrial tRNA^leu(UUR), tRNA^val, and tRNA^phe or 5S RNA as a loading control. Densitometric measurements of mt-tRNA^leu(UUR), tRNA^val, and tRNA^phe in uninduced and induced T1V1 and T1L1 transfectants were normalised to the 5S RNA. Data are expressed as percentages of mt-tRNA in induced cells over mean uninduced cell values and presented below * P < 0.02.

4. Synthesis of mitochondrially encoded proteins increases upon aaRS overexpression. Mitochondrially encoded proteins in 143B.206 Rho⁺, T1V1 and T1L1 cells with and without aaRS overexpression, were pulse labeled with ³⁵S-methionine and separated by 15% SDS–PAGE. Below the autoradiogram is a western blot of the same membrane probed for β-actin as loading control. Increases in protein synthesis post induction are indicated (right panel) by densitometric quantification of individual RC subunits (designated by *), relative to-Tet and normalized to β-actin.

Figure 3

In each case overexpression of the LARS2 FLAG tagged C-terminus was induced by 3 days tetracycline (Tet) treatment. Samples presented in panels A and B were separated on a single 16% Tricine PAG. Post transfer the membrane was cut to allow probing with different antibodies, positions of the molecular weight markers (MWM) are indicated. Samples in C, D and E represent RNA binding determined by crosslinking/immunoprecipitation (CLIP). Derived data is presented as IonTorrent reads aligned against the mtRNA genes, which are depicted as in circular mtDNA. Concentric circles indicate a log scale of number of reads / site.

1. A

   Proteinase K (PK) shaving of mitoplasts confirms loss of OMM and IMS but retention of IMM and matrix fractions. Mitochondria (50μg, lane 1), mitoplast (75μg, lane 2) and shaved mitoplast (50μg, lane 3) fractions were analysed by western with the antibodies indicated.

2. B

   LARS2 C-terminal peptide is localized to the inner mitochondrial compartment. The shaved mitoplasts (˜1mg of preparation presented in panel A; lanes 1 and 4) and TCA precipitated postmitochondrial supernatant (PMS; lanes 3 and 5) were probed for the presence of the LARS2 C-terminus. COXI was used as a marker to confirm presence of inner mitochondrial proteins that were absent from the PMS fraction. The poor resolution is due to the large amount of protein loaded in this well. Left Panel directly visualized by BioRad Chemidoc MP, right panel visualized by autoradiography.

3. C–E

   CLIP derived mt-RNA binding capacity. (C) Physiological RNA binding by the LARS2 C-terminal peptide; (D) Physiological RNA binding by the full length LARS2; (E) As a control, RNA binding by the mitochondrially targeted FLAG tagged luciferase was also determined.

Figure 4

In each case aaRS overexpression was induced by tetracycline (Tet) treatment, indicated by + or − symbols. All data and images represent a minimum of 3 independent experiments. Statistically significant P values are as indicated.

1. Growth rates for cell lines with or without aaRS overexpression. 143B.206 Rho⁺, T1 cells and T1 transfectants for VARS2 (T1V1), LARS2 (T1L1), AARS2 (T1A2) and FARS2 (T1F2) were seeded at 2 × 10⁴ (indicated by dashed line) in medium containing galactose. Cells were counted at 72 h with (+) or without (−) aaRS overexpression. Data are represented as mean ± s.d.

2. Overexpression of neither alanyl-nor phenylalanyl tRNA synthetase changes steady state levels of respiratory protein. Western blots of cell lysates (25 μg) from T1A2 and T1F2 cells with or without 3 days aaRS overexpression were probed with antibodies directed against FLAG tag, OXPHOS proteins and β-actin as a loading control. Lengthy exposures were required to ensure detection of COX2 signals.

3. Overexpression of neither alanyl-nor phenylalanyl tRNA synthetase rescues respiratory defect. Basal respiration rates (mean ± s.d.) were determined for 143B.206 Rho⁺, T1 cells and T1V1, T1L1 and T1F2 transfectants with or without aaRS 2 days overexpression.