A Compendium of Genetic Modifiers of Mitochondrial Dysfunction Reveals Intra-organelle Buffering.

Mitochondrial dysfunction is associated with a spectrum of human conditions, ranging from rare, inborn errors of metabolism to the aging process. To identify pathways that modify mitochondrial dysfunction, we performed genome-wide CRISPR screens in the presence of small-molecule mitochondrial inhibitors. We report a compendium of chemical-genetic interactions involving 191 distinct genetic modifiers, including 38 that are synthetic sick/lethal and 63 that are suppressors. Genes involved in glycolysis (PFKP), pentose phosphate pathway (G6PD), and defense against lipid peroxidation (GPX4) scored high as synthetic sick/lethal. A surprisingly large fraction of suppressors are pathway intrinsic and encode mitochondrial proteins. A striking example of such "intra-organelle" buffering is the alleviation of a chemical defect in complex V by simultaneous inhibition of complex I, which benefits cells by rebalancing redox cofactors, increasing reductive carboxylation, and promoting glycolysis. Perhaps paradoxically, certain forms of mitochondrial dysfunction may best be buffered with "second site" inhibitors to the organelle.

In situ architecture of Opa1-dependent mitochondrial cristae remodeling.

Cristae membrane state plays a central role in regulating mitochondrial function and cellular metabolism. The protein Optic atrophy 1 (Opa1) is an important crista remodeler that exists as two forms in the mitochondrion, a membrane-anchored long form (l-Opa1) and a processed short form (s-Opa1). The mechanisms for how Opa1 influences cristae shape have remained unclear due to lack of native three-dimensional views of cristae. We perform in situ cryo-electron tomography of cryo-focused ion beam milled mouse embryonic fibroblasts with defined Opa1 states to understand how each form of Opa1 influences cristae architecture. In our tomograms, we observe a variety of cristae shapes with distinct trends dependent on s-Opa1:l-Opa1 balance. Increased l-Opa1 levels promote cristae stacking and elongated mitochondria, while increased s-Opa1 levels correlated with irregular cristae packing and round mitochondria shape. Functional assays indicate a role for l-Opa1 in wild-type apoptotic and calcium handling responses, and show a compromised respiratory function under Opa1 imbalance. In summary, we provide three-dimensional visualization of cristae architecture to reveal relationships between mitochondrial ultrastructure and cellular function dependent on Opa1-mediated membrane remodeling.

Concerted 2-5A-Mediated mRNA Decay and Transcription Reprogram Protein Synthesis in the dsRNA Response.

Viral and endogenous double-stranded RNA (dsRNA) is a potent trigger for programmed RNA degradation by the 2-5A/RNase L complex in cells of all mammals. This 2-5A-mediated decay (2-5AMD) is a conserved stress response switching global protein synthesis from homeostasis to production of interferons (IFNs). To understand this mechanism, we examined 2-5AMD in human cells and found that it triggers polysome collapse characteristic of inhibited translation initiation. We determined that translation initiation complexes and ribosomes purified from translation-arrested cells remain functional. However, spike-in RNA sequencing (RNA-seq) revealed cell-wide decay of basal mRNAs accompanied by rapid accumulation of mRNAs encoding innate immune proteins. Our data attribute this 2-5AMD evasion to better stability of defense mRNAs and positive feedback in the IFN response amplified by RNase L-resistant molecules. We conclude that 2-5AMD and transcription act in concert to refill mammalian cells with defense mRNAs, thereby "prioritizing" the synthesis of innate immune proteins.

Introns encode dsRNAs undetected by RIG-I/MDA5/interferons and sensed via RNase L.

Double-stranded RNA (dsRNA), a hallmark viral material that activates antiviral interferon (IFN) responses, can appear in human cells also in the absence of viruses. We identify phosphorothioate DNAs (PS DNAs) as triggers of such endogenous dsRNA (endo-dsRNA). PS DNAs inhibit decay of nuclear RNAs and induce endo-dsRNA via accumulation of high levels of intronic and intergenic inverted retroelements (IIIR). IIIRs activate endo-dsRNA responses distinct from antiviral defense programs. IIIRs do not turn on transcriptional RIG-I/MDA5/IFN signaling, but they trigger the dsRNA-sensing pathways of OAS3/RNase L and PKR. Thus, nuclear RNA decay and nuclear-cytosolic RNA sorting actively protect from these innate immune responses to self. Our data suggest that the OAS3/RNase L and PKR arms of innate immunity diverge from antiviral IFN responses and monitor nuclear RNA decay by sensing cytosolic escape of IIIRs. OAS3 provides a receptor for IIIRs, whereas RNase L cleaves IIIR-carrying introns and intergenic RNAs.

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.

Real-time 2-5A kinetics suggest that interferons ß and λ evade global arrest of translation by RNase L.

Cells of all mammals recognize double-stranded RNA (dsRNA) as a foreign material. In response, they release interferons (IFNs) and activate a ubiquitously expressed pseudokinase/endoribonuclease RNase L. RNase L executes regulated RNA decay and halts global translation. Here, we developed a biosensor for 2',5'-oligoadenylate (2-5A), the natural activator of RNase L. Using this biosensor, we found that 2-5A was acutely synthesized by cells in response to dsRNA sensing, which immediately triggered cellular RNA cleavage by RNase L and arrested host protein synthesis. However, translation-arrested cells still transcribed IFN-stimulated genes and secreted IFNs of types I and III (IFN-ß and IFN-λ). Our data suggest that IFNs escape from the action of RNase L on translation. We propose that the 2-5A/RNase L pathway serves to rapidly and accurately suppress basal protein synthesis, preserving privileged production of defense proteins of the innate immune system.

Rapid RNase L-driven arrest of protein synthesis in the dsRNA response without degradation of translation machinery.

Mammalian cells respond to double-stranded RNA (dsRNA) by activating a translation-inhibiting endoribonuclease, RNase L. Consensus in the field indicates that RNase L arrests protein synthesis by degrading ribosomal RNAs (rRNAs) and messenger RNAs (mRNAs). However, here we provide evidence for a different and far more efficient mechanism. By sequencing abundant RNA fragments generated by RNase L in human cells, we identify site-specific cleavage of two groups of noncoding RNAs: Y-RNAs, whose function is poorly understood, and cytosolic tRNAs, which are essential for translation. Quantitative analysis of human RNA cleavage versus nascent protein synthesis in lung carcinoma cells shows that RNase L stops global translation when tRNAs, as well as rRNAs and mRNAs, are still intact. Therefore, RNase L does not have to degrade the translation machinery to stop protein synthesis. Our data point to a rapid mechanism that transforms a subtle RNA cleavage into a cell-wide translation arrest.

Structural mechanism of sensing long dsRNA via a noncatalytic domain in human oligoadenylate synthetase 3.

The mammalian innate immune system uses several sensors of double-stranded RNA (dsRNA) to develop the interferon response. Among these sensors are dsRNA-activated oligoadenylate synthetases (OAS), which produce signaling 2',5'-linked RNA molecules (2-5A) that activate regulated RNA decay in mammalian tissues. Different receptors from the OAS family contain one, two, or three copies of the 2-5A synthetase domain, which in several instances evolved into pseudoenzymes. The structures of the pseudoenzymatic domains and their roles in sensing dsRNA are unknown. Here we present the crystal structure of the first catalytically inactive domain of human OAS3 (hOAS3.DI) in complex with a 19-bp dsRNA, determined at 2.0-Å resolution. The conformation of hOAS3.DI is different from the apo- and the dsRNA-bound states of the catalytically active homolog, OAS1, reported previously. The unique conformation of hOAS3.DI disables 2-5A synthesis by placing the active site residues nonproductively, but favors the binding of dsRNA. Biochemical data show that hOAS3.DI is essential for activation of hOAS3 and serves as a dsRNA-binding module, whereas the C-terminal domain DIII carries out catalysis. The location of the dsRNA-binding domain (DI) and the catalytic domain (DIII) at the opposite protein termini makes hOAS3 selective for long dsRNA. This mechanism relies on the catalytic inactivity of domain DI, revealing a surprising role of pseudoenzyme evolution in dsRNA surveillance.

Human RNase L tunes gene expression by selectively destabilizing the microRNA-regulated transcriptome.

Double-stranded RNA (dsRNA) activates the innate immune system of mammalian cells and triggers intracellular RNA decay by the pseudokinase and endoribonuclease RNase L. RNase L protects from pathogens and regulates cell growth and differentiation by destabilizing largely unknown mammalian RNA targets. We developed an approach for transcriptome-wide profiling of RNase L activity in human cells and identified hundreds of direct RNA targets and nontargets. We show that this RNase L-dependent decay selectively affects transcripts regulated by microRNA (miR)-17/miR-29/miR-200 and other miRs that function as suppressors of mammalian cell adhesion and proliferation. RNase L mimics the effects of these miRs and acts as a suppressor of proliferation and adhesion in mammalian cells. Our data suggest that RNase L-dependent decay serves to establish an antiproliferative state via destabilization of the miR-regulated transcriptome.

Control of apoptosis by Drosophila DCAF12.

Regulated Apoptosis (Programmed Cell Death, PCD) maintains tissue homeostasis in adults, and ensures proper growth and morphogenesis of tissues during development of metazoans. Accordingly, defects in cellular processes triggering or executing apoptotic programs have been implicated in a variety of degenerative and neoplastic diseases. Here, we report the identification of DCAF12, an evolutionary conserved member of the WD40-motif repeat family of proteins, as a new regulator of apoptosis in Drosophila. We find that DCAF12 is required for Diap1 cleavage in response to pro-apoptotic signals, and is thus necessary and sufficient for RHG (Reaper, Hid, and Grim)-mediated apoptosis. Loss of DCAF12 perturbs the elimination of supernumerary or proliferation-impaired cells during development, and enhances tumor growth induced by loss of neoplastic tumor suppressors, highlighting the wide requirement for DCAF12 in PCD.

In situ architecture of Opa1-dependent mitochondrial cristae remodeling.

Cristae membrane state plays a central role in regulating mitochondrial function and cellular metabolism. The protein Optic atrophy 1 (Opa1) is an important crista remodeler that exists as two forms in the mitochondrion, a membrane-anchored long form (l-Opa1) and a processed short form (s-Opa1). The mechanisms for how Opa1 influences cristae shape have remained unclear due to lack of native three-dimensional views of cristae. We perform in situ cryo-electron tomography of cryo-focused ion beam milled mouse embryonic fibroblasts with defined Opa1 states to understand how each form of Opa1 influences cristae architecture. In our tomograms, we observe a variety of cristae shapes with distinct trends dependent on s-Opa1:l-Opa1 balance. Increased l-Opa1 levels promote cristae stacking and elongated mitochondria while increased s-Opa1 levels correlated with irregular cristae packing and round mitochondria shape. Functional assays indicate a role for l-Opa1 in wild-type apoptotic and calcium handling responses, and compromised respiratory function under Opa1 imbalance. In summary, we provide three-dimensional visualization of cristae architecture to reveal relationships between mitochondrial ultrastructure and cellular function dependent on Opa1-mediated membrane remodeling.

Effectors Enabling Adaptation to Mitochondrial Complex I Loss in Hürthle Cell Carcinoma.

Oncocytic (Hürthle cell) carcinoma of the thyroid (HCC) is genetically characterized by complex I mitochondrial DNA mutations and widespread chromosomal losses. Here, we utilize RNA sequencing and metabolomics to identify candidate molecular effectors activated by these genetic drivers. We find glutathione biosynthesis, amino acid metabolism, mitochondrial unfolded protein response, and lipid peroxide scavenging to be increased in HCC. A CRISPR-Cas9 knockout screen in a new HCC model reveals which pathways are key for fitness, and highlights loss of GPX4, a defense against lipid peroxides and ferroptosis, as a strong liability. Rescuing complex I redox activity with the yeast NADH dehydrogenase (NDI1) in HCC cells diminishes ferroptosis sensitivity, while inhibiting complex I in normal thyroid cells augments ferroptosis induction. Our work demonstrates unmitigated lipid peroxide stress to be an HCC vulnerability that is mechanistically coupled to the genetic loss of mitochondrial complex I activity. SIGNIFICANCE: HCC harbors abundant mitochondria, mitochondrial DNA mutations, and chromosomal losses. Using a CRISPR-Cas9 screen inspired by transcriptomic and metabolomic profiling, we identify molecular effectors essential for cell fitness. We uncover lipid peroxide stress as a vulnerability coupled to mitochondrial complex I loss in HCC. See related article by Frank et al., p. 1884. This article is highlighted in the In This Issue feature, p. 1749.

The metabolites NADP+ and NADPH are the targets of the circadian protein Nocturnin (Curled).

Nocturnin (NOCT) is a rhythmically expressed protein that regulates metabolism under the control of circadian clock. It has been proposed that NOCT deadenylates and regulates metabolic enzyme mRNAs. However, in contrast to other deadenylases, purified NOCT lacks the deadenylase activity. To identify the substrate of NOCT, we conducted a mass spectrometry screen and report that NOCT specifically and directly converts the dinucleotide NADP+ into NAD+ and NADPH into NADH. Further, we demonstrate that the Drosophila NOCT ortholog, Curled, has the same enzymatic activity. We obtained the 2.7 Šcrystal structure of the human NOCT•NADPH complex, which revealed that NOCT recognizes the chemically unique ribose-phosphate backbone of the metabolite, placing the 2'-terminal phosphate productively for removal. We provide evidence for NOCT targeting to mitochondria and propose that NADP(H) regulation, which takes place at least in part in mitochondria, establishes the molecular link between circadian clock and metabolism.

Ribonuclease L mediates the cell-lethal phenotype of double-stranded RNA editing enzyme ADAR1 deficiency in a human cell line.

ADAR1 isoforms are adenosine deaminases that edit and destabilize double-stranded RNA reducing its immunostimulatory activities. Mutation of ADAR1 leads to a severe neurodevelopmental and inflammatory disease of children, Aicardi-Goutiéres syndrome. In mice, Adar1 mutations are embryonic lethal but are rescued by mutation of the Mda5 or Mavs genes, which function in IFN induction. However, the specific IFN regulated proteins responsible for the pathogenic effects of ADAR1 mutation are unknown. We show that the cell-lethal phenotype of ADAR1 deletion in human lung adenocarcinoma A549 cells is rescued by CRISPR/Cas9 mutagenesis of the RNASEL gene or by expression of the RNase L antagonist, murine coronavirus NS2 accessory protein. Our result demonstrate that ablation of RNase L activity promotes survival of ADAR1 deficient cells even in the presence of MDA5 and MAVS, suggesting that the RNase L system is the primary sensor pathway for endogenous dsRNA that leads to cell death.

Structure of human RNase L reveals the basis for regulated RNA decay in the IFN response.

One of the hallmark mechanisms activated by type I interferons (IFNs) in human tissues involves cleavage of intracellular RNA by the kinase homology endoribonuclease RNase L. We report 2.8 and 2.1 angstrom crystal structures of human RNase L in complexes with synthetic and natural ligands and a fragment of an RNA substrate. RNase L forms a crossed homodimer stabilized by ankyrin (ANK) and kinase homology (KH) domains, which positions two kinase extension nuclease (KEN) domains for asymmetric RNA recognition. One KEN protomer recognizes an identity nucleotide (U), whereas the other protomer cleaves RNA between nucleotides +1 and +2. The coordinated action of the ANK, KH, and KEN domains thereby provides regulated, sequence-specific cleavage of viral and host RNA targets by RNase L.