{"gene":"ALKBH8","run_date":"2026-04-28T17:12:37","timeline":{"discoveries":[{"year":2010,"finding":"ALKBH8 is a tRNA methyltransferase required for the final step in biogenesis of 5-methoxycarbonylmethyluridine (mcm5U) at the wobble position of tRNA. Interaction with the small accessory protein TRM112 is required to form a functional tRNA methyltransferase complex. Prior ALKBH8-mediated methylation is a prerequisite for subsequent thiolation (mcm5s2U) and 2'-O-ribose methylation (mcm5Um). Loss of these modifications in Alkbh8-/- mice causes aberrant modification of selenocysteine tRNA (tRNASec) and reduced UGA stop codon recoding to selenocysteine for Gpx1.","method":"Alkbh8-/- mouse knockout, tRNA modification analysis (HPLC/MS), in vitro methyltransferase assay, co-immunoprecipitation of ALKBH8-TRM112 complex","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1–2 — KO mouse model, in vitro assay, multiple orthogonal methods; independently validated by subsequent studies","pmids":["20123966"],"is_preprint":false},{"year":2011,"finding":"The AlkB oxygenase domain of ALKBH8 specifically hydroxylates mcm5U to (S)-mchm5U in tRNA-Gly(UCC), generating a novel diastereomeric pair of wobble nucleosides. The mammalian ALKBH8 AlkB domain acts as an RNA hydroxylase rather than a DNA repair enzyme, expanding ALKBH oxygenase function beyond nucleic acid repair.","method":"In vitro hydroxylation assay with recombinant ALKBH8 AlkB domain, tRNA modification analysis by mass spectrometry, in vivo analysis using Alkbh8-/- mouse tRNA","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with domain-specific mutagenesis context plus in vivo validation in KO mice","pmids":["21285950"],"is_preprint":false},{"year":2011,"finding":"Trm9 (yeast ortholog of ALKBH8) and Trm112 physically interact and function together as a complex for the final methylation step (cm5U → mcm5U) in wobble uridine modification. Co-expression of His-tagged Trm9 with native Trm112 in E. coli yielded a purified complex, and Trm112 dramatically improves Trm9 methyltransferase activity in vitro.","method":"Co-expression and co-purification from E. coli, in vitro methyltransferase assay, tRNA modification analysis by HPLC from trm9Δ and trm112Δ yeast strains","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 — reconstituted complex in vitro with activity assay, confirmed by in vivo genetic knockouts","pmids":["21687733"],"is_preprint":false},{"year":2015,"finding":"Crystal structure of the Trm9-Trm112 complex reveals the structural basis for mcm5U modification of tRNA anticodon wobble position. Trm112 acts as an obligate activating platform, interacting with Trm9 through a structurally plastic interface that is shared across multiple Trm112-methyltransferase complexes despite low sequence identity among partners.","method":"X-ray crystallography, structure-function analysis with mutagenesis","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with functional validation","pmids":["26438534"],"is_preprint":false},{"year":2007,"finding":"Yeast Trm9 (ALKBH8 ortholog) methylates the wobble uridine of tRNAARG(UCU) and tRNAGLU(UUC), and this modification enhances codon-specific translation elongation, promoting increased protein levels of DNA damage response proteins (Yef3, Rnr1, Rnr3) that are enriched in cognate arginine and glutamic acid codons.","method":"Genetic deletion (trm9Δ), tRNA modification analysis, reporter assays, protein level measurement by western blot, computational codon usage analysis","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods in yeast ortholog; highly cited foundational study replicated in subsequent work","pmids":["18082610"],"is_preprint":false},{"year":2012,"finding":"Trm9-catalyzed wobble uridine modifications (mcm5U and mcm5s2U) are required for translational fidelity; loss of these two modifications in trm9Δ yeast causes translational errors at specific arginine and glutamic acid codons, leading to protein misfolding and activation of unfolded protein and heat shock responses.","method":"Phenotypic assays, translational fidelity reporters, quantitative tRNA modification analysis (HPLC), protein-based fidelity assays in trm9Δ yeast, codon reengineering","journal":"RNA biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods establishing pathway position and phenotypic consequence","pmids":["22832247"],"is_preprint":false},{"year":2009,"finding":"ALKBH8 silencing in bladder cancer cells reduces ROS production via downregulation of NOX-1 and induces apoptosis through activation of JNK and p38, which cause γH2AX phosphorylation. ALKBH8 knockdown also suppresses invasion and angiogenesis in vivo.","method":"siRNA knockdown, ROS measurement, western blot for pathway components (JNK, p38, γH2AX, NOX-1), chorioallantoic membrane assay, orthotopic mouse model","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — clean KD with defined cellular phenotype and pathway placement, single lab","pmids":["19293182"],"is_preprint":false},{"year":2016,"finding":"ALKBH8 promotes bladder cancer cell survival by maintaining protein expression of survivin (an anti-apoptotic factor); ALKBH8 knockdown induces apoptosis via downregulation of survivin, and ALKBH8 transgenic mice show accelerated bladder tumor growth and invasiveness.","method":"siRNA knockdown, western blot for survivin, transgenic mouse model with carcinogen-induced bladder cancer (N-butyl-N-(4-hydroxybutyl)-nitrosamine)","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — defined molecular target (survivin) with in vivo transgenic validation, single lab","pmids":["27329810"],"is_preprint":false},{"year":2014,"finding":"Protozoan ALKBH8 proteins display dual activity: DNA repair (dealkylation) and tRNA wobble uridine hydroxylation (mcm5U modification in tRNAGly(UCC)), demonstrating functional duality within the ALKBH8 family. Bacterial ALKBH8 shows DNA repair activity in vitro but does not modify tRNAGly(UCC) wobble uridine in vivo.","method":"In vitro DNA repair assay, in vitro tRNA modification assay, in vivo tRNA modification analysis in Agrobacterium ALKBH8 mutant","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 1 — in vitro biochemical assays with in vivo genetic confirmation, single study","pmids":["24914785"],"is_preprint":false},{"year":2021,"finding":"A missense variant in the ALKBH8 methyltransferase domain causes complete absence of ALKBH8-dependent tRNA modifications in patient cells (confirmed by targeted proteomics), establishing that loss of methyltransferase activity is the disease mechanism for MRT71 intellectual disability, without loss of ALKBH8 protein expression.","method":"Patient cell analysis, mass spectrometry-based detection of tRNA modifications, targeted proteomics of ALKBH8 protein levels","journal":"Human genetics","confidence":"Medium","confidence_rationale":"Tier 2 — patient-derived cells with direct biochemical confirmation of enzymatic loss-of-function","pmids":["34757492"],"is_preprint":false},{"year":2022,"finding":"HITS-CLIP and RIP-seq analysis demonstrates that ALKBH8 binds fully processed, CCA-modified substrate tRNAs (the known wobble U-containing tRNAs) and also interacts with several noncoding RNAs, particularly C/D box snoRNAs, in human cells.","method":"HITS-CLIP, RIP-seq","journal":"RNA (New York, N.Y.)","confidence":"Medium","confidence_rationale":"Tier 2 — transcriptome-wide binding analysis with two orthogonal methods, single lab","pmids":["36192131"],"is_preprint":false},{"year":2020,"finding":"ALKBH8 modifies the wobble uridine of selenocysteine tRNA to promote selenoprotein translation; Alkbh8-deficient mice show increased oxidative stress markers and decreased thioredoxin reductase protein levels under basal conditions, and fail to develop naphthalene tolerance, establishing ALKBH8 as protective against oxidative lung damage via selenoprotein-dependent antioxidant mechanisms.","method":"Alkbh8-/- mouse model, western blot for oxidative stress markers and thioredoxin reductase, naphthalene exposure challenge experiments","journal":"Epigenetics","confidence":"Medium","confidence_rationale":"Tier 2 — defined molecular mechanism (selenoprotein translation via tRNA modification) linked to in vivo phenotype","pmids":["32303148"],"is_preprint":false},{"year":2024,"finding":"In Alkbh8-/- mice, mcm5U modification is reduced in most tissues but partially compensated in the brain. Loss of ALKBH8 reduces tRNA protein translation efficiency, impairs red blood cell differentiation and embryogenesis (shown by proteome analysis linking downregulated factors to red blood cell and protoporphyrin metabolism), and causes neural dysfunction with oxidative stress and reduced mitochondrial membrane potential in neurons and glial cells.","method":"Alkbh8-/- mouse model, UPLC-MS/MS for tRNA modification quantification, proteomics, behavioral tests (novel object recognition, rotarod, forced swim), brain histopathology, mitochondrial membrane potential assay","journal":"iScience / PNAS nexus","confidence":"Medium","confidence_rationale":"Tier 2 — KO mouse with multiple orthogonal phenotypic readouts, single lab","pmids":["39280612","38550277"],"is_preprint":false},{"year":2025,"finding":"ALKBH8 is a direct transcriptional target of Wnt/β-catenin signaling. Its methyltransferase activity promotes translation elongation at adenine-ending codons by modifying U34 tRNA, specifically regulating KRAS translation in a codon-dependent manner. Rescue experiments with methyltransferase-dead ALKBH8 fail to restore KRAS translation, confirming catalytic activity requirement. Loss of ALKBH8 induces ribosome pausing at adenine-ending codons.","method":"CRISPR knockout, ribosome profiling (ribosome pausing analysis), rescue with wildtype vs. catalytically-dead ALKBH8 mutant, Apcmin/+ and AOM/DSS mouse tumor models, xenograft, reporter assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 — multiple in vivo models, ribosome profiling, mutagenesis rescue, identifies specific substrate (KRAS) and upstream regulator (Wnt/β-catenin)","pmids":["41083459"],"is_preprint":false},{"year":2025,"finding":"During ZIKV infection, ALKBH8 is required for mcm5s2U34 tRNA modification; CRISPR/shRNA-mediated knockdown of ALKBH8 significantly reduces ZIKV replication, indicating that ALKBH8-mediated U34 tRNA modification is exploited by the virus to optimize translation of A-ending codon-biased viral proteins.","method":"CRISPR/Cas9 and shRNA knockdown of ALKBH8, mass spectrometry for tRNA modification quantification, codon-biased GFP reporters, viral replication assays","journal":"bioRxiv (preprint)","confidence":"Medium","confidence_rationale":"Tier 2 — multiple KD approaches with viral replication readout, preprint","pmids":[],"is_preprint":true}],"current_model":"ALKBH8 is a bifunctional enzyme containing a methyltransferase domain (requiring TRM112 as an obligate activating partner) that converts cm5U to mcm5U at the wobble position (U34) of specific tRNAs, and an AlkB oxygenase domain that further hydroxylates mcm5U to (S)-mchm5U in tRNA-Gly(UCC); these wobble uridine modifications promote codon-specific translation elongation—particularly at A-ending codons—thereby regulating the translational output of codon-biased mRNAs including selenoprotein mRNAs (via tRNASec modification) and oncogenes such as KRAS, with ALKBH8 itself being transcriptionally regulated by Wnt/β-catenin, and its loss causing translational infidelity, oxidative stress, and neurodevelopmental defects."},"narrative":{"teleology":[{"year":2007,"claim":"Establishing that wobble uridine methylation by the ALKBH8 ortholog Trm9 has a translational output—it selectively enhances expression of codon-biased proteins involved in the DNA damage response—moved the modification from a biochemical curiosity to a gene-expression regulatory mechanism.","evidence":"Yeast trm9Δ deletion with tRNA modification analysis, codon usage computation, and western blot for target proteins","pmids":["18082610"],"confidence":"High","gaps":["Whether the same codon-specific translational control operates in mammalian systems was untested","Direct ribosome-level evidence of elongation rate change was lacking"]},{"year":2010,"claim":"Generation of Alkbh8-knockout mice demonstrated that ALKBH8 is the mammalian enzyme responsible for the cm5U-to-mcm5U methylation step, that TRM112 is its obligate activating partner, and that loss of this modification disrupts selenoprotein translation by impairing tRNASec modification.","evidence":"Alkbh8−/− mouse, HPLC/MS tRNA modification profiling, in vitro methyltransferase assay, co-immunoprecipitation of ALKBH8–TRM112","pmids":["20123966"],"confidence":"High","gaps":["Function of the AlkB oxygenase domain remained unknown","Whether translational fidelity is affected in mammalian cells was not assessed"]},{"year":2011,"claim":"Identification of the AlkB domain as an RNA hydroxylase that converts mcm5U to (S)-mchm5U on tRNA-Gly(UCC) revealed that ALKBH8 is a true bifunctional enzyme and expanded the ALKBH family beyond DNA repair functions.","evidence":"In vitro hydroxylation assay with recombinant AlkB domain, mass spectrometry, validation in Alkbh8−/− mouse tRNA","pmids":["21285950"],"confidence":"High","gaps":["Biological consequence of the hydroxylation product (S)-mchm5U was not defined","No structural information for the mammalian enzyme"]},{"year":2012,"claim":"Demonstrating that loss of Trm9-dependent modifications causes translational infidelity and triggers the unfolded protein response established that these wobble modifications are required for accurate decoding, not merely for efficiency.","evidence":"Translational fidelity reporters and phenotypic assays in trm9Δ yeast","pmids":["22832247"],"confidence":"High","gaps":["Whether translational infidelity occurs in mammalian ALKBH8-null cells was unknown","Specific miscoded codons in vivo were not fully mapped"]},{"year":2015,"claim":"The crystal structure of the Trm9–Trm112 complex revealed how TRM112 serves as a structurally plastic activating platform shared among multiple methyltransferases, explaining the obligate requirement for this partner.","evidence":"X-ray crystallography with structure-guided mutagenesis","pmids":["26438534"],"confidence":"High","gaps":["No structure of full-length mammalian ALKBH8 including the AlkB domain","How substrate tRNA is recognized by the complex was not resolved"]},{"year":2020,"claim":"Linking ALKBH8-dependent selenoprotein translation to an in vivo oxidative stress phenotype established that wobble uridine modification is physiologically required for antioxidant defense, specifically through thioredoxin reductase expression.","evidence":"Alkbh8−/− mice challenged with naphthalene; western blot for thioredoxin reductase and oxidative stress markers","pmids":["32303148"],"confidence":"Medium","gaps":["Which specific selenoproteins are most affected was not comprehensively profiled","Single-lab study without independent replication"]},{"year":2021,"claim":"Patient-derived evidence that a missense variant in the ALKBH8 methyltransferase domain abolishes all ALKBH8-dependent tRNA modifications without reducing protein levels established enzymatic loss-of-function as the disease mechanism for MRT71 intellectual disability.","evidence":"Patient cell analysis with mass spectrometry-based tRNA modification profiling and targeted proteomics","pmids":["34757492"],"confidence":"Medium","gaps":["Only a single variant/family was studied","Neuronal-specific consequences of modification loss were not mechanistically dissected"]},{"year":2022,"claim":"Transcriptome-wide binding studies revealed that ALKBH8 directly binds its known substrate tRNAs in their mature CCA-modified form and also associates with C/D box snoRNAs, suggesting possible non-tRNA functions.","evidence":"HITS-CLIP and RIP-seq in human cells","pmids":["36192131"],"confidence":"Medium","gaps":["Functional significance of snoRNA interaction is unknown","Whether snoRNA binding reflects a catalytic or regulatory role is unresolved"]},{"year":2024,"claim":"Comprehensive phenotyping of Alkbh8−/− mice revealed tissue-specific compensation of tRNA modification (partial rescue in brain), impaired erythropoiesis, and neural dysfunction with reduced mitochondrial membrane potential, connecting wobble uridine modification to mitochondrial and neurodevelopmental physiology.","evidence":"Alkbh8−/− mouse model with UPLC-MS/MS tRNA modification profiling, proteomics, behavioral testing, and mitochondrial assays","pmids":["39280612","38550277"],"confidence":"Medium","gaps":["Mechanism of tissue-specific compensation is unknown","Identity of the compensating enzyme(s) in brain is not established"]},{"year":2025,"claim":"Identifying ALKBH8 as a direct Wnt/β-catenin target gene whose methyltransferase activity specifically promotes KRAS translation through codon-dependent ribosome elongation provided the first mechanistic link between an epitranscriptomic modifier and oncogenic signaling in colorectal cancer.","evidence":"CRISPR knockout, ribosome profiling showing ribosome pausing at A-ending codons, rescue with catalytically dead mutant, Apcmin/+ and AOM/DSS mouse tumor models","pmids":["41083459"],"confidence":"High","gaps":["Whether other oncogenes with similar codon bias are co-regulated was not systematically addressed","Therapeutic targetability of ALKBH8 catalytic activity is untested"]},{"year":null,"claim":"Key unresolved questions include the biological function of ALKBH8's hydroxylation product (S)-mchm5U, the mechanism of substrate tRNA recognition by the full-length mammalian enzyme, the functional significance of snoRNA interactions, and the basis for tissue-specific compensation of tRNA modification loss.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structure of full-length mammalian ALKBH8","Biological role of (S)-mchm5U hydroxylation uncharacterized","snoRNA interaction function unknown","Tissue-specific compensation mechanism undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,2,3,4,9,13]},{"term_id":"GO:0016491","term_label":"oxidoreductase activity","supporting_discovery_ids":[1,8]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[0,10]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[10]}],"pathway":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[0]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,1,4,5,13]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,4,5,13]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[13]}],"complexes":["ALKBH8–TRM112 methyltransferase complex"],"partners":["TRM112"],"other_free_text":[]},"mechanistic_narrative":"ALKBH8 is a bifunctional tRNA-modifying enzyme that regulates codon-specific translation elongation by installing wobble uridine (U34) modifications on select tRNAs. Its methyltransferase domain, activated by the obligate partner TRM112, catalyzes the conversion of cm5U to mcm5U—a prerequisite for downstream thiolation (mcm5s2U) and 2'-O-ribose methylation—while its AlkB oxygenase domain hydroxylates mcm5U to (S)-mchm5U specifically on tRNA-Gly(UCC) [PMID:20123966, PMID:21285950, PMID:26438534]. These modifications promote efficient decoding of adenine-ending codons, thereby controlling translation of codon-biased mRNAs including selenoprotein mRNAs (via tRNASec modification to support UGA recoding) and oncogenes such as KRAS, whose translation depends on ALKBH8 catalytic activity downstream of Wnt/β-catenin transcriptional activation [PMID:20123966, PMID:32303148, PMID:41083459]. Loss-of-function mutations in the ALKBH8 methyltransferase domain cause intellectual disability (MRT71) through complete abrogation of ALKBH8-dependent tRNA modifications [PMID:34757492]."},"prefetch_data":{"uniprot":{"accession":"Q96BT7","full_name":"tRNA (carboxymethyluridine(34)-5-O)-methyltransferase ALKBH8","aliases":["Alkylated DNA repair protein alkB homolog 8","Alpha-ketoglutarate-dependent dioxygenase ALKBH8","S-adenosyl-L-methionine-dependent tRNA methyltransferase ALKBH8"],"length_aa":664,"mass_kda":75.2,"function":"Catalyzes the methylation of 5-carboxymethyl uridine to 5-methylcarboxymethyl uridine at the wobble position of the anticodon loop in tRNA via its methyltransferase domain (PubMed:20123966, PubMed:20308323, PubMed:31079898). Catalyzes the last step in the formation of 5-methylcarboxymethyl uridine at the wobble position of the anticodon loop in target tRNA (PubMed:20123966, PubMed:20308323). Has a preference for tRNA(Arg) and tRNA(Glu), and does not bind tRNA(Lys) (PubMed:20308323). Binds tRNA and catalyzes the iron and alpha-ketoglutarate dependent hydroxylation of 5-methylcarboxymethyl uridine at the wobble position of the anticodon loop in tRNA via its dioxygenase domain, giving rise to 5-(S)-methoxycarbonylhydroxymethyluridine; has a preference for tRNA(Gly) (PubMed:21285950). Required for normal survival after DNA damage (PubMed:20308323). May inhibit apoptosis and promote cell survival and angiogenesis (PubMed:19293182)","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q96BT7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ALKBH8","classification":"Not Classified","n_dependent_lines":14,"n_total_lines":1208,"dependency_fraction":0.011589403973509934},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ALKBH8","total_profiled":1310},"omim":[{"mim_id":"618630","title":"tRNA METHYLTRANSFERASE SUBUNIT 11-2; TRMT112","url":"https://www.omim.org/entry/618630"},{"mim_id":"618504","title":"INTELLECTUAL DEVELOPMENTAL DISORDER, AUTOSOMAL RECESSIVE 71; MRT71","url":"https://www.omim.org/entry/618504"},{"mim_id":"613306","title":"AlkB HOMOLOG 8, tRNA METHYLTRANSFERASE; ALKBH8","url":"https://www.omim.org/entry/613306"},{"mim_id":"610396","title":"TRAFFICKING PROTEIN PARTICLE COMPLEX, SUBUNIT 6A; TRAPPC6A","url":"https://www.omim.org/entry/610396"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Microtubules","reliability":"Additional"},{"location":"Mitotic spindle","reliability":"Additional"},{"location":"Primary cilium","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ALKBH8"},"hgnc":{"alias_symbol":["MGC10235","TRM9","TRMT9A"],"prev_symbol":[]},"alphafold":{"accession":"Q96BT7","domains":[{"cath_id":"3.30.70.330","chopping":"14-120","consensus_level":"high","plddt":85.3351,"start":14,"end":120},{"cath_id":"2.60.120.1520","chopping":"138-349","consensus_level":"high","plddt":85.4132,"start":138,"end":349},{"cath_id":"3.40.50.150","chopping":"365-510_635-664","consensus_level":"high","plddt":95.1862,"start":365,"end":664}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96BT7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96BT7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96BT7-F1-predicted_aligned_error_v6.png","plddt_mean":80.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ALKBH8","jax_strain_url":"https://www.jax.org/strain/search?query=ALKBH8"},"sequence":{"accession":"Q96BT7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96BT7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96BT7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96BT7"}},"corpus_meta":[{"pmid":"18082610","id":"PMC_18082610","title":"Trm9-catalyzed tRNA modifications link translation to the DNA damage response.","date":"2007","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/18082610","citation_count":259,"is_preprint":false},{"pmid":"20123966","id":"PMC_20123966","title":"Mammalian ALKBH8 possesses tRNA methyltransferase activity required for the biogenesis of multiple wobble uridine modifications implicated in translational decoding.","date":"2010","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/20123966","citation_count":199,"is_preprint":false},{"pmid":"21285950","id":"PMC_21285950","title":"ALKBH8-mediated formation of a novel diastereomeric pair of wobble nucleosides in mammalian tRNA.","date":"2011","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/21285950","citation_count":138,"is_preprint":false},{"pmid":"19293182","id":"PMC_19293182","title":"A novel human AlkB homologue, ALKBH8, contributes to human bladder cancer 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Interaction with the small accessory protein TRM112 is required to form a functional tRNA methyltransferase complex. Prior ALKBH8-mediated methylation is a prerequisite for subsequent thiolation (mcm5s2U) and 2'-O-ribose methylation (mcm5Um). Loss of these modifications in Alkbh8-/- mice causes aberrant modification of selenocysteine tRNA (tRNASec) and reduced UGA stop codon recoding to selenocysteine for Gpx1.\",\n      \"method\": \"Alkbh8-/- mouse knockout, tRNA modification analysis (HPLC/MS), in vitro methyltransferase assay, co-immunoprecipitation of ALKBH8-TRM112 complex\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — KO mouse model, in vitro assay, multiple orthogonal methods; independently validated by subsequent studies\",\n      \"pmids\": [\"20123966\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The AlkB oxygenase domain of ALKBH8 specifically hydroxylates mcm5U to (S)-mchm5U in tRNA-Gly(UCC), generating a novel diastereomeric pair of wobble nucleosides. The mammalian ALKBH8 AlkB domain acts as an RNA hydroxylase rather than a DNA repair enzyme, expanding ALKBH oxygenase function beyond nucleic acid repair.\",\n      \"method\": \"In vitro hydroxylation assay with recombinant ALKBH8 AlkB domain, tRNA modification analysis by mass spectrometry, in vivo analysis using Alkbh8-/- mouse tRNA\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with domain-specific mutagenesis context plus in vivo validation in KO mice\",\n      \"pmids\": [\"21285950\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Trm9 (yeast ortholog of ALKBH8) and Trm112 physically interact and function together as a complex for the final methylation step (cm5U → mcm5U) in wobble uridine modification. Co-expression of His-tagged Trm9 with native Trm112 in E. coli yielded a purified complex, and Trm112 dramatically improves Trm9 methyltransferase activity in vitro.\",\n      \"method\": \"Co-expression and co-purification from E. coli, in vitro methyltransferase assay, tRNA modification analysis by HPLC from trm9Δ and trm112Δ yeast strains\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted complex in vitro with activity assay, confirmed by in vivo genetic knockouts\",\n      \"pmids\": [\"21687733\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Crystal structure of the Trm9-Trm112 complex reveals the structural basis for mcm5U modification of tRNA anticodon wobble position. Trm112 acts as an obligate activating platform, interacting with Trm9 through a structurally plastic interface that is shared across multiple Trm112-methyltransferase complexes despite low sequence identity among partners.\",\n      \"method\": \"X-ray crystallography, structure-function analysis with mutagenesis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with functional validation\",\n      \"pmids\": [\"26438534\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Yeast Trm9 (ALKBH8 ortholog) methylates the wobble uridine of tRNAARG(UCU) and tRNAGLU(UUC), and this modification enhances codon-specific translation elongation, promoting increased protein levels of DNA damage response proteins (Yef3, Rnr1, Rnr3) that are enriched in cognate arginine and glutamic acid codons.\",\n      \"method\": \"Genetic deletion (trm9Δ), tRNA modification analysis, reporter assays, protein level measurement by western blot, computational codon usage analysis\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods in yeast ortholog; highly cited foundational study replicated in subsequent work\",\n      \"pmids\": [\"18082610\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Trm9-catalyzed wobble uridine modifications (mcm5U and mcm5s2U) are required for translational fidelity; loss of these two modifications in trm9Δ yeast causes translational errors at specific arginine and glutamic acid codons, leading to protein misfolding and activation of unfolded protein and heat shock responses.\",\n      \"method\": \"Phenotypic assays, translational fidelity reporters, quantitative tRNA modification analysis (HPLC), protein-based fidelity assays in trm9Δ yeast, codon reengineering\",\n      \"journal\": \"RNA biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods establishing pathway position and phenotypic consequence\",\n      \"pmids\": [\"22832247\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"ALKBH8 silencing in bladder cancer cells reduces ROS production via downregulation of NOX-1 and induces apoptosis through activation of JNK and p38, which cause γH2AX phosphorylation. ALKBH8 knockdown also suppresses invasion and angiogenesis in vivo.\",\n      \"method\": \"siRNA knockdown, ROS measurement, western blot for pathway components (JNK, p38, γH2AX, NOX-1), chorioallantoic membrane assay, orthotopic mouse model\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KD with defined cellular phenotype and pathway placement, single lab\",\n      \"pmids\": [\"19293182\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ALKBH8 promotes bladder cancer cell survival by maintaining protein expression of survivin (an anti-apoptotic factor); ALKBH8 knockdown induces apoptosis via downregulation of survivin, and ALKBH8 transgenic mice show accelerated bladder tumor growth and invasiveness.\",\n      \"method\": \"siRNA knockdown, western blot for survivin, transgenic mouse model with carcinogen-induced bladder cancer (N-butyl-N-(4-hydroxybutyl)-nitrosamine)\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — defined molecular target (survivin) with in vivo transgenic validation, single lab\",\n      \"pmids\": [\"27329810\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Protozoan ALKBH8 proteins display dual activity: DNA repair (dealkylation) and tRNA wobble uridine hydroxylation (mcm5U modification in tRNAGly(UCC)), demonstrating functional duality within the ALKBH8 family. Bacterial ALKBH8 shows DNA repair activity in vitro but does not modify tRNAGly(UCC) wobble uridine in vivo.\",\n      \"method\": \"In vitro DNA repair assay, in vitro tRNA modification assay, in vivo tRNA modification analysis in Agrobacterium ALKBH8 mutant\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — in vitro biochemical assays with in vivo genetic confirmation, single study\",\n      \"pmids\": [\"24914785\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"A missense variant in the ALKBH8 methyltransferase domain causes complete absence of ALKBH8-dependent tRNA modifications in patient cells (confirmed by targeted proteomics), establishing that loss of methyltransferase activity is the disease mechanism for MRT71 intellectual disability, without loss of ALKBH8 protein expression.\",\n      \"method\": \"Patient cell analysis, mass spectrometry-based detection of tRNA modifications, targeted proteomics of ALKBH8 protein levels\",\n      \"journal\": \"Human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — patient-derived cells with direct biochemical confirmation of enzymatic loss-of-function\",\n      \"pmids\": [\"34757492\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"HITS-CLIP and RIP-seq analysis demonstrates that ALKBH8 binds fully processed, CCA-modified substrate tRNAs (the known wobble U-containing tRNAs) and also interacts with several noncoding RNAs, particularly C/D box snoRNAs, in human cells.\",\n      \"method\": \"HITS-CLIP, RIP-seq\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — transcriptome-wide binding analysis with two orthogonal methods, single lab\",\n      \"pmids\": [\"36192131\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ALKBH8 modifies the wobble uridine of selenocysteine tRNA to promote selenoprotein translation; Alkbh8-deficient mice show increased oxidative stress markers and decreased thioredoxin reductase protein levels under basal conditions, and fail to develop naphthalene tolerance, establishing ALKBH8 as protective against oxidative lung damage via selenoprotein-dependent antioxidant mechanisms.\",\n      \"method\": \"Alkbh8-/- mouse model, western blot for oxidative stress markers and thioredoxin reductase, naphthalene exposure challenge experiments\",\n      \"journal\": \"Epigenetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — defined molecular mechanism (selenoprotein translation via tRNA modification) linked to in vivo phenotype\",\n      \"pmids\": [\"32303148\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In Alkbh8-/- mice, mcm5U modification is reduced in most tissues but partially compensated in the brain. Loss of ALKBH8 reduces tRNA protein translation efficiency, impairs red blood cell differentiation and embryogenesis (shown by proteome analysis linking downregulated factors to red blood cell and protoporphyrin metabolism), and causes neural dysfunction with oxidative stress and reduced mitochondrial membrane potential in neurons and glial cells.\",\n      \"method\": \"Alkbh8-/- mouse model, UPLC-MS/MS for tRNA modification quantification, proteomics, behavioral tests (novel object recognition, rotarod, forced swim), brain histopathology, mitochondrial membrane potential assay\",\n      \"journal\": \"iScience / PNAS nexus\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse with multiple orthogonal phenotypic readouts, single lab\",\n      \"pmids\": [\"39280612\", \"38550277\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ALKBH8 is a direct transcriptional target of Wnt/β-catenin signaling. Its methyltransferase activity promotes translation elongation at adenine-ending codons by modifying U34 tRNA, specifically regulating KRAS translation in a codon-dependent manner. Rescue experiments with methyltransferase-dead ALKBH8 fail to restore KRAS translation, confirming catalytic activity requirement. Loss of ALKBH8 induces ribosome pausing at adenine-ending codons.\",\n      \"method\": \"CRISPR knockout, ribosome profiling (ribosome pausing analysis), rescue with wildtype vs. catalytically-dead ALKBH8 mutant, Apcmin/+ and AOM/DSS mouse tumor models, xenograft, reporter assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple in vivo models, ribosome profiling, mutagenesis rescue, identifies specific substrate (KRAS) and upstream regulator (Wnt/β-catenin)\",\n      \"pmids\": [\"41083459\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"During ZIKV infection, ALKBH8 is required for mcm5s2U34 tRNA modification; CRISPR/shRNA-mediated knockdown of ALKBH8 significantly reduces ZIKV replication, indicating that ALKBH8-mediated U34 tRNA modification is exploited by the virus to optimize translation of A-ending codon-biased viral proteins.\",\n      \"method\": \"CRISPR/Cas9 and shRNA knockdown of ALKBH8, mass spectrometry for tRNA modification quantification, codon-biased GFP reporters, viral replication assays\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple KD approaches with viral replication readout, preprint\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"ALKBH8 is a bifunctional enzyme containing a methyltransferase domain (requiring TRM112 as an obligate activating partner) that converts cm5U to mcm5U at the wobble position (U34) of specific tRNAs, and an AlkB oxygenase domain that further hydroxylates mcm5U to (S)-mchm5U in tRNA-Gly(UCC); these wobble uridine modifications promote codon-specific translation elongation—particularly at A-ending codons—thereby regulating the translational output of codon-biased mRNAs including selenoprotein mRNAs (via tRNASec modification) and oncogenes such as KRAS, with ALKBH8 itself being transcriptionally regulated by Wnt/β-catenin, and its loss causing translational infidelity, oxidative stress, and neurodevelopmental defects.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"ALKBH8 is a bifunctional tRNA-modifying enzyme that regulates codon-specific translation elongation by installing wobble uridine (U34) modifications on select tRNAs. Its methyltransferase domain, activated by the obligate partner TRM112, catalyzes the conversion of cm5U to mcm5U—a prerequisite for downstream thiolation (mcm5s2U) and 2'-O-ribose methylation—while its AlkB oxygenase domain hydroxylates mcm5U to (S)-mchm5U specifically on tRNA-Gly(UCC) [PMID:20123966, PMID:21285950, PMID:26438534]. These modifications promote efficient decoding of adenine-ending codons, thereby controlling translation of codon-biased mRNAs including selenoprotein mRNAs (via tRNASec modification to support UGA recoding) and oncogenes such as KRAS, whose translation depends on ALKBH8 catalytic activity downstream of Wnt/β-catenin transcriptional activation [PMID:20123966, PMID:32303148, PMID:41083459]. Loss-of-function mutations in the ALKBH8 methyltransferase domain cause intellectual disability (MRT71) through complete abrogation of ALKBH8-dependent tRNA modifications [PMID:34757492].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Establishing that wobble uridine methylation by the ALKBH8 ortholog Trm9 has a translational output—it selectively enhances expression of codon-biased proteins involved in the DNA damage response—moved the modification from a biochemical curiosity to a gene-expression regulatory mechanism.\",\n      \"evidence\": \"Yeast trm9Δ deletion with tRNA modification analysis, codon usage computation, and western blot for target proteins\",\n      \"pmids\": [\"18082610\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the same codon-specific translational control operates in mammalian systems was untested\", \"Direct ribosome-level evidence of elongation rate change was lacking\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Generation of Alkbh8-knockout mice demonstrated that ALKBH8 is the mammalian enzyme responsible for the cm5U-to-mcm5U methylation step, that TRM112 is its obligate activating partner, and that loss of this modification disrupts selenoprotein translation by impairing tRNASec modification.\",\n      \"evidence\": \"Alkbh8−/− mouse, HPLC/MS tRNA modification profiling, in vitro methyltransferase assay, co-immunoprecipitation of ALKBH8–TRM112\",\n      \"pmids\": [\"20123966\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Function of the AlkB oxygenase domain remained unknown\", \"Whether translational fidelity is affected in mammalian cells was not assessed\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identification of the AlkB domain as an RNA hydroxylase that converts mcm5U to (S)-mchm5U on tRNA-Gly(UCC) revealed that ALKBH8 is a true bifunctional enzyme and expanded the ALKBH family beyond DNA repair functions.\",\n      \"evidence\": \"In vitro hydroxylation assay with recombinant AlkB domain, mass spectrometry, validation in Alkbh8−/− mouse tRNA\",\n      \"pmids\": [\"21285950\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Biological consequence of the hydroxylation product (S)-mchm5U was not defined\", \"No structural information for the mammalian enzyme\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Demonstrating that loss of Trm9-dependent modifications causes translational infidelity and triggers the unfolded protein response established that these wobble modifications are required for accurate decoding, not merely for efficiency.\",\n      \"evidence\": \"Translational fidelity reporters and phenotypic assays in trm9Δ yeast\",\n      \"pmids\": [\"22832247\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether translational infidelity occurs in mammalian ALKBH8-null cells was unknown\", \"Specific miscoded codons in vivo were not fully mapped\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"The crystal structure of the Trm9–Trm112 complex revealed how TRM112 serves as a structurally plastic activating platform shared among multiple methyltransferases, explaining the obligate requirement for this partner.\",\n      \"evidence\": \"X-ray crystallography with structure-guided mutagenesis\",\n      \"pmids\": [\"26438534\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure of full-length mammalian ALKBH8 including the AlkB domain\", \"How substrate tRNA is recognized by the complex was not resolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Linking ALKBH8-dependent selenoprotein translation to an in vivo oxidative stress phenotype established that wobble uridine modification is physiologically required for antioxidant defense, specifically through thioredoxin reductase expression.\",\n      \"evidence\": \"Alkbh8−/− mice challenged with naphthalene; western blot for thioredoxin reductase and oxidative stress markers\",\n      \"pmids\": [\"32303148\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Which specific selenoproteins are most affected was not comprehensively profiled\", \"Single-lab study without independent replication\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Patient-derived evidence that a missense variant in the ALKBH8 methyltransferase domain abolishes all ALKBH8-dependent tRNA modifications without reducing protein levels established enzymatic loss-of-function as the disease mechanism for MRT71 intellectual disability.\",\n      \"evidence\": \"Patient cell analysis with mass spectrometry-based tRNA modification profiling and targeted proteomics\",\n      \"pmids\": [\"34757492\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Only a single variant/family was studied\", \"Neuronal-specific consequences of modification loss were not mechanistically dissected\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Transcriptome-wide binding studies revealed that ALKBH8 directly binds its known substrate tRNAs in their mature CCA-modified form and also associates with C/D box snoRNAs, suggesting possible non-tRNA functions.\",\n      \"evidence\": \"HITS-CLIP and RIP-seq in human cells\",\n      \"pmids\": [\"36192131\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional significance of snoRNA interaction is unknown\", \"Whether snoRNA binding reflects a catalytic or regulatory role is unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Comprehensive phenotyping of Alkbh8−/− mice revealed tissue-specific compensation of tRNA modification (partial rescue in brain), impaired erythropoiesis, and neural dysfunction with reduced mitochondrial membrane potential, connecting wobble uridine modification to mitochondrial and neurodevelopmental physiology.\",\n      \"evidence\": \"Alkbh8−/− mouse model with UPLC-MS/MS tRNA modification profiling, proteomics, behavioral testing, and mitochondrial assays\",\n      \"pmids\": [\"39280612\", \"38550277\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of tissue-specific compensation is unknown\", \"Identity of the compensating enzyme(s) in brain is not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identifying ALKBH8 as a direct Wnt/β-catenin target gene whose methyltransferase activity specifically promotes KRAS translation through codon-dependent ribosome elongation provided the first mechanistic link between an epitranscriptomic modifier and oncogenic signaling in colorectal cancer.\",\n      \"evidence\": \"CRISPR knockout, ribosome profiling showing ribosome pausing at A-ending codons, rescue with catalytically dead mutant, Apcmin/+ and AOM/DSS mouse tumor models\",\n      \"pmids\": [\"41083459\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other oncogenes with similar codon bias are co-regulated was not systematically addressed\", \"Therapeutic targetability of ALKBH8 catalytic activity is untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the biological function of ALKBH8's hydroxylation product (S)-mchm5U, the mechanism of substrate tRNA recognition by the full-length mammalian enzyme, the functional significance of snoRNA interactions, and the basis for tissue-specific compensation of tRNA modification loss.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structure of full-length mammalian ALKBH8\", \"Biological role of (S)-mchm5U hydroxylation uncharacterized\", \"snoRNA interaction function unknown\", \"Tissue-specific compensation mechanism undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 2, 3, 4, 9, 13]},\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [1, 8]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [0, 10]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 1, 4, 5, 13]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 4, 5, 13]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [13]}\n    ],\n    \"complexes\": [\n      \"ALKBH8–TRM112 methyltransferase complex\"\n    ],\n    \"partners\": [\n      \"TRM112\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}