✕
Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 May;36(9):3065-74.
doi: 10.1093/nar/gkn147. Epub 2008 Apr 8.

Overexpression of human mitochondrial valyl tRNA synthetase can partially restore levels of cognate mt-tRNAVal carrying the pathogenic C25U mutation

Joanna Rorbach  1 , Abdul Aziz Yusoff, Helen Tuppen, Dyg P Abg-Kamaludin, Zofia M A Chrzanowska-Lightowlers, Robert W Taylor, Douglass M Turnbull, Robert McFarland, Robert N Lightowlers
Affiliations

Overexpression of human mitochondrial valyl tRNA synthetase can partially restore levels of cognate mt-tRNAVal carrying the pathogenic C25U mutation

Joanna Rorbach et al. Nucleic Acids Res. 2008 May.

Abstract

Phenotypic diversity associated with pathogenic mutations of the human mitochondrial genome (mtDNA) has often been explained by unequal segregation of the mutated and wild-type genomes (heteroplasmy). However, this simple hypothesis cannot explain the tissue specificity of disorders caused by homoplasmic mtDNA mutations. We have previously associated a homoplasmic point mutation (1624C>T) in MTTV with a profound metabolic disorder that resulted in the neonatal deaths of numerous siblings. Affected tissues harboured a marked biochemical defect in components of the mitochondrial respiratory chain, presumably due to the extremely low (<1%) steady-state levels of mt-tRNA(Val). In primary myoblasts and transmitochondrial cybrids established from the proband (index case) and offspring, the marked respiratory deficiency was lost and steady-state levels of the mutated mt-tRNA(Val) were greater than in the biopsy material, but were still an order of magnitude lower than in control myoblasts. We present evidence that the generalized decrease in steady-state mt-tRNA(Val) observed in the homoplasmic 1624C>T-cell lines is caused by a rapid degradation of the deacylated form of the abnormal mt-tRNA(Val). By both establishing the identity of the human mitochondrial valyl-tRNA synthetase then inducing its overexpression in transmitochondrial cell lines, we have been able to partially restore steady-state levels of the mutated mt-tRNA(Val), consistent with an increased stability of the charged mt-tRNA. These data indicate that variations in the levels of VARS2L between tissue types and patients could underlie the difference in clinical presentation between individuals homoplasmic for the 1624C>T mutation.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Steady-state levels of mt-tRNAVal are decreased in primary cell lines from the patient and proband but do not show the dramatically low levels evident in tissues. (A) Schematic representation of the human MTTV gene sequence, highlighting the 1624C>T mutation, corresponding to C25U substitution in the tRNA (http://mamit-trna.u-strasbg.fr.) This mutation occurs in the D-stem of the tRNA secondary structure that is predicted to weaken the interaction with G10, a base pairing that has previously been shown to modulate interaction with class I-type synthetases (27,28). (B and C) High-resolution northern blots were performed on RNA isolated from muscle homogenate (B) or myoblasts (C) of the proband (II-1), offspring (III-10) and two unaffected independent controls C1 and C2 from which myoblast cultures were. Differences in specific activity of the various probes are normalized by calculating ratios of signals for the mt-tRNAVal and -tRNAPhe species (B, upper panel) or –tRNALeu(UUR) (B, lower panel; C) in each sample and comparing these ratios between samples on the same blot. Ratios of mt-tRNA levels in homogenate and myoblast sample are compared to C2 (B) or C1 (C) that is set to 100% and are reported under each lane. Representative experiments are shown. RNA was isolated from cell lines on at least five separate occasions over a 2-year period. Variance in ratios did not differ by >2%.
Figure 2.
Figure 2.
Decreased levels of mt-tRNAVal C25U could not be shown to affect mitochondrial protein synthesis. Myoblasts from patient and control were grown in culture prior to in vivo mitochondrial protein labelling as described in Materials and methods section and (29). Following inhibition of cytosolic protein synthesis, radiolabelled methionine/cysteine was added for 2 h and whole-cell lysates (20 μg total protein) separated by SDS–PAGE. A representative PhosphorImage is shown. Attribution of products is determined by reference to 29.
Figure 3.
Figure 3.
Stability of mt-tRNAVal C25U is impaired. To compare the stability of mt-tRNAVal in patients (II-1 and III-10) and control, mitochondrial RNA synthesis was inhibited by the addition of ethidium bromide (250 ng/ml) and the remaining RNA was isolated at the indicated time points. High-resolution blots were then performed with probes specific for mt-tRNAVal and –tRNALeu(UUR). Estimates for t1/2 were calculated by measuring pixel intensity for each mt-tRNA at each time point. For each set of experiments, 5 μg of low molecular weight RNA was separated and equal loading confirmed by reference to the nuclear encoded 5S RNA (data not shown). For ease of comparison, the specific activity of mt-tRNALeu(UUR) probe was decreased to allow both signals to be visualized on the same blot and time course. A representative time course is shown for RNA isolated from the three cell lines.
Figure 4.
Figure 4.
Inhibition of mitochondrial protein synthesis partially restores the levels of mt-tRNAVal. Myoblast cell lines from the patient (III-10) and control were treated with 50 μg/ml of the mitochondrial protein synthesis inhibitor thiamphenicol (+TAP, A), various concentrations of doxycycline (5 or 10 μM, B) or untreated (–TAP, A or 0, B) and RNA were isolated after 2 days. High-resolution northern blots were probed for mt-tRNAVal or –tRNALeu(UUR) and the relative level of –tRNAVal estimated by normalizing the ratio of the two signals for the untreated controls.
Figure 5.
Figure 5.
VARS2L is a mitochondrial protein. (A) HeLa cells were transiently transfected with a construct expressing the N-terminal 232 residues of VARS2L fused to GFP. Following transfection (24 h), cells were stained with the mitochondriotropic dye Mitotracker Red CM-H2XROS. Transfected cells were visualized by fluorescence microscopy. Images were captured and mitochondrial co-localization of the fusion protein was confirmed by superimposition of the green and red signals of a linescan of the image (dashed line visible on image). An image typical of three independent transfections is shown. (B) Transmitochondrial HEK293T Flip-in T-REx cybrids inducibly expressing full-length VARS2L were produced as detailed in Materials and methods section. Mitochondrial protein was isolated from control HEK293T Flip-in T-REx cells (293) and the cybrid transfectants (HEK1624 Clone 17) with (+) or without (−) 48 h of induction as described in the Materials and methods section. Aliquots (100 μg) were subjected to western analysis using anti-VARS2L antibodies. To confirm equal loading of mitochondrial protein, an antibody to mitochondrial ribosome recycling factor (mtRRF) was used.
Figure 6.
Figure 6.
Over-expression of human VARS2L can increase the steady-state level of mutated mt-tRNAVal. (A) HEK293T1624C>T clone 17 transfected cybrids, uninduced (−) or induced (+) were grown for 3 days prior to isolation of RNA. High-resolution northern blots were performed with probes to mt-tRNAVal and –tRNALeu(UUR) as indicated. Natural levels for both tRNA species are shown in the control untransfected HEK293 cell line (293). Signal was quantified by ImageQuant after PhosphorImager exposure and the ratio of mt-tRNAVal to –tRNALeu(UUR) given for each sample. (B) Extended over-expression of VARS2L maintains the increased steady-state level of mutated mt-tRNAVal. A similar experiment to (A) was performed, with RNA isolated after the indicated induction time in days. Ratios of the mt-tRNAs are shown for control untreated (293) and the 143B1624C>T homoplasmic cybrids (143B).
See this image and copyright information in PMC

References

    1. Taylor RW, Turnbull DM. Mitochondrial DNA mutations in human disease. Nat. Rev. Genet. 2005;6:389–402. - PMC - PubMed
    1. Zeviani M, Carelli V. Mitochondrial disorders. Curr. Opin. Neurol. 2007;20:564–571. - PubMed
    1. Wallace DC, Singh G, Lott MT, Hodge JA, Schurr TG, Lezza A.MS, Elsas L.J., II., Nikoskelainen EK. Mitochondrial DNA mutation associated with Leber's hereditary optic neuropathy. Science. 1988;242:1427–1430. - PubMed
    1. Carelli V, Giordano C, d’Amati G. Pathogenic expression of homoplasmic mtDNA mutations needs a complex nuclear-mitochondrial interaction. Trends Genet. 2003;19:257–262. - PubMed
    1. Prezant TR, Agapian JV, Bohlman MC, Bu X, Oztas S, Al E. Mitochondrial ribosomal RNA mutation associated with both antibiotic-induced and non-syndromic deafness. Nat. Genet. 1993;4:289–294. - PubMed

Publication types