Stress signaling and cellular proliferation reverse the effects of mitochondrial mistranslation.

Translation fidelity is crucial for prokaryotes and eukaryotic nuclear-encoded proteins; however, little is known about the role of mistranslation in mitochondria and its potential effects on metabolism. We generated yeast and mouse models with error-prone and hyper-accurate mitochondrial translation, and found that translation rate is more important than translational accuracy for cell function in mammals. Specifically, we found that mitochondrial mistranslation causes reduced overall mitochondrial translation and respiratory complex assembly rates. In mammals, this effect is compensated for by increased mitochondrial protein stability and upregulation of the citric acid cycle. Moreover, this induced mitochondrial stress signaling, which enables the recovery of mitochondrial translation via mitochondrial biogenesis, telomerase expression, and cell proliferation, and thereby normalizes metabolism. Conversely, we show that increased fidelity of mitochondrial translation reduces the rate of protein synthesis without eliciting a mitochondrial stress response. Consequently, the rate of translation cannot be recovered and this leads to dilated cardiomyopathy in mice. In summary, our findings reveal mammalian-specific signaling pathways that respond to changes in the fidelity of mitochondrial protein synthesis and affect metabolism.

Biallelic Mutations in MRPS34 Lead to Instability of the Small Mitoribosomal Subunit and Leigh Syndrome.

The synthesis of all 13 mitochondrial DNA (mtDNA)-encoded protein subunits of the human oxidative phosphorylation (OXPHOS) system is carried out by mitochondrial ribosomes (mitoribosomes). Defects in the stability of mitoribosomal proteins or mitoribosome assembly impair mitochondrial protein translation, causing combined OXPHOS enzyme deficiency and clinical disease. Here we report four autosomal-recessive pathogenic mutations in the gene encoding the small mitoribosomal subunit protein, MRPS34, in six subjects from four unrelated families with Leigh syndrome and combined OXPHOS defects. Whole-exome sequencing was used to independently identify all variants. Two splice-site mutations were identified, including homozygous c.321+1G>T in a subject of Italian ancestry and homozygous c.322-10G>A in affected sibling pairs from two unrelated families of Puerto Rican descent. In addition, compound heterozygous MRPS34 mutations were identified in a proband of French ancestry; a missense (c.37G>A [p.Glu13Lys]) and a nonsense (c.94C>T [p.Gln32∗]) variant. We demonstrated that these mutations reduce MRPS34 protein levels and the synthesis of OXPHOS subunits encoded by mtDNA. Examination of the mitoribosome profile and quantitative proteomics showed that the mitochondrial translation defect was caused by destabilization of the small mitoribosomal subunit and impaired monosome assembly. Lentiviral-mediated expression of wild-type MRPS34 rescued the defect in mitochondrial translation observed in skin fibroblasts from affected subjects, confirming the pathogenicity of MRPS34 mutations. Our data establish that MRPS34 is required for normal function of the mitoribosome in humans and furthermore demonstrate the power of quantitative proteomic analysis to identify signatures of defects in specific cellular pathways in fibroblasts from subjects with inherited disease.

Concerted regulation of mitochondrial and nuclear non-coding RNAs by a dual-targeted RNase Z.

The molecular roles of the dually targeted ElaC domain protein 2 (ELAC2) during nuclear and mitochondrial RNA processing in vivo have not been distinguished. We generated conditional knockout mice of ELAC2 to identify that it is essential for life and its activity is non-redundant. Heart and skeletal muscle-specific loss of ELAC2 causes dilated cardiomyopathy and premature death at 4 weeks. Transcriptome-wide analyses of total RNAs, small RNAs, mitochondrial RNAs, and miRNAs identified the molecular targets of ELAC2 in vivo We show that ELAC2 is required for processing of tRNAs and for the balanced maintenance of C/D box snoRNAs, miRNAs, and a new class of tRNA fragments. We identify that correct biogenesis of regulatory non-coding RNAs is essential for both cytoplasmic and mitochondrial protein synthesis and the assembly of mitochondrial ribosomes and cytoplasmic polysomes. We show that nuclear tRNA processing is required for the balanced production of snoRNAs and miRNAs for gene expression and that 3' tRNA processing is an essential step in the production of all mature mitochondrial RNAs and the majority of nuclear tRNAs.

Mutation in MRPS34 compromises protein synthesis and causes mitochondrial dysfunction.

The evolutionary divergence of mitochondrial ribosomes from their bacterial and cytoplasmic ancestors has resulted in reduced RNA content and the acquisition of mitochondria-specific proteins. The mitochondrial ribosomal protein of the small subunit 34 (MRPS34) is a mitochondria-specific ribosomal protein found only in chordates, whose function we investigated in mice carrying a homozygous mutation in the nuclear gene encoding this protein. The Mrps34 mutation causes a significant decrease of this protein, which we show is required for the stability of the 12S rRNA, the small ribosomal subunit and actively translating ribosomes. The synthesis of all 13 mitochondrially-encoded polypeptides is compromised in the mutant mice, resulting in reduced levels of mitochondrial proteins and complexes, which leads to decreased oxygen consumption and respiratory complex activity. The Mrps34 mutation causes tissue-specific molecular changes that result in heterogeneous pathology involving alterations in fractional shortening of the heart and pronounced liver dysfunction that is exacerbated with age. The defects in mitochondrial protein synthesis in the mutant mice are caused by destabilization of the small ribosomal subunit that affects the stability of the mitochondrial ribosome with age.

A bifunctional protein regulates mitochondrial protein synthesis.

Mitochondrial gene expression is predominantly regulated at the post-transcriptional level and mitochondrial ribonucleic acid (RNA)-binding proteins play a key role in RNA metabolism and protein synthesis. The AU-binding homolog of enoyl-coenzyme A (CoA) hydratase (AUH) is a bifunctional protein with RNA-binding activity and a role in leucine catabolism. AUH has a mitochondrial targeting sequence, however, its role in mitochondrial function has not been investigated. Here, we found that AUH localizes to the inner mitochondrial membrane and matrix where it associates with mitochondrial ribosomes and regulates protein synthesis. Decrease or overexpression of the AUH protein in cells causes defects in mitochondrial translation that lead to changes in mitochondrial morphology, decreased mitochondrial RNA stability, biogenesis and respiratory function. Because of its role in leucine metabolism, we investigated the importance of the catalytic activity of AUH and found that it affects the regulation of mitochondrial translation and biogenesis in response to leucine.

Mitochondrial gene expression is required for platelet function and blood clotting.

Platelets are anucleate blood cells that contain mitochondria and regulate blood clotting in response to injury. Mitochondria contain their own gene expression machinery that relies on nuclear-encoded factors for the biogenesis of the oxidative phosphorylation system to produce energy required for thrombosis. The autonomy of the mitochondrial gene expression machinery from the nucleus is unclear, and platelets provide a valuable model to understand its importance in anucleate cells. Here, we conditionally delete Elac2, Ptcd1, or Mtif3 in platelets, which are essential for mitochondrial gene expression at the level of RNA processing, stability, or translation, respectively. Loss of ELAC2, PTCD1, or MTIF3 leads to increased megakaryocyte ploidy, elevated circulating levels of reticulated platelets, thrombocytopenia, and consequent extended bleeding time. Impaired mitochondrial gene expression reduces agonist-induced platelet activation. Transcriptomic and proteomic analyses show that mitochondrial gene expression is required for fibrinolysis, hemostasis, and blood coagulation in response to injury.

Mitochondrial mistranslation modulated by metabolic stress causes cardiovascular disease and reduced lifespan.

Changes in the rate and fidelity of mitochondrial protein synthesis impact the metabolic and physiological roles of mitochondria. Here we explored how environmental stress in the form of a high-fat diet modulates mitochondrial translation and affects lifespan in mutant mice with error-prone (Mrps12ep/ep ) or hyper-accurate (Mrps12ha/ha ) mitochondrial ribosomes. Intriguingly, although both mutations are metabolically beneficial in reducing body weight, decreasing circulating insulin and increasing glucose tolerance during a high-fat diet, they manifest divergent (either deleterious or beneficial) outcomes in a tissue-specific manner. In two distinct organs that are commonly affected by the metabolic disease, the heart and the liver, Mrps12ep/ep mice were protected against heart defects but sensitive towards lipid accumulation in the liver, activating genes involved in steroid and amino acid metabolism. In contrast, enhanced translational accuracy in Mrps12ha/ha mice protected the liver from a high-fat diet through activation of liver proliferation programs, but enhanced the development of severe hypertrophic cardiomyopathy and led to reduced lifespan. These findings reflect the complex transcriptional and cell signalling responses that differ between post-mitotic (heart) and highly proliferative (liver) tissues. We show trade-offs between the rate and fidelity of mitochondrial protein synthesis dictate tissue-specific outcomes due to commonly encountered stressful environmental conditions or aging.

A common genetic variant of a mitochondrial RNA processing enzyme predisposes to insulin resistance.

Mitochondrial energy metabolism plays an important role in the pathophysiology of insulin resistance. Recently, a missense N437S variant was identified in the MRPP3 gene, which encodes a mitochondrial RNA processing enzyme within the RNase P complex, with predicted impact on metabolism. We used CRISPR-Cas9 genome editing to introduce this variant into the mouse Mrpp3 gene and show that the variant causes insulin resistance on a high-fat diet. The variant did not influence mitochondrial gene expression markedly, but instead, it reduced mitochondrial calcium that lowered insulin release from the pancreatic islet ß cells of the Mrpp3 variant mice. Reduced insulin secretion resulted in lower insulin levels that contributed to imbalanced metabolism and liver steatosis in the Mrpp3 variant mice on a high-fat diet. Our findings reveal that the MRPP3 variant may be a predisposing factor to insulin resistance and metabolic disease in the human population.

Reduced mitochondrial translation prevents diet-induced metabolic dysfunction but not inflammation.

The contribution of dysregulated mitochondrial gene expression and consequent imbalance in biogenesis is not well understood in metabolic disorders such as insulin resistance and obesity. The ribosomal RNA maturation protein PTCD1 is essential for mitochondrial protein synthesis and its reduction causes adult-onset obesity and liver steatosis. We used haploinsufficient Ptcd1 mice fed normal or high fat diets to understand how changes in mitochondrial biogenesis can lead to metabolic dysfunction. We show that Akt-stimulated reduction in lipid content and upregulation of mitochondrial biogenesis effectively protected mice with reduced mitochondrial protein synthesis from excessive weight gain on a high fat diet, resulting in improved glucose and insulin tolerance and reduced lipid accumulation in the liver. However, inflammation of the white adipose tissue and early signs of fibrosis in skeletal muscle, as a consequence of reduced protein synthesis, were exacerbated with the high fat diet. We identify that reduced mitochondrial protein synthesis and OXPHOS biogenesis can be recovered in a tissue-specific manner via Akt-mediated increase in insulin sensitivity and transcriptional activation of the mitochondrial stress response.

Fidelity of translation initiation is required for coordinated respiratory complex assembly.

Mammalian mitochondrial ribosomes are unique molecular machines that translate 11 leaderless mRNAs; however, it is not clear how mitoribosomes initiate translation, since mitochondrial mRNAs lack untranslated regions. Mitochondrial translation initiation shares similarities with prokaryotes, such as the formation of a ternary complex of fMet-tRNAMet, mRNA and the 28S subunit, but differs in the requirements for initiation factors. Mitochondria have two initiation factors: MTIF2, which closes the decoding center and stabilizes the binding of the fMet-tRNAMet to the leaderless mRNAs, and MTIF3, whose role is not clear. We show that MTIF3 is essential for survival and that heart- and skeletal muscle-specific loss of MTIF3 causes cardiomyopathy. We identify increased but uncoordinated mitochondrial protein synthesis in mice lacking MTIF3, resulting in loss of specific respiratory complexes. Ribosome profiling shows that MTIF3 is required for recognition and regulation of translation initiation of mitochondrial mRNAs and for coordinated assembly of OXPHOS complexes in vivo.

Tighter Ligand Binding Can Compensate for Impaired Stability of an RNA-Binding Protein.

It has been widely shown that ligand-binding residues, by virtue of their orientation, charge, and solvent exposure, often have a net destabilizing effect on proteins that is offset by stability conferring residues elsewhere in the protein. This structure-function trade-off can constrain possible adaptive evolutionary changes of function and may hamper protein engineering efforts to design proteins with new functions. Here, we present evidence from a large randomized mutant library screen that, in the case of PUF RNA-binding proteins, this structural relationship may be inverted and that active-site mutations that increase protein activity are also able to compensate for impaired stability. We show that certain mutations in RNA-protein binding residues are not necessarily destabilizing and that increased ligand-binding can rescue an insoluble, unstable PUF protein. We hypothesize that these mutations restabilize the protein via thermodynamic coupling of protein folding and RNA binding.

Adult-onset obesity is triggered by impaired mitochondrial gene expression.

Mitochondrial gene expression is essential for energy production; however, an understanding of how it can influence physiology and metabolism is lacking. Several proteins from the pentatricopeptide repeat (PPR) family are essential for the regulation of mitochondrial gene expression, but the functions of the remaining members of this family are poorly understood. We created knockout mice to investigate the role of the PPR domain 1 (PTCD1) protein and show that loss of PTCD1 is embryonic lethal, whereas haploinsufficient, heterozygous mice develop age-induced obesity. The molecular defects and metabolic consequences of mitochondrial protein haploinsufficiency in vivo have not been investigated previously. We show that PTCD1 haploinsufficiency results in increased RNA metabolism, in response to decreased protein synthesis and impaired RNA processing that affect the biogenesis of the respiratory chain, causing mild uncoupling and changes in mitochondrial morphology. We demonstrate that with age, these effects lead to adult-onset obesity that results in liver steatosis and cardiac hypertrophy in response to tissue-specific differential regulation of the mammalian target of rapamycin pathways. Our findings indicate that changes in mitochondrial gene expression have long-term consequences on energy metabolism, providing evidence that haploinsufficiency of PTCD1 can be a major predisposing factor for the development of metabolic syndrome.

Loss of the RNA-binding protein TACO1 causes late-onset mitochondrial dysfunction in mice.

The recognition and translation of mammalian mitochondrial mRNAs are poorly understood. To gain further insights into these processes in vivo, we characterized mice with a missense mutation that causes loss of the translational activator of cytochrome oxidase subunit I (TACO1). We report that TACO1 is not required for embryonic survival, although the mutant mice have substantially reduced COXI protein, causing an isolated complex IV deficiency. We show that TACO1 specifically binds the mt-Co1 mRNA and is required for translation of COXI through its association with the mitochondrial ribosome. We determined the atomic structure of TACO1, revealing three domains in the shape of a hook with a tunnel between domains 1 and 3. Mutations in the positively charged domain 1 reduce RNA binding by TACO1. The Taco1 mutant mice develop a late-onset visual impairment, motor dysfunction and cardiac hypertrophy and thus provide a useful model for future treatment trials for mitochondrial disease.

The Role of the L-Type Ca2+ Channel in Altered Metabolic Activity in a Murine Model of Hypertrophic Cardiomyopathy.

Heterozygous mice (αMHC403/+ ) expressing the human disease-causing mutation Arg403Gln exhibit cardinal features of hypertrophic cardiomyopathy (HCM) including hypertrophy, myocyte disarray, and increased myocardial fibrosis. Treatment of αMHC403/+ mice with the L-type calcium channel (ICa-L) antagonist diltiazem has been shown to decrease left ventricular anterior wall thickness, cardiac myocyte hypertrophy, disarray, and fibrosis. However, the role of the ICa-L in the development of HCM is not known. In addition to maintaining cardiac excitation and contraction in myocytes, the ICa-L also regulates mitochondrial function through transmission of movement of ICa-L via cytoskeletal proteins to mitochondrial voltage-dependent anion channel. Here, the authors investigated the role of ICa-L in regulating mitochondrial function in αMHC403/+ mice. Whole-cell patch clamp studies showed that ICa-L current inactivation kinetics were significantly increased in αMHC403/+ cardiac myocytes, but that current density and channel expression were similar to wild-type cardiac myocytes. Activation of ICa-L caused a significantly greater increase in mitochondrial membrane potential and metabolic activity in αMHC403/+ . These increases were attenuated with ICa-L antagonists and following F-actin or ß-tubulin depolymerization. The authors observed increased levels of fibroblast growth factor-21 in αMHC403/+ mice, and altered mitochondrial DNA copy number consistent with altered mitochondrial activity and the development of cardiomyopathy. These studies suggest that the Arg403Gln mutation leads to altered functional communication between ICa-L and mitochondria that is associated with increased metabolic activity, which may contribute to the development of cardiomyopathy. ICa-L antagonists may be effective in reducing the cardiomyopathy in HCM by altering metabolic activity.

Hierarchical RNA Processing Is Required for Mitochondrial Ribosome Assembly.

The regulation of mitochondrial RNA processing and its importance for ribosome biogenesis and energy metabolism are not clear. We generated conditional knockout mice of the endoribonuclease component of the RNase P complex, MRPP3, and report that it is essential for life and that heart and skeletal-muscle-specific knockout leads to severe cardiomyopathy, indicating that its activity is non-redundant. Transcriptome-wide parallel analyses of RNA ends (PARE) and RNA-seq enabled us to identify that in vivo 5' tRNA cleavage precedes 3' tRNA processing, and this is required for the correct biogenesis of the mitochondrial ribosomal subunits. We identify that mitoribosomal biogenesis proceeds co-transcriptionally because large mitoribosomal proteins can form a subcomplex on an unprocessed RNA containing the 16S rRNA. Taken together, our data show that RNA processing links transcription to translation via assembly of the mitoribosome.

A mutation in MT-TW causes a tRNA processing defect and reduced mitochondrial function in a family with Leigh syndrome.

Leigh syndrome (LS) is a progressive mitochondrial neurodegenerative disorder, whose symptoms most commonly include psychomotor delay with regression, lactic acidosis and a failure to thrive. Here we describe three siblings with LS, but with additional manifestations including hypertrophic cardiomyopathy, hepatosplenomegaly, cholestatic hepatitis, and seizures. All three affected siblings were found to be homoplasmic for an m. 5559A>G mutation in the T stem of the mitochondrial DNA-encoded MT-TW by next generation sequencing. The m.5559A>G mutation causes a reduction in the steady state levels of tRNA(Trp) and this decrease likely affects the stability of other mitochondrial RNAs in the patient fibroblasts. We observe accumulation of an unprocessed transcript containing tRNA(Trp), decreased de novo protein synthesis and consequently lowered steady state levels of mitochondrial DNA-encoded proteins that compromise mitochondrial respiration. Our results show that the m.5559A>G mutation at homoplasmic levels causes LS in association with severe multi-organ disease (LS-plus) as a consequence of dysfunctional mitochondrial RNA metabolism.

Mitochondria: Unusual features of the mammalian mitoribosome.

Mitochondria are responsible for generating most of the energy required by the cell. The oxidative phosphorylation (OXPHOS) system that produces the energy is composed of nuclear and mitochondrial encoded polypeptides. The 13 polypeptides encoded by the mitochondrial genome are synthesized by mitochondrial ribosomes (mitoribosomes). The evolutionary divergence of mitoribosomes has seen a reduction in their rRNA content and an increase in ribosomal proteins compared to their bacterial and cytoplasmic counterparts. Recent advances in cryo-electron microscopy (cryo-EM) mapping have revealed not all of these proteins simply replace the roles of the rRNA and that many have new roles. The mitoribosome has unique features that include a gatelike structure at the mRNA entrance that may facilitate recruitment of leaderless mitochondrial mRNAs and also a polypeptide exit tunnel that has an unusual nascent-polypeptide exit mechanism. Defects in the mitochondrial translation machinery are a common contributor to multi-system disorders known as mitochondrial diseases for which currently there are no cures or effective treatments.

Evidence for age-related and individual-specific changes in DNA methylation profile of mononuclear cells during early immune development in humans.

Environment induced epigenetic effects on gene expression in early life are likely to play important roles in mediating the risk of several immune-related diseases. In order to investigate this fully, it is essential to first document temporal changes in epigenetic profile in disease-free individuals as a prelude to defining environmentally mediated changes. Mononuclear cells (MC) were collected longitudinally from a small number of females at birth, 1 year, 2.5 years and 5 years of age and examined for changes in genome-scale DNA methylation profiles using the Illumina Infinium HumanMethylation27 BeadChip array platform. MC from two males were included for comparative purposes. Flow cytometry was used to define MC cell populations in each sample in order to exclude this as the major driver of epigenetic change. The data underwent quality control and normalization within the R programming environment. Unsupervised hierarchical clustering of samples clearly delineated neonatal MC from all other ages. A further clear distinction was observed between 1 year and 5 year samples, with 2.5 year samples showing a mixed distribution between the 1 and 5 year groups. Gene ontology of probes significantly variable over the neonatal period revealed methylation changes in genes associated with cell surface receptor and signal transduction events. In the postnatal period, methylation changes were mostly associated with the development of effector immune responses and homeostasis. Unlike all other chromosomes tested, a predominantly genetic effect was identified as controlling maintenance of X-chromosome methylation profile in females, largely refractory to change over time. This data suggests that the primary driver of neonatal epigenome is determined in utero, whilst postnatally, multiple genetic and environmental factors are implicated in the development of MC epigenetic profile, particularly between the ages of 1-5 years, when the highest level of inter individual variation is apparent. This supports a model for differential sensitivity of specific individuals to disruption in the developing epigenome during the first years of life. Further studies are now needed to examine evolving epigenetic variations in specific cell populations in relation to environmental exposures, immune phenotype and subsequent disease susceptibility.

RNA processing in human mitochondria.

Mammalian mitochondrial DNA is transcribed as precursor polycistronic transcripts containing 13 mRNAs, 2 rRNAs, punctuated by 22 tRNAs. The mechanisms involved in the excision of mitochondrial tRNAs from these polycistronic transcripts have remained largely unknown. We have investigated the roles of ELAC2, mitochondrial RNase P proteins 1 and 3, and pentatricopeptide repeat domain protein 1 in the processing of mitochondrial polycistronic transcripts. We used a deep sequencing approach to characterize the 5' and 3' ends of processed mitochondrial transcripts and provide a detailed map of mitochondrial tRNA processing sites affected by these proteins. We show that MRPP1 and MRPP3 process the 5' ends of tRNAs and the 5' unconventional, non tRNA containing site of the CO1 transcript. By contrast, we find that ELAC2 and PTCD1 affect the 3' end processing of tRNAs. Finally, we found that MRPP1 is essential for transcript processing, RNA modification, translation and mitochondrial respiration.