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Translation of mitochondrial messenger RNA (mt-mRNA) is performed by distinct mitoribosomes comprising at least 36 mitochondria-specific proteins. How these mitoribosomal proteins assist in the binding of mt-mRNA and to what extent they are involved in the translocation of transfer RNA (mt-tRNA) is unclear. To visualize the process of translation in human mitochondria, we report ~3.0 Å resolution structure of the human mitoribosome, including the L7/L12 stalk, and eight structures of its functional complexes with mt-mRNA, mt-tRNAs, recycling factor and additional trans factors. The study reveals a transacting protein module LRPPRC-SLIRP that delivers mt-mRNA to the mitoribosomal small subunit through a dedicated platform formed by the mitochondria-specific protein mS39. Mitoribosomal proteins of the large subunit mL40, mL48, and mL64 coordinate translocation of mt-tRNA. The comparison between those structures shows dynamic interactions between the mitoribosome and its ligands, suggesting a sequential mechanism of conformational changes.

BackgroundNatural killer (NK) cells exhibit potent cytotoxic activity against various cancer cell types. Over the past five decades, numerous methodologies have been employed to elucidate the intricate molecular mechanisms underlying NK cell-mediated tumor control. While significant progress has been made in elucidating the interactions between NK cells and tumor cells, the regulatory factors governing NK cell-mediated tumor cell destruction are not yet fully understood. This includes the diverse array of tumor ligands recognized by NK cells and the mechanisms that NK cells employ to eliminate tumor cells.MethodsIn this study, we employed a genome-wide CRISPR/Cas9 screening approach in conjunction with functional cytotoxicity assays to delineate the pathways modulating the susceptibility of colon adenocarcinoma HCT-116 cells to NK cell-mediated cytotoxicity.ResultsAnalysis of guide RNA distribution in HCT-116 cells that survived co-incubation with NK cells identified ICAM-1 as a pivotal player in the NKp44-mediated immune synapse, with NKp44 serving as an activating receptor crucial for the elimination of HCT-116 tumor cells by NK cells. Furthermore, disruption of genes involved in the apoptosis or interferon (IFN)-γ signaling pathways conferred resistance to NK cell attack. We further dissected that NK cell-derived IFN-γ promotes mitochondrial apoptosis in vitro and exerts control over B16-F10 lung metastases in vivo.ConclusionMonitoring ICAM-1 levels on the surface of tumor cells or modulating its expression should be considered in the context of NK cell-based therapy. Furthermore, promoting FasL expression on the NK cell surface is reaffirmed as an important strategy to enhance NK cell-mediated tumor killing, offering an additional avenue for therapeutic optimization. Additionally, considering the diffusion properties of IFN-γ, our findings highlight the potential of leveraging NK cell-derived IFN-γ to enhance direct tumor cell killing and facilitate bystander effects via cytokine diffusion, warranting further investigation.

Aggressive tumors often display mitochondrial dysfunction. Upon oxidative stress, mitochondria undergo fission through OMA1-mediated cleavage of the fusion effector OPA1. In yeast, a redox-sensing switch participates in OMA1 activation. 3D modeling of OMA1 comforted the notion that cysteine 403 might participate in a similar sensor in mammalian cells. Using prime editing, we developed a mouse sarcoma cell line in which OMA1 cysteine 403 was mutated in alanine. Mutant cells showed impaired mitochondrial responses to stress including ATP production, reduced fission, resistance to apoptosis, and enhanced mitochondrial DNA release. This mutation prevented tumor development in immunocompetent, but not nude or cDC1 dendritic cell–deficient, mice. These cells prime CD8 + lymphocytes that accumulate in mutant tumors, whereas their depletion delays tumor control. Thus, OMA1 inactivation increased the development of anti-tumor immunity. Patients with complex genomic soft tissue sarcoma showed variations in the level of OMA1 and OPA1 transcripts. High expression of OPA1 in primary tumors was associated with shorter metastasis-free survival after surgery, and low expression of OPA1, with anti-tumor immune signatures. Targeting OMA1 activity may enhance sarcoma immunogenicity.

The human genome expresses thousands of natural antisense transcripts (NAT) that can regulate epigenetic state, transcription, RNA stability or translation of their overlapping genes 1 , 2 . Here we describe MAPT-AS1, a brain-enriched NAT that is conserved in primates and contains an embedded mammalian-wide interspersed repeat (MIR), which represses tau translation by competing for ribosomal RNA pairing with the MAPT mRNA internal ribosome entry site 3 . MAPT encodes tau, a neuronal intrinsically disordered protein (IDP) that stabilizes axonal microtubules. Hyperphosphorylated, aggregation-prone tau forms the hallmark inclusions of tauopathies 4 . Mutations in MAPT cause familial frontotemporal dementia, and common variations forming the MAPT H1 haplotype are a significant risk factor in many tauopathies 5 and Parkinson’s disease. Notably, expression of MAPT-AS1 or minimal essential sequences from MAPT-AS1 (including MIR) reduces—whereas silencing MAPT-AS1 expression increases—neuronal tau levels, and correlate with tau pathology in human brain. Moreover, we identified many additional NATs with embedded MIRs (MIR-NATs), which are overrepresented at coding genes linked to neurodegeneration and/or encoding IDPs, and confirmed MIR-NAT-mediated translational control of one such gene, PLCG1 . These results demonstrate a key role for MAPT-AS1 in tauopathies and reveal a potentially broad contribution of MIR-NATs to the tightly controlled translation of IDPs 6 , with particular relevance for proteostasis in neurodegeneration. The natural antisense transcript MAPT-AS1 interferes with translation of mRNA transcript into tau protein in the brain and may represent a general mechanism for controlling levels of intrinsically disordered proteins, with particular relevance for neurodegeneration.