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.

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.

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.DOI: http://dx.doi.org/10.7554/eLife.25687.001

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.

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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.I nterferon (IFN)-inducible oligoadenylate synthetases (OASs) are mammalian sensors of double-stranded RNA (dsRNA) that are transcriptionally up-regulated during infections with pathogens, such as Staphylococcus aureus (1) or H1N1 swine flu virus (2). Human cells express four related OAS family members: OAS1, OAS2, OAS3, and OASL. Whereas OASL is catalytically inactive, the remaining family members are dsRNA-activated enzymes synthesizing 2′,5′-linked oligoadenylates (2-5A). The 2-5A serve as chemically unique second messengers that induce regulated RNA decay via RNase L (3, 4) and mediate antiviral and antibacterial innate immunity (5, 6). Here we report the structural and functional mechanism of dsRNA surveillance by the largest 2-5A synthetase, OAS3.The core building unit of the OAS family is a polymerase beta (pol-β)-like nucleotidyl transferase domain, which shares structural similarity with poly-A polymerase, CCA-adding enzyme, and cytosolic dsDNA sensor cyclic GAMP synthetase (cGAS) (7,8). Similar to these polymerases, OAS1 and OASL contain a single pol-β domain. However, OAS2 and OAS3 are unusual and contain two and three copies, respectively, acquired apparently by gene duplication (Fig. 1A) (9). The N-terminal pol-β-like domains of OAS2 and OAS3 are thought to have lost their enzymatic activity during evolution (3). In agreement with this hypothesis, mutagenesis of the C-terminal domains in OAS2 and OAS3 has been reported to inactivate these enzymes (10, 11).The functions of the nonenzymatic domains in OAS2 and OAS3 are largely unknown. Experimental ...

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 miRregulated transcriptome.R Nase L is a mammalian endoribonuclease regulated by the action of dsRNA and IFNs α/β/λ, which induce the intracellular synthesis of a specific RNase L activator, 2-5A (1). RNA cleavage is thought to account for all biological functions of the RNase L·2-5A complex, including innate immunity during the IFN response (2, 3), and regulation of cell cycle (4), proliferation (5), adipocyte differentiation (6), and apoptosis (7). RNase L inhibits translation by site-specific cleavage of 18S and 28S rRNA (8) and activates transcription and the inflammasome NLRP3 by releasing signaling RNA fragments (2, 9, 10). These mechanisms complement or operate in parallel with posttranscriptional gene control via regulated decay of some mRNAs, including myogenic regulatory factor MyoD (11), components of IFN signaling ISG43 and ISG15 (12), translation-inhibiting kinase PKR (13), cathepsin E gastric protease (3), 3′-UTRbinding protein HuR (4), as well as ribosomal and mitochondrial protein-encoding mRNAs (14-16). Although the repression of these transcripts depends on RNase L, it remains unknown how many of them are cleaved physically and how many are downregulated indirectly-for example, via transcription.A recent RNA-sequencing (RNA-seq) study described some of the direct targets of RNase L (8). Cleavages were reported for 18S rRNA and U6 snRNA, however the experiment was designed to detect predominantly ribosomal reads and the direct impact of RNase L on mRNAs was not defined. Structural and biochemical studies found that RNase L cleaves RNA at the consensus sequence UN^N (N = A, U, G, or C;^is the cleavage location) (17-19). The UN^N motifs are abundant in all mammalian RNAs, suggesting that RNase L may degrade every mRNA it encounters, which surprisingly contrasts regulation of RNase L by the highly specific stimuli dsRNA, IFNs, and 2-5A.Mammalian cells contain a transmembrane RNase L homolog, a kinase/RNase Ire1, which drives the unfolded protein response and regulated Ire1-dependent decay (RIDD) (18,20). The cleavage consensus sequence of Ire1 (UĜC) is similarly relaxed, however Ire1 targets only specific mRNAs....

Highlights d DsRNA rapidly arrests translation using 2-5A/RNase-Lmediated mRNA decay d Defense mRNAs preferentially accumulate due to positive feedback in the IFN response d RNase L-cleaved ribosomes are translationally competent d Human cells have RNase-L-inaccessible poly(A) + mRNA pools that are not translating