Publications by authors named "Robert N Lightowlers"

106 Publications

High-resolution imaging reveals compartmentalization of mitochondrial protein synthesis in cultured human cells.

Proc Natl Acad Sci U S A 2021 Feb;118(6)

Faculty of Medical Sciences, Newcastle University Biosciences Institute, Newcastle upon Tyne NE2 4HH, United Kingdom;

Human mitochondria contain their own genome, mitochondrial DNA, that is expressed in the mitochondrial matrix. This genome encodes 13 vital polypeptides that are components of the multisubunit complexes that couple oxidative phosphorylation (OXPHOS). The inner mitochondrial membrane that houses these complexes comprises the inner boundary membrane that runs parallel to the outer membrane, infoldings that form the cristae membranes, and the cristae junctions that separate the two. It is in these cristae membranes that the OXPHOS complexes have been shown to reside in various species. The majority of the OXPHOS subunits are nuclear-encoded and must therefore be imported from the cytosol through the outer membrane at contact sites with the inner boundary membrane. As the mitochondrially encoded components are also integral members of these complexes, where does protein synthesis occur? As transcription, mRNA processing, maturation, and at least part of the mitoribosome assembly process occur at the nucleoid and the spatially juxtaposed mitochondrial RNA granules, is protein synthesis also performed at the RNA granules close to these entities, or does it occur distal to these sites? We have adapted a click chemistry-based method coupled with stimulated emission depletion nanoscopy to address these questions. We report that, in human cells in culture, within the limits of our methodology, the majority of mitochondrial protein synthesis is detected at the cristae membranes and is spatially separated from the sites of RNA processing and maturation.
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http://dx.doi.org/10.1073/pnas.2008778118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8017971PMC
February 2021

Visualizing Mitochondrial Ribosomal RNA and Mitochondrial Protein Synthesis in Human Cell Lines.

Methods Mol Biol 2021 ;2192:159-181

Wellcome Centre for Mitochondrial Research, Newcastle University Biosciences Institute, Newcastle University, Medical School, Newcastle Upon Tyne, UK.

Human mitochondria contain their own DNA (mtDNA) that encodes 13 proteins all of which are core subunits of oxidative phosphorylation (OXPHOS) complexes. To form functional complexes, these 13 components need to be correctly assembled with approximately 70 nuclear-encoded subunits that are imported following synthesis in the cytosol. How this complicated coordinated translation and assembly is choreographed is still not clear. Methods are being developed to determine whether all members of a particular complex are translated in close proximity, whether protein synthesis is clustered in submitochondrial factories, whether these align with incoming polypeptides, and if there is evidence for co-translational translation that is regulated and limited by the interaction of the incoming proteins with synthesis of their mtDNA-encoded partners. Two methods are described in this chapter to visualize the distribution of mitochondrial ribosomal RNAs in conjunction with newly synthesized mitochondrial proteins. The first combines RNA Fluorescent In Situ Hybridization (FISH) and super-resolution immunocytochemistry to pinpoint mitochondrial ribosomal RNA. The second localizes nascent translation within the mitochondrial network through non-canonical amino acid labeling, click chemistry and fluorescent microscopy.
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http://dx.doi.org/10.1007/978-1-0716-0834-0_13DOI Listing
March 2021

Mitochondrial transplantation-a possible therapeutic for mitochondrial dysfunction?: Mitochondrial transfer is a potential cure for many diseases but proof of efficacy and safety is still lacking.

EMBO Rep 2020 09 27;21(9):e50964. Epub 2020 Aug 27.

Faculty of Medical Sciences, Translational and Clinical Research Institute, Newcastle University, Newcastle upon Tyne, UK.

Transplantation of functional mitochondria directly into defective cells is a novel approach that has recently caught the attention of scientists and the general public alike. Could this be too good to be true?
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http://dx.doi.org/10.15252/embr.202050964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7507022PMC
September 2020

Redecorating the Mitochondrial Inner Membrane: A Treatment for mtDNA Disorders.

Mol Ther 2020 08 16;28(8):1749-1751. Epub 2020 Jul 16.

Newcastle University Biosciences Institute, Wellcome Centre for Mitochondrial Research, Faculty of Medical Sciences, Newcastle upon Tyne, UK. Electronic address:

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http://dx.doi.org/10.1016/j.ymthe.2020.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7403454PMC
August 2020

Messenger RNA delivery to mitoribosomes - hints from a bacterial toxin.

FEBS J 2021 Jan 11;288(2):437-451. Epub 2020 May 11.

The Wellcome Centre for Mitochondrial Research, Institute of Neuroscience, Newcastle University, UK.

In mammalian mitochondria, messenger RNA is processed and matured from large primary transcripts in structures known as RNA granules. The identity of the factors and process transferring the matured mRNA to the mitoribosome for translation is unclear. Nascent mature transcripts are believed to associate initially with the small mitoribosomal subunit prior to recruitment of the large subunit to form the translationally active monosome. When the small subunit fails to assemble, however, the stability of mt-mRNA is only marginally affected, and under these conditions, the LRPPRC/SLIRP RNA-binding complex has been implicated in maintaining mt-mRNA stability. Here, we exploit the activity of a bacterial ribotoxin, VapC20, to show that in the absence of the large mitoribosomal subunit, mt-mRNA species are selectively lost. Further, if the small subunit is also depleted, the mt-mRNA levels are recovered. As a consequence of these data, we suggest a natural pathway for loading processed mt-mRNA onto the mitoribosome.
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http://dx.doi.org/10.1111/febs.15342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891357PMC
January 2021

Mitochondrial Diseases: Hope for the Future.

Cell 2020 04 26;181(1):168-188. Epub 2020 Mar 26.

Wellcome Centre for Mitochondrial Research, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne NE2 4HH, UK; Clinical and Translational Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne NE2 4HH, UK. Electronic address:

Mitochondrial diseases are clinically heterogeneous disorders caused by a wide spectrum of mutations in genes encoded by either the nuclear or the mitochondrial genome. Treatments for mitochondrial diseases are currently focused on symptomatic management rather than improving the biochemical defect caused by a particular mutation. This review focuses on the latest advances in the development of treatments for mitochondrial disease, both small molecules and gene therapies, as well as methods to prevent transmission of mitochondrial disease through the germline.
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http://dx.doi.org/10.1016/j.cell.2020.02.051DOI Listing
April 2020

Rescuing stalled mammalian mitoribosomes - what can we learn from bacteria?

J Cell Sci 2020 01 2;133(1). Epub 2020 Jan 2.

The Wellcome Centre for Mitochondrial Research, Newcastle University, The Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK

In the canonical process of translation, newly completed proteins escape from the ribosome following cleavage of the ester bond that anchors the polypeptide to the P-site tRNA, after which the ribosome can be recycled to initiate a new round of translation. Not all protein synthesis runs to completion as various factors can impede the progression of ribosomes. Rescuing of stalled ribosomes in mammalian mitochondria, however, does not share the same mechanisms that many bacteria use. The classic method for rescuing bacterial ribosomes is -translation. The key components of this system are absent from mammalian mitochondria; however, four members of a translation termination factor family are present, with some evidence of homology to members of a bacterial back-up rescue system. To date, there is no definitive demonstration of any other member of this family functioning in mitoribosome rescue. Here, we provide an overview of the processes and key players of canonical translation termination in both bacteria and mammalian mitochondria, followed by a perspective of the bacterial systems used to rescue stalled ribosomes. We highlight any similarities or differences with the mitochondrial translation release factors, and suggest potential roles for these proteins in ribosome rescue in mammalian mitochondria.
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http://dx.doi.org/10.1242/jcs.231811DOI Listing
January 2020

Mammalian mitochondrial translation - revealing consequences of divergent evolution.

Biochem Soc Trans 2019 10;47(5):1429-1436

The Wellcome Centre for Mitochondrial Research, Institute of Neuroscience, Newcastle University, Newcastle upon Tyne NE2 4HH, U.K.

Mitochondria are ubiquitous organelles present in the cytoplasm of all nucleated eukaryotic cells. These organelles are described as arising from a common ancestor but a comparison of numerous aspects of mitochondria between different organisms provides remarkable examples of divergent evolution. In humans, these organelles are of dual genetic origin, comprising ∼1500 nuclear-encoded proteins and thirteen that are encoded by the mitochondrial genome. Of the various functions that these organelles perform, it is only oxidative phosphorylation, which provides ATP as a source of chemical energy, that is dependent on synthesis of these thirteen mitochondrially encoded proteins. A prerequisite for this process of translation are the mitoribosomes. The recent revolution in cryo-electron microscopy has generated high-resolution mitoribosome structures and has undoubtedly revealed some of the most distinctive molecular aspects of the mitoribosomes from different organisms. However, we still lack a complete understanding of the mechanistic aspects of this process and many of the factors involved in post-transcriptional gene expression in mitochondria. This review reflects on the current knowledge and illustrates some of the striking differences that have been identified between mitochondria from a range of organisms.
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http://dx.doi.org/10.1042/BST20190265DOI Listing
October 2019

M2I-1 disrupts the in vivo interaction between CDC20 and MAD2 and increases the sensitivities of cancer cell lines to anti-mitotic drugs via MCL-1s.

Cell Div 2019 15;14. Epub 2019 Jun 15.

1Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH UK.

Background: Drugs such as taxanes, epothilones, and vinca alkaloids are widely used in the treatment of breast, ovarian, and lung cancers but come with major side effects such as neuropathy and loss of neutrophils and as single agents have a lack of efficacy. M2I-1 (MAD2 inhibitor-1) has been shown to disrupt the CDC20-MAD2 interaction, and consequently, the assembly of the mitotic checkpoint complex (MCC).

Results: We report here that M2I-1 can significantly increase the sensitivity of several cancer cell lines to anti-mitotic drugs, with cell death occurring after a prolonged mitotic arrest. In the presence of nocodazole or taxol combined with M2I-1 cell death is triggered by the premature degradation of Cyclin B1, the perturbation of the microtubule network, and an increase in the level of the pro-apoptotic protein MCL-1s combined with a marginal increase in the level of NOXA. The elevated level of MCL-1s and the marginally increased NOXA antagonized the increased level of MCL-1, a pro-survival protein of the Bcl-2 family.

Conclusion: Our results provide some important molecular mechanisms for understanding the relationship between the mitotic checkpoint and programmed cell death and demonstrate that M2I-1 exhibits antitumor activity in the presence of current anti-mitotic drugs such as taxol and nocodazole and has the potential to be developed as an anticancer agent.
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http://dx.doi.org/10.1186/s13008-019-0049-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6570884PMC
June 2019

How much does a disrupted mitochondrial network influence neuronal dysfunction?

EMBO Mol Med 2019 01;11(1)

Wellcome Centre for Mitochondrial Research, Institute for Cell and Molecular Biosciences, Medical School, Newcastle University, Newcastle upon Tyne, UK.

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http://dx.doi.org/10.15252/emmm.201809899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6328938PMC
January 2019

mutations cause mitochondrial encephalopathy and a combined oxidative phosphorylation defect.

EMBO Mol Med 2018 11;10(11)

Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK

OXA1, the mitochondrial member of the YidC/Alb3/Oxa1 membrane protein insertase family, is required for the assembly of oxidative phosphorylation complexes IV and V in yeast. However, depletion of human OXA1 (OXA1L) was previously reported to impair assembly of complexes I and V only. We report a patient presenting with severe encephalopathy, hypotonia and developmental delay who died at 5 years showing complex IV deficiency in skeletal muscle. Whole exome sequencing identified biallelic variants (c.500_507dup, p.(Ser170Glnfs*18) and c.620G>T, p.(Cys207Phe)) that segregated with disease. Patient muscle and fibroblasts showed decreased OXA1L and subunits of complexes IV and V. Crucially, expression of wild-type human in patient fibroblasts rescued the complex IV and V defects. Targeted depletion of OXA1L in human cells or caused defects in the assembly of complexes I, IV and V, consistent with patient data. Immunoprecipitation of OXA1L revealed the enrichment of mtDNA-encoded subunits of complexes I, IV and V. Our data verify the pathogenicity of these variants and demonstrate that OXA1L is required for the assembly of multiple respiratory chain complexes.
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http://dx.doi.org/10.15252/emmm.201809060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6220311PMC
November 2018

Advances in methods for reducing mitochondrial DNA disease by replacing or manipulating the mitochondrial genome.

Essays Biochem 2018 07 20;62(3):455-465. Epub 2018 Jul 20.

Wellcome Centre for Mitochondrial Research, Newcastle upon Tyne, U.K.

Mitochondrial DNA (mtDNA) is a multi-copy genome whose cell copy number varies depending on tissue type. Mutations in mtDNA can cause a wide spectrum of diseases. Mutated mtDNA is often found as a subset of the total mtDNA population in a cell or tissue, a situation known as heteroplasmy. As mitochondrial dysfunction only presents after a certain level of heteroplasmy has been acquired, ways to artificially reduce or replace the mutated species have been attempted. This review addresses recent approaches and advances in this field, focusing on the prevention of pathogenic mtDNA transfer via mitochondrial donation techniques such as maternal spindle transfer and pronuclear transfer in which mutated mtDNA in the oocyte or fertilized embryo is substituted with normal copies of the mitochondrial genome. This review also discusses the molecular targeting and cleavage of pathogenic mtDNA to shift heteroplasmy using antigenomic therapy and genome engineering techniques including Zinc-finger nucleases and transcription activator-like effector nucleases. Finally, it considers CRISPR technology and the unique difficulties that mitochondrial genome editing presents.
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http://dx.doi.org/10.1042/EBC20170113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6056713PMC
July 2018

Defective mitochondrial protease LonP1 can cause classical mitochondrial disease.

Hum Mol Genet 2018 05;27(10):1743-1753

Wellcome Centre for Mitochondrial Research, Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne, UK.

LonP1 is a mitochondrial matrix protease whose selective substrate specificity is essential for maintaining mitochondrial homeostasis. Recessively inherited, pathogenic defects in LonP1 have been previously reported to underlie cerebral, ocular, dental, auricular and skeletal anomalies (CODAS) syndrome, a complex multisystemic and developmental disorder. Intriguingly, although classical mitochondrial disease presentations are well-known to exhibit marked clinical heterogeneity, the skeletal and dental features associated with CODAS syndrome are pathognomonic. We have applied whole exome sequencing to a patient with congenital lactic acidosis, muscle weakness, profound deficiencies in mitochondrial oxidative phosphorylation associated with loss of mtDNA copy number and MRI abnormalities consistent with Leigh syndrome, identifying biallelic variants in the LONP1 (NM_004793.3) gene; c.1693T > C predicting p.(Tyr565His) and c.2197G > A predicting p.(Glu733Lys); no evidence of the classical skeletal or dental defects observed in CODAS syndrome patients were noted in our patient. In vitro experiments confirmed the p.(Tyr565His) LonP1 mutant alone could not bind or degrade a substrate, consistent with the predicted function of Tyr565, whilst a second missense [p.(Glu733Lys)] variant had minimal effect. Mixtures of p.(Tyr565His) mutant and wild-type LonP1 retained partial protease activity but this was severely depleted when the p.(Tyr565His) mutant was mixed with the p.(Glu733Lys) mutant, data consistent with the compound heterozygosity detected in our patient. In summary, we conclude that pathogenic LONP1 variants can lead to a classical mitochondrial disease presentations associated with severe biochemical defects in oxidative phosphorylation in clinically relevant tissues.
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http://dx.doi.org/10.1093/hmg/ddy080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5932559PMC
May 2018

Using mitoribosomal profiling to investigate human mitochondrial translation.

Wellcome Open Res 2017 11;2:116. Epub 2017 Dec 11.

The Wellcome Trust Centre for Mitochondrial Research, Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, UK.

: Gene expression in human mitochondria has various idiosyncratic features. One of these was recently revealed as the unprecedented recruitment of a mitochondrially-encoded tRNA as a structural component of the large mitoribosomal subunit. In porcine particles this is mt-tRNA whilst in humans it is mt-tRNA . We have previously shown that when a mutation in mt-tRNA causes very low steady state levels, there is preferential recruitment of mt-tRNA . We have investigated whether this altered mitoribosome affects intra-organellar protein synthesis. : By using mitoribosomal profiling we have revealed aspects of mitoribosome behaviour with its template mt-mRNA under both normal conditions as well as those where the mitoribosome has incorporated mt-tRNA . : Analysis of the mitoribosome residency on transcripts under control conditions reveals that although mitochondria employ only 22 mt-tRNAs for protein synthesis, the use of non-canonical wobble base pairs at codon position 3 does not cause any measurable difference in mitoribosome occupancy irrespective of the codon. Comparison of the profile of aberrant mt-tRNA containing mitoribosomes with those of controls that integrate mt-tRNA revealed that the impaired translation seen in the latter was not due to stalling on triplets encoding either of these amino acids. The alterations in mitoribosome interactions with start codons was not directly attributable to the either the use of non-cognate initiation codons or the presence or absence of 5' leader sequences, except in the two bicistronic RNA units, and where the initiation sites are internal. : These data report the power of mitoribosomal profiling in helping to understand the subtleties of mammalian mitochondrial protein synthesis. Analysis of profiles from the mutant mt-tRNA cell line suggest that despite mt-tRNA being preferred in the porcine mitoribosome, its integration into the human counterpart results in a suboptimal structure that modifies its interaction with mt-mRNAs.
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http://dx.doi.org/10.12688/wellcomeopenres.13119.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5771143PMC
December 2017

Preferential amplification of a human mitochondrial DNA deletion in vitro and in vivo.

Sci Rep 2018 01 29;8(1):1799. Epub 2018 Jan 29.

Novartis Institutes for BioMedical Research, Novartis Campus, Basel, CH-4056, Switzerland.

We generated induced pluripotent stem cells (iPSCs) from patient fibroblasts to yield cell lines containing varying degrees of heteroplasmy for a m.13514 A > G mtDNA point mutation (2 lines) and for a ~6 kb single, large scale mtDNA deletion (3 lines). Long term culture of the iPSCs containing a single, large-scale mtDNA deletion showed consistent increase in mtDNA deletion levels with time. Higher levels of mtDNA heteroplasmy correlated with increased respiratory deficiency. To determine what changes occurred in deletion level during differentiation, teratomas comprising all three embryonic germ layers were generated from low (20%) and intermediate heteroplasmy (55%) mtDNA deletion clones. Regardless of whether iPSCs harbouring low or intermediate mtDNA heteroplasmy were used, the final levels of heteroplasmy in all teratoma germ layers increased to a similar high level (>60%). Thus, during human stem cell division, cells not only tolerate high mtDNA deletion loads but seem to preferentially replicate deleted mtDNA genomes. This has implications for the involvement of mtDNA deletions in both disease and ageing.
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http://dx.doi.org/10.1038/s41598-018-20064-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5789095PMC
January 2018

Biallelic C1QBP Mutations Cause Severe Neonatal-, Childhood-, or Later-Onset Cardiomyopathy Associated with Combined Respiratory-Chain Deficiencies.

Am J Hum Genet 2017 Oct 21;101(4):525-538. Epub 2017 Sep 21.

Institute of Human Genetics, Technische Universität München, 81675 Munich, Germany; Institute of Human Genetics, Helmholtz Zentrum München, 85764 Neuherberg, Germany. Electronic address:

Complement component 1 Q subcomponent-binding protein (C1QBP; also known as p32) is a multi-compartmental protein whose precise function remains unknown. It is an evolutionary conserved multifunctional protein localized primarily in the mitochondrial matrix and has roles in inflammation and infection processes, mitochondrial ribosome biogenesis, and regulation of apoptosis and nuclear transcription. It has an N-terminal mitochondrial targeting peptide that is proteolytically processed after import into the mitochondrial matrix, where it forms a homotrimeric complex organized in a doughnut-shaped structure. Although C1QBP has been reported to exert pleiotropic effects on many cellular processes, we report here four individuals from unrelated families where biallelic mutations in C1QBP cause a defect in mitochondrial energy metabolism. Infants presented with cardiomyopathy accompanied by multisystemic involvement (liver, kidney, and brain), and children and adults presented with myopathy and progressive external ophthalmoplegia. Multiple mitochondrial respiratory-chain defects, associated with the accumulation of multiple deletions of mitochondrial DNA in the later-onset myopathic cases, were identified in all affected individuals. Steady-state C1QBP levels were decreased in all individuals' samples, leading to combined respiratory-chain enzyme deficiency of complexes I, III, and IV. C1qbp mouse embryonic fibroblasts (MEFs) resembled the human disease phenotype by showing multiple defects in oxidative phosphorylation (OXPHOS). Complementation with wild-type, but not mutagenized, C1qbp restored OXPHOS protein levels and mitochondrial enzyme activities in C1qbp MEFs. C1QBP deficiency represents an important mitochondrial disorder associated with a clinical spectrum ranging from infantile lactic acidosis to childhood (cardio)myopathy and late-onset progressive external ophthalmoplegia.
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http://dx.doi.org/10.1016/j.ajhg.2017.08.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5630164PMC
October 2017

The human RNA-binding protein RBFA promotes the maturation of the mitochondrial ribosome.

Biochem J 2017 06 13;474(13):2145-2158. Epub 2017 Jun 13.

Institute for Cell and Molecular Bioscience, Newcastle University, Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, U.K.

Accurate assembly and maturation of human mitochondrial ribosomes is essential for synthesis of the 13 polypeptides encoded by the mitochondrial genome. This process requires the correct integration of 80 proteins, 1 mt (mitochondrial)-tRNA and 2 mt-rRNA species, the latter being post-transcriptionally modified at many sites. Here, we report that human ribosome-binding factor A (RBFA) is a mitochondrial RNA-binding protein that exerts crucial roles in mitoribosome biogenesis. Unlike its bacterial orthologue, RBFA associates mainly with helices 44 and 45 of the 12S rRNA in the mitoribosomal small subunit to promote dimethylation of two highly conserved consecutive adenines. Characterization of RBFA-depleted cells indicates that this dimethylation is not a prerequisite for assembly of the small ribosomal subunit. However, the RBFA-facilitated modification is necessary for completing mt-rRNA maturation and regulating association of the small and large subunits to form a functional monosome implicating RBFA in the quality control of mitoribosome formation.
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http://dx.doi.org/10.1042/BCJ20170256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5468982PMC
June 2017

ClickIn: a flexible protocol for quantifying mitochondrial uptake of nucleobase derivatives.

Interface Focus 2017 Apr;7(2):20160117

The Wellcome Trust Centre for Mitochondrial Research, Institute for Cell and Molecular Biosciences , The Medical School, Newcastle University , Newcastle upon Tyne , UK.

There is an increasing interest in targeting molecules to the mitochondrial matrix. Many proteins are naturally imported through the translocase complexes found in the outer and inner mitochondrial membranes. One possible means for importing molecules is therefore to use a mitochondrial pre-protein as a vector and assess what forms of molecules can be attached to the pre-protein as cargo. A major difficulty with this approach is to ensure that any chimaeric molecule does indeed access the mitochondrial matrix and does not merely associate with the mitochondrial membranes. We have recently demonstrated that click chemistry can be used both to demonstrate convincingly mitochondrial import of a peptide-peptide nucleic acid conjugate and also to quantify the mitochondrial uptake for specific synthetic conjugates. We now report an adaptation of the synthesis to facilitate simple quantification of multiple molecules and hence to calculate the efficiency of their mitochondrial import.
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http://dx.doi.org/10.1098/rsfs.2016.0117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5311907PMC
April 2017

The Pseudouridine Synthase RPUSD4 Is an Essential Component of Mitochondrial RNA Granules.

J Biol Chem 2017 03 12;292(11):4519-4532. Epub 2017 Jan 12.

From the Department of Cell Biology, University of Geneva, 30 quai Ernest-Ansermet, 1211 Genève 4, Switzerland,

Mitochondrial gene expression is a fundamental process that is largely dependent on nuclear-encoded proteins. Several steps of mitochondrial RNA processing and maturation, including RNA post-transcriptional modification, appear to be spatially organized into distinct foci, which we have previously termed mitochondrial RNA granules (MRGs). Although an increasing number of proteins have been localized to MRGs, a comprehensive analysis of the proteome of these structures is still lacking. Here, we have applied a microscopy-based approach that has allowed us to identify novel components of the MRG proteome. Among these, we have focused our attention on RPUSD4, an uncharacterized mitochondrial putative pseudouridine synthase. We show that RPUSD4 depletion leads to a severe reduction of the steady-state level of the 16S mitochondrial (mt) rRNA with defects in the biogenesis of the mitoribosome large subunit and consequently in mitochondrial translation. We report that RPUSD4 binds 16S mt-rRNA, mt-tRNA, and mt-tRNA, and we demonstrate that it is responsible for pseudouridylation of the latter. These data provide new insights into the relevance of RNA pseudouridylation in mitochondrial gene expression.
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http://dx.doi.org/10.1074/jbc.M116.771105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5377769PMC
March 2017

Human mitochondrial nucleases.

FEBS J 2017 06 1;284(12):1767-1777. Epub 2017 Feb 1.

The Wellcome Trust Centre for Mitochondrial Research, The Medical School, Newcastle University, UK.

Mitochondria are cytosolic organelles that have many essential roles including ATP production via oxidative phosphorylation, apoptosis, iron-sulfur cluster biogenesis, heme and steroid synthesis, calcium homeostasis, and regulation of cellular redox state. One of the unique features of these organelles is the presence of an extrachromosomal mitochondrial genome (mtDNA), together with all the machinery required to replicate and transcribe mtDNA. The accurate maintenance of mitochondrial gene expression is essential for correct organellar metabolism, and is in part dependent on the levels of mtDNA and mtRNA, which are regulated by balancing synthesis against degradation. It is clear that although a number of mitochondrial nucleases have been identified, not all those responsible for the degradation of DNA or RNA have been characterized. Recent investigations, however, have revealed the contribution that mutations in the genes coding for these enzymes has made to causing pathogenic mitochondrial diseases.
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http://dx.doi.org/10.1111/febs.13981DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5484287PMC
June 2017

Adult Onset Leigh Syndrome in the Intensive Care Setting: A Novel Presentation of a C12orf65 Related Mitochondrial Disease.

J Neuromuscul Dis 2015 Oct;2(4):409-419

Wellcome Trust Centre for Mitochondrial Research, Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, UK.

Background: Mitochondrial disease can present at any age, with dysfunction in almost any tissue making diagnosis a challenge. It can result from inherited or sporadic mutations in either the mitochondrial or the nuclear genome, many of which affect intraorganellar gene expression. The estimated prevalence of 1/4300 indicates these to be amongst the commonest inherited neuromuscular disorders, emphasising the importance of recognition of the diagnostic clinical features.

Objective: Despite major advances in our understanding of the molecular basis of mitochondrial diseases, accurate and early diagnoses are critically dependent on the fastidious clinical and biochemical characterisation of patients. Here we describe a patient harbouring a previously reported homozygous mutation in C12orf65, a mitochondrial protein of unknown function, which does not adhere to the proposed distinct genotype-phenotype relationship.

Methods: We performed clinical, biochemical and molecular analysis including whole exome sequencing on patient samples and cell lines.

Results: We report an extremely rare case of an adult presenting with Leigh-like disease, in intensive care, in the 5th decade of life, harbouring a recessively inherited mutation previously reported in children. A global reduction in intra-mitochondrial protein synthesis was observed despite normal or elevated levels of mt-RNA, leading to an isolated complex IV deficiency.

Conclusions: All the reported C12orf65 mutations have shown an autosomal recessive pattern of inheritance. Mitochondrial disease causing mutations inherited in this manner are usually of early onset and associated with a severe, often fatal clinical phenotype. Presentations in adulthood are usually less severe. This patient's late adulthood presentation is in sharp contrast emphasising the clinical variability that is characteristic of mitochondrial disease and illustrates why making a definitive diagnosis remains a formidable challenge.
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http://dx.doi.org/10.3233/JND-150121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5240610PMC
October 2015

Cl-out is a novel cooperative optogenetic tool for extruding chloride from neurons.

Nat Commun 2016 11 17;7:13495. Epub 2016 Nov 17.

Institute of Neuroscience, Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.

Chloride regulation affects brain function in many ways, for instance, by dictating the GABAergic reversal potential, and thereby influencing neuronal excitability and spike timing. Consistent with this, there is increasing evidence implicating chloride in a range of neurological conditions. Investigations about these conditions, though, are made difficult by the limited range of tools available to manipulate chloride levels. In particular, there has been no way to actively remove chloride from neurons; we now describe an optogenetic strategy, 'Cl-out', to do exactly this. Cl-out achieves its effect by the cooperative action of two different component opsins: the proton pump, Archaerhodopsin and a chloride channel opsin. The removal of chloride happens when both are activated together, using Archaerhodopsin as an optical voltage clamp to provide the driving force for chloride removal through the concurrently opened, chloride channels. We further show that this novel optogenetic strategy can reverse an in vitro epileptogenic phenotype.
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http://dx.doi.org/10.1038/ncomms13495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5118542PMC
November 2016

Human mitochondrial ribosomes can switch their structural RNA composition.

Proc Natl Acad Sci U S A 2016 10 11;113(43):12198-12201. Epub 2016 Oct 11.

The Wellcome Trust Centre for Mitochondrial Research, Institute of Neuroscience, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom; The Wellcome Trust Centre for Mitochondrial Research, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom

The recent developments in cryo-EM have revolutionized our access to previously refractory structures. In particular, such studies of mammalian mitoribosomes have confirmed the absence of any 5S rRNA species and revealed the unexpected presence of a mitochondrially encoded tRNA (mt-tRNA) that usurps this position. Although the cryo-EM structures resolved the conundrum of whether mammalian mitoribosomes contain a 5S rRNA, they introduced a new dilemma: Why do human and porcine mitoribosomes integrate contrasting mt-tRNAs? Human mitoribosomes have been shown to integrate mt-tRNA compared with the porcine use of mt-tRNA We have explored this observation further. Our studies examine whether a range of mt-tRNAs are used by different mammals, or whether the mt-tRNA selection is strictly limited to only these two species of the 22 tRNAs encoded by the mitochondrial genome (mtDNA); whether there is tissue-specific variation within a single organism; and what happens to the human mitoribosome when levels of the mt-tRNA are depleted. Our data demonstrate that only mt-tRNA or mt-tRNA are found in the mitoribosomes of five different mammals, each mammal favors the same mt-tRNA in all tissue types, and strikingly, when steady-state levels of mt-tRNA are reduced, human mitoribosome biogenesis displays an adaptive response by switching to the incorporation of mt-tRNA to generate translationally competent machinery.
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http://dx.doi.org/10.1073/pnas.1609338113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087001PMC
October 2016

Pathogenic variants in HTRA2 cause an early-onset mitochondrial syndrome associated with 3-methylglutaconic aciduria.

J Inherit Metab Dis 2017 01 30;40(1):121-130. Epub 2016 Sep 30.

Wellcome Trust Centre for Mitochondrial Research, Institute of Neuroscience, The Medical School, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK.

Mitochondrial diseases collectively represent one of the most heterogeneous group of metabolic disorders. Symptoms can manifest at any age, presenting with isolated or multiple-organ involvement. Advances in next-generation sequencing strategies have greatly enhanced the diagnosis of patients with mitochondrial disease, particularly where a mitochondrial aetiology is strongly suspected yet OXPHOS activities in biopsied tissue samples appear normal. We used whole exome sequencing (WES) to identify the molecular basis of an early-onset mitochondrial syndrome-pathogenic biallelic variants in the HTRA2 gene, encoding a mitochondria-localised serine protease-in five subjects from two unrelated families characterised by seizures, neutropenia, hypotonia and cardio-respiratory problems. A unifying feature in all affected children was 3-methylglutaconic aciduria (3-MGA-uria), a common biochemical marker observed in some patients with mitochondrial dysfunction. Although functional studies of HTRA2 subjects' fibroblasts and skeletal muscle homogenates showed severely decreased levels of mutant HTRA2 protein, the structural subunits and complexes of the mitochondrial respiratory chain appeared normal. We did detect a profound defect in OPA1 processing in HTRA2-deficient fibroblasts, suggesting a role for HTRA2 in the regulation of mitochondrial dynamics and OPA1 proteolysis. In addition, investigated subject fibroblasts were more susceptible to apoptotic insults. Our data support recent studies that described important functions for HTRA2 in programmed cell death and confirm that patients with genetically-unresolved 3-MGA-uria should be screened by WES with pathogenic variants in the HTRA2 gene prioritised for further analysis.
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http://dx.doi.org/10.1007/s10545-016-9977-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5203855PMC
January 2017

The process of mammalian mitochondrial protein synthesis.

Cell Tissue Res 2017 Jan 14;367(1):5-20. Epub 2016 Jul 14.

Wellcome Trust Centre for Mitochondrial Research, Institute for Cell and Molecular Biosciences, Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.

Oxidative phosphorylation (OXPHOS) is the mechanism whereby ATP, the major energy source for the cell, is produced by harnessing cellular respiration in the mitochondrion. This is facilitated by five multi-subunit complexes housed within the inner mitochondrial membrane. These complexes, with the exception of complex II, are of a dual genetic origin, requiring expression from nuclear and mitochondrial genes. Mitochondrially encoded mRNA is translated on the mitochondrial ribosome (mitoribosome) and the recent release of the near atomic resolution structure of the mammalian mitoribosome has highlighted its peculiar features. However, whereas some aspects of mitochondrial translation are understood, much is to be learnt about the presentation of mitochondrial mRNA to the mitoribosome, the biogenesis of the machinery, the exact role of the membrane, the constitution of the translocon/insertion machinery and the regulation of translation in the mitochondrion. This review addresses our current knowledge of mammalian mitochondrial gene expression, highlights key questions and indicates how defects in this process can result in profound mitochondrial disease.
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http://dx.doi.org/10.1007/s00441-016-2456-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5203842PMC
January 2017

SLIRP stabilizes LRPPRC via an RRM-PPR protein interface.

Nucleic Acids Res 2016 08 28;44(14):6868-82. Epub 2016 Jun 28.

Department of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, D-50931 Cologne, Germany Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 171 77 Stockholm, Sweden

LRPPRC is a protein that has attracted interest both for its role in post-transcriptional regulation of mitochondrial gene expression and more recently because numerous mutated variants have been characterized as causing severe infantile mitochondrial neurodegeneration. LRPPRC belongs to the pentatricopeptide repeat (PPR) protein family, originally defined by their RNA binding capacity, and forms a complex with SLIRP that harbours an RNA recognition motif (RRM) domain. We show here that LRPPRC displays a broad and strong RNA binding capacity in vitro in contrast to SLIRP that associates only weakly with RNA. The LRPPRC-SLIRP complex comprises a hetero-dimer via interactions by polar amino acids in the single RRM domain of SLIRP and three neighbouring PPR motifs in the second quarter of LRPPRC, which critically contribute to the LRPPRC-SLIRP binding interface to enhance its stability. Unexpectedly, specific amino acids at this interface are located within the PPRs of LRPPRC at positions predicted to interact with RNA and within the RNP1 motif of SLIRP's RRM domain. Our findings thus unexpectedly establish that despite the prediction that these residues in LRPPRC and SLIRP should bind RNA, they are instead used to facilitate protein-protein interactions, enabling the formation of a stable complex between these two proteins.
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http://dx.doi.org/10.1093/nar/gkw575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5001613PMC
August 2016

Assessing the Delivery of Molecules to the Mitochondrial Matrix Using Click Chemistry.

Chembiochem 2016 07 25;17(14):1312-6. Epub 2016 May 25.

The Wellcome Trust Centre for Mitochondrial Research, Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne, UK.

Mitochondria are central to health and disease, hence there is considerable interest in developing mitochondria-targeted therapies that require the delivery of peptides or nucleic acid oligomers. However, progress has been impeded by the lack of a measure of mitochondrial import of these molecules. Here, we address this need by quantitatively detecting molecules within the mitochondrial matrix. We used a mitochondria- targeted cyclooctyne (MitoOct) that accumulates several- hundredfold in the matrix, driven by the membrane potential. There, MitoOct reacts through click chemistry with an azide on the target molecule to form a diagnostic product that can be quantified by mass spectrometry. Because the membrane potential-dependent MitoOct concentration in the matrix is essential for conjugation, we can now determine definitively whether a putative mitochondrion-targeted molecule reaches the matrix. This "ClickIn" approach will facilitate development of mitochondria-targeted therapies.
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http://dx.doi.org/10.1002/cbic.201600188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4973694PMC
July 2016

Applying the Airbrakes: Treating Mitochondrial Disease with Hypoxia.

Mol Cell 2016 Apr;62(1):5-6

Wellcome Trust Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK. Electronic address:

A paper from Jain et al. (2016) using whole-genome CRISPR knockout libraries in human cells and models of mitochondrial disease suggests chronic hypoxia could be an unexpected treatment for disorders of mitochondrial respiration.
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http://dx.doi.org/10.1016/j.molcel.2016.03.027DOI Listing
April 2016

LRPPRC mutations cause early-onset multisystem mitochondrial disease outside of the French-Canadian population.

Brain 2015 Dec 27;138(Pt 12):3503-19. Epub 2015 Oct 27.

1 Wellcome Trust Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK

Mitochondrial Complex IV [cytochrome c oxidase (COX)] deficiency is one of the most common respiratory chain defects in humans. The clinical phenotypes associated with COX deficiency include liver disease, cardiomyopathy and Leigh syndrome, a neurodegenerative disorder characterized by bilateral high signal lesions in the brainstem and basal ganglia. COX deficiency can result from mutations affecting many different mitochondrial proteins. The French-Canadian variant of COX-deficient Leigh syndrome is unique to the Saguenay-Lac-Saint-Jean region of Québec and is caused by a founder mutation in the LRPPRC gene. This encodes the leucine-rich pentatricopeptide repeat domain protein (LRPPRC), which is involved in post-transcriptional regulation of mitochondrial gene expression. Here, we present the clinical and molecular characterization of novel, recessive LRPPRC gene mutations, identified using whole exome and candidate gene sequencing. The 10 patients come from seven unrelated families of UK-Caucasian, UK-Pakistani, UK-Indian, Turkish and Iraqi origin. They resemble the French-Canadian Leigh syndrome patients in having intermittent severe lactic acidosis and early-onset neurodevelopmental problems with episodes of deterioration. In addition, many of our patients have had neonatal cardiomyopathy or congenital malformations, most commonly affecting the heart and the brain. All patients who were tested had isolated COX deficiency in skeletal muscle. Functional characterization of patients' fibroblasts and skeletal muscle homogenates showed decreased levels of mutant LRPPRC protein and impaired Complex IV enzyme activity, associated with abnormal COX assembly and reduced steady-state levels of numerous oxidative phosphorylation subunits. We also identified a Complex I assembly defect in skeletal muscle, indicating different roles for LRPPRC in post-transcriptional regulation of mitochondrial mRNAs between tissues. Patient fibroblasts showed decreased steady-state levels of mitochondrial mRNAs, although the length of poly(A) tails of mitochondrial transcripts were unaffected. Our study identifies LRPPRC as an important disease-causing gene in an early-onset, multisystem and neurological mitochondrial disease, which should be considered as a cause of COX deficiency even in patients originating outside of the French-Canadian population.
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http://dx.doi.org/10.1093/brain/awv291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4655343PMC
December 2015

Mutations causing mitochondrial disease: What is new and what challenges remain?

Science 2015 Sep 24;349(6255):1494-9. Epub 2015 Sep 24.

Wellcome Trust Centre for Mitochondrial Research, Institute for Cell and Molecular Biosciences and Institute of Neuroscience, Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, UK.

Mitochondrial diseases are among the most common and most complex of all inherited genetic diseases. The involvement of both the mitochondrial and nuclear genome presents unique challenges, but despite this there have been some remarkable advances in our knowledge of mitochondrial diseases over the past few years. A greater understanding of mitochondrial genetics has led to improved diagnosis as well as novel ways to prevent transmission of severe mitochondrial disease. These and other advances have had a major impact on patient care, but considerable challenges remain, particularly in the areas of therapies for those patients manifesting clinical symptoms associated with mitochondrial dysfunction and the tissue specificity seen in many mitochondrial disorders. This review highlights some important recent advances in mitochondrial disease but also stresses the areas where progress is essential.
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http://dx.doi.org/10.1126/science.aac7516DOI Listing
September 2015