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1.  Human mitochondrial leucyl tRNA synthetase can suppress non cognate pathogenic mt-tRNA mutations 
EMBO Molecular Medicine  2014;6(2):183-193.
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.
doi:10.1002/emmm.201303202
PMCID: PMC3927954  PMID: 24413189
aminoacyl tRNA synthetase; disease; mitochondria; protein synthesis; therapy
2.  Interactions between peptidyl tRNA hydrolase homologs and the ribosomal release factor Mrf1 in S. pombe mitochondria 
Mitochondrion  2013;13(6):871-880.
Mitochondrial translation synthesizes key subunits of the respiratory complexes. In Schizosaccharomyces pombe, strains lacking Mrf1, the mitochondrial stop codon recognition factor, are viable, suggesting that other factors can play a role in translation termination. S. pombe contains four predicted peptidyl tRNA hydrolases, two of which (Pth3 and Pth4), have a GGQ motif that is conserved in class I release factors. We show that high dosage of Pth4 can compensate for the absence of Mrf1 and loss of Pth4 exacerbates the lack of Mrf1. Also Pth4 is a component of the mitochondrial ribosome, suggesting that it could help recycling stalled ribosomes.
Highlights
•In S. pombe the peptidyl tRNA hydrolases Pth3 and Pth4 are mitochondrial proteins.•Pth3 and Pth4 are associated with the mitochondrial ribosome and the large subunit.•Deletion of pth4 and mrf1, encoding the mitochondrial release factor, is co-lethal.•Over-expression of pth4 compensates for the deletion of mrf1.•Pth4 can act as a release factor in S. pombe mitochondria.
doi:10.1016/j.mito.2013.07.115
PMCID: PMC3919214  PMID: 23892058
Fission yeast; Mitochondria; Translation; Peptidyl tRNA hydrolase; Release factor; Mitochondrial ribosome tagging
3.  The role of the mitochondrial ribosome in human disease: searching for mutations in 12S mitochondrial rRNA with high disruptive potential 
Human Molecular Genetics  2013;23(4):949-967.
Mutations of mitochondrial DNA are linked to many human diseases. Despite the identification of a large number of variants in the mitochondrially encoded rRNA (mt-rRNA) genes, the evidence supporting their pathogenicity is, at best, circumstantial. Establishing the pathogenicity of these variations is of major diagnostic importance. Here, we aim to estimate the disruptive effect of mt-rRNA variations on the function of the mitochondrial ribosome. In the absence of direct biochemical methods to study the effect of mt-rRNA variations, we relied on the universal conservation of the rRNA fold to infer their disruptive potential. Our method, named heterologous inferential analysis or HIA, combines conservational information with functional and structural data obtained from heterologous ribosomal sources. Thus, HIA's predictive power is superior to the traditional reliance on simple conservation indexes. By using HIA, we have been able to evaluate the disruptive potential for a subset of uncharacterized 12S mt-rRNA variations. Our analysis revealed the existence of variations in the rRNA component of the human mitoribosome with different degrees of disruptive power. In cases where sufficient information regarding the genetic and pathological manifestation of the mitochondrial phenotype is available, HIA data can be used to predict the pathogenicity of mt-rRNA mutations. In other cases, HIA analysis will allow the prioritization of variants for additional investigation. Eventually, HIA-inspired analysis of potentially pathogenic mt-rRNA variations, in the context of a scoring system specifically designed for these variants, could lead to a powerful diagnostic tool.
doi:10.1093/hmg/ddt490
PMCID: PMC3900107  PMID: 24092330
4.  REXO2 Is an Oligoribonuclease Active in Human Mitochondria 
PLoS ONE  2013;8(5):e64670.
The Escherichia coli oligoribonuclease, ORN, has a 3′ to 5′ exonuclease activity specific for small oligomers that is essential for cell viability. The human homologue, REXO2, has hitherto been incompletely characterized, with only its in vitro ability to degrade small single-stranded RNA and DNA fragments documented. Here we show that the human enzyme has clear dual cellular localization being present both in cytosolic and mitochondrial fractions. Interestingly, the mitochondrial form localizes to both the intermembrane space and the matrix. Depletion of REXO2 by RNA interference causes a strong morphological phenotype in human cells, which show a disorganized network of punctate and granular mitochondria. Lack of REXO2 protein also causes a substantial decrease of mitochondrial nucleic acid content and impaired de novo mitochondrial protein synthesis. Our data constitute the first in vivo evidence for an oligoribonuclease activity in human mitochondria.
doi:10.1371/journal.pone.0064670
PMCID: PMC3669425  PMID: 23741365
5.  GRSF1 Regulates RNA Processing in Mitochondrial RNA Granules 
Cell Metabolism  2013;17(3):399-410.
Summary
Various specialized domains have been described in the cytosol and the nucleus; however, little is known about compartmentalization within the mitochondrial matrix. GRSF1 (G-rich sequence factor 1) is an RNA binding protein that was previously reported to localize in the cytosol. We found that an isoform of GRSF1 accumulates in discrete foci in the mitochondrial matrix. These foci are composed of nascent mitochondrial RNA and also contain RNase P, an enzyme that participates in mitochondrial RNA processing. GRSF1 was found to interact with RNase P and to be required for processing of both classical and tRNA-less RNA precursors. In its absence, cleavage of primary RNA transcripts is abnormal, leading to decreased expression of mitochondrially encoded proteins and mitochondrial dysfunction. Our findings suggest that the foci containing GRSF1 and RNase P correspond to sites where primary RNA transcripts converge to be processed. We have termed these large ribonucleoprotein structures “mitochondrial RNA granules.”
Graphical Abstract
Highlights
► GRSF1 resides in the mitochondrial matrix and is required for mitochondrial function ► GRSF1 is required for the processing of tRNA-containing and tRNA-lacking precursors ► GRSF1, RNase P, and nascent RNA are part of “mitochondrial RNA granules” ► Mitochondrial RNA granules are functionally linked to RNA processing
doi:10.1016/j.cmet.2013.02.005
PMCID: PMC3593211  PMID: 23473034
6.  Structure based hypothesis of a mitochondrial ribosome rescue mechanism 
Biology Direct  2012;7:14.
Background
mtRF1 is a vertebrate mitochondrial protein with an unknown function that arose from a duplication of the mitochondrial release factor mtRF1a. To elucidate the function of mtRF1, we determined the positions that are conserved among mtRF1 sequences but that are different in their mtRF1a paralogs. We subsequently modeled the 3D structure of mtRF1a and mtRF1 bound to the ribosome, highlighting the structural implications of these differences to derive a hypothesis for the function of mtRF1.
Results
Our model predicts, in agreement with the experimental data, that the 3D structure of mtRF1a allows it to recognize the stop codons UAA and UAG in the A-site of the ribosome. In contrast, we show that mtRF1 likely can only bind the ribosome when the A-site is devoid of mRNA. Furthermore, while mtRF1a will adopt its catalytic conformation, in which it functions as a peptidyl-tRNA hydrolase in the ribosome, only upon binding of a stop codon in the A-site, mtRF1 appears specifically adapted to assume this extended, peptidyl-tRNA hydrolyzing conformation in the absence of mRNA in the A-site.
Conclusions
We predict that mtRF1 specifically recognizes ribosomes with an empty A-site and is able to function as a peptidyl-tRNA hydrolase in those situations. Stalled ribosomes with empty A-sites that still contain a tRNA bound to a peptide chain can result from the translation of truncated, stop-codon less mRNAs. We hypothesize that mtRF1 recycles such stalled ribosomes, performing a function that is analogous to that of tmRNA in bacteria.
Reviewers
This article was reviewed by Dr. Eugene Koonin, Prof. Knud H. Nierhaus (nominated by Dr. Sarah Teichmann) and Dr. Shamil Sunyaev.
doi:10.1186/1745-6150-7-14
PMCID: PMC3418547  PMID: 22569235
Class I release factor; mtRF1; mtRF1a; Mitochondrial genetic code; Translation termination; Stalled ribosome
7.  Nuclear factors involved in mitochondrial translation cause a subgroup of combined respiratory chain deficiency 
Brain  2010;134(1):183-195.
Mutations in several mitochondrial DNA and nuclear genes involved in mitochondrial protein synthesis have recently been reported in combined respiratory chain deficiency, indicating a generalized defect in mitochondrial translation. However, the number of patients with pathogenic mutations is small, implying that nuclear defects of mitochondrial translation are either underdiagnosed or intrauterine lethal. No comprehensive studies have been reported on large cohorts of patients with combined respiratory chain deficiency addressing the role of nuclear genes affecting mitochondrial protein synthesis to date. We investigated a cohort of 52 patients with combined respiratory chain deficiency without causative mitochondrial DNA mutations, rearrangements or depletion, to determine whether a defect in mitochondrial translation defines the pathomechanism of their clinical disease. We followed a combined approach of sequencing known nuclear genes involved in mitochondrial protein synthesis (EFG1, EFTu, EFTs, MRPS16, TRMU), as well as performing in vitro functional studies in 22 patient cell lines. The majority of our patients were children (<15 years), with an early onset of symptoms <1 year of age (65%). The most frequent clinical presentation was mitochondrial encephalomyopathy (63%); however, a number of patients showed cardiomyopathy (33%), isolated myopathy (15%) or hepatopathy (13%). Genomic sequencing revealed compound heterozygous mutations in the mitochondrial transfer ribonucleic acid modifying factor (TRMU) in a single patient only, presenting with early onset, reversible liver disease. No pathogenic mutation was detected in any of the remaining 51 patients in the other genes analysed. In vivo labelling of mitochondrial polypeptides in 22 patient cell lines showed overall (three patients) or selective (four patients) defects of mitochondrial translation. Immunoblotting for mitochondrial proteins revealed decreased steady state levels of proteins in some patients, but normal or increased levels in others, indicating a possible compensatory mechanism. In summary, candidate gene sequencing in this group of patients has a very low detection rate (1/52), although in vivo labelling of mitochondrial translation in 22 patient cell lines indicate that a nuclear defect affecting mitochondrial protein synthesis is responsible for about one-third of combined respiratory chain deficiencies (7/22). In the remaining patients, the impaired respiratory chain activity is most likely the consequence of several different events downstream of mitochondrial translation. Clinical classification of patients with biochemical analysis, genetic testing and, more importantly, in vivo labelling and immunoblotting of mitochondrial proteins show incoherent results, but a systematic review of these data in more patients may reveal underlying mechanisms, and facilitate the identification of novel factors involved in combined respiratory chain deficiency.
doi:10.1093/brain/awq320
PMCID: PMC3707321  PMID: 21169334
mitochondrial translation; combined respiratory chain deficiency; early-onset encephalomyopathy
8.  The p.M292T NDUFS2 mutation causes complex I-deficient Leigh syndrome in multiple families 
Brain  2010;133(10):2952-2963.
Isolated complex I deficiency is the most frequently observed oxidative phosphorylation defect in children with mitochondrial disease, leading to a diverse range of clinical presentations, including Leigh syndrome. For most patients the genetic cause of the biochemical defect remains unknown due to incomplete understanding of the complex I assembly process. Nonetheless, a plethora of pathogenic mutations have been described to date in the seven mitochondrial-encoded subunits of complex I as well as in 12 of the nuclear-encoded subunits and in six assembly factors. Whilst several mitochondrial DNA mutations are recurrent, the majority of these mutations are reported in single families. We have sequenced core structural and functional nuclear-encoded subunits of complex I in a cohort of 34 paediatric patients with isolated complex I deficiency, identifying pathogenic mutations in 6 patients. These included a novel homozygous NDUFS1 mutation in an Asian child with Leigh syndrome, a previously identified NDUFS8 mutation (c.236C>T, p.P79L) in a second Asian child with Leigh-like syndrome and six novel, compound heterozygous NDUFS2 mutations in four white Caucasian patients with Leigh or Leigh-like syndrome. Three of these children harboured an identical NDUFS2 mutation (c.875T>C, p.M292T), which was also identified in conjunction with a novel NDUFS2 splice site mutation (c.866+4A>G) in a fourth Caucasian child who presented to a different diagnostic centre, with microsatellite and single nucleotide polymorphism analyses indicating that this was due to an ancient common founder event. Our results confirm that NDUFS2 is a mutational hotspot in Caucasian children with isolated complex I deficiency and recommend the routine diagnostic investigation of this gene in patients with Leigh or Leigh-like phenotypes.
doi:10.1093/brain/awq232
PMCID: PMC2947428  PMID: 20819849
mitochondrial disease; Leigh syndrome; complex I deficiency; NDUFS2; recurrent mutation
9.  Human ERAL1 is a mitochondrial RNA chaperone involved in the assembly of the 28S small mitochondrial ribosomal subunit 
Biochemical Journal  2010;430(Pt 3):551-558.
The bacterial Ras-like protein Era has been reported previously to bind 16S rRNA within the 30S ribosomal subunit and to play a crucial role in ribosome assembly. An orthologue of this essential GTPase ERAL1 (Era G-protein-like 1) exists in higher eukaryotes and although its exact molecular function and cellular localization is unknown, its absence has been linked to apoptosis. In the present study we show that human ERAL1 is a mitochondrial protein important for the formation of the 28S small mitoribosomal subunit. We also show that ERAL1 binds in vivo to the rRNA component of the small subunit [12S mt (mitochondrial)-rRNA]. Bacterial Era associates with a 3′ unstructured nonanucleotide immediately downstream of the terminal stem–loop (helix 45) of 16S rRNA. This site contains an AUCA sequence highly conserved across all domains of life, immediately upstream of the anti-Shine–Dalgarno sequence, which is conserved in bacteria. Strikingly, this entire region is absent from 12S mt-rRNA. We have mapped the ERAL1-binding site to a 33 nucleotide section delineating the 3′ terminal stem–loop region of 12S mt-rRNA. This loop contains two adenine residues that are reported to be dimethylated on mitoribosome maturation. Furthermore, and also in contrast with the bacterial orthologue, loss of ERAL1 leads to rapid decay of nascent 12S mt-rRNA, consistent with a role as a mitochondrial RNA chaperone. Finally, whereas depletion of ERAL1 leads to apoptosis, cell death occurs prior to any appreciable loss of mitochondrial protein synthesis or reduction in the stability of mitochondrial mRNA.
doi:10.1042/BJ20100757
PMCID: PMC2995420  PMID: 20604745
Era G-protein-like 1 (ERAL1); mitoribosome; ribosome assembly; rRNA; translation; CLIP, cytoplasmic linker protein; COX, cyclo-oxygenase; DAP3, death-associated protein 3; ERAL1, Era G-protein-like 1; FBS, fetal bovine serum; GDH, glutamate dehydrogenase; HEK-293T, HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40); HSP70, heat-shock protein 70; ICT1, immature colon carcinoma transcript 1; IP, immunoprecipitation; KH, K homology; LSU, large subunit; MEM, minimal essential medium; MGC, mammalian gene collection; MRP, mammalian ribosomal protein; mt, mitochondrial; MTND, mitochondrially encoded NADH dehydrogenase; NDUFA9, NADH dehydrogenase (ubiquinone) 1 α subcomplex 9; NDUFB8, NADH dehydrogenase (ubiquinone) 1 β subcomplex 8; NEAA, non-essential amino acids; NT, non-targeting; RNP, ribonucleoprotein; ROS, reactive oxygen species; SD, Shine–Dalgarno; siRNA, small interfering RNA; SSU, small subunit; TFB1M, transcription factor B1, mitochondrial; UTR, untranslated region
10.  Human mitochondrial mRNAs—like members of all families, similar but different 
Biochimica et Biophysica Acta  2010;1797(6-7):1081-1085.
The messenger RNAs containing the thirteen protein coding sequences of the human mitochondrial genome have frequently been regarded as a single functional category, alike in arrangement and hence in mode of expression. The “generic” mitochondrial mRNA is perceived as having (i) an arrangement within the polycistronic unit that permits its liberation following mt-tRNA processing, (ii) no 5′ cap structure or introns, (iii) essentially no untranslated regions, and (iv) a poly(A) tail of approximately fifty nucleotides that is required in part to complete the termination codon. Closer inspection reveals that only two molecules fit this pattern. This article examines the extent to which human mitochondrial mRNA species differ from one another.
doi:10.1016/j.bbabio.2010.02.036
PMCID: PMC3003153  PMID: 20211597
mt-mRNA, mitochondrial messenger RNA; mtDNA, mitochondrial genome; ORF, open reading frame; UTR, untranslated region; nt, nucleotide; Mitochondria; RNA; Poly(A); Processing; Transcript; Translation; Termination
11.  A functional peptidyl-tRNA hydrolase, ICT1, has been recruited into the human mitochondrial ribosome 
The EMBO Journal  2010;29(6):1116-1125.
Bioinformatic analysis classifies the human protein encoded by immature colon carcinoma transcript-1 (ICT1) as one of a family of four putative mitochondrial translation release factors. However, this has not been supported by any experimental evidence. As only a single member of this family, mtRF1a, is required to terminate the synthesis of all 13 mitochondrially encoded polypeptides, the true physiological function of ICT1 was unclear. Here, we report that ICT1 is an essential mitochondrial protein, but unlike the other family members that are matrix-soluble, ICT1 has become an integral component of the human mitoribosome. Release-factor assays show that although ICT1 has retained its ribosome-dependent PTH activity, this is codon-independent; consistent with its loss of both domains that promote codon recognition in class-I release factors. Mutation of the GGQ domain common to ribosome-dependent PTHs causes a loss of activity in vitro and, crucially, a loss of cell viability, in vivo. We suggest that ICT1 may be essential for hydrolysis of prematurely terminated peptidyl-tRNA moieties in stalled mitoribosomes.
doi:10.1038/emboj.2010.14
PMCID: PMC2845271  PMID: 20186120
mitoribosomes; peptidyl-tRNA hydrolase; translation release factor
12.  Targeting of the cytosolic poly(A) binding protein PABPC1 to mitochondria causes mitochondrial translation inhibition 
Nucleic Acids Research  2010;38(11):3732-3742.
Mammalian mitochondria contain their own genome that is almost fully transcribed from both strands, generating polycistronic RNA units that are processed and matured. The mitochondrial mRNA is modified by oligo- or polyadenylation at the 3′ termini, but the exact function of this post-transcriptional addition is unclear. Current debate focuses on the role of polyadenylation in transcript stability. An equally likely function that has received little attention is that, as in the cytosol of eukaryotes, polyadenylation facilitates translation in the mitochondrion. To address this issue, we have targeted cytosolic proteins to the mitochondrion, a poly(A) specific 3′ exoribonuclease, mtPARN, and a poly(A)binding protein, mtPABP1. Removal of the 3′ adenylyl extensions had a variable effect on mt-mRNA steady-state levels, increasing (MTND1, 2, 5) or decreasing (MTCO1, 2, RNA14) certain species with minimal effect on others (RNA7, MTND3). Translation was markedly affected, but interpretation of this was complicated by the concomitant 3′ truncation of the open reading frame in most cases. Coating of the poly(A) tail by mtPABP1, however, did not lead to transcript decay but caused a marked inhibition of mitochondrial translation. These data are consistent with endogenous RNA-binding factor(s) interacting with the poly(A) to optimize mitochondrial protein synthesis.
doi:10.1093/nar/gkq068
PMCID: PMC2887948  PMID: 20144953
13.  Fine mapping of interactions between eEF1α protein and 3′UTR of metallothionein-1 mRNA 
The localization of metallothionein-1 (MT-1) mRNA to the perinuclear cytoskeleton is determined by a signal in the 3′untranslated region (3′UTR) and trans-acting binding proteins. The present study carried out detailed mapping of this signal and further characterized the binding to elongation factor 1 alpha (eEF1α) and other interacting proteins. Electrophoresis mobility shift assays demonstrated that shortening of a stem region proximal to nucleotides 66–76 abrogated binding. Full length recombinant rat eEF1α, and independently domains I and III, formed complexes with the mRNA. Proteins binding to biotinylated MT-1 3′UTR sequences were isolated using RNA-affinity techniques, and mass spectrometry identified histidine-tRNA ligase as one of the major MT-1 3′UTR binding proteins. We conclude that a 5-bp internal stem in the MT-1 3′UTR is critical for binding of eEF1α and histidine-tRNA ligase, and that binding of eEF1α is facilitated through domains I and III.
doi:10.1016/j.bbrc.2009.05.146
PMCID: PMC2719692  PMID: 19501573
Metallothionein-1; Perinuclear mRNA localisation; 3′Untranslated region; Elongation factor 1α; RNA binding protein; RNA secondary structure; Histidine-tRNA ligase
14.  The human mitochondrial ribosome recycling factor is essential for cell viability 
Nucleic Acids Research  2008;36(18):5787-5799.
The molecular mechanism of human mitochondrial translation has yet to be fully described. We are particularly interested in understanding the process of translational termination and ribosome recycling in the mitochondrion. Several candidates have been implicated, for which subcellular localization and characterization have not been reported. Here, we show that the putative mitochondrial recycling factor, mtRRF, is indeed a mitochondrial protein. Expression of human mtRRF in fission yeast devoid of endogenous mitochondrial recycling factor suppresses the respiratory phenotype. Further, human mtRRF is able to associate with Escherichia coli ribosomes in vitro and can associate with mitoribosomes in vivo. Depletion of mtRRF in human cell lines is lethal, initially causing profound mitochondrial dysmorphism, aggregation of mitoribosomes, elevated mitochondrial superoxide production and eventual loss of OXPHOS complexes. Finally, mtRRF was shown to co-immunoprecipitate a large number of mitoribosomal proteins attached to other mitochondrial proteins, including putative members of the mitochondrial nucleoid.
doi:10.1093/nar/gkn576
PMCID: PMC2566884  PMID: 18782833
15.  Overexpression of human mitochondrial valyl tRNA synthetase can partially restore levels of cognate mt-tRNAVal carrying the pathogenic C25U mutation 
Nucleic Acids Research  2008;36(9):3065-3074.
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-tRNAVal. 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-tRNAVal 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-tRNAVal observed in the homoplasmic 1624C>T-cell lines is caused by a rapid degradation of the deacylated form of the abnormal mt-tRNAVal. 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-tRNAVal, 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.
doi:10.1093/nar/gkn147
PMCID: PMC2396425  PMID: 18400783
16.  Production of transmitochondrial cybrids containing naturally occurring pathogenic mtDNA variants 
Nucleic Acids Research  2006;34(13):e95.
The human mitochondrial genome (mtDNA) encodes polypeptides that are critical for coupling oxidative phosphorylation. Our detailed understanding of the molecular processes that mediate mitochondrial gene expression and the structure–function relationships of the OXPHOS components could be greatly improved if we were able to transfect mitochondria and manipulate mtDNA in vivo. Increasing our knowledge of this process is not merely of fundamental importance, as mutations of the mitochondrial genome are known to cause a spectrum of clinical disorders and have been implicated in more common neurodegenerative disease and the ageing process. In organellar or in vitro reconstitution studies have identified many factors central to the mechanisms of mitochondrial gene expression, but being able to investigate the molecular aetiology of a limited number of cell lines from patients harbouring mutated mtDNA has been enormously beneficial. In the absence of a mechanism for manipulating mtDNA, a much larger pool of pathogenic mtDNA mutations would increase our knowledge of mitochondrial gene expression. Colonic crypts from ageing individuals harbour mutated mtDNA. Here we show that by generating cytoplasts from colonocytes, standard fusion techniques can be used to transfer mtDNA into rapidly dividing immortalized cells and, thereby, respiratory-deficient transmitochondrial cybrids can be isolated. A simple screen identified clones that carried putative pathogenic mutations in MTRNR1, MTRNR2, MTCOI and MTND2, MTND4 and MTND6. This method can therefore be exploited to produce a library of cell lines carrying pathogenic human mtDNA for further study.
doi:10.1093/nar/gkl516
PMCID: PMC1540737  PMID: 16885236
17.  Serum-deprivation stimulates cap-binding by PARN at the expense of eIF4E, consistent with the observed decrease in mRNA stability 
Nucleic Acids Research  2005;33(1):376-387.
PARN, a poly(A)-specific ribonuclease, binds the 5′ cap-structure of mRNA and initiates deadenylation-dependent decay. Eukaryotic initiation factor 4E (eIF4E) also binds to the cap structure, an interaction that is critical for initiating cap-dependent translation. The stability of various mRNA transcripts in human cell lines is reduced under conditions of serum starvation as determined by both functional and chemical half-lives. Serum starvation also leads to enhanced cap association by PARN. In contrast, the 5′ cap occupancy by eIF4E decreases under serum-deprivation, as does the translation of reporter transcripts. Further, we show that PARN is a phosphoprotein and that this modification can be modulated by serum status. Taken together, these data are consistent with a natural competition existing at the 5′ cap structure between PARN and eIF4E that may be regulated by changes in post-translational modifications. These phosphorylation-induced changes in the interplay of PARN and eIF4E may determine whether the mRNA is translated or decayed.
doi:10.1093/nar/gki169
PMCID: PMC546156  PMID: 15653638
18.  Mutation of the human mitochondrial phenylalanine-tRNA synthetase causes infantile-onset epilepsy and cytochrome c oxidase deficiency☆ 
Biochimica et Biophysica Acta  2014;1842(1):56-64.
Mitochondrial aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in protein synthesis since they charge tRNAs with their cognate amino acids. Mutations in the genes encoding mitochondrial aaRSs have been associated with a wide spectrum of human mitochondrial diseases. Here we report the identification of pathogenic mutations (a partial genomic deletion and a highly conserved p. Asp325Tyr missense variant) in FARS2, the gene encoding mitochondrial phenylalanyl-tRNA synthetase, in a patient with early-onset epilepsy and isolated complex IV deficiency in muscle. The biochemical defect was expressed in myoblasts but not in fibroblasts and associated with decreased steady state levels of COXI and COXII protein and reduced steady state levels of the mt-tRNAPhe transcript. Functional analysis of the recombinant mutant p. Asp325Tyr FARS2 protein showed an inability to bind ATP and consequently undetectable aminoacylation activity using either bacterial tRNA or human mt-tRNAPhe as substrates. Lentiviral transduction of cells with wildtype FARS2 restored complex IV protein levels, confirming that the p.Asp325Tyr mutation is pathogenic, causing respiratory chain deficiency and neurological deficits on account of defective aminoacylation of mt-tRNAPhe.
Highlights
•The tissue specific variation in presentation of aaRS mutations is discussed.•A further clinical presentation of a FARS2 defect.•Molecular mechanism of FARS2 defect is characterised in vitro.
doi:10.1016/j.bbadis.2013.10.008
PMCID: PMC3898479  PMID: 24161539
OXPHOS, oxidative phosphorylation; aaRS, aminoacyl-tRNA synthetase; mt-, mitochondrial; mtDNA, mitochondrial DNA; MRI, magnetic resonance imaging; LBSL, leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation; PCH6, pontocerebellar hypoplasia type 6; MLASA, myopathy, lactic acidosis and sideroblastic anaemia; Mitochondria; Mitochondrial disease; Aminoacyl-tRNA synthetase; Aminoacylation; Mitochondrial translation; Protein synthesis
19.  Mutations in mitochondrial ribosomal protein MRPL12 leads to growth retardation, neurological deterioration and mitochondrial translation deficiency☆ 
Biochimica et Biophysica Acta  2013;1832(8):1304-1312.
Multiple respiratory chain deficiencies represent a common cause of mitochondrial diseases and are associated with a wide range of clinical symptoms. We report a subject, born to consanguineous parents, with growth retardation and neurological deterioration. Multiple respiratory chain deficiency was found in muscle and fibroblasts of the subject as well as abnormal assembly of complexes I and IV. A microsatellite genotyping of the family members detected only one region of homozygosity on chromosome 17q24.2–q25.3 in which we focused our attention to genes involved in mitochondrial translation. We sequenced MRPL12, encoding the mitochondrial ribosomal protein L12 and identified a c.542C>T transition in exon 5 changing a highly conserved alanine into a valine (p.Ala181Val). This mutation resulted in a decreased steady-state level of MRPL12 protein, with altered integration into the large ribosomal subunit. Moreover, an overall mitochondrial translation defect was observed in the subject's fibroblasts with a significant reduction of synthesis of COXI, COXII and COXIII subunits. Modeling of MRPL12 shows Ala181 positioned in a helix potentially involved in an interface of interaction suggesting that the p.Ala181Val change might be predicted to alter interactions with the elongation factors. These results contrast with the eubacterial orthologues of human MRPL12, where L7/L12 proteins do not appear to have a selective effect on translation. Therefore, analysis of the mutated version found in the subject presented here suggests that the mammalian protein does not function in an entirely analogous manner to the eubacterial L7/L12 equivalent.
Highlights
•MRPL12 function is not entirely analogous to the eubacterial L7/L12 equivalent.•Mutations in MRPL12 cause translation defects.•Mutations in apparently universal translation factors can affect different OXPHOS complexes.
doi:10.1016/j.bbadis.2013.04.014
PMCID: PMC3787750  PMID: 23603806
MRP, mitoribosomal protein; OXPHOS, oxidative phosphorylation; COX, cytochrome c oxidase; POLRMT, mitochondrial RNA polymerase; Mitochondria; Mitoribosome; Protein synthesis; Disease; OXPHOS defect
20.  Molecular basis of infantile reversible cytochrome c oxidase deficiency myopathy 
Brain  2009;132(11):3165-3174.
Childhood-onset mitochondrial encephalomyopathies are usually severe, relentlessly progressive conditions that have a fatal outcome. However, a puzzling infantile disorder, long known as ‘benign cytochrome c oxidase deficiency myopathy’ is an exception because it shows spontaneous recovery if infants survive the first months of life. Current investigations cannot distinguish those with a good prognosis from those with terminal disease, making it very difficult to decide when to continue intensive supportive care. Here we define the principal molecular basis of the disorder by identifying a maternally inherited, homoplasmic m.14674T>C mt-tRNAGlu mutation in 17 patients from 12 families. Our results provide functional evidence for the pathogenicity of the mutation and show that tissue-specific mechanisms downstream of tRNAGlu may explain the spontaneous recovery. This study provides the rationale for a simple genetic test to identify infants with mitochondrial myopathy and good prognosis.
doi:10.1093/brain/awp221
PMCID: PMC2768660  PMID: 19720722
mitochondrial myopathy; reversible COX deficiency; homoplasmic tRNA mutation

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