Hypoxia and intra-complex genetic suppressors rescue complex I mutants by a shared mechanism.

The electron transport chain (ETC) of mitochondria, bacteria, and archaea couples electron flow to proton pumping and is adapted to diverse oxygen environments. Remarkably, in mice, neurological disease due to ETC complex I dysfunction is rescued by hypoxia through unknown mechanisms. Here, we show that hypoxia rescue and hyperoxia sensitivity of complex I deficiency are evolutionarily conserved to C. elegans and are specific to mutants that compromise the electron-conducting matrix arm. We show that hypoxia rescue does not involve the hypoxia-inducible factor pathway or attenuation of reactive oxygen species. To discover the mechanism, we use C. elegans genetic screens to identify suppressor mutations in the complex I accessory subunit NDUFA6/nuo-3 that phenocopy hypoxia rescue. We show that NDUFA6/nuo-3(G60D) or hypoxia directly restores complex I forward activity, with downstream rescue of ETC flux and, in some cases, complex I levels. Additional screens identify residues within the ubiquinone binding pocket as being required for the rescue by NDUFA6/nuo-3(G60D) or hypoxia. This reveals oxygen-sensitive coupling between an accessory subunit and the quinone binding pocket of complex I that can restore forward activity in the same manner as hypoxia.

Recovering from the stress of the COVID-19 pandemic.

For our special issue on stress, we asked scientists about recovering from the stress of the pandemic, including some who shared insights with us in mid-2020. They discuss the importance of teamwork, reassessing priorities, and the added stresses of the cost-of-living crisis, funding cuts, and retaining scientists in academia.

Loss of LUC7L2 and U1 snRNP subunits shifts energy metabolism from glycolysis to OXPHOS.

Oxidative phosphorylation (OXPHOS) and glycolysis are the two major pathways for ATP production. The reliance on each varies across tissues and cell states, and can influence susceptibility to disease. At present, the full set of molecular mechanisms governing the relative expression and balance of these two pathways is unknown. Here, we focus on genes whose loss leads to an increase in OXPHOS activity. Unexpectedly, this class of genes is enriched for components of the pre-mRNA splicing machinery, in particular for subunits of the U1 snRNP. Among them, we show that LUC7L2 represses OXPHOS and promotes glycolysis by multiple mechanisms, including (1) splicing of the glycolytic enzyme PFKM to suppress glycogen synthesis, (2) splicing of the cystine/glutamate antiporter SLC7A11 (xCT) to suppress glutamate oxidation, and (3) secondary repression of mitochondrial respiratory supercomplex formation. Our results connect LUC7L2 expression and, more generally, the U1 snRNP to cellular energy metabolism.

MitoCarta3.0: an updated mitochondrial proteome now with sub-organelle localization and pathway annotations.

The mammalian mitochondrial proteome is under dual genomic control, with 99% of proteins encoded by the nuclear genome and 13 originating from the mitochondrial DNA (mtDNA). We previously developed MitoCarta, a catalogue of over 1000 genes encoding the mammalian mitochondrial proteome. This catalogue was compiled using a Bayesian integration of multiple sequence features and experimental datasets, notably protein mass spectrometry of mitochondria isolated from fourteen murine tissues. Here, we introduce MitoCarta3.0. Beginning with the MitoCarta2.0 inventory, we performed manual review to remove 100 genes and introduce 78 additional genes, arriving at an updated inventory of 1136 human genes. We now include manually curated annotations of sub-mitochondrial localization (matrix, inner membrane, intermembrane space, outer membrane) as well as assignment to 149 hierarchical 'MitoPathways' spanning seven broad functional categories relevant to mitochondria. MitoCarta3.0, including sub-mitochondrial localization and MitoPathway annotations, is freely available at http://www.broadinstitute.org/mitocarta and should serve as a continued community resource for mitochondrial biology and medicine.

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

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.

FASTKD1 and FASTKD4 have opposite effects on expression of specific mitochondrial RNAs, depending upon their endonuclease-like RAP domain.

FASTK family proteins have been identified as regulators of mitochondrial RNA homeostasis linked to mitochondrial diseases, but much remains unknown about these proteins. We show that CRISPR-mediated disruption of FASTKD1 increases ND3 mRNA level, while disruption of FASTKD4 reduces the level of ND3 and of other mature mRNAs including ND5 and CYB, and causes accumulation of ND5-CYB precursor RNA. Disrupting both FASTKD1 and FASTKD4 in the same cell results in decreased ND3 mRNA similar to the effect of depleting FASTKD4 alone, indicating that FASTKD4 loss is epistatic. Interestingly, very low levels of FASTKD4 are sufficient to prevent ND3 loss and ND5-CYB precursor accumulation, suggesting that FASTKD4 may act catalytically. Furthermore, structural modeling predicts that each RAP domain of FASTK proteins contains a nuclease fold with a conserved aspartate residue at the putative active site. Accordingly, mutation of this residue in FASTKD4 abolishes its function. Experiments with FASTK chimeras indicate that the RAP domain is essential for the function of the FASTK proteins, while the region upstream determines RNA targeting and protein localization. In conclusion, this paper identifies new aspects of FASTK protein biology and suggests that the RAP domain function depends on an intrinsic nucleolytic activity.

The FASTK family of proteins: emerging regulators of mitochondrial RNA biology.

The FASTK family proteins have recently emerged as key post-transcriptional regulators of mitochondrial gene expression. FASTK, the founding member and its homologs FASTKD1-5 are architecturally related RNA-binding proteins, each having a different function in the regulation of mitochondrial RNA biology, from mRNA processing and maturation to ribosome assembly and translation. In this review, we outline the structure, evolution and function of these FASTK proteins and discuss the individual role that each has in mitochondrial RNA biology. In addition, we highlight the aspects of FASTK research that still require more attention.

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

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-tRNAMet, and mt-tRNAPhe, 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.

CLIC, a tool for expanding biological pathways based on co-expression across thousands of datasets.

In recent years, there has been a huge rise in the number of publicly available transcriptional profiling datasets. These massive compendia comprise billions of measurements and provide a special opportunity to predict the function of unstudied genes based on co-expression to well-studied pathways. Such analyses can be very challenging, however, since biological pathways are modular and may exhibit co-expression only in specific contexts. To overcome these challenges we introduce CLIC, CLustering by Inferred Co-expression. CLIC accepts as input a pathway consisting of two or more genes. It then uses a Bayesian partition model to simultaneously partition the input gene set into coherent co-expressed modules (CEMs), while assigning the posterior probability for each dataset in support of each CEM. CLIC then expands each CEM by scanning the transcriptome for additional co-expressed genes, quantified by an integrated log-likelihood ratio (LLR) score weighted for each dataset. As a byproduct, CLIC automatically learns the conditions (datasets) within which a CEM is operative. We implemented CLIC using a compendium of 1774 mouse microarray datasets (28628 microarrays) or 1887 human microarray datasets (45158 microarrays). CLIC analysis reveals that of 910 canonical biological pathways, 30% consist of strongly co-expressed gene modules for which new members are predicted. For example, CLIC predicts a functional connection between protein C7orf55 (FMC1) and the mitochondrial ATP synthase complex that we have experimentally validated. CLIC is freely available at www.gene-clic.org. We anticipate that CLIC will be valuable both for revealing new components of biological pathways as well as the conditions in which they are active.

Role of FAST Kinase Domains 3 (FASTKD3) in Post-transcriptional Regulation of Mitochondrial Gene Expression.

The Fas-activated serine/threonine kinase (FASTK) family of proteins has recently emerged as a central regulator of mitochondrial gene expression through the function of an unusual RNA-binding domain named RAP (for RNA-binding domain abundant in Apicomplexans), shared by all six members of the family. Here we describe the role of one of the less characterized members, FASTKD3, in mitochondrial RNA metabolism. First, we show that, in contrast to FASTK, FASTKD2, and FASTKD5, FASTKD3 does not localize in mitochondrial RNA granules, which are sites of processing and maturation of mtRNAs and ribosome biogenesis. Second, we generated FASTKD3 homozygous knock-out cell lines by homologous recombination and observed that the absence of FASTKD3 resulted in increased steady-state levels and half-lives of a subset of mature mitochondrial mRNAs: ND2, ND3, CYTB, COX2, and ATP8/6. No aberrant processing of RNA precursors was observed. Rescue experiments demonstrated that RAP domain is required for FASTKD3 function in mRNA stability. Besides, we describe that FASTKD3 is required for efficient COX1 mRNA translation without altering mRNA levels, which results in a decrease in the steady-state levels of COX1 protein. This finding is associated with reduced mitochondrial complex IV assembly and activity. Our observations suggest that the function of this family of proteins goes beyond RNA processing and ribosome assembly and includes RNA stability and translation regulation within mitochondria.

Diabetogenic milieus induce specific changes in mitochondrial transcriptome and differentiation of human pancreatic islets.

In pancreatic ß-cells, mitochondria play a central role in coupling glucose metabolism to insulin secretion. Chronic exposure of ß-cells to metabolic stresses impairs their function and potentially induces apoptosis. Little is known on mitochondrial adaptation to metabolic stresses, i.e. high glucose, fatty acids or oxidative stress; being all highlighted in the pathogenesis of type 2 diabetes. Here, human islets were exposed for 3 days to 25 mm glucose, 0.4 mm palmitate, 0.4 mm oleate and transiently to H2O2. Culture at physiological 5.6 mm glucose served as no-stress control. Expression of mitochondrion-associated genes was quantified, including the transcriptome of mitochondrial inner membrane carriers. Targets of interest were further evaluated at the protein level. Three days after acute oxidative stress, no significant alteration in ß-cell function or apoptosis was detected in human islets. Palmitate specifically increased expression of the pyruvate carriers MPC1 and MPC2, whereas the glutamate carrier GC1 and the aspartate/glutamate carrier AGC1 were down-regulated by palmitate and oleate, respectively. High glucose decreased mRNA levels of key transcription factors (HNF4A, IPF1, PPARA and TFAM) and energy-sensor SIRT1. High glucose also reduced expression of 11 mtDNA-encoded respiratory chain subunits. Interestingly, transcript levels of the carriers for aspartate/glutamate AGC2, malate DIC and malate/oxaloacetate/aspartate UCP2 were increased by high glucose, a profile suggesting important mitochondrial anaplerotic/cataplerotic activities and NADPH-generating shuttles. Chronic exposure to high glucose impaired glucose-stimulated insulin secretion, decreased insulin content, promoted caspase-3 cleavage and cell death, revealing glucotoxicity. Overall, expression profile of mitochondrion-associated genes was selectively modified by glucose, delineating a glucotoxic-specific signature.

A human mitochondrial poly(A) polymerase mutation reveals the complexities of post-transcriptional mitochondrial gene expression.

The p.N478D missense mutation in human mitochondrial poly(A) polymerase (mtPAP) has previously been implicated in a form of spastic ataxia with optic atrophy. In this study, we have investigated fibroblast cell lines established from family members. The homozygous mutation resulted in the loss of polyadenylation of all mitochondrial transcripts assessed; however, oligoadenylation was retained. Interestingly, this had differential effects on transcript stability that were dependent on the particular species of transcript. These changes were accompanied by a severe loss of oxidative phosphorylation complexes I and IV, and perturbation of de novo mitochondrial protein synthesis. Decreases in transcript polyadenylation and in respiratory chain complexes were effectively rescued by overexpression of wild-type mtPAP. Both mutated and wild-type mtPAP localized to the mitochondrial RNA-processing granules thereby eliminating mislocalization as a cause of defective polyadenylation. In vitro polyadenylation assays revealed severely compromised activity by the mutated protein, which generated only short oligo(A) extensions on RNA substrates, irrespective of RNA secondary structure. The addition of LRPPRC/SLIRP, a mitochondrial RNA-binding complex, enhanced activity of the wild-type mtPAP resulting in increased overall tail length. The LRPPRC/SLIRP effect although present was less marked with mutated mtPAP, independent of RNA secondary structure. We conclude that (i) the polymerase activity of mtPAP can be modulated by the presence of LRPPRC/SLIRP, (ii) N478D mtPAP mutation decreases polymerase activity and (iii) the alteration in poly(A) length is sufficient to cause dysregulation of post-transcriptional expression and the pathogenic lack of respiratory chain complexes.

Nucleosides are overlooked fuels in central carbon metabolism.

From our daily nutrition and synthesis within cells, nucleosides enter the bloodstream and circulate throughout the body and tissues. Nucleosides and nucleotides are classically viewed as precursors of nucleic acids, but recently they have emerged as a novel energy source for central carbon metabolism. Through catabolism by nucleoside phosphorylases, the ribose sugar group is released and can provide substrates for lower steps in glycolysis. In environments with limited glucose, such as at sites of infection or in the tumor microenvironment (TME), cells can use, and may even require, this alternative energy source. Here, we discuss the implications of these new findings in health and disease and speculate on the potential new roles of nucleosides and nucleic acids in energy metabolism.

Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification.

Most physiological and disease processes, from central metabolism to immune response to neurodegeneration, involve mitochondria. The mitochondrial proteome is composed of more than 1,000 proteins, and the abundance of each can vary dynamically in response to external stimuli or during disease progression. Here, we describe a protocol for isolating high-quality mitochondria from primary cells and tissues. The two-step procedure comprises (1) mechanical homogenization and differential centrifugation to isolate crude mitochondria, and (2) tag-free immune capture of mitochondria to isolate pure organelles and eliminate contaminants. Mitochondrial proteins from each purification stage are analyzed by quantitative mass spectrometry, and enrichment yields are calculated, allowing the discovery of novel mitochondrial proteins by subtractive proteomics. Our protocol provides a sensitive and comprehensive approach to studying mitochondrial content in cell lines, primary cells, and tissues.

Dead-Seq: Discovering Synthetic Lethal Interactions from Dead Cells Genomics.

Pooled genetic screens have revolutionized the field of functional genomics, yet perturbations that decrease fitness, such as those leading to synthetic lethality, have remained difficult to quantify at the genomic level. We and colleagues previously developed "death screening," a protocol based on the purification of dead cells in genetic screens, and used it to identify a set of genes necessary for mitochondrial gene expression, translation, and oxidative phosphorylation (OXPHOS), thus offering new possibilities for the diagnosis of mitochondrial disorders. Here, we describe Dead-Seq, a refined protocol for death screening that is compatible with most pooled screening protocols, including genome-wide CRISPR/Cas9 screening. Dead-Seq converts negative-selection screens into positive-selection screens and generates high-quality data directly from dead cells, at limited sequencing costs.

Salvage of ribose from uridine or RNA supports glycolysis in nutrient-limited conditions.

Glucose is vital for life, serving as both a source of energy and carbon building block for growth. When glucose is limiting, alternative nutrients must be harnessed. To identify mechanisms by which cells can tolerate complete loss of glucose, we performed nutrient-sensitized genome-wide genetic screens and a PRISM growth assay across 482 cancer cell lines. We report that catabolism of uridine from the medium enables the growth of cells in the complete absence of glucose. While previous studies have shown that uridine can be salvaged to support pyrimidine synthesis in the setting of mitochondrial oxidative phosphorylation deficiency1, our work demonstrates that the ribose moiety of uridine or RNA can be salvaged to fulfil energy requirements via a pathway based on: (1) the phosphorylytic cleavage of uridine by uridine phosphorylase UPP1/UPP2 into uracil and ribose-1-phosphate (R1P), (2) the conversion of uridine-derived R1P into fructose-6-P and glyceraldehyde-3-P by the non-oxidative branch of the pentose phosphate pathway and (3) their glycolytic utilization to fuel ATP production, biosynthesis and gluconeogenesis. Capacity for glycolysis from uridine-derived ribose appears widespread, and we confirm its activity in cancer lineages, primary macrophages and mice in vivo. An interesting property of this pathway is that R1P enters downstream of the initial, highly regulated steps of glucose transport and upper glycolysis. We anticipate that 'uridine bypass' of upper glycolysis could be important in the context of disease and even exploited for therapeutic purposes.

The mitochondrial succinate dehydrogenase complex controls the STAT3-IL-10 pathway in inflammatory macrophages.

The functions of macrophages are tightly regulated by their metabolic state. However, the role of the mitochondrial electron transport chain (ETC) in macrophage functions remains understudied. Here, we provide evidence that the succinate dehydrogenase (SDH)/complex II (CII) is required for respiration and plays a role in controlling effector responses in macrophages. We find that the absence of the catalytic subunits Sdha and Sdhb in macrophages impairs their ability to effectively stabilize HIF-1α and produce the pro-inflammatory cytokine IL-1ß in response to LPS stimulation. We also arrive at the novel result that both subunits are essential for the LPS-driven production of IL-10, a potent negative feedback regulator of the macrophage inflammatory response. This phenomenon is explained by the fact that the absence of Sdha and Sdhb leads to the inhibition of Stat3 tyrosine phosphorylation, caused partially by the excessive accumulation of mitochondrial reactive oxygen species (mitoROS) in the knockout cells.

Distinct mitochondrial defects trigger the integrated stress response depending on the metabolic state of the cell.

Mitochondrial dysfunction is associated with activation of the integrated stress response (ISR) but the underlying triggers remain unclear. We systematically combined acute mitochondrial inhibitors with genetic tools for compartment-specific NADH oxidation to trace mechanisms linking different forms of mitochondrial dysfunction to the ISR in proliferating mouse myoblasts and in differentiated myotubes. In myoblasts, we find that impaired NADH oxidation upon electron transport chain (ETC) inhibition depletes asparagine, activating the ISR via the eIF2α kinase GCN2. In myotubes, however, impaired NADH oxidation following ETC inhibition neither depletes asparagine nor activates the ISR, reflecting an altered metabolic state. ATP synthase inhibition in myotubes triggers the ISR via a distinct mechanism related to mitochondrial inner-membrane hyperpolarization. Our work dispels the notion of a universal path linking mitochondrial dysfunction to the ISR, instead revealing multiple paths that depend both on the nature of the mitochondrial defect and on the metabolic state of the cell.

Mitochondrial Reprogramming Underlies Resistance to BCL-2 Inhibition in Lymphoid Malignancies.

Mitochondrial apoptosis can be effectively targeted in lymphoid malignancies with the FDA-approved B cell lymphoma 2 (BCL-2) inhibitor venetoclax, but resistance to this agent is emerging. We show that venetoclax resistance in chronic lymphocytic leukemia is associated with complex clonal shifts. To identify determinants of resistance, we conducted parallel genome-scale screens of the BCL-2-driven OCI-Ly1 lymphoma cell line after venetoclax exposure along with integrated expression profiling and functional characterization of drug-resistant and engineered cell lines. We identified regulators of lymphoid transcription and cellular energy metabolism as drivers of venetoclax resistance in addition to the known involvement by BCL-2 family members, which were confirmed in patient samples. Our data support the implementation of combinatorial therapy with metabolic modulators to address venetoclax resistance.