{"gene":"ALKBH8","run_date":"2026-06-09T22:02:43","timeline":{"discoveries":[{"year":2010,"finding":"Mammalian ALKBH8 is a tRNA methyltransferase required for the final biosynthetic step of 5-methoxycarbonylmethyluridine (mcm5U) at the wobble position of tRNA; this activity requires interaction with the small accessory protein TRM112 to form a functional complex. Furthermore, prior ALKBH8-mediated mcm5U methylation is a prerequisite for subsequent thiolation (forming mcm5s2U) and 2'-O-ribose methylation (forming mcm5Um). In Alkbh8-/- mice, the selenocysteine-specific tRNA (tRNASec) is aberrantly modified, resulting in reduced UGA stop codon recoding to selenocysteine for the selenoprotein Gpx1.","method":"Knockout mouse tRNA analysis, biochemical complementation, mass spectrometry of tRNA modifications, Co-IP of ALKBH8-TRM112 complex, selenoprotein recoding assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — knockout mouse with multiple orthogonal biochemical readouts (tRNA mass spectrometry, protein complex pulldown, selenoprotein assay), replicated in subsequent studies","pmids":["20123966"],"is_preprint":false},{"year":2011,"finding":"The AlkB oxygenase domain of ALKBH8 specifically hydroxylates mcm5U to generate (S)-5-methoxycarbonylhydroxymethyluridine (mchm5U) in tRNA-Gly(UCC), creating a novel diastereomeric pair of wobble nucleosides (both (R)- and (S)-mchm5U are present in mammalian tRNA). This hydroxylation activity is distinct from the methyltransferase domain activity and expands ALKBH8's function beyond tRNA methylation to include tRNA hydroxylation.","method":"In vitro enzymatic assay of purified ALKBH8 AlkB domain, mass spectrometry of tRNA modifications from wild-type and Alkbh8-/- mice, NMR-based stereochemical characterization","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzymatic reconstitution combined with in vivo knockout validation and rigorous chemical characterization","pmids":["21285950"],"is_preprint":false},{"year":2007,"finding":"Yeast Trm9 (ortholog of ALKBH8) methylates the wobble uridine of tRNAARG(UCU) and tRNAGLU(UUC), and this modification enhances translational elongation of transcripts enriched in specific arginine and glutamic acid codons, resulting in increased protein levels of DNA damage response proteins (Yef3, Rnr1, Rnr3), thus linking tRNA modification to the DNA damage response.","method":"Genetic deletion of TRM9, computational codon usage analysis, quantitative protein-level assays (immunoblot), phenotypic cell death assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic knockout with multiple orthogonal readouts (protein levels, codon usage analysis, phenotypic rescue), replicated in subsequent studies","pmids":["18082610"],"is_preprint":false},{"year":2011,"finding":"Yeast Trm9 (ALKBH8 ortholog) and Trm112 function together at the final step in formation of mcm5U by methylating the cm5U intermediate substrate; co-expression and purification of His-tagged Trm9 with native Trm112 from E. coli showed they form a stable complex, and Trm112 dramatically improves the methyltransferase activity of Trm9 in vitro. In trm9Δ or trm112Δ mutants, ncm5U and ncm5s2U accumulate rather than the expected cm5U intermediates.","method":"Recombinant protein co-expression and purification, in vitro methyltransferase activity assay, HPLC analysis of tRNA modifications from yeast mutants","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of complex with enzymatic activity assay, corroborated by in vivo tRNA modification analysis in deletion mutants","pmids":["21687733"],"is_preprint":false},{"year":2015,"finding":"Crystal structure of the yeast Trm9-Trm112 complex (ortholog of mammalian ALKBH8-TRM112) reveals the structural basis for their interaction in mediating mcm5U modification at the tRNA anticodon wobble position; Trm112 uses a similar interaction mode to bind multiple methyltransferase partners despite low sequence identity (<20%) among them, demonstrating molecular plasticity.","method":"X-ray crystallography, structure-function mutagenesis analysis, comparison with other Trm112-MTase complex structures","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with functional validation and structure-function analysis","pmids":["26438534"],"is_preprint":false},{"year":2006,"finding":"Yeast Trm9 (ALKBH8 ortholog) acts downstream of Elongator in the tRNA wobble uridine modification pathway; genetic epistasis (tot3Δtrm9Δ double mutant) shows that Elongator-dependent tRNA modification acts upstream of Trm9-mediated methylation, and that Trm9 specifically methylates the wobble uridine of tRNAGlu.","method":"Genetic epistasis analysis (double mutants), zymocin sensitivity assays, tRNA overexpression suppressor analysis","journal":"Molecular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic epistasis in yeast with multiple phenotypic readouts, single laboratory","pmids":["16390459"],"is_preprint":false},{"year":2012,"finding":"Loss of Trm9-catalyzed mcm5U and mcm5s2U wobble modifications (in trm9Δ yeast) leads to increased translational infidelity specifically at arginine and glutamic acid codons from mixed codon boxes, resulting in protein errors and activation of unfolded protein and heat shock responses.","method":"Reporter assays for translational infidelity, quantitative tRNA modification analysis, codon re-engineering experiments, phenotypic assays for stress response activation","journal":"RNA biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal functional assays (reporter, proteomics, stress markers) in single laboratory","pmids":["22832247"],"is_preprint":false},{"year":2014,"finding":"The mammalian ALKBH8 protein possesses two separable enzymatic activities encoded in its two domains: tRNA hydroxylase activity (AlkB domain, converting mcm5U to mchm5U in tRNA-Gly(UCC)) and DNA repair demethylase activity. Protozoan ALKBH8 orthologs show both DNA repair and tRNA modification activities in vitro, while bacterial ALKBH8 orthologs show DNA repair but not tRNA modification, indicating domain-specific functional divergence during evolution.","method":"In vitro enzymatic assays (tRNA modification, DNA repair), mass spectrometry of tRNA modifications, analysis of ALKBH8-deficient Agrobacterium mutant","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro biochemical characterization across multiple organisms, single laboratory","pmids":["24914785"],"is_preprint":false},{"year":2009,"finding":"Silencing of ALKBH8 in human bladder cancer cells reduces ROS production via down-regulation of NOX-1, which then activates JNK and p38, leading to H2AX phosphorylation (γH2AX) and apoptosis; ALKBH8 knockdown also suppresses tumor invasion and angiogenesis in vivo in chorioallantoic membrane and orthotopic mouse models.","method":"siRNA knockdown, ROS assay, immunoblot for signaling components (JNK, p38, γH2AX), chorioallantoic membrane assay, orthotopic mouse tumor model","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean knockdown with multiple pathway readouts and in vivo validation, but mechanistic connection to tRNA modification activity not established","pmids":["19293182"],"is_preprint":false},{"year":2016,"finding":"ALKBH8 knockdown in bladder cancer cells induces apoptosis via downregulation of the anti-apoptotic protein survivin; ALKBH8 transgenic mice show accelerated bladder tumor growth and invasiveness in a carcinogen-induced model.","method":"siRNA knockdown, immunoblot for survivin, ALKBH8 transgenic mouse with N-butyl-N-(4-hydroxybutyl)-nitrosamine-induced bladder cancer model","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro knockdown with protein-level readout combined with in vivo transgenic mouse model, single laboratory","pmids":["27329810"],"is_preprint":false},{"year":2020,"finding":"ALKBH8-deficient mice show increased markers of oxidative stress, decreased thioredoxin reductase protein levels (consistent with impaired selenoprotein translation), reprogrammed stress response gene expression under basal conditions, and hypersensitivity to naphthalene-induced lung damage; wild-type mice develop naphthalene tolerance after repeated exposures, which is absent in Alkbh8-deficient mice.","method":"Alkbh8 knockout mouse, oxidative stress marker assays, immunoblot for thioredoxin reductase, naphthalene exposure paradigm, histological lung analysis","journal":"Epigenetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockout mouse with multiple biochemical and phenotypic readouts, single laboratory","pmids":["32303148"],"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 showing protein is still expressed), establishing that the methyltransferase domain activity is essential and that loss-of-function of the tRNA methyltransferase activity is the disease mechanism for MRT71 intellectual disability syndrome.","method":"Patient cell-based tRNA modification analysis, targeted proteomics to confirm protein expression, clinical genetics","journal":"Human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct biochemical confirmation in patient cells using targeted proteomics, single laboratory","pmids":["34757492"],"is_preprint":false},{"year":2022,"finding":"HITS-CLIP and RIP-seq analyses of human ALKBH8 show that it binds fully processed and CCA-modified tRNAs as substrates, including the known wobble U-containing tRNAs. Additionally, ALKBH8 binds several types of noncoding RNAs including C/D box snoRNAs, suggesting substrate interactions beyond the canonical tRNAs.","method":"HITS-CLIP, RIP-seq, transcriptome-wide RNA-protein interaction mapping","journal":"RNA (New York, N.Y.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — transcriptome-wide binding analysis with two orthogonal methods (HITS-CLIP and RIP-seq), single laboratory","pmids":["36192131"],"is_preprint":false},{"year":2024,"finding":"Alkbh8-knockout mice show reduced mcm5U and (S)-mchm5U levels in tRNA; in the brain, mcm5U levels are partially compensated. Alkbh8-/- mice exhibit cognitive and motor behavioral deficits, reduced brain weight, ischemic pathological changes in cerebral cortex and hippocampus, and differential expression of oxidative stress-related proteins and metabolites; neurons and glial cells from knockout mice show reduced mitochondrial membrane potential, indicating ALKBH8 maintains neural function through oxidative stress regulation.","method":"Alkbh8 knockout mouse, mass spectrometry of tRNA modifications, behavioral tests (novel object recognition, rotarod, forced swim), histology, proteomics, metabolomics, mitochondrial membrane potential assay","journal":"PNAS nexus","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockout mouse with multiple orthogonal phenotypic, proteomic, and metabolomic readouts, single laboratory","pmids":["38550277"],"is_preprint":false},{"year":2024,"finding":"In Alkbh8-knockout mice, tRNA protein translation efficiency is reduced and proteome analysis reveals downregulation of factors associated with red blood cell differentiation and protoporphyrin metabolism, suggesting ALKBH8-mediated mcm5U modification is essential for normal erythropoiesis during embryogenesis.","method":"Alkbh8 knockout mouse, UPLC-MS/MS quantification of RNA modifications at embryonic stages, proteome analysis","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockout mouse with proteomics and modification analysis, single laboratory","pmids":["39280612"],"is_preprint":false},{"year":2025,"finding":"ALKBH8 is a direct transcriptional target of Wnt/β-catenin signaling and is upregulated in colorectal cancer; genetic ablation of ALKBH8 inhibits intestinal tumor development in multiple mouse models (Apcmin/+, AOM/DSS, xenograft). Loss of ALKBH8 causes ribosome pausing at adenine-ending codons, impairing translation elongation of mRNAs enriched with these codons including KRAS proto-oncogene; rescue experiments confirm that the methyltransferase activity of ALKBH8 is required for its translation-promoting function.","method":"Genetic ablation in multiple mouse tumor models, ribosome profiling (ribosome pausing analysis), codon-specific translation reporter assays, methyltransferase-dead mutant rescue experiments, Wnt/β-catenin pathway reporter assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal approaches (ribosome profiling, mouse models, mutagenesis rescue) in single study with rigorous controls","pmids":["41083459"],"is_preprint":false},{"year":2025,"finding":"CRISPR/Cas9 knockout of ALKBH8 significantly reduces Zika virus (ZIKV) replication in human cells; ZIKV infection increases mcm5s2U34 tRNA modification content in host cells, and ALKBH8 loss impairs the preferential decoding of AA-ending codons that ZIKV genome exploits for protein synthesis.","method":"CRISPR/Cas9 knockout, viral replication assay, mass spectrometry of tRNA modifications, codon-biased GFP sensor assays","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 2 / Weak — preprint, single laboratory, clean CRISPR knockout with viral phenotype but limited mechanistic follow-up specific to ALKBH8","pmids":["bio_10.1101_2025.06.03.657606"],"is_preprint":true},{"year":2025,"finding":"ALKBH8 deletion reduces chikungunya virus (CHIKV) replication by impairing sulfur relay, recapitulating effects of methionine-cysteine deprivation; CHIKV upregulates ALKBH8 under sulfur-depleted conditions, suggesting ALKBH8-mediated U34-tRNA modification is exploited by the virus to support its replication.","method":"ALKBH8 deletion cell line, viral replication assay, metabolic supplementation experiments","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 2 / Weak — preprint, single laboratory, genetic deletion with viral phenotype but mechanistic details are limited in abstract","pmids":["bio_10.1101_2025.07.11.664323"],"is_preprint":true}],"current_model":"ALKBH8 is a dual-domain enzyme that functions as both a tRNA methyltransferase (Trm9-like domain, requiring TRM112 as an obligate co-activator) and an AlkB-type oxygenase: the methyltransferase domain catalyzes the final step of mcm5U formation at the wobble position (U34) of specific tRNAs including tRNASec, while the AlkB domain subsequently hydroxylates mcm5U to (S)-mchm5U specifically in tRNA-Gly(UCC); ALKBH8-mediated mcm5U is a prerequisite for further wobble modifications (thiolation to mcm5s2U and 2'-O-methylation to mcm5Um), and the resulting modified tRNAs promote codon-specific translation elongation—particularly at adenine-ending and glutamic acid/arginine codons—thereby regulating selenoprotein synthesis, DNA damage response, neuronal function, erythropoiesis, and oncogenic translation programs including KRAS in colorectal cancer."},"narrative":{"mechanistic_narrative":"ALKBH8 is a dual-domain tRNA-modifying enzyme that catalyzes wobble-uridine (U34) modifications which tune codon-specific translation elongation and downstream cellular stress, neural, and oncogenic programs [PMID:20123966, PMID:41083459]. Its Trm9-like methyltransferase domain performs the final step of 5-methoxycarbonylmethyluridine (mcm5U) formation at U34, an activity that requires the accessory protein TRM112 as an obligate co-activator forming a stable complex; conservation of this partnership is established by the yeast Trm9–Trm112 orthologs, whose reconstitution and crystal structure define the interaction mode and show that Trm112 strongly stimulates methyltransferase activity [PMID:20123966, PMID:21687733, PMID:26438534]. ALKBH8-generated mcm5U is a prerequisite for further wobble maturation, including thiolation to mcm5s2U and 2'-O-methylation to mcm5Um [PMID:20123966]. A second, separable AlkB-type oxygenase domain hydroxylates mcm5U to (S)-mchm5U specifically in tRNA-Gly(UCC) [PMID:21285950, PMID:24914785]. ALKBH8 binds fully processed, CCA-modified tRNAs as substrates [PMID:36192131]. Functionally, mcm5U modification of tRNA-Sec is required for UGA recoding and selenoprotein (Gpx1, thioredoxin reductase) synthesis, linking ALKBH8 loss to oxidative-stress dysregulation [PMID:20123966, PMID:32303148], and the modification promotes elongation at adenine-ending codons such that ALKBH8 loss causes ribosome pausing and impaired translation of KRAS in colorectal cancer, where ALKBH8 is a Wnt/β-catenin transcriptional target whose ablation suppresses intestinal tumorigenesis [PMID:41083459]. ALKBH8 also supports neural function and erythropoiesis through its translation role [PMID:38550277, PMID:39280612]. A loss-of-function missense variant in the methyltransferase domain abolishes ALKBH8-dependent tRNA modifications and causes the MRT71 intellectual disability syndrome [PMID:34757492].","teleology":[{"year":2007,"claim":"Established that the ALKBH8 ortholog Trm9 links wobble-uridine tRNA methylation to codon-biased translation, answering how a tRNA modification influences specific protein outputs.","evidence":"Genetic deletion of yeast TRM9 with codon-usage analysis and quantitative protein assays for DNA damage response proteins","pmids":["18082610"],"confidence":"High","gaps":["Performed in yeast; mammalian ALKBH8 substrates not yet defined","Mechanism of elongation enhancement at the ribosome not directly measured"]},{"year":2006,"claim":"Placed Trm9-mediated methylation downstream of Elongator in the wobble-uridine modification pathway, ordering the biosynthetic steps.","evidence":"Genetic epistasis (double mutants) and zymocin sensitivity assays in yeast","pmids":["16390459"],"confidence":"Medium","gaps":["Pathway order inferred genetically, not biochemically reconstituted","Yeast-only; mammalian pathway hierarchy untested here"]},{"year":2009,"claim":"First implicated mammalian ALKBH8 in cancer cell survival via ROS/NOX-1 signaling, raising the question of how its enzymatic activity connects to tumor phenotypes.","evidence":"siRNA knockdown in bladder cancer cells with ROS, signaling immunoblots, and in vivo tumor models","pmids":["19293182"],"confidence":"Medium","gaps":["Mechanistic link to tRNA modification activity not established","NOX-1 regulation mechanism unresolved"]},{"year":2010,"claim":"Defined mammalian ALKBH8 as the methyltransferase completing mcm5U formation, requiring TRM112, and showed it is a prerequisite for downstream wobble modifications and selenoprotein recoding.","evidence":"Alkbh8 knockout mouse tRNA mass spectrometry, ALKBH8-TRM112 Co-IP, biochemical complementation, and Gpx1 selenoprotein recoding assay","pmids":["20123966"],"confidence":"High","gaps":["AlkB domain function not yet defined here","Substrate tRNA repertoire not exhaustively mapped"]},{"year":2011,"claim":"Assigned the AlkB oxygenase domain a distinct hydroxylation activity, revealing ALKBH8 as a bifunctional enzyme generating mchm5U.","evidence":"In vitro assay of purified AlkB domain plus knockout-mouse tRNA mass spectrometry and NMR stereochemistry","pmids":["21285950"],"confidence":"High","gaps":["Functional consequence of mchm5U on translation not determined","Why hydroxylation is restricted to tRNA-Gly(UCC) unexplained"]},{"year":2011,"claim":"Demonstrated that Trm112 forms a stable complex with Trm9/ALKBH8 and is required to activate its methyltransferase activity, defining the co-activator relationship biochemically.","evidence":"Recombinant co-expression/purification, in vitro methyltransferase assay, and HPLC of tRNA modifications in deletion mutants","pmids":["21687733"],"confidence":"High","gaps":["Structural basis of activation not resolved here","Mammalian complex stoichiometry not addressed"]},{"year":2014,"claim":"Showed the two ALKBH8 domains carry separable activities (tRNA hydroxylase and DNA repair demethylase) and that these diverged across evolution.","evidence":"In vitro tRNA-modification and DNA-repair assays across protozoan and bacterial orthologs plus an Agrobacterium deletion mutant","pmids":["24914785"],"confidence":"Medium","gaps":["Physiological relevance of mammalian ALKBH8 DNA-repair activity not demonstrated in vivo","Single laboratory"]},{"year":2015,"claim":"Resolved the structural basis of the Trm9-Trm112 interaction, explaining Trm112's plasticity in partnering multiple methyltransferases.","evidence":"X-ray crystallography of the yeast complex with structure-function mutagenesis","pmids":["26438534"],"confidence":"High","gaps":["Structure is of yeast orthologs, not mammalian ALKBH8","tRNA-bound complex not captured"]},{"year":2016,"claim":"Extended ALKBH8 tumor-promoting function to survivin regulation and in vivo carcinogen-induced bladder cancer, reinforcing an oncogenic role.","evidence":"siRNA knockdown with survivin immunoblot and ALKBH8 transgenic carcinogen-induced mouse model","pmids":["27329810"],"confidence":"Medium","gaps":["Link between survivin and tRNA modification activity not established","Single laboratory"]},{"year":2020,"claim":"Connected ALKBH8 loss to systemic oxidative-stress dysregulation and impaired chemical tolerance, consistent with defective selenoprotein translation.","evidence":"Alkbh8 knockout mouse oxidative-stress markers, thioredoxin reductase immunoblot, and naphthalene exposure paradigm","pmids":["32303148"],"confidence":"Medium","gaps":["Direct ribosome-level translation defect not measured","Single laboratory"]},{"year":2021,"claim":"Established that loss of ALKBH8 methyltransferase activity is the disease mechanism for MRT71 intellectual disability, tying enzyme function to human pathology.","evidence":"Patient cell tRNA-modification analysis with targeted proteomics confirming protein expression and clinical genetics","pmids":["34757492"],"confidence":"Medium","gaps":["Neuronal cell-type-specific consequences not defined","Single family/laboratory"]},{"year":2022,"claim":"Mapped ALKBH8's RNA substrate landscape transcriptome-wide, confirming mature tRNA binding and revealing additional noncoding RNA interactions.","evidence":"HITS-CLIP and RIP-seq in human cells","pmids":["36192131"],"confidence":"Medium","gaps":["Functional significance of snoRNA binding unknown","Binding does not establish catalytic modification of all bound RNAs"]},{"year":2024,"claim":"Linked ALKBH8-dependent wobble modification to neural function and erythropoiesis through reduced translation efficiency in vivo.","evidence":"Alkbh8 knockout mice with tRNA mass spectrometry, behavioral/histological analysis, mitochondrial assays, proteomics, and embryonic-stage proteome/modification analysis","pmids":["38550277","39280612"],"confidence":"Medium","gaps":["Codon-level translation defects in brain/erythroid cells not resolved","Brain mcm5U partial compensation mechanism unknown"]},{"year":2025,"claim":"Defined the codon-specific translation mechanism of ALKBH8 oncogenesis: it is a Wnt/β-catenin target whose mcm5U activity prevents ribosome pausing at adenine-ending codons, sustaining KRAS translation in colorectal cancer.","evidence":"Ribosome profiling, codon reporter assays, methyltransferase-dead rescue, and multiple genetic mouse tumor models","pmids":["41083459"],"confidence":"High","gaps":["Full repertoire of A-ending-codon-dependent oncogenic transcripts not catalogued","Therapeutic targetability not tested"]},{"year":2025,"claim":"Implicated ALKBH8-mediated U34 modification in viral exploitation of host codon-biased translation.","evidence":"CRISPR/Cas9 and deletion cell lines with viral replication assays, tRNA mass spectrometry, and codon sensor assays for ZIKV and CHIKV (preprints)","pmids":["bio_10.1101_2025.06.03.657606","bio_10.1101_2025.07.11.664323"],"confidence":"Low","gaps":["Preprints, not peer-reviewed","ALKBH8-specific mechanism in viral replication only partially defined"]},{"year":null,"claim":"How ALKBH8 substrate selectivity is achieved (why hydroxylation is restricted to tRNA-Gly(UCC), the role of noncoding RNA binding, and whether mammalian DNA-repair activity is physiologically relevant) remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of the substrate-bound mammalian ALKBH8-TRM112 complex","Functional role of ALKBH8 snoRNA binding unknown","In vivo relevance of the AlkB-domain DNA demethylase activity untested in mammals"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,3,11,15]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[0,1,7]},{"term_id":"GO:0016491","term_label":"oxidoreductase activity","supporting_discovery_ids":[1,7]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[12]},{"term_id":"GO:0045182","term_label":"translation regulator activity","supporting_discovery_ids":[2,15]}],"localization":[],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,1,3]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[2,15]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[10]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[11,15]}],"complexes":["ALKBH8-TRM112 methyltransferase complex"],"partners":["TRM112"],"other_free_text":[]}},"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":261,"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":200,"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":139,"is_preprint":false},{"pmid":"19293182","id":"PMC_19293182","title":"A novel human AlkB homologue, ALKBH8, contributes to human bladder cancer progression.","date":"2009","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/19293182","citation_count":130,"is_preprint":false},{"pmid":"16390459","id":"PMC_16390459","title":"tRNAGlu wobble uridine methylation by Trm9 identifies Elongator's key role for zymocin-induced cell death in yeast.","date":"2006","source":"Molecular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/16390459","citation_count":88,"is_preprint":false},{"pmid":"22832247","id":"PMC_22832247","title":"Translational infidelity-induced protein stress results from a deficiency in Trm9-catalyzed tRNA modifications.","date":"2012","source":"RNA biology","url":"https://pubmed.ncbi.nlm.nih.gov/22832247","citation_count":83,"is_preprint":false},{"pmid":"21687733","id":"PMC_21687733","title":"Unexpected accumulation of ncm(5)U and ncm(5)S(2) (U) in a trm9 mutant suggests an additional step in the synthesis of mcm(5)U and mcm(5)S(2)U.","date":"2011","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/21687733","citation_count":59,"is_preprint":false},{"pmid":"26438534","id":"PMC_26438534","title":"Insights into molecular plasticity in protein complexes from Trm9-Trm112 tRNA modifying enzyme crystal structure.","date":"2015","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/26438534","citation_count":35,"is_preprint":false},{"pmid":"27329810","id":"PMC_27329810","title":"ALKBH8 promotes bladder cancer growth and progression through regulating the expression of survivin.","date":"2016","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/27329810","citation_count":34,"is_preprint":false},{"pmid":"24914785","id":"PMC_24914785","title":"Protozoan ALKBH8 oxygenases display both DNA repair and tRNA modification activities.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/24914785","citation_count":27,"is_preprint":false},{"pmid":"33544954","id":"PMC_33544954","title":"Neurodevelopmental disorder in an Egyptian family with a biallelic ALKBH8 variant.","date":"2021","source":"American journal of medical genetics. Part A","url":"https://pubmed.ncbi.nlm.nih.gov/33544954","citation_count":24,"is_preprint":false},{"pmid":"32303148","id":"PMC_32303148","title":"The epitranscriptomic writer ALKBH8 drives tolerance and protects mouse lungs from the environmental pollutant naphthalene.","date":"2020","source":"Epigenetics","url":"https://pubmed.ncbi.nlm.nih.gov/32303148","citation_count":18,"is_preprint":false},{"pmid":"34757492","id":"PMC_34757492","title":"Insight into ALKBH8-related intellectual developmental disability based on the first pathogenic missense variant.","date":"2021","source":"Human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/34757492","citation_count":15,"is_preprint":false},{"pmid":"37119026","id":"PMC_37119026","title":"ALKBH8 as a potential N6 -methyladenosine (m6 A) eraser in insects.","date":"2023","source":"Insect molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/37119026","citation_count":12,"is_preprint":false},{"pmid":"38550277","id":"PMC_38550277","title":"ALKBH8 contributes to neurological function through oxidative stress regulation.","date":"2024","source":"PNAS nexus","url":"https://pubmed.ncbi.nlm.nih.gov/38550277","citation_count":11,"is_preprint":false},{"pmid":"39280612","id":"PMC_39280612","title":"RNA-modifying enzyme Alkbh8 is involved in mouse embryonic development.","date":"2024","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/39280612","citation_count":5,"is_preprint":false},{"pmid":"36192131","id":"PMC_36192131","title":"HITS-CLIP analysis of human ALKBH8 reveals interactions with fully processed substrate tRNAs and with specific noncoding RNAs.","date":"2022","source":"RNA (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/36192131","citation_count":5,"is_preprint":false},{"pmid":"38189198","id":"PMC_38189198","title":"The first Turkish family with a novel biallelic missense variant of the ALKBH8 gene: A study on the clinical and variant spectrum of ALKBH8-related intellectual developmental disorders.","date":"2024","source":"American journal of medical genetics. Part A","url":"https://pubmed.ncbi.nlm.nih.gov/38189198","citation_count":4,"is_preprint":false},{"pmid":"41083459","id":"PMC_41083459","title":"ALKBH8-mediated codon-specific translation promotes colorectal tumorigenesis.","date":"2025","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/41083459","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.07.11.664323","title":"Metabolic reprogramming of methylthioadenosine-dependent sulfur recycling is a major driver of CHIKV infection","date":"2025-07-11","source":"bioRxiv","url":"https://doi.org/10.1101/2025.07.11.664323","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.06.03.657606","title":"Zika virus reprograms the host tRNA epitranscriptome to adapt translation to A-ending codon bias","date":"2025-06-03","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.03.657606","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.03.12.642825","title":"WEE1 inhibitors synergise with mRNA translation defects via activation of the kinase GCN2","date":"2025-03-13","source":"bioRxiv","url":"https://doi.org/10.1101/2025.03.12.642825","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":12582,"output_tokens":4877,"usd":0.05545,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":13199,"output_tokens":4623,"usd":0.090785,"stage2_stop_reason":"end_turn"},"total_usd":0.146235,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2010,\n      \"finding\": \"Mammalian ALKBH8 is a tRNA methyltransferase required for the final biosynthetic step of 5-methoxycarbonylmethyluridine (mcm5U) at the wobble position of tRNA; this activity requires interaction with the small accessory protein TRM112 to form a functional complex. Furthermore, prior ALKBH8-mediated mcm5U methylation is a prerequisite for subsequent thiolation (forming mcm5s2U) and 2'-O-ribose methylation (forming mcm5Um). In Alkbh8-/- mice, the selenocysteine-specific tRNA (tRNASec) is aberrantly modified, resulting in reduced UGA stop codon recoding to selenocysteine for the selenoprotein Gpx1.\",\n      \"method\": \"Knockout mouse tRNA analysis, biochemical complementation, mass spectrometry of tRNA modifications, Co-IP of ALKBH8-TRM112 complex, selenoprotein recoding assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — knockout mouse with multiple orthogonal biochemical readouts (tRNA mass spectrometry, protein complex pulldown, selenoprotein assay), replicated in 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 generate (S)-5-methoxycarbonylhydroxymethyluridine (mchm5U) in tRNA-Gly(UCC), creating a novel diastereomeric pair of wobble nucleosides (both (R)- and (S)-mchm5U are present in mammalian tRNA). This hydroxylation activity is distinct from the methyltransferase domain activity and expands ALKBH8's function beyond tRNA methylation to include tRNA hydroxylation.\",\n      \"method\": \"In vitro enzymatic assay of purified ALKBH8 AlkB domain, mass spectrometry of tRNA modifications from wild-type and Alkbh8-/- mice, NMR-based stereochemical characterization\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzymatic reconstitution combined with in vivo knockout validation and rigorous chemical characterization\",\n      \"pmids\": [\"21285950\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Yeast Trm9 (ortholog of ALKBH8) methylates the wobble uridine of tRNAARG(UCU) and tRNAGLU(UUC), and this modification enhances translational elongation of transcripts enriched in specific arginine and glutamic acid codons, resulting in increased protein levels of DNA damage response proteins (Yef3, Rnr1, Rnr3), thus linking tRNA modification to the DNA damage response.\",\n      \"method\": \"Genetic deletion of TRM9, computational codon usage analysis, quantitative protein-level assays (immunoblot), phenotypic cell death assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic knockout with multiple orthogonal readouts (protein levels, codon usage analysis, phenotypic rescue), replicated in subsequent studies\",\n      \"pmids\": [\"18082610\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Yeast Trm9 (ALKBH8 ortholog) and Trm112 function together at the final step in formation of mcm5U by methylating the cm5U intermediate substrate; co-expression and purification of His-tagged Trm9 with native Trm112 from E. coli showed they form a stable complex, and Trm112 dramatically improves the methyltransferase activity of Trm9 in vitro. In trm9Δ or trm112Δ mutants, ncm5U and ncm5s2U accumulate rather than the expected cm5U intermediates.\",\n      \"method\": \"Recombinant protein co-expression and purification, in vitro methyltransferase activity assay, HPLC analysis of tRNA modifications from yeast mutants\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of complex with enzymatic activity assay, corroborated by in vivo tRNA modification analysis in deletion mutants\",\n      \"pmids\": [\"21687733\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Crystal structure of the yeast Trm9-Trm112 complex (ortholog of mammalian ALKBH8-TRM112) reveals the structural basis for their interaction in mediating mcm5U modification at the tRNA anticodon wobble position; Trm112 uses a similar interaction mode to bind multiple methyltransferase partners despite low sequence identity (<20%) among them, demonstrating molecular plasticity.\",\n      \"method\": \"X-ray crystallography, structure-function mutagenesis analysis, comparison with other Trm112-MTase complex structures\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with functional validation and structure-function analysis\",\n      \"pmids\": [\"26438534\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Yeast Trm9 (ALKBH8 ortholog) acts downstream of Elongator in the tRNA wobble uridine modification pathway; genetic epistasis (tot3Δtrm9Δ double mutant) shows that Elongator-dependent tRNA modification acts upstream of Trm9-mediated methylation, and that Trm9 specifically methylates the wobble uridine of tRNAGlu.\",\n      \"method\": \"Genetic epistasis analysis (double mutants), zymocin sensitivity assays, tRNA overexpression suppressor analysis\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic epistasis in yeast with multiple phenotypic readouts, single laboratory\",\n      \"pmids\": [\"16390459\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Loss of Trm9-catalyzed mcm5U and mcm5s2U wobble modifications (in trm9Δ yeast) leads to increased translational infidelity specifically at arginine and glutamic acid codons from mixed codon boxes, resulting in protein errors and activation of unfolded protein and heat shock responses.\",\n      \"method\": \"Reporter assays for translational infidelity, quantitative tRNA modification analysis, codon re-engineering experiments, phenotypic assays for stress response activation\",\n      \"journal\": \"RNA biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal functional assays (reporter, proteomics, stress markers) in single laboratory\",\n      \"pmids\": [\"22832247\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The mammalian ALKBH8 protein possesses two separable enzymatic activities encoded in its two domains: tRNA hydroxylase activity (AlkB domain, converting mcm5U to mchm5U in tRNA-Gly(UCC)) and DNA repair demethylase activity. Protozoan ALKBH8 orthologs show both DNA repair and tRNA modification activities in vitro, while bacterial ALKBH8 orthologs show DNA repair but not tRNA modification, indicating domain-specific functional divergence during evolution.\",\n      \"method\": \"In vitro enzymatic assays (tRNA modification, DNA repair), mass spectrometry of tRNA modifications, analysis of ALKBH8-deficient Agrobacterium mutant\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro biochemical characterization across multiple organisms, single laboratory\",\n      \"pmids\": [\"24914785\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Silencing of ALKBH8 in human bladder cancer cells reduces ROS production via down-regulation of NOX-1, which then activates JNK and p38, leading to H2AX phosphorylation (γH2AX) and apoptosis; ALKBH8 knockdown also suppresses tumor invasion and angiogenesis in vivo in chorioallantoic membrane and orthotopic mouse models.\",\n      \"method\": \"siRNA knockdown, ROS assay, immunoblot for signaling components (JNK, p38, γH2AX), chorioallantoic membrane assay, orthotopic mouse tumor model\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean knockdown with multiple pathway readouts and in vivo validation, but mechanistic connection to tRNA modification activity not established\",\n      \"pmids\": [\"19293182\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ALKBH8 knockdown in bladder cancer cells induces apoptosis via downregulation of the anti-apoptotic protein survivin; ALKBH8 transgenic mice show accelerated bladder tumor growth and invasiveness in a carcinogen-induced model.\",\n      \"method\": \"siRNA knockdown, immunoblot for survivin, ALKBH8 transgenic mouse with N-butyl-N-(4-hydroxybutyl)-nitrosamine-induced bladder cancer model\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro knockdown with protein-level readout combined with in vivo transgenic mouse model, single laboratory\",\n      \"pmids\": [\"27329810\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ALKBH8-deficient mice show increased markers of oxidative stress, decreased thioredoxin reductase protein levels (consistent with impaired selenoprotein translation), reprogrammed stress response gene expression under basal conditions, and hypersensitivity to naphthalene-induced lung damage; wild-type mice develop naphthalene tolerance after repeated exposures, which is absent in Alkbh8-deficient mice.\",\n      \"method\": \"Alkbh8 knockout mouse, oxidative stress marker assays, immunoblot for thioredoxin reductase, naphthalene exposure paradigm, histological lung analysis\",\n      \"journal\": \"Epigenetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockout mouse with multiple biochemical and phenotypic readouts, single laboratory\",\n      \"pmids\": [\"32303148\"],\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 showing protein is still expressed), establishing that the methyltransferase domain activity is essential and that loss-of-function of the tRNA methyltransferase activity is the disease mechanism for MRT71 intellectual disability syndrome.\",\n      \"method\": \"Patient cell-based tRNA modification analysis, targeted proteomics to confirm protein expression, clinical genetics\",\n      \"journal\": \"Human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct biochemical confirmation in patient cells using targeted proteomics, single laboratory\",\n      \"pmids\": [\"34757492\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"HITS-CLIP and RIP-seq analyses of human ALKBH8 show that it binds fully processed and CCA-modified tRNAs as substrates, including the known wobble U-containing tRNAs. Additionally, ALKBH8 binds several types of noncoding RNAs including C/D box snoRNAs, suggesting substrate interactions beyond the canonical tRNAs.\",\n      \"method\": \"HITS-CLIP, RIP-seq, transcriptome-wide RNA-protein interaction mapping\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transcriptome-wide binding analysis with two orthogonal methods (HITS-CLIP and RIP-seq), single laboratory\",\n      \"pmids\": [\"36192131\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Alkbh8-knockout mice show reduced mcm5U and (S)-mchm5U levels in tRNA; in the brain, mcm5U levels are partially compensated. Alkbh8-/- mice exhibit cognitive and motor behavioral deficits, reduced brain weight, ischemic pathological changes in cerebral cortex and hippocampus, and differential expression of oxidative stress-related proteins and metabolites; neurons and glial cells from knockout mice show reduced mitochondrial membrane potential, indicating ALKBH8 maintains neural function through oxidative stress regulation.\",\n      \"method\": \"Alkbh8 knockout mouse, mass spectrometry of tRNA modifications, behavioral tests (novel object recognition, rotarod, forced swim), histology, proteomics, metabolomics, mitochondrial membrane potential assay\",\n      \"journal\": \"PNAS nexus\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockout mouse with multiple orthogonal phenotypic, proteomic, and metabolomic readouts, single laboratory\",\n      \"pmids\": [\"38550277\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In Alkbh8-knockout mice, tRNA protein translation efficiency is reduced and proteome analysis reveals downregulation of factors associated with red blood cell differentiation and protoporphyrin metabolism, suggesting ALKBH8-mediated mcm5U modification is essential for normal erythropoiesis during embryogenesis.\",\n      \"method\": \"Alkbh8 knockout mouse, UPLC-MS/MS quantification of RNA modifications at embryonic stages, proteome analysis\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockout mouse with proteomics and modification analysis, single laboratory\",\n      \"pmids\": [\"39280612\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ALKBH8 is a direct transcriptional target of Wnt/β-catenin signaling and is upregulated in colorectal cancer; genetic ablation of ALKBH8 inhibits intestinal tumor development in multiple mouse models (Apcmin/+, AOM/DSS, xenograft). Loss of ALKBH8 causes ribosome pausing at adenine-ending codons, impairing translation elongation of mRNAs enriched with these codons including KRAS proto-oncogene; rescue experiments confirm that the methyltransferase activity of ALKBH8 is required for its translation-promoting function.\",\n      \"method\": \"Genetic ablation in multiple mouse tumor models, ribosome profiling (ribosome pausing analysis), codon-specific translation reporter assays, methyltransferase-dead mutant rescue experiments, Wnt/β-catenin pathway reporter assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal approaches (ribosome profiling, mouse models, mutagenesis rescue) in single study with rigorous controls\",\n      \"pmids\": [\"41083459\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CRISPR/Cas9 knockout of ALKBH8 significantly reduces Zika virus (ZIKV) replication in human cells; ZIKV infection increases mcm5s2U34 tRNA modification content in host cells, and ALKBH8 loss impairs the preferential decoding of AA-ending codons that ZIKV genome exploits for protein synthesis.\",\n      \"method\": \"CRISPR/Cas9 knockout, viral replication assay, mass spectrometry of tRNA modifications, codon-biased GFP sensor assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 2 / Weak — preprint, single laboratory, clean CRISPR knockout with viral phenotype but limited mechanistic follow-up specific to ALKBH8\",\n      \"pmids\": [\"bio_10.1101_2025.06.03.657606\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ALKBH8 deletion reduces chikungunya virus (CHIKV) replication by impairing sulfur relay, recapitulating effects of methionine-cysteine deprivation; CHIKV upregulates ALKBH8 under sulfur-depleted conditions, suggesting ALKBH8-mediated U34-tRNA modification is exploited by the virus to support its replication.\",\n      \"method\": \"ALKBH8 deletion cell line, viral replication assay, metabolic supplementation experiments\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 2 / Weak — preprint, single laboratory, genetic deletion with viral phenotype but mechanistic details are limited in abstract\",\n      \"pmids\": [\"bio_10.1101_2025.07.11.664323\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"ALKBH8 is a dual-domain enzyme that functions as both a tRNA methyltransferase (Trm9-like domain, requiring TRM112 as an obligate co-activator) and an AlkB-type oxygenase: the methyltransferase domain catalyzes the final step of mcm5U formation at the wobble position (U34) of specific tRNAs including tRNASec, while the AlkB domain subsequently hydroxylates mcm5U to (S)-mchm5U specifically in tRNA-Gly(UCC); ALKBH8-mediated mcm5U is a prerequisite for further wobble modifications (thiolation to mcm5s2U and 2'-O-methylation to mcm5Um), and the resulting modified tRNAs promote codon-specific translation elongation—particularly at adenine-ending and glutamic acid/arginine codons—thereby regulating selenoprotein synthesis, DNA damage response, neuronal function, erythropoiesis, and oncogenic translation programs including KRAS in colorectal cancer.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ALKBH8 is a dual-domain tRNA-modifying enzyme that catalyzes wobble-uridine (U34) modifications which tune codon-specific translation elongation and downstream cellular stress, neural, and oncogenic programs [#0, #15]. Its Trm9-like methyltransferase domain performs the final step of 5-methoxycarbonylmethyluridine (mcm5U) formation at U34, an activity that requires the accessory protein TRM112 as an obligate co-activator forming a stable complex; conservation of this partnership is established by the yeast Trm9–Trm112 orthologs, whose reconstitution and crystal structure define the interaction mode and show that Trm112 strongly stimulates methyltransferase activity [#0, #3, #4]. ALKBH8-generated mcm5U is a prerequisite for further wobble maturation, including thiolation to mcm5s2U and 2'-O-methylation to mcm5Um [#0]. A second, separable AlkB-type oxygenase domain hydroxylates mcm5U to (S)-mchm5U specifically in tRNA-Gly(UCC) [#1, #7]. ALKBH8 binds fully processed, CCA-modified tRNAs as substrates [#12]. Functionally, mcm5U modification of tRNA-Sec is required for UGA recoding and selenoprotein (Gpx1, thioredoxin reductase) synthesis, linking ALKBH8 loss to oxidative-stress dysregulation [#0, #10], and the modification promotes elongation at adenine-ending codons such that ALKBH8 loss causes ribosome pausing and impaired translation of KRAS in colorectal cancer, where ALKBH8 is a Wnt/\\u03b2-catenin transcriptional target whose ablation suppresses intestinal tumorigenesis [#15]. ALKBH8 also supports neural function and erythropoiesis through its translation role [#13, #14]. A loss-of-function missense variant in the methyltransferase domain abolishes ALKBH8-dependent tRNA modifications and causes the MRT71 intellectual disability syndrome [#11].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Established that the ALKBH8 ortholog Trm9 links wobble-uridine tRNA methylation to codon-biased translation, answering how a tRNA modification influences specific protein outputs.\",\n      \"evidence\": \"Genetic deletion of yeast TRM9 with codon-usage analysis and quantitative protein assays for DNA damage response proteins\",\n      \"pmids\": [\"18082610\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Performed in yeast; mammalian ALKBH8 substrates not yet defined\", \"Mechanism of elongation enhancement at the ribosome not directly measured\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Placed Trm9-mediated methylation downstream of Elongator in the wobble-uridine modification pathway, ordering the biosynthetic steps.\",\n      \"evidence\": \"Genetic epistasis (double mutants) and zymocin sensitivity assays in yeast\",\n      \"pmids\": [\"16390459\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Pathway order inferred genetically, not biochemically reconstituted\", \"Yeast-only; mammalian pathway hierarchy untested here\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"First implicated mammalian ALKBH8 in cancer cell survival via ROS/NOX-1 signaling, raising the question of how its enzymatic activity connects to tumor phenotypes.\",\n      \"evidence\": \"siRNA knockdown in bladder cancer cells with ROS, signaling immunoblots, and in vivo tumor models\",\n      \"pmids\": [\"19293182\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic link to tRNA modification activity not established\", \"NOX-1 regulation mechanism unresolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined mammalian ALKBH8 as the methyltransferase completing mcm5U formation, requiring TRM112, and showed it is a prerequisite for downstream wobble modifications and selenoprotein recoding.\",\n      \"evidence\": \"Alkbh8 knockout mouse tRNA mass spectrometry, ALKBH8-TRM112 Co-IP, biochemical complementation, and Gpx1 selenoprotein recoding assay\",\n      \"pmids\": [\"20123966\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"AlkB domain function not yet defined here\", \"Substrate tRNA repertoire not exhaustively mapped\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Assigned the AlkB oxygenase domain a distinct hydroxylation activity, revealing ALKBH8 as a bifunctional enzyme generating mchm5U.\",\n      \"evidence\": \"In vitro assay of purified AlkB domain plus knockout-mouse tRNA mass spectrometry and NMR stereochemistry\",\n      \"pmids\": [\"21285950\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of mchm5U on translation not determined\", \"Why hydroxylation is restricted to tRNA-Gly(UCC) unexplained\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrated that Trm112 forms a stable complex with Trm9/ALKBH8 and is required to activate its methyltransferase activity, defining the co-activator relationship biochemically.\",\n      \"evidence\": \"Recombinant co-expression/purification, in vitro methyltransferase assay, and HPLC of tRNA modifications in deletion mutants\",\n      \"pmids\": [\"21687733\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of activation not resolved here\", \"Mammalian complex stoichiometry not addressed\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showed the two ALKBH8 domains carry separable activities (tRNA hydroxylase and DNA repair demethylase) and that these diverged across evolution.\",\n      \"evidence\": \"In vitro tRNA-modification and DNA-repair assays across protozoan and bacterial orthologs plus an Agrobacterium deletion mutant\",\n      \"pmids\": [\"24914785\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological relevance of mammalian ALKBH8 DNA-repair activity not demonstrated in vivo\", \"Single laboratory\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Resolved the structural basis of the Trm9-Trm112 interaction, explaining Trm112's plasticity in partnering multiple methyltransferases.\",\n      \"evidence\": \"X-ray crystallography of the yeast complex with structure-function mutagenesis\",\n      \"pmids\": [\"26438534\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure is of yeast orthologs, not mammalian ALKBH8\", \"tRNA-bound complex not captured\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Extended ALKBH8 tumor-promoting function to survivin regulation and in vivo carcinogen-induced bladder cancer, reinforcing an oncogenic role.\",\n      \"evidence\": \"siRNA knockdown with survivin immunoblot and ALKBH8 transgenic carcinogen-induced mouse model\",\n      \"pmids\": [\"27329810\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Link between survivin and tRNA modification activity not established\", \"Single laboratory\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Connected ALKBH8 loss to systemic oxidative-stress dysregulation and impaired chemical tolerance, consistent with defective selenoprotein translation.\",\n      \"evidence\": \"Alkbh8 knockout mouse oxidative-stress markers, thioredoxin reductase immunoblot, and naphthalene exposure paradigm\",\n      \"pmids\": [\"32303148\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ribosome-level translation defect not measured\", \"Single laboratory\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Established that loss of ALKBH8 methyltransferase activity is the disease mechanism for MRT71 intellectual disability, tying enzyme function to human pathology.\",\n      \"evidence\": \"Patient cell tRNA-modification analysis with targeted proteomics confirming protein expression and clinical genetics\",\n      \"pmids\": [\"34757492\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Neuronal cell-type-specific consequences not defined\", \"Single family/laboratory\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Mapped ALKBH8's RNA substrate landscape transcriptome-wide, confirming mature tRNA binding and revealing additional noncoding RNA interactions.\",\n      \"evidence\": \"HITS-CLIP and RIP-seq in human cells\",\n      \"pmids\": [\"36192131\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional significance of snoRNA binding unknown\", \"Binding does not establish catalytic modification of all bound RNAs\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Linked ALKBH8-dependent wobble modification to neural function and erythropoiesis through reduced translation efficiency in vivo.\",\n      \"evidence\": \"Alkbh8 knockout mice with tRNA mass spectrometry, behavioral/histological analysis, mitochondrial assays, proteomics, and embryonic-stage proteome/modification analysis\",\n      \"pmids\": [\"38550277\", \"39280612\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Codon-level translation defects in brain/erythroid cells not resolved\", \"Brain mcm5U partial compensation mechanism unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined the codon-specific translation mechanism of ALKBH8 oncogenesis: it is a Wnt/\\u03b2-catenin target whose mcm5U activity prevents ribosome pausing at adenine-ending codons, sustaining KRAS translation in colorectal cancer.\",\n      \"evidence\": \"Ribosome profiling, codon reporter assays, methyltransferase-dead rescue, and multiple genetic mouse tumor models\",\n      \"pmids\": [\"41083459\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full repertoire of A-ending-codon-dependent oncogenic transcripts not catalogued\", \"Therapeutic targetability not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Implicated ALKBH8-mediated U34 modification in viral exploitation of host codon-biased translation.\",\n      \"evidence\": \"CRISPR/Cas9 and deletion cell lines with viral replication assays, tRNA mass spectrometry, and codon sensor assays for ZIKV and CHIKV (preprints)\",\n      \"pmids\": [\"bio_10.1101_2025.06.03.657606\", \"bio_10.1101_2025.07.11.664323\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Preprints, not peer-reviewed\", \"ALKBH8-specific mechanism in viral replication only partially defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How ALKBH8 substrate selectivity is achieved (why hydroxylation is restricted to tRNA-Gly(UCC), the role of noncoding RNA binding, and whether mammalian DNA-repair activity is physiologically relevant) remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of the substrate-bound mammalian ALKBH8-TRM112 complex\", \"Functional role of ALKBH8 snoRNA binding unknown\", \"In vivo relevance of the AlkB-domain DNA demethylase activity untested in mammals\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 3, 11, 15]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [0, 1, 7]},\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [1, 7]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [12]},\n      {\"term_id\": \"GO:0045182\", \"supporting_discovery_ids\": [2, 15]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 1, 3]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [2, 15]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [11, 15]}\n    ],\n    \"complexes\": [\"ALKBH8-TRM112 methyltransferase complex\"],\n    \"partners\": [\"TRM112\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}