A Human Disease-causing Point Mutation in Mitochondrial Threonyl-tRNA Synthetase Induces Both Structural and Functional Defects

Abstract

Mitochondria require all translational components, including aminoacyl-tRNA synthetases (aaRSs), to complete organelle protein synthesis. Some aaRS mutations cause mitochondrial disorders, including human mitochondrial threonyl-tRNA synthetase (hmtThrRS) (encoded by TARS2), the P282L mutation of which causes mitochondrial encephalomyopathies. However, its catalytic and structural consequences remain unclear. Herein, we cloned TARS2 and purified the wild-type and P282L mutant hmtThrRS. hmtThrRS misactivates non-cognate Ser and uses post-transfer editing to clear erroneously synthesized products. In vitro and in vivo analyses revealed that the mutation induces a decrease in Thr activation, aminoacylation, and proofreading activities and a change in the protein structure and/or stability, which might cause reduced catalytic efficiency. We also identified a splicing variant of TARS2 mRNA lacking exons 8 and 9, the protein product of which is targeted into mitochondria. In HEK293T cells, the variant does not dimerize and cannot complement the ThrRS knock-out strain in yeast, suggesting that the truncated protein is inactive and might have a non-canonical function, as observed for other aaRS fragments. The present study describes the aminoacylation and editing properties of hmtThrRS, clarifies the molecular consequences of the P282L mutation, and shows that the yeast ThrRS-deletion model is suitable to test pathology-associated point mutations or alternative splicing variants of mammalian aaRS mRNAs.

Keywords: alternative splicing; aminoacyl-tRNA synthetase; enzyme kinetics; mitochondria; mitochondrial disease; threonyl-tRNA synthetase.

FIGURE 1.

Purification of an active hmtThrRS. A, SDS-PAGE analysis of purified hmtThrRS. B, time course curve of aminoacylation of hmttRNA^Thr by hmtThrRS. The control was performed without enzyme addition. Error bars, S.D.

FIGURE 2.

hmttRNA^Thr determinants for aminoacylation and cross-species recognition of tRNA^Thr by hmtThrRS. A, cloverleaf structure of hmttRNA^Thr(UGU) with mutations indicated. B, aminoacylation of hmttRNA^Thr and its mutants, including G35C, U36C, and A73C, by hmtThrRS. The control curve was obtained without tRNA addition. C, aminoacylation of hmttRNA^Thr(UGU), hctRNA^Thr(AGU), SctRNA^Thr(AGU), SctRNA^Thr(CGU), SctRNA^Thr(UGU), and control (without adding tRNA) by hmtThrRS. D, cloverleaf structures of SctRNA^Thr(AGU) and hctRNA^Thr(AGU). E, complementation phenotypes of hmtThrRS mutations with hctRNA^Thr(AGU). Drop test of ScΔthrS strain expressing hmtThrRS-Δ_N19 and hmtThrRS-Δ_N38 with hctRNA^Thr(AGU). Strains harboring empty p425TEF or empty p413GPD were used as negative controls. Error bars, S.D.

FIGURE 3.

Editing and mischarging properties of hmtThrRS and the mutated derivative hmtThrRS-H133A/H137A. A, AMP formation by hmtThrRS and the double mutant in the presence of non-cognate Ser and in the presence or absence of tRNA. [³²P]AMP was quantified after TLC separation on PEI plates. B, deacylation of Ser-[³²P]hmttRNA^Thr by hmtThrRS and the double mutant hmtThrRS-H133A/H137A. The control without enzyme represents the spontaneous hydrolysis of mischarged tRNA. After incubation with ThrRS, the samples were treated by nuclease S1, and Ser-[³²P]AMP was quantified after TLC separation. C, mischarging of hmttRNAThr by [¹⁴C]Ser catalyzed by hmtThrRS and the double mutant. The control curve was obtained without enzyme addition. Error bars, S.D.

FIGURE 4.

Analyses of the structure and stability of hmtThrRS-P282L. A, sequence alignment of different ThrRS with the position of amino acid 282 indicated. Hs, Homo sapiens; Mm, Mus musculus; Sc, Saccharomyces cerevisiae; Ca, Candida albicans; Ec, Escherichia coli. B, putative location of Pro²⁸² in hmtThrRS. The arrow shows the corresponding residue in the EcThrRS crystallographic structure (Protein Data Bank code 1QF6). C, measurements of protein levels of hmtThrRS and hmtThrRS-P282L by Western blotting after overexpression in HEK293T cells. GAPDH was used as the internal control. D, CD analysis of hmtThrRS and hmtThrRS-P282L. The CD spectra of the two proteins are presented as molar residue ellipticity (deg.cm²dmol⁻¹). E and F, measurement of equilibrium dissociation constants for hmtThrRS-P282L (E) and hmtThrRS (F) by filter binding assays. G, comparison of the thermal resistance of hmtThrRS and hmtThrRS-P282L. Aminoacylation activities were measured after increasing incubation times of heating at 50 °C. Activities were represented as a percentage of the initial activity before heating. Error bars, S.D.

FIGURE 5.

Editing properties and in vivo activity of hmtThrRS-P282L. A, complementation phenotypes of hmtThrRS and its mutations with hctRNA^Thr(AGU). Drop test shows ScΔthrS strains expressing hmtThrRS, hmtThrRS-P282L, and hmtThrRS-SV with hctRNA^Thr(AGU). B, complementation phenotypes of ScThrRS and ScThrRS-P310L. C, detection of the amounts of ScThrRS and ScThrRS-P310L by Western blotting. GAPDH was used as the internal control. D, quantification of AMP formation in the presence of non-cognate Ser by hmtThrRS with or without tRNA or by hmtThrRS-P282L with or without tRNA. E, deacylation of Ser-[³²P]hmttRNA^Thr by hmtThrRS and hmtThrRS-P282L and the control without the addition of enzyme. Error bars, S.D.

FIGURE 6.

Detection of a splicing variant of TARS2 in different cell lines. A, cloning of TARS2 cDNA led to two different plasmid constructs. DNA sequencing revealed the whole sequence of native hmtThrRS (lane 1) and a truncated sequence lacking 246 nucleotides of exons 8 and 9, termed hmtThrRS-SV (lane 2). B, schematic representation of the exon composition of hmtThrRS and hmtThrRS-SV. The domain structure of both proteins is also indicated at the top. C, detection of the hmtThrRS-SV in different cell lines. PCR amplification with oligonucleotides pairing with exon 7 (forward) and exon 10 (reverse) shows that the native (upper bands) and splice variant hmtThrRS (lower bands) co-exist in five different human cell lines. D, relative amounts of hmtThrRS-SV mRNA to hmtThrRS mRNA in different cell lines. qPCR experiments were performed with one set of primers spanning exons 8 and 9 (detecting the native hmtThrRS) and the other recognizing the exon 7 to 10 boundary (detecting the hmtThrRS-SV). qPCR values obtained for each cell line were normalized to GAPDH mRNA levels. Relative mRNA levels of hmtThrRS-SV were calculated using ΔΔCt method based on three independent qPCR assays. Error bars, S.D. E, Western blotting detection of the native full-length hmtThrRS and truncated hmtThrRS corresponding to the splice variant mRNA. Full-length hmtThrRS and hmtThrRS-SV were cloned in pCMV-3Tag-3A, and HEK293T cells were transfected.

FIGURE 7.

Mitochondrial import of hmtThrRS-SV and in vivo structural and functional properties. A, both native hmtThrRS and hmtThrRS-SV localized in the mitochondria. HEK293T cells were transfected with the FLAG-tagged constructs of pCMV-3Tag-3A containing the coding sequences for native hmtThrRS or hmtThrRS-SV, respectively. The cells were stained with MitoTracker and immunostained with anti-FLAG. Fluorescent images of MitoTracker (red) and FLAG (green) and the nuclear counterstain DAPI (blue) were captured by a confocal microscope. Scale, 20 μm. B, HEK293T cells were co-transfected with pCMV-3Tag-4A-hmtThrRS (c-Myc-tagged hmtThrRS) in the presence of pCMV-3Tag-3A-hmtThrRS (FLAG-tagged hmtThrRS) or control vector pCMV-3Tag-3A. The same procedures were performed for hmtThrRS-SV. Cell lysates were immunoprecipitated with anti-FLAG antibodies conjugated to agarose beads. Western blotting was used to detect whether c-Myc-tagged proteins were co-immunoprecipitated with FLAG-tagged proteins. C, a shuffle assay showing that the ScThrRS-SV could not complement the ScΔthrS strain. Wild-type ScThrRS and empty vector p425TEF were used as controls.