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.

Murine cytomegalovirus infection exacerbates complex IV deficiency in a model of mitochondrial disease.

The influence of environmental insults on the onset and progression of mitochondrial diseases is unknown. To evaluate the effects of infection on mitochondrial disease we used a mouse model of Leigh Syndrome, where a missense mutation in the Taco1 gene results in the loss of the translation activator of cytochrome c oxidase subunit I (TACO1) protein. The mutation leads to an isolated complex IV deficiency that mimics the disease pathology observed in human patients with TACO1 mutations. We infected Taco1 mutant and wild-type mice with a murine cytomegalovirus and show that a common viral infection exacerbates the complex IV deficiency in a tissue-specific manner. We identified changes in neuromuscular morphology and tissue-specific regulation of the mammalian target of rapamycin pathway in response to viral infection. Taken together, we report for the first time that a common stress condition, such as viral infection, can exacerbate mitochondrial dysfunction in a genetic model of mitochondrial disease.

Cardiolipin is required for membrane docking of mitochondrial ribosomes and protein synthesis.

The mitochondrial inner membrane contains a unique phospholipid known as cardiolipin (CL), which stabilises the protein complexes embedded in the membrane and supports its overall structure. Recent evidence indicates that the mitochondrial ribosome may associate with the inner membrane to facilitate co-translational insertion of the hydrophobic oxidative phosphorylation (OXPHOS) proteins into the inner membrane. We generated three mutant knockout cell lines for the CL biosynthesis gene Crls1 to investigate the effects of CL loss on mitochondrial protein synthesis. Reduced CL levels caused altered mitochondrial morphology and transcriptome-wide changes that were accompanied by uncoordinated mitochondrial translation rates and impaired respiratory chain supercomplex formation. Aberrant protein synthesis was caused by impaired formation and distribution of mitochondrial ribosomes. Reduction or loss of CL resulted in divergent mitochondrial and endoplasmic reticulum stress responses. We show that CL is required to stabilise the interaction of the mitochondrial ribosome with the membrane via its association with OXA1 (also known as OXA1L) during active translation. This interaction facilitates insertion of newly synthesised mitochondrial proteins into the inner membrane and stabilises the respiratory supercomplexes.

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.

Simultaneous processing and degradation of mitochondrial RNAs revealed by circularized RNA sequencing.

Mammalian mitochondrial RNAs are unique as they are derived from primary transcripts that encompass almost the entire mitochondrial genome. This necessitates extensive processing to release the individual mRNAs, rRNAs and tRNAs required for gene expression. Recent studies have revealed many of the proteins required for mitochondrial RNA processing, however the rapid turnover of precursor RNAs has made it impossible to analyze their composition and the hierarchy of processing. Here, we find that circularization of RNA prior to deep sequencing enables the discovery and characterization of unprocessed RNAs. Using this approach, we identify the most stable processing intermediates and the presence of intermediate processing products that are partially degraded and polyadenylated. Analysis of libraries constructed using RNA from mice lacking the nuclease subunit of the mitochondrial RNase P reveals the identities of stalled processing intermediates, their order of cleavage, and confirms the importance of RNase P in generating mature mitochondrial RNAs. Using RNA circularization prior to library preparation should provide a generally useful approach to studying RNA processing in many different biological systems.

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.

Multi-omic profiling reveals an RNA processing rheostat that predisposes to prostate cancer.

Prostate cancer is the most commonly diagnosed malignancy and the third leading cause of cancer deaths. GWAS have identified variants associated with prostate cancer susceptibility; however, mechanistic and functional validation of these mutations is lacking. We used CRISPR-Cas9 genome editing to introduce a missense variant identified in the ELAC2 gene, which encodes a dually localised nuclear and mitochondrial RNA processing enzyme, into the mouse Elac2 gene as well as to generate a prostate-specific knockout of Elac2. These mutations caused enlargement and inflammation of the prostate and nodule formation. The Elac2 variant or knockout mice on the background of the transgenic adenocarcinoma of the mouse prostate (TRAMP) model show that Elac2 mutation with a secondary genetic insult exacerbated the onset and progression of prostate cancer. Multiomic profiling revealed defects in energy metabolism that activated proinflammatory and tumorigenic pathways as a consequence of impaired noncoding RNA processing and reduced protein synthesis. Our physiologically relevant models show that the ELAC2 variant is a predisposing factor for prostate cancer and identify changes that underlie the pathogenesis of this cancer.

Copy number variation in tRNA isodecoder genes impairs mammalian development and balanced translation.

The number of tRNA isodecoders has increased dramatically in mammals, but the specific molecular and physiological reasons for this expansion remain elusive. To address this fundamental question we used CRISPR editing to knockout the seven-membered phenylalanine tRNA gene family in mice, both individually and combinatorially. Using ATAC-Seq, RNA-seq, ribo-profiling and proteomics we observed distinct molecular consequences of single tRNA deletions. We show that tRNA-Phe-1-1 is required for neuronal function and its loss is partially compensated by increased expression of other tRNAs but results in mistranslation. In contrast, the other tRNA-Phe isodecoder genes buffer the loss of each of the remaining six tRNA-Phe genes. In the tRNA-Phe gene family, the expression of at least six tRNA-Phe alleles is required for embryonic viability and tRNA-Phe-1-1 is most important for development and survival. Our results reveal that the multi-copy configuration of tRNA genes is required to buffer translation and viability in mammals.

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.

PTCD1 Is Required for 16S rRNA Maturation Complex Stability and Mitochondrial Ribosome Assembly.

The regulation of mitochondrial RNA life cycles and their roles in ribosome biogenesis and energy metabolism are not fully understood. We used CRISPR/Cas9 to generate heart- and skeletal-muscle-specific knockout mice of the pentatricopeptide repeat domain protein 1, PTCD1, and show that its loss leads to severe cardiomyopathy and premature death. Our detailed transcriptome-wide and functional analyses of these mice enabled us to identify the molecular role of PTCD1 as a 16S rRNA-binding protein essential for its stability, pseudouridylation, and correct biogenesis of the mitochondrial large ribosomal subunit. We show that impaired mitoribosome biogenesis can have retrograde signaling effects on nuclear gene expression through the transcriptional activation of the mTOR pathway and upregulation of cytoplasmic protein synthesis and pro-survival factors in the absence of mitochondrial translation. Taken together, our data show that impaired assembly of the mitoribosome exerts its consequences via differential regulation of mitochondrial and cytoplasmic protein synthesis.

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.

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.