Unique architectural features of mammalian mitochondrial protein synthesis.

Mitochondria rely on coordinated expression of their own mitochondrial DNA (mtDNA) with that of the nuclear genome for their biogenesis. The bacterial ancestry of mitochondria has given rise to unique and idiosyncratic features of the mtDNA and its expression machinery that can be specific to different organisms. In animals, the mitochondrial protein synthesis machinery has acquired many new components and mechanisms over evolution. These include several new ribosomal proteins, new stop codons and ways to recognise them, and new mechanisms to deliver nascent proteins into the mitochondrial inner membrane. Here we describe the mitochondrial protein synthesis machinery in mammals and its unique mechanisms of action elucidated to date and highlight the technologies poised to reveal the next generation of discoveries in mitochondrial translation.

Illuminating mitochondrial translation through mouse models.

Mitochondria are hubs of metabolic activity with a major role in ATP conversion by oxidative phosphorylation (OXPHOS). The mammalian mitochondrial genome encodes 11 mRNAs encoding 13 OXPHOS proteins along with 2 rRNAs and 22 tRNAs, that facilitate their translation on mitoribosomes. Maintaining the internal production of core OXPHOS subunits requires modulation of the mitochondrial capacity to match the cellular requirements and correct insertion of particularly hydrophobic proteins into the inner mitochondrial membrane. The mitochondrial translation system is essential for energy production and defects result in severe, phenotypically diverse diseases, including mitochondrial diseases that typically affect postmitotic tissues with high metabolic demands. Understanding the complex mechanisms that underlie the pathologies of diseases involving impaired mitochondrial translation is key to tailoring specific treatments and effectively targeting the affected organs. Disease mutations have provided a fundamental, yet limited, understanding of mitochondrial protein synthesis, since effective modification of the mitochondrial genome has proven challenging. However, advances in next generation sequencing, cryoelectron microscopy, and multi-omic technologies have revealed unexpected and unusual features of the mitochondrial protein synthesis machinery in the last decade. Genome editing tools have generated unique models that have accelerated our mechanistic understanding of mitochondrial translation and its physiological importance. Here we review the most recent mouse models of disease pathogenesis caused by defects in mitochondrial protein synthesis and discuss their value for preclinical research and therapeutic development.

Mutational rescue of the activity of high-fidelity Cas9 enzymes.

Programmable DNA endonucleases derived from bacterial genetic defense systems, exemplified by CRISPR-Cas9, have made it significantly easier to perform genomic modifications in living cells. However, unprogrammed, off-target modifications can have serious consequences, as they often disrupt the function or regulation of non-targeted genes and compromise the safety of therapeutic gene editing applications. High-fidelity mutants of Cas9 have been established to enable more accurate gene editing, but these are typically less efficient. Here, we merge the strengths of high-fidelity Cas9 and hyperactive Cas9 variants to provide an enzyme, which we dub HyperDriveCas9, that yields the desirable properties of both parents. HyperDriveCas9 functions efficiently in mammalian cells and introduces insertion and deletion mutations into targeted genomic regions while maintaining a favorable off-target profile. HyperDriveCas9 is a precise and efficient tool for gene editing applications in science and medicine.

Quantitative subcellular reconstruction reveals a lipid mediated inter-organelle biogenesis network.

The structures and functions of organelles in cells depend on each other but have not been systematically explored. We established stable knockout cell lines of peroxisomal, Golgi and endoplasmic reticulum genes identified in a whole-genome CRISPR knockout screen for inducers of mitochondrial biogenesis stress, showing that defects in peroxisome, Golgi and endoplasmic reticulum metabolism disrupt mitochondrial structure and function. Our quantitative total-organelle profiling approach for focussed ion beam scanning electron microscopy revealed in unprecedented detail that specific organelle dysfunctions precipitate multi-organelle biogenesis defects, impair mitochondrial morphology and reduce respiration. Multi-omics profiling showed a unified proteome response and global shifts in lipid and glycoprotein homeostasis that are elicited when organelle biogenesis is compromised, and that the resulting mitochondrial dysfunction can be rescued with precursors for ether-glycerophospholipid metabolic pathways. This work defines metabolic and morphological interactions between organelles and how their perturbation can cause disease.

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.

ATFS-1 counteracts mitochondrial DNA damage by promoting repair over transcription.

The ability to balance conflicting functional demands is critical for ensuring organismal survival. The transcription and repair of the mitochondrial genome (mtDNA) requires separate enzymatic activities that can sterically compete1, suggesting a life-long trade-off between these two processes. Here in Caenorhabditis elegans, we find that the bZIP transcription factor ATFS-1/Atf5 (refs. 2,3) regulates this balance in favour of mtDNA repair by localizing to mitochondria and interfering with the assembly of the mitochondrial pre-initiation transcription complex between HMG-5/TFAM and RPOM-1/mtRNAP. ATFS-1-mediated transcriptional inhibition decreases age-dependent mtDNA molecular damage through the DNA glycosylase NTH-1/NTH1, as well as the helicase TWNK-1/TWNK, resulting in an enhancement in the functional longevity of cells and protection against decline in animal behaviour caused by targeted and severe mtDNA damage. Together, our findings reveal that ATFS-1 acts as a molecular focal point for the control of balance between genome expression and maintenance in the mitochondria.

Molecular basis of translation termination at noncanonical stop codons in human mitochondria.

The genetic code that specifies the identity of amino acids incorporated into proteins during protein synthesis is almost universally conserved. Mitochondrial genomes feature deviations from the standard genetic code, including the reassignment of two arginine codons to stop codons. The protein required for translation termination at these noncanonical stop codons to release the newly synthesized polypeptides is not currently known. In this study, we used gene editing and ribosomal profiling in combination with cryo-electron microscopy to establish that mitochondrial release factor 1 (mtRF1) detects noncanonical stop codons in human mitochondria by a previously unknown mechanism of codon recognition. We discovered that binding of mtRF1 to the decoding center of the ribosome stabilizes a highly unusual conformation in the messenger RNA in which the ribosomal RNA participates in specific recognition of the noncanonical stop codons.

Digital RNase Footprinting of RNA-Protein Complexes and Ribosomes in Mitochondria.

RNA-binding proteins and mitochondrial ribosomes have been found to be linchpins of mitochondrial gene expression in health and disease. The expanding repertoire of proteins that bind and regulate the mitochondrial transcriptome has necessitated the development of new tools and methods to examine their molecular functions. Next-generation sequencing technologies have advanced the RNA biology field through application of high-throughput methods to study RNA-protein interactions. Here we describe a digital RNase footprinting method to analyze protein and ribosome interactions with mitochondrially encoded transcripts that provides insight into their mechanisms and minimal binding sites. We provide details on RNase digestion and next-generation sequencing, along with computational analyses and visualization of the binding targets within the mitochondrial transcriptome.

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.

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.

Frankenstein Cas9: engineering improved gene editing systems.

The discovery of CRISPR-Cas9 and its widespread use has revolutionised and propelled research in biological sciences. Although the ability to target Cas9's nuclease activity to specific sites via an easily designed guide RNA (gRNA) has made it an adaptable gene editing system, it has many characteristics that could be improved for use in biotechnology. Cas9 exhibits significant off-target activity and low on-target nuclease activity in certain contexts. Scientists have undertaken ambitious protein engineering campaigns to bypass these limitations, producing several promising variants of Cas9. Cas9 variants with improved and alternative activities provide exciting new tools to expand the scope and fidelity of future CRISPR applications.

ANGEL2 phosphatase activity is required for non-canonical mitochondrial RNA processing.

Canonical RNA processing in mammalian mitochondria is defined by tRNAs acting as recognition sites for nucleases to release flanking transcripts. The relevant factors, their structures, and mechanism are well described, but not all mitochondrial transcripts are punctuated by tRNAs, and their mode of processing has remained unsolved. Using Drosophila and mouse models, we demonstrate that non-canonical processing results in the formation of 3' phosphates, and that phosphatase activity by the carbon catabolite repressor 4 domain-containing family member ANGEL2 is required for their hydrolysis. Furthermore, our data suggest that members of the FAST kinase domain-containing protein family are responsible for these 3' phosphates. Our results therefore propose a mechanism for non-canonical RNA processing in metazoan mitochondria, by identifying the role of ANGEL2.

Computationally designed hyperactive Cas9 enzymes.

The ability to alter the genomes of living cells is key to understanding how genes influence the functions of organisms and will be critical to modify living systems for useful purposes. However, this promise has long been limited by the technical challenges involved in genetic engineering. Recent advances in gene editing have bypassed some of these challenges but they are still far from ideal. Here we use FuncLib to computationally design Cas9 enzymes with substantially higher donor-independent editing activities. We use genetic circuits linked to cell survival in yeast to quantify Cas9 activity and discover synergistic interactions between engineered regions. These hyperactive Cas9 variants function efficiently in mammalian cells and introduce larger and more diverse pools of insertions and deletions into targeted genomic regions, providing tools to enhance and expand the possible applications of CRISPR-based gene editing.

Organization and expression of the mammalian mitochondrial genome.

The mitochondrial genome encodes core subunits of the respiratory chain that drives oxidative phosphorylation and is, therefore, essential for energy conversion. Advances in high-throughput sequencing technologies and cryoelectron microscopy have shed light on the structure and organization of the mitochondrial genome and revealed unique mechanisms of mitochondrial gene regulation. New animal models of impaired mitochondrial protein synthesis have shown how the coordinated regulation of the cytoplasmic and mitochondrial translation machineries ensures the correct assembly of the respiratory chain complexes. These new technologies and disease models are providing a deeper understanding of mitochondrial genome organization and expression and of the diseases caused by impaired energy conversion, including mitochondrial, neurodegenerative, cardiovascular and metabolic diseases. They also provide avenues for the development of treatments for these conditions.

In silico evolution of nucleic acid-binding proteins from a nonfunctional scaffold.

Directed evolution emulates the process of natural selection to produce proteins with improved or altered functions. These approaches have proven to be very powerful but are technically challenging and particularly time and resource intensive. To bypass these limitations, we constructed a system to perform the entire process of directed evolution in silico. We employed iterative computational cycles of mutation and evaluation to predict mutations that confer high-affinity binding activities for DNA and RNA to an initial de novo designed protein with no inherent function. Beneficial mutations revealed modes of nucleic acid recognition not previously observed in natural proteins, highlighting the ability of computational directed evolution to access new molecular functions. Furthermore, the process by which new functions were obtained closely resembles natural evolution and can provide insights into the contributions of mutation rate, population size and selective pressure on functionalization of macromolecules in nature.

Deleterious variants in CRLS1 lead to cardiolipin deficiency and cause an autosomal recessive multi-system mitochondrial disease.

Mitochondrial diseases are a group of inherited diseases with highly varied and complex clinical presentations. Here, we report four individuals, including two siblings, affected by a progressive mitochondrial encephalopathy with biallelic variants in the cardiolipin biosynthesis gene CRLS1. Three affected individuals had a similar infantile presentation comprising progressive encephalopathy, bull's eye maculopathy, auditory neuropathy, diabetes insipidus, autonomic instability, cardiac defects and early death. The fourth affected individual presented with chronic encephalopathy with neurodevelopmental regression, congenital nystagmus with decreased vision, sensorineural hearing loss, failure to thrive and acquired microcephaly. Using patient-derived fibroblasts, we characterized cardiolipin synthase 1 (CRLS1) dysfunction that impaired mitochondrial morphology and biogenesis, providing functional evidence that the CRLS1 variants cause mitochondrial disease. Lipid profiling in fibroblasts from two patients further confirmed the functional defect demonstrating reduced cardiolipin levels, altered acyl-chain composition and significantly increased levels of phosphatidylglycerol, the substrate of CRLS1. Proteomic profiling of patient cells and mouse Crls1 knockout cell lines identified both endoplasmic reticular and mitochondrial stress responses, and key features that distinguish between varying degrees of cardiolipin insufficiency. These findings support that deleterious variants in CRLS1 cause an autosomal recessive mitochondrial disease, presenting as a severe encephalopathy with multi-systemic involvement. Furthermore, we identify key signatures in cardiolipin and proteome profiles across various degrees of cardiolipin loss, facilitating the use of omics technologies to guide future diagnosis of mitochondrial diseases.

The FASTK family proteins fine-tune mitochondrial RNA processing.

Transcription of the human mitochondrial genome and correct processing of the two long polycistronic transcripts are crucial for oxidative phosphorylation. According to the tRNA punctuation model, nucleolytic processing of these large precursor transcripts occurs mainly through the excision of the tRNAs that flank most rRNAs and mRNAs. However, some mRNAs are not punctuated by tRNAs, and it remains largely unknown how these non-canonical junctions are resolved. The FASTK family proteins are emerging as key players in non-canonical RNA processing. Here, we have generated human cell lines carrying single or combined knockouts of several FASTK family members to investigate their roles in non-canonical RNA processing. The most striking phenotypes were obtained with loss of FASTKD4 and FASTKD5 and with their combined double knockout. Comprehensive mitochondrial transcriptome analyses of these cell lines revealed a defect in processing at several canonical and non-canonical RNA junctions, accompanied by an increase in specific antisense transcripts. Loss of FASTKD5 led to the most severe phenotype with marked defects in mitochondrial translation of key components of the electron transport chain complexes and in oxidative phosphorylation. We reveal that the FASTK protein family members are crucial regulators of non-canonical junction and non-coding mitochondrial RNA processing.

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.