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Deciphering the logic of mammalian mitochondrially-localised translation

ABSTRACT: Localised translation broadly enables spatiotemporal control of gene expression yet currently there are limited tools for monitoring subcellular translation in mammalian cells. Here we present LOCL-TL (LOV-domain Controlled Ligase for Translation Localisation) an optogenetic approach for labelling ribosomes at any defined subcellular location and quantitatively determining their translation with codon precision through ribosome profiling. Application of LOCL-TL to mitochondrially localised translation revealed that ~20% of human nuclear-encoded mitochondrial genes are robustly translated on the outer mitochondrial membrane (OMM). Mitochondrially-translated messages form two classes distinguished by the length of proteins they encode, and the mechanism, and function of their recruitment. An evolutionarily ancient mechanism allows nascent chains to drive the cotranslational recruitment of long proteins via a bipartite targeting signal, which acts on mostly mitochondrial matrix proteins of bacterial origin. Conversely, mRNAs of short proteins, especially eukaryotic origin electron transport chain (ETC) components, are directly recruited to the OMM, in a manner that depends on the 3’UTR, mRNA splicing and the OMM protein A-Kinase Anchoring Protein 1 (AKAP1) and disruption of localised translation through the loss of AKAP1 lowers ETC production. Our studies thus reveal a hierarchical strategy used to enable preferential translation of a subset of proteins on the OMM.

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ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Kelsey Hickey

LAB HEAD: J. Wade Harper

PROVIDER: PXD054646 | Pride | 2025-07-29

REPOSITORIES: Pride

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Project description:Tumor antigens are central to antitumor immunity. Recent evidence suggests that peptides from non-canonical (nonC) aberrantly translated proteins can be presented on HLA-I by tumor cells. Here, we investigated the presentation and immunogenicity of nonC antigens across different cancer types to better understand their contribution to cancer immunosurveillance and to address whether they could be exploited therapeutically. To this end, we employed a proteogenomics pipeline to identify nonC HLA-I ligands derived from off-frame translation of coding sequences and non-coding regions (UTR, ncRNA, intronic and intergenic) in patient-derived tumor cell lines (TCL) of different histological types (4 gynecological cancer, 3 melanoma and 2 head and neck cancer patients). First, peptides bound to HLA-I were isolated and analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using state-of-the-art procedures. Amino acid (Aa) sequences were identified through the previously described pipeline Peptide-PRISM, with some modifications. Briefly, for each MS spectrum, the top 10 candidates were first identified by de novo sequencing and later mapped to a database including the 3-frame transcriptome and 6-frame genome. Additionally, whole-exome sequencing (WES) information of each TCL was included to interrogate the presentation of mutated peptides derived cancer-specific NSM. The false-discovery rate (FDR) was calculated independently for each category considering the search space and peptide length in a stratified mixture model as previously described. Next, in order to select nonC peptides preferentially presented by tumor cells, immunopeptidomics data from several healthy tissues and samples available from the HLA ligand atlas was used to filter out peptides presented in healthy tissues. To overcome a potential bias toward frequent alleles, the peptides were excluded at the ORF level rather than the Aa sequence. As a result, we found that from a total of 839 unique nonC peptides detected in our tumor samples, 38.5% were predicted to derive from ORFs also present in healthy tissue (nonC-HL). Hence, 61.6% (n=517) were considered preferentially presented on tumor HLA-I, referred to as non-canonical tumor ligands (nonC-TL). Out results showed that, although nonC-TL constitued the most abundant source of candidate tumor antigens, as compared to neoantigens, cancer-germline or melanoma-associated antigens, pre-existing antitumor T cells in cancer patients preferentially recognized neoantigens rather than nonC-TL. Nonetehless, nonC-TL elicited de novo T-cell responses via in vitro sensitization of donor lymphocytes. We identified TCRs specific to three nonC-TL, two of which mapped to the 5’ UTR regions of HOXC13 and ZKSCAN1, and one mapping to a non-coding spliced variant of the C5orf22C. These immunogenic nonC-TL were expressed across tumor types but were barely or not detected in healthy cells. Our findings predict a limited contribution of nonC-TL to cancer immunosurveillance but demonstrate they are attractive novel targets for widely applicable immunotherapies.

Project description:Although metabolic benefits of glycolysis have been extensively described in tumor cells, the extra-metabolic functions linked to this energetic pathway in tumor growth and cell proliferation have not been clearly established yet. Recently, some key glycolytic enzymes, such as GAPDH and PKM2, were reported to regulate mRNA translation. Translational control of gene expression is considered as a critical effector in cancer biology, representing a highly promising area of research. Here, we report that Hexokinase 2 (HK2), a glucose kinase that catalyzes the first step of glycolysis at the outer mitochondrial membrane (OMM), is an RNA-binding protein (RBP) that regulates mRNA translation in melanoma cell lines. Polysome profiling experiments followed by RNA sequencing indicate that the translational regulation exerted by HK2 is partly independent of the metabolic status or the glycolytic pathway. We found that HK2 specifically regulates translation of the mRNA encoding SOX10, a transcription factor implicated in the regulation of tumor initiation, maintenance, and progression in melanoma. RNA-protein interaction assays, including crosslinking immunoprecipitation (CLIP), indicate that HK2 is an RBP whose interaction with RNA is independent of its enzymatic activity, its ability to bind glucose or its association with the OMM. HK2 directly interacts with the 5’ untranslated region (5’UTR) of the SOX10 mRNA through a stem-loop RNA secondary structure. Using RNA-protein proximity ligation assays and a fluorescence-based ribosome-bound mRNA mapping method (RIBOmap), we found that high glucose conditions, which promotes the release of HK2 from the OMM, induces an increase in HK2-SOX10 mRNA interaction and SOX10 mRNA translation in the cytoplasm. We further showed that HK2-dependent SOX10 mRNA translation is involved in melanoma cell proliferation and colony formation. Collectively, our data highlight a non-metabolic function of HK2 acting as an RBP and translation regulator.

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Deciphering the logic of mammalian mitochondrially-localised translation

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