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Case Reports
. 2015 Aug 6;97(2):319-28.
doi: 10.1016/j.ajhg.2015.06.011. Epub 2015 Jul 16.

TRMT5 Mutations Cause a Defect in Post-transcriptional Modification of Mitochondrial tRNA Associated with Multiple Respiratory-Chain Deficiencies

Christopher A Powell  1 , Robert Kopajtich  2 , Aaron R D'Souza  1 , Joanna Rorbach  1 , Laura S Kremer  2 , Ralf A Husain  3 , Cristina Dallabona  4 , Claudia Donnini  4 , Charlotte L Alston  5 , Helen Griffin  6 , Angela Pyle  6 , Patrick F Chinnery  6 , Tim M Strom  2 , Thomas Meitinger  7 , Richard J Rodenburg  8 , Gudrun Schottmann  9 , Markus Schuelke  9 , Nadine Romain  10 , Ronald G Haller  10 , Ileana Ferrero  4 , Tobias B Haack  2 , Robert W Taylor  5 , Holger Prokisch  11 , Michal Minczuk  12
Affiliations
Case Reports

TRMT5 Mutations Cause a Defect in Post-transcriptional Modification of Mitochondrial tRNA Associated with Multiple Respiratory-Chain Deficiencies

Christopher A Powell et al. Am J Hum Genet. .

Abstract

Deficiencies in respiratory-chain complexes lead to a variety of clinical phenotypes resulting from inadequate energy production by the mitochondrial oxidative phosphorylation system. Defective expression of mtDNA-encoded genes, caused by mutations in either the mitochondrial or nuclear genome, represents a rapidly growing group of human disorders. By whole-exome sequencing, we identified two unrelated individuals carrying compound heterozygous variants in TRMT5 (tRNA methyltransferase 5). TRMT5 encodes a mitochondrial protein with strong homology to members of the class I-like methyltransferase superfamily. Both affected individuals presented with lactic acidosis and evidence of multiple mitochondrial respiratory-chain-complex deficiencies in skeletal muscle, although the clinical presentation of the two affected subjects was remarkably different; one presented in childhood with failure to thrive and hypertrophic cardiomyopathy, and the other was an adult with a life-long history of exercise intolerance. Mutations in TRMT5 were associated with the hypomodification of a guanosine residue at position 37 (G37) of mitochondrial tRNA; this hypomodification was particularly prominent in skeletal muscle. Deficiency of the G37 modification was also detected in human cells subjected to TRMT5 RNAi. The pathogenicity of the detected variants was further confirmed in a heterologous yeast model and by the rescue of the molecular phenotype after re-expression of wild-type TRMT5 cDNA in cells derived from the affected individuals. Our study highlights the importance of post-transcriptional modification of mitochondrial tRNAs for faithful mitochondrial function.

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Figures

Figure 1
Figure 1
TRMT5 Variants and Gene Structure (A) Pedigrees of the two families identified with recessively inherited TRMT5 variants. “D” indicates anonymous sperm donor. (B) Gene structure of TRMT5 with known protein domains of the gene product and location and conservation of amino-acid residues affected by mutations (in red). Intronic regions are not drawn to scale. Shadowing in the sequence alignment represents the homology of amino-acid residues.
Figure 2
Figure 2
3D Model of Human TRMT5 (A) Ribbon presentation of the Methanocaldococcus jannaschii aTrm5–tRNACys(GCA)–AdoMet complex structure. The fragment of structure typical for the AdoMet-dependent methyltransferases superfamily (SCOP: SSF53335) is colored gray (the remaining portion of aTrm5 is colored cyan). tRNACys is colored light orange, and G37 is presented as a stick model. SAM are depicted as stick models and carbon atoms are yellow. The residues that are key for catalysis are indicated in green. (B) Detailed view of the structural model of the catalytic site of aTrm5. Color coding is the same as in panel (A). (C) Detailed view of the 3D model of the predicted catalytic site of human TRMT5. TRMT5 residues corresponding to the catalytically important amino acids in aTrm5 are indicated in dark green. Seven out of nine catalytically important residues are absolutely conserved in TRMT5 and aTrm5, and the remaining two, His289 (Methanocaldococcus jannaschii Arg186) and Arg454 (Methanocaldococcus jannaschii Lys318), share very similar chemical properties between the two proteins. The residues mutated in individuals 73901 and 65205, Arg291 and Met389, respectively, are indicated in red.
Figure 3
Figure 3
Mitochondrial Localization of TRMT5 (A) Localization of TRMT5 by immunofluorescence in HeLa cells following the transient expression of a C-terminal FLAG-tagged TRMT5 construct (TRMT5-FLAG). Cell nuclei were stained with DAPI (top left). TRMT5-FLAG was detected by an anti-FLAG antibody (top right). We detected mitochondria by targeting a known mitochondrial protein, TOM20, with an anti-TOM20 antibody (bottom left). We digitally merged the preceding images, and signal overlap between TRMT5-FLAG and TOM20 appears yellow (bottom right). (B) Localization of TRMT5 in sub-cellular fractions. HeLa cells were fractionated into debris (D, lane 2), cytosol (C, lane 3), and mitochondria (C, lanes 4–6). The mitochondrial fraction was treated with 25 μg/ml proteinase K in the absence (C, lane 5) or presence (C, lane 6) of 1% Triton X-100. The fractions were analyzed by western blotting with antibodies against TRMT5 (HPA000943, Sigma). The distribution of TRMT5 was compared with that of the following marker proteins: mtSSB1 (mitochondrial matrix), TOM22 (mitochondrial outer membrane), GAPDH (cytosol), and Histone H4 (nucleus). Abbreviations are as follows: T, total cell lysate; fl, full-length TOM22; tr, truncated TOM22.
Figure 4
Figure 4
Hypomodification of G37 in Mitochondrial tRNA in Affected Individuals and upon Downregulation of TRMT5 Expression (A) A radioactively labeled, complementary primer (red) is annealed to mt-tRNALeu(CUN) and subjected to a RT-PEx reaction. The presence of m1G37 results in RT-PEx pausing, producing a shorter product (dark blue). However, in the absence of m1G37, the extension is able to progress until stalling due to the lack of a dNTP (dTTP in the above case, light blue), producing a longer product. (B) Separation and detection of products of RT-PEx reactions preformed on RNA extracted from skin fibroblasts (FB) of healthy controls (C1 and C2) and the affected individuals (65205 and 73901; left panel). The gel shows a representative result of at least three biological replicates (right panel). Quantification values represent percentage of RT-PEx reaction readthrough the G37 site as the average of the dTTP-induced stalling intensity to the total stalling (dTTP-induced stalling + m1G37-induced stalling). Error bars = 1 SEM; n = 5, ∗p < 0.05, ∗∗∗p < 0.001, unpaired two-tailed Student’s t test with C1. (C) RT-PEx experiment performed as in (B) on RNA extracted from skeletal muscle (SKM) of healthy control individuals (C1 and C2) and affected individuals (65205 and 73901). Error bars = 1 SEM; n = 3, ∗∗∗p < 0.001, unpaired two-tailed Student’s t test with C1. (D) Separation and detection of RT-PEx reactions on HeLa cells-derived RNA following a 6-day siRNA-mediated depletion of TRMT5. Two different siRNA were used (HSS126475 and HSS126476, Life Technologies). Untransfected cells and cells transfected with siRNA against GFP (Dsc-1001, Lonza) were used as controls. Quantification values displayed as per (B).
Figure 5
Figure 5
Causal Role for p.Arg291His and p.Met386Val TRMT5 Variants in mt-tRNA Modification and Oxidative Metabolism Deficiency (A) RT-PEx assay performed on RNA derived from individual 73901 and control (C2) skin fibroblasts (FB) with and without the expression of wild-type TRMT5. Quantification values are displayed as per Figure 4B. Error bars = 1 SEM; n = 4, ∗∗p < 0.01, unpaired two-tailed Student’s t test. (B) Complementation assays in yeast. TRM5 was cloned under its natural promoter upon PCR amplification. PCR-based mutagenesis was performed to obtain the trm5R270H and trm5M368V mutant alleles. The trm5Δ1-33 allele was constructed as previously described. Disruption of genomic TRM5 gene was performed in the presence of TRM5 on the pFL38 plasmid, given that the deletion is lethal. The TRM5, trm5R270H, trm5M368V, and trm5Δ1-33 alleles cloned into the pFL39 vector were introduced into this strain, and then pFL38-TRM5 was lost through plasmid shuffling. Oxygen consumption rate was recorded on intact cells grown at 28°C in synthetic complete medium without tryptophan, supplemented with 0.5% glucose. Values were normalized to the rate of oxygen consumption of the TRM5 transformant and represented as the mean of at least three values. Error bars = 1 SD; ∗p < 0.05, paired Student’s t test.
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