Mitochondrial myopathy associated with a novel 5522G>A mutation in the mitochondrial tRNATrp gene

We report a novel pathogenic mutation of the mitochondrial transfer RNA (tRNA) gene for tryptophan in a patient with isolated myopathy and persistently elevated creatine kinase. Muscle studies revealed ragged red fibres and decreased activity of respiratory chain complex I and cytochrome c oxidase (COX). Sequencing of the 22 mitochondrial tRNA genes revealed a mutation m.5522G>A, which alters a conserved base pairing in the D-stem of the tRNA for tryptophan. The mutation was heteroplasmic with a mutational load between 88 and 99% in COX-negative fibres. This case contributes to the genetic heterogeneity of mitochondrial diseases caused by mutations in mitochondrial tRNA genes.     

Mutation in subdomain G' of mitochondrial elongation factor G1 is associated with combined OXPHOS deficiency in fibroblasts but not in muscle

The mitochondrial translation system is responsible for the synthesis of 13 proteins required for oxidative phosphorylation (OXPHOS), the major energy-generating process of our cells. Mitochondrial translation is controlled by various nuclear encoded proteins. In 27 patients with combined OXPHOS deficiencies, in whom complex II (the only complex that is entirely encoded by the nuclear DNA) showed normal activities, and mutations in the mitochondrial genome as well as polymerase gamma were excluded, we screened all mitochondrial translation factors for mutations. Here, we report a mutation in mitochondrial elongation factor G1 (GFM1) in a patient affected by severe, rapidly progressive mitochondrial encephalopathy. This mutation is predicted to result in an Arg250Trp substitution in subdomain G' of the elongation factor G1 protein and is presumed to hamper ribosome-dependent GTP hydrolysis. Strikingly, the decrease in enzyme activities of complex I, III and IV detected in patient fibroblasts was not found in muscle tissue. The OXPHOS system defects and the impairment in mitochondrial translation in fibroblasts were rescued by overexpressing wild-type GFM1, establishing the GFM1 defect as the cause of the fatal mitochondrial disease. Furthermore, this study evinces the importance of a thorough diagnostic biochemical analysis of both muscle tissue and fibroblasts in patients suspected to suffer from a mitochondrial disorder, as enzyme deficiencies can be selectively expressed.     

A novel mutation in the mitochondrial tRNAPro gene associated with late-onset ataxia, retinitis pigmentosa, deafness, leukoencephalopathy and complex I deficiency

We present a patient with ataxia, retinitis pigmentosa, dysarthria, neurosensorial deafness, nystagmus and leukoencephalopathy. A novel heteroplasmic G to A transition at nucleotide 15 975 was found, affecting the T arm of the mitochondrial (mt) tRNA^Pro gene. A biochemical analysis of respiratory chain enzymes in muscle revealed isolated complex I deficiency. This is the fourth pathogenic tRNA^Pro point mutation to be associated with an mt disorder. The result highlights the importance of molecular dissection of mtDNA in patients with defined mt disorder and confirms the clinical and biochemical heterogeneity associated with tRNA^Pro mutations.     

Biallelic variants in WARS2 encoding mitochondrial tryptophanyl‐tRNA synthase in six individuals with mitochondrial encephalopathy

Mitochondrial protein synthesis involves an intricate interplay between mitochondrial DNA encoded RNAs and nuclear DNA encoded proteins, such as ribosomal proteins and aminoacyl‐tRNA synthases. Eukaryotic cells contain 17 mitochondria‐specific aminoacyl‐tRNA synthases. WARS2 encodes mitochondrial tryptophanyl‐tRNA synthase (mtTrpRS), a homodimeric class Ic enzyme (mitochondrial tryptophan‐tRNA ligase; EC 6.1.1.2). Here, we report six individuals from five families presenting with either severe neonatal onset lactic acidosis, encephalomyopathy and early death or a later onset, more attenuated course of disease with predominating intellectual disability. Respiratory chain enzymes were usually normal in muscle and fibroblasts, while a severe combined respiratory chain deficiency was found in the liver of a severely affected individual. Exome sequencing revealed rare biallelic variants in WARS2 in all affected individuals. An increase of uncharged mitochondrial tRNA^Trp and a decrease of mtTrpRS protein content were found in fibroblasts of affected individuals. We hereby define the clinical, neuroradiological, and metabolic phenotype of WARS2 defects. This confidently implicates that mutations in WARS2 cause mitochondrial disease with a broad spectrum of clinical presentation.     

Expression pattern of mitochondrial respiratory chain enzymes in skeletal muscle of patients with mitochondrial myopathy associated with the homoplasmic m.14674T>C variant

Two homoplasmic variants in tRNA^Glu (m.14674T>C/G) are associated with reversible infantile respiratory chain deficiency. This study sought to further characterize the expression of the individual mitochondrial respiratory chain complexes and to describe the natural history of the disease. Seven patients from four families with mitochondrial myopathy associated with the homoplasmic m.14674T>C variant were investigated. All patients underwent skeletal muscle biopsy and mtDNA sequencing. Whole‐genome sequencing was performed in one family. Western blot and immunohistochemical analyses were used to characterize the expression of the individual respiratory chain complexes. Patients presented with hypotonia and feeding difficulties within the first weeks or months of life, except for one patient who first showed symptoms at 4 years of age. Histopathological findings in muscle included lipid accumulation, numerous COX‐deficient fibers, and mitochondrial proliferation. Ultrastructural abnormalities included enlarged mitochondria with concentric cristae and dense mitochondrial matrix. The m.14674T>C variant in MT‐TE was identified in all patients. Immunohistochemistry and immunoblotting demonstrated pronounced deficiency of the complex I subunit NDUFB8. The expression of MTCO1, a complex IV subunit, was also decreased, but not to the same extent as NDUFB8. Longitudinal follow‐up data demonstrated that not all features of the disorder are entirely transient, that the disease may be progressive, and that signs and symptoms of myopathy may develop during childhood. This study sheds new light on the involvement of complex I in reversible infantile respiratory chain deficiency, it shows that the disorder may be progressive, and that myopathy can develop without an infantile episode.     

Yeast mitochondrial Gln-tRNAGln is generated by a GatFAB-mediated transamidation pathway involving Arc1p-controlled subcellular sorting of cytosolic GluRS

It is impossible to predict which pathway, direct glutaminylation of tRNA^Gln or tRNA-dependent transamidation of glutamyl-tRNA^Gln, generates mitochondrial glutaminyl-tRNA^Gln for protein synthesis in a given species. The report that yeast mitochondria import both cytosolic glutaminyl-tRNA synthetase and tRNA^Gln has challenged the widespread use of the transamidation pathway in organelles. Here we demonstrate that yeast mitochondrial glutaminyl-tRNA^Gln is in fact generated by a transamidation pathway involving a novel type of trimeric tRNA-dependent amidotransferase (AdT). More surprising is the fact that cytosolic glutamyl-tRNA synthetase (_cERS) is imported into mitochondria, where it constitutes the mitochondrial nondiscriminating ERS that generates the mitochondrial mischarged glutamyl-tRNA^Gln substrate for the AdT. We show that dual localization of _cERS is controlled by binding to Arc1p, a tRNA nuclear export cofactor that behaves as a cytosolic anchoring platform for _cERS. Expression of Arc1p is down-regulated when yeast cells are switched from fermentation to respiratory metabolism, thus allowing increased import of _cERS to satisfy a higher demand of mitochondrial glutaminyl-tRNA^Gln for mitochondrial protein synthesis. This novel strategy that enables a single protein to be localized in both the cytosol and mitochondria provides a new paradigm for regulation of the dynamic subcellular distribution of proteins between membrane-separated compartments.     

Analysis of a DNA segment from rat liver mitochondria containing the genes for the cytochrome oxidase subunits I,II and III,ATPase subunit 6, and several tRNA genes

Summary The sequence of a 6.24 kb DNA segment of the mitochondrial genome from rat liver has been determined. It comprises several genes coding for mitochondrial protein subunits and five tRNA genes in the following order: cytochrome oxidase subunit I — tRNA _(UCN) ^Ser —tRNA^Asp — cytochrome oxidase subunit II — tRNALys —ATPase subunit — cytochrome oxidase subunit III —tRNA^Gly — potential open reading frame — tRNA^Arg —two potential open reading frames. The tRNA genes were detected by a computer search programme. The assignments for the protein coding sequences were made through comparison with known sequences, mainly from the yeast mitochondrial proteins (e.g. Bonitz et al. 1980). Our data are discussed with regard to the features of gene arrangement, codon usage, and tRNA structure in mammalian mitochondria (Anderson et al. 1981).Abbreviations COX I, COX II, COX III mitochondrial cytochrome oxidase subunits I, II, and III - ATPase mitochondrial ATPase subunit 6 - U.R.F. unidentified reading frame (Anderson et al. 1981). Other abbreviations follow IUB-IUPAC conventions.     

Homology in the region containing a tRNATrp gene and a (complete or partial) tRNAPro gene in wheat mitochondrial and chloroplast genomes

Summary We have used bean mitochondrial (mt) and chloroplast (cp) tRNA^Trp as probes to locate the corresponding genes on the mt and cp genomes of wheat and we have determined the nucleotide sequences of the wheat mt and cp tRNATrp genes and of the flanking regions. Sequence comparisons show that the wheat mt and cp tRNA^Trp genes are 97% homologous.On the wheat cp DNA, a tRNA _Pro ^UGG gene was found 139 by upstream of the cp tRNA^Trp gene. On the wheat mt DNA, a sequence of 23 nucleotides completely homologous with the 3' end of this cp tRNA^Pro gene was found 136 by upstream of the rut tRNA^Trp gene, but there is only 38% homology between cp and mt wheat genomes in the intergenic regions. The overall organization of this region in the chloroplast genome (a tRNA^Trp gene separated by about 140 by from a tRNA^Pro gene) is also found in the mitochondrial genome, suggesting that this mitochondrial fragment might have originated from a chloroplast DNA insertion. A comparison of the genes and of the intergenic regions located between the tRNA^Trp gene and the tRNA^Pro (or partial tRNA^Pro) gene shows that there is an almost complete conservation of these sequences in the mitochondrial DNA of wheat and maize, whereas wheat mt and cp intergenic regions show more sequence divergence.Wheat mt tRNA^Trp gene is encoded by the main mt genome (accounted for by the master chromosome) but, in the case of maize mitochondria, this gene was found to be encoded by the 2.3 kb linear plasmid, indicating that this plasmid is not dispensable in maize mitochondria.     

A novel mutation in the mitochondrial tRNA(Ser(AGY)) gene associated with mitochondrial myopathy, encephalopathy, and complex I deficiency

Purpose

To identify molecular defects in a girl with clinical features of MELAS (mitochondrial encephalomyopathy and lactic acidosis) and MERRF (ragged‐red fibres) syndromes.

Methods

The enzyme complex activities of the mitochondrial respiratory chain were assayed. Temporal temperature gradient gel electrophoresis was used to scan the entire mitochondrial genome for unknown mitochondrial DNA (mtDNA) alterations, which were then identified by direct DNA sequencing.

Results

A novel heteroplasmic mtDNA mutation, G12207A, in the tRNA^Ser(AGY) gene was identified in the patient who had a history of developmental delay, feeding difficulty, lesions within her basal ganglia, cerebral atrophy, proximal muscle weakness, increased blood lactate, liver dysfunction, and fatty infiltration of her muscle. Muscle biopsy revealed ragged red fibres and pleomorphic mitochondria. Study of skeletal muscle mitochondria revealed complex I deficiency associated with mitochondrial proliferation. Real time quantitative PCR analysis showed elevated mtDNA content, 2.5 times higher than normal. The tRNA^Ser(AGY) mutation was found in heteroplasmic state (92%) in the patient''s skeletal muscle. It was not present in her unaffected mother''s blood or in 200 healthy controls. This mutation occurs at the first nucleotide of the 5′ end of tRNA, which is involved in the formation of the stem region of the amino acid acceptor arm. Mutation at this position may affect processing of the precursor RNA, the stability and amino acid charging efficiency of the tRNA, and overall efficiency of protein translation.

Conclusion

This case underscores the importance of comprehensive mutational analysis of the entire mitochondrial genome when a mtDNA defect is strongly suggested.     

Presence of a chloroplast-like elongator tRNAMet gene in the mitochondrial genomes of soybean and Arabidopsis thaliana

Summary The nucleotide sequence of elongator tRNA^Met genes from soybean chloroplast and mitochondria and Arabidopsis thaliana mitochondria have been determined. The mitochondrial tRNA^Met genes from soybean and A. thaliana are identical, and they differ from the soybean chloroplast tRNA^Met gene by only four nucleotides. Analysis of the flanking regions indicates that the mitochondrial tRNA^Met gene is not present on a large chloroplast DNA insertion in the mitochondrial genome, but it suggests that they have a common origin. Comparison of the three genes and the evolutionary implications are discussed.     

Two Siblings with Homozygous Pathogenic Splice‐Site Variant in Mitochondrial Asparaginyl–tRNA Synthetase (NARS2)

A homozygous missense mutation (c.822G>C) was found in the gene encoding the mitochondrial asparaginyl–tRNA synthetase (NARS2) in two siblings born to consanguineous parents. These siblings presented with different phenotypes: one had mild intellectual disability and epilepsy in childhood, whereas the other had severe myopathy. Biochemical analysis of the oxidative phosphorylation (OXPHOS) complexes in both siblings revealed a combined complex I and IV deficiency in skeletal muscle. In‐gel activity staining after blue native‐polyacrylamide gel electrophoresis confirmed the decreased activity of complex I and IV, and, in addition, showed the presence of complex V subcomplexes. Considering the consanguineous descent, homozygosity mapping and whole‐exome sequencing were combined revealing the presence of one single missense mutation in the shared homozygous region. The c.822G>C variant affects the 3′ splice site of exon 7, leading to skipping of the whole exon 7 and a part of exon 8 in the NARS2 mRNA. In EBV‐transformed lymphoblasts, a specific decrease in the amount of charged mt‐tRNA^Asn was demonstrated as compared with controls. This confirmed the pathogenic nature of the variant. To conclude, the reported variant in NARS2 results in a combined OXPHOS complex deficiency involving complex I and IV, making NARS2 a new member of disease‐associated aaRS2.     

Differences in assembly or stability of complex I and other mitochondrial OXPHOS complexes in inherited complex I deficiency

NADH-ubiquinone oxidoreductase (complex I) deficiency is amongst the most encountered defects of the mitochondrial oxidative phosphorylation (OXPHOS) system and is associated with a wide variety of clinical signs and symptoms. Mutations in complex I nuclear structural genes are the most common cause of isolated complex I enzyme deficiencies. The cell biological consequences of such mutations are poorly understood. In this paper we have used blue native electrophoresis in order to study how different nuclear mutations affect the integrity of mitochondrial OXPHOS complexes in fibroblasts from 15 complex I-deficient patients. Our results show an important decrease in the levels of intact complex I in patients harboring mutations in nuclear-encoded complex I subunits, indicating that complex I assembly and/or stability is compromised. Different patterns of low molecular weight subcomplexes are present in these patients, suggesting that the formation of the peripheral arm is affected at an early assembly stage. Mutations in complex I genes can also affect the stability of other mitochondrial complexes, with a specific decrease of fully-assembled complex III in patients with mutations in NDUFS2 and NDUFS4. We have extended this analysis to patients with an isolated complex I deficiency in which no mutations in structural subunits have been found. In this group, we can discriminate between complex I assembly and catalytic defects attending to the fact whether there is a correlation between assembly/activity levels or not. This will help us to point more selectively to candidate genes for pathogenic mutations that could lead to an isolated complex I defect.     

Different mitochondrial gene orders among insects: exchanged tRNA gene positions in the COII/COIII region between an orthopteran and a dipteran species

Summary We have cloned and sequenced a 2.65 kb segment of the mtDNA molecule of the orthopteran insect Locusta migratoria. It harbors the genes for four mitochondrial tRNAs, for cytochrome c oxidase subunits II and III and for ATPase subunits 6 and 8. The order of the locust genes resembles that of Drosophila yakuba: in both insects the genes for C0II and ATPase 8 are separated from each other by the genes encoding tRNA^lys and tRNA^asp, but in the locust, the positions of the two tRNA genes are reversed. This leads to a different mitochondrial gene order in the two insects.     

Two dimensional polyacrylamide gel electrophoresis analysis of Tetrahymena mitochondrial tRNA

Summary Two dimensional (2D) urea-polyacrylamide gel electrophoresis of tRNA isolated from Tetrahymena mitochondria separated at least 36 spots, while more than 45 major and minor spots were resolved with cytosolic tRNA. Co-electrophoresis of mitochondrial and cytosolic tRNAs revealed that many spots co-migrate. When radioactive mitochondrial tRNA was hybridized to mtDNA under various conditions and tRNA melted from the hybrid was analyzed by 2D gel electrophoresis, only 10 tRNA spots were found. Identified as mtDNA-encoded were 2 spots for tRNA^leu, 2 for tRNA^met, and 1 each for tRNA^phe, tRNAtrp and tRNA^tyr. The remaining three were unidentified. Mitochondrial tRNA spots that correspond to the tRNAs for arg, gly, ile, lys, ser, and val do not hybridize with mtDNA, and in gel positions they correspond to the cytoplasmic tRNA spots for the same respective amino acids. These mitochondrial tRNAs isolated from the gel can be acylated either by the mitochondrial or cytostolic enzymes. Mitochondrial tRNA isolated from a Tetrahymena cell homogenate which was pretreated with RNase A and Micrococcus nuclease exhibited the same 2D gel pattern as a nontreated control. Mitochondrial tRNAs from old and young cells showed generally similar tRNA spots in 2D gels, though more variable spots were seen with old cells. ³H-labeled whole-cell tRNA added to the cell homogenate prior to the mitochondrial isolation procedure did not remain associated with the final mitochondrial tRNA preparation. The present studies also showed mitochondrial tRNAs bound to the mitochondrial 80S monosome and polysome fractions. Radioactive tRNA added to the mitochondrial lysate does not adhere to the ribosomes, suggesting that the ribosome-bound tRNAs are not contaminating cytoplasmic tRNAs. These results are generally in good agreement with our previous data showing that only a small number of tRNAs are coded for by the mitochondrial DNA, while the others are a selected set of imported cytoplasmic tRNAs.     

Sequences in the U3 region of human immunodeficiency virus 1 improve efficiency of minus strand transfer in infected cells

A tRNA gene-like sequence has been identified near the 3′ end of HIV-1. Two segments of this sequence (motif 9 and segment 1) promoted minus strand transfer in vitro. The segments are complementary to the tRNA₃^Lys primer, and apparently act by binding the tRNA, thereby bringing the 3′ and 5′ ends of viral RNA into proximity for strand transfer. In this report, we used full-length HIV-1 to demonstrate biological relevance of these segments. We constructed HIV-1 genomes capable of single cycle infection and altered in one or both of two segments. We devised a real time PCR method for quantifying the amount of (−)ssDNA that completes transfer. Results showed that depending on the mutation the efficiency of transfer decreased from 9% to 26%. Alteration of segment 1 had the greatest effect. Alteration of motif 9 or both sequences also caused a reduction, but smaller than alteration of segment 1 alone.     

Different states of avian myeloblastosis virus DNA polymerase and their binding capacity to primer rRNATrp.

Three different DNA polymerase species can be identified by phosphocellulose chromatography following treatment of the avian myeloblastosis virus αβ DNA polymerase with 1,4-dioxane: (1) α DNA polymerase, (2) residual αβ DNA polymerase, and (3) a species enriched for β DNA polymerase. Binding studies with the major species of primer RNA (tRNA^TrP) involved in the initiation of RNA-directed DNA synthesis in vitro, were carried out with these three DNA polymerase species. Both αβ and the β DNA polymerase enriched species bound tightly to [³²P]tRNA^Trp as demonstrated by the ability of both enzymes to exclude tRNA^Trp on Sephadex G-75 columns and by increasing the sedimentation rate of tRNA^Trp in glycerol gradients. Neither enzyme required divalent metal ion for binding to tRNA^Trp at 0°. The binding capacity of these two enzymes to tRNA^Trp was significantly reduced in the presence of MgCl₂. This reduction in binding capacity was most likely due to conformational changes of tRNA^Trp resulting from selective binding with Mg²⁺. α DNA polymerase, even when present in a 1000-fold molar excess with respect to tRNA^Trp, did not bind to this primer RNA irrespective of whether or not divalent metal ion was present. These results suggest that the β subunit in αβ DNA polymerase is required for effective binding of the holoenzyme to tRNA^Trp.