{"gene":"ALKBH3","run_date":"2026-06-09T22:02:43","timeline":{"discoveries":[{"year":2011,"finding":"ALKBH3 forms a complex with the Activating Signal Cointegrator Complex (ASCC), and ASCC3 (the largest subunit) encodes a 3'-5' DNA helicase whose activity generates single-stranded DNA upon which ALKBH3 preferentially performs dealkylation. Loss of either ALKBH3 or ASCC3 leads to increased 3-methylcytosine levels, reduced cell proliferation, and pH2A.X/53BP1 foci formation.","method":"Protein complex purification, co-immunoprecipitation, helicase activity assay, siRNA knockdown with DNA damage readouts (pH2A.X, 53BP1 foci), cell proliferation assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — complex purified biochemically, helicase activity demonstrated in vitro, functional epistasis confirmed by orthogonal cellular assays with rigorous controls","pmids":["22055184"],"is_preprint":false},{"year":2019,"finding":"ALKBH3 is a 1-methyladenosine (m1A) and 3-methylcytidine (m3C) demethylase of tRNA. ALKBH3-demethylated tRNA is more sensitive to angiogenin (ANG) cleavage, generating tRNA-derived small RNAs (tDRs) around anticodon regions that strengthen ribosome assembly and prevent cytochrome c-triggered apoptosis.","method":"In vitro demethylation assay, tDR profiling, ribosome assembly assay, apoptosis assay (cytochrome c), siRNA knockdown, xenograft tumor model","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro enzymatic assay combined with multiple orthogonal cellular and in vivo readouts in a single study","pmids":["30541109"],"is_preprint":false},{"year":2018,"finding":"ALKBH3-mediated m1A demethylation of CSF-1 mRNA increases CSF-1 mRNA stability (half-life) in breast and ovarian cancer cells, promoting cancer cell invasiveness. The m1A site is mapped to the 5'UTR near the translation initiation site, and YTHDF2 (an m6A reader) is not the reader of m1A-containing CSF-1 mRNA.","method":"ALKBH3 overexpression/knockdown, mRNA stability assay (half-life measurement), m1A mapping, invasion assay, YTHDF2 interaction test","journal":"Biochimica et biophysica acta. Gene regulatory mechanisms","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — single lab with multiple orthogonal methods (stability assay, m1A mapping, functional invasion assay) but no in vitro reconstitution","pmids":["30342176"],"is_preprint":false},{"year":2019,"finding":"ALKBH2 and ALKBH3 (and E. coli AlkB) can oxidize 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine in vitro, demonstrating capacity to oxidize a methyl group attached to carbon rather than nitrogen.","method":"In vitro enzymatic assay with purified proteins, mass spectrometry detection of oxidized products, computational docking","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro reconstitution with purified enzymes, single lab, no cellular validation reported","pmids":["31114894"],"is_preprint":false},{"year":2022,"finding":"ALKBH3 removes m1A from Aurora A mRNA, stabilizing it and promoting its translation; depletion of ALKBH3 enhances Aurora A mRNA decay and inhibits its translation, leading to inhibition of ciliogenesis. The catalytically inactive ALKBH3 mutant cannot rescue ciliary defects in alkbh3 morphant zebrafish, confirming the demethylation activity is required.","method":"ALKBH3 knockdown/overexpression, Aurora A mRNA stability and translation assays, catalytic mutant rescue, zebrafish alkbh3 morpholino knockdown with phenotypic rescue experiments","journal":"Cell discovery","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (mRNA decay, translation, catalytic mutant, in vivo zebrafish rescue) establishing pathway and catalytic requirement","pmids":["35277482"],"is_preprint":false},{"year":2019,"finding":"ALKBH3 directly interacts with human RAD51 paralogue RAD51C via protein-protein interaction, and RAD51C-ALKBH3 interaction stimulates ALKBH3-mediated repair of methyl-adducts within 3'-tailed DNA substrates; disruption of this interaction impairs ALKBH3 function both in vitro and in vivo.","method":"Co-immunoprecipitation, pulldown assay, in vitro demethylation assay with 3'-tailed DNA substrates, cellular alkylation damage resistance assay","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — reciprocal interaction confirmed, in vitro stimulation assay, cellular validation, single lab","pmids":["31642493"],"is_preprint":false},{"year":2021,"finding":"ASCC3, the ALKBH3 binding partner, mediates P-body formation and promotes selective removal of chemically induced m1A and m3C from mRNA; ASCC3-deficient cells show delayed clearance of MMS-induced m1A and m3C from mRNA and impaired P-body formation, consistent with a model where ASCC3-mediated ribosome disassembly allows ALKBH3-dependent mRNA demethylation.","method":"Quantitative mass spectrometry of mRNA methylation, SILAC proteomics of mRNA-binding proteins, ASCC3 knockout cells, P-body imaging","journal":"Journal of translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (MS quantitation, KO cells, imaging) in single lab","pmids":["34217309"],"is_preprint":false},{"year":2015,"finding":"ALKBH3 binds to transcription-associated genomic locations including promoter-proximal paused RNA Pol II sites and enhancers in prostate cancer cells; it strongly binds to transcription initiation sites of a small number of highly active promoters characterized by high levels of Mediator, cohesin, and active histone marks. ALKBH3 depletion does not directly alter transcription of its target genes but induces upregulation of ALKBH3-non-bound inflammatory genes.","method":"ChIP-seq (endogenous ALKBH3), microarray gene expression after ALKBH3 depletion","journal":"Genome medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-seq with expression analysis, single lab, two orthogonal genome-wide methods","pmids":["26221185"],"is_preprint":false},{"year":2016,"finding":"A fluorogenic probe (MAQ) exploiting fluorescence quenching of 1-methyladenine enables direct measurement of ALKBH3 repair activity in vitro and in cells; the probe is specific for ALKBH3 over ALKBH2 and shows Km and kcat values equivalent to the native substrate. ALKBH3 activity was imaged and quantified in live cells by microscopy and flow cytometry.","method":"Fluorogenic substrate assay, enzyme kinetics (Km, kcat), specificity comparison with ALKBH2, live-cell imaging, flow cytometry","journal":"Journal of the American Chemical Society","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic reconstitution with quantitative kinetics, cellular imaging validation, specificity controls; single lab but multiple orthogonal methods","pmids":["26967262"],"is_preprint":false},{"year":2024,"finding":"Crystal structures of ALKBH3 crosslinked to oligonucleotide substrates (obtained with a synthetic antibody chaperone) reveal that ALKBH3 uses two β-hairpins (β4-loop-β5 and β'-loop-β'') and an α2 helix for single-stranded substrate binding. Residue Thr133 in the active pocket is required for specific recognition of m1A and m3C; mutation of Thr133 to the corresponding FTO or ALKBH5 residue converts ALKBH3 substrate selectivity from m1A to m6A. Asp194 forms a bubble-like region also critical for substrate recognition.","method":"X-ray crystallography of ALKBH3-oligo crosslinked complexes, site-directed mutagenesis (Thr133, Asp194), in vitro demethylation assay with mutants","journal":"Angewandte Chemie (International ed. in English)","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with mutagenesis and in vitro enzymatic validation in a single study, multiple orthogonal methods","pmids":["38158383"],"is_preprint":false},{"year":2024,"finding":"Biochemical and mutagenesis analysis identifies Tyr143, Leu177, and His191 as key residues for ALKBH3 secondary structure and catalytic activity toward methylated single-stranded DNA. Tyr143 is critical for binding the flipped-out methylated base and stabilizing its everted conformation; Leu177 and His191 are required for secondary structure integrity. Stopped-flow fluorescence spectroscopy revealed a transient kinetic mechanism comprising substrate binding, base eversion, and anchoring steps.","method":"Site-directed mutagenesis, stopped-flow fluorescence spectroscopy, CD spectroscopy, in vitro activity assays","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro reconstitution with mutagenesis, single lab, no structural coordinates reported","pmids":["38256217"],"is_preprint":false},{"year":2021,"finding":"The ASCC2 CUE domain selectively binds K63-linked polyubiquitin chains (diubiquitin) by contacting both the distal and proximal ubiquitin, thereby localizing the ASCC-ALKBH3 repair complex to alkylation damage sites in the nucleus. Mutation of residues in the N-terminal portion of the ASCC2 α1 helix that contact the proximal ubiquitin decreases ASCC2 nuclear recruitment in response to DNA alkylation.","method":"Co-crystallography/structural analysis, mutagenesis of ASCC2 CUE domain, cellular ASCC2 recruitment assay upon DNA alkylation damage","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — structural and mutagenesis data combined with cellular recruitment assay, single lab","pmids":["34971705"],"is_preprint":false},{"year":2016,"finding":"Alkbh3 (and Alkbh2), but not alkyladenine DNA glycosylase (Aag), can repair N3-ethylthymidine (N3-EtdT) in mammalian cells, as shown by transcription-based lesion bypass assays. Purified human Alkbh2 directly reverses N3-EtdT in vitro. N3-CMdT, O2-EtdT, O4-EtdT, and O4-CMdT are not repaired by Alkbh2 or Alkbh3.","method":"Site-specific lesion in non-replicative vectors, transcription bypass assay in mammalian cells, in vitro repair assay with purified Alkbh2","journal":"ACS chemical biology","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — cell-based repair assay and in vitro enzymatic assay (for Alkbh2), Alkbh3 cellular data without in vitro reconstitution for Alkbh3 specifically","pmids":["26930515"],"is_preprint":false},{"year":2024,"finding":"ALKBH3-mediated m1A demethylation of SP100A mRNA prevents its recognition by YTHDF1 (an m1A reader that promotes RNA stability and translation), thereby reducing SP100A protein levels and attenuating formation of tumor-suppressive PML nuclear condensates. YTHDF1 is identified as a reader of m1A-methylated SP100A mRNA.","method":"Multiomics (m1A epitranscriptomics + proteomics), ALKBH3 knockdown with SP100A rescue, YTHDF1 interaction with m1A-containing mRNA, in vitro/in vivo functional assays","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiomics identification with functional rescue and reader identification, single lab","pmids":["38118002"],"is_preprint":false},{"year":2025,"finding":"ALKBH3 demethylates m1A on METTL3 mRNA, preventing YTHDF2-dependent mRNA decay of METTL3 transcript and thereby increasing METTL3 protein levels. Elevated METTL3 then stabilizes COL1A1 and FN1 mRNAs via m6A modification, promoting pathological skin fibrosis (hypertrophic scars).","method":"m1A epitranscriptomics, ALKBH3 knockdown/overexpression, METTL3 rescue experiments, in vitro and in vivo fibrosis models, YTHDF2 interaction assay","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic pathway established through multiple functional assays and rescue experiments, single lab","pmids":["40019372"],"is_preprint":false},{"year":2024,"finding":"m1A demethylase Alkbh3 promotes neurogenesis by demethylating m1A on Mmp15 mRNA, improving its RNA stability and translational efficacy; depletion of Alkbh3 in neural stem cells decreases neuronal differentiation and proliferation while increasing gliogenesis, and reduces hippocampal neurogenesis and spatial memory in adult mice.","method":"Alkbh3 knockdown/overexpression in neural stem cells, Mmp15 mRNA stability and translation assay, m1A profiling, in vivo hippocampal neurogenesis and behavioral testing","journal":"Cell & bioscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (mRNA stability, translation, in vivo mouse model), single lab","pmids":["39004750"],"is_preprint":false},{"year":2025,"finding":"ALKBH3 demethylates m1A on HK2 mRNA in retinal pigment epithelial cells, activating glycolysis and excess lactate production. This lactate promotes H3K18 histone lactylation, which binds the ALKBH3 promoter to amplify its transcription, establishing a positive feedback loop. ALKBH3 also directly demethylates VEGFA mRNA to promote choroidal neovascularization.","method":"dm1ACRISPR system for site-specific m1A demethylation, gene knockout mice, ChIP for histone lactylation at ALKBH3 promoter, m1A epitranscriptomics, ALKBH3 inhibitor (HUHS015), in vivo AMD model","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-specific editing tool, in vivo model, ChIP, inhibitor validation; single lab","pmids":["40493193"],"is_preprint":false},{"year":2025,"finding":"ALKBH3-mediated m1A demethylation of ALDOA mRNA at the 3'UTR stabilizes ALDOA mRNA by preventing recruitment of the YTHDF2/PAN2-PAN3 complex that drives mRNA degradation; this stabilization potentiates glycolysis and doxorubicin resistance in triple-negative breast cancer cells.","method":"ALKBH3 knockdown/overexpression, m1A site mapping in ALDOA 3'UTR, mRNA stability assay, YTHDF2/PAN2-PAN3 interaction assay, glycolysis metabolite measurement, in vivo xenograft","journal":"Acta pharmaceutica Sinica. B","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — m1A site mapped, reader/degradation complex identified, functional in vitro and in vivo validation; single lab","pmids":["40654364"],"is_preprint":false},{"year":2026,"finding":"ALKBH3 removes m1A from PINK1 mRNA, promoting its stability and translation; elevated ALKBH3 in Alzheimer's disease models impairs PINK1-dependent mitophagy, leading to mitochondrial dysfunction and neuronal damage. Alkbh3 reduction decreases amyloid-β plaques and restores cognition in 5xFAD mice.","method":"m1A epitranscriptomics, Alkbh3 knockout/reduction in 5xFAD mice, PINK1 mRNA stability and translation assays, mitophagy and mitochondrial function assays, behavioral testing","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — m1A on PINK1 mRNA identified, in vivo Alzheimer's model with multiple readouts; single lab","pmids":["41816968"],"is_preprint":false},{"year":2026,"finding":"ALKBH3 demethylates m1A on ZBED6 mRNA, enhancing ZBED6 translation; ZBED6 then physically interacts with STAT1 (confirmed by co-immunoprecipitation and ChIP) and represses STAT1-driven AIM2 transcription, thereby suppressing PANoptosis (pyroptosis/apoptosis/necroptosis) in cardiomyocytes during ischemia/reperfusion injury.","method":"m1A epitranscriptomics, ALKBH3 overexpression/siRNA in cells and in vivo I/R model, co-immunoprecipitation (ZBED6-STAT1), ChIP, dual-luciferase reporter (AIM2 promoter), loss- and gain-of-function for ZBED6, STAT1, AIM2","journal":"Clinical and translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pathway established by multiple orthogonal methods including co-IP, ChIP, reporter assay, and in vivo model; single lab","pmids":["41816893"],"is_preprint":false},{"year":2024,"finding":"PUS7-dependent pseudouridylation of ALKBH3 mRNA at position U696 enhances its translation efficiency, thereby increasing ALKBH3 protein levels and suppressing gastric cancer progression.","method":"Locus-specific pseudouridine detection assay, polysome profiling, RT-qPCR, Western blotting, 3D colony formation assay, xenograft model","journal":"Clinical and translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-specific modification mapped with functional validation via polysome profiling, in vivo xenograft; single lab","pmids":["39175405"],"is_preprint":false},{"year":2025,"finding":"ALKBH3-mediated m1A demethylation of ATF4 mRNA increases ATF4 expression, which inhibits ferroptosis (by upregulating SLC7A11, GPX4, FTH1) and promotes AML cell survival; ALKBH3 knockdown promotes ferroptosis in KG-1 cells.","method":"ALKBH3 knockdown, ATF4 knockdown rescue experiments, ferroptosis markers (ROS, MDA, iron, SOD, GSH), flow cytometry, xenograft model","journal":"Hematology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — functional assays performed but m1A site on ATF4 mRNA not directly mapped; mechanistic link inferred from knockdown epistasis, single lab","pmids":["39803678"],"is_preprint":false}],"current_model":"ALKBH3 is a Fe(II)/α-ketoglutarate-dependent dioxygenase that oxidatively demethylates N1-methyladenosine (m1A) and N3-methylcytidine (m3C) in both single-stranded DNA and RNA (including tRNA, mRNA, and mitochondrial RNA); it operates in a complex with ASCC (particularly the ASCC3 helicase that generates ssDNA substrate and ASCC2 that recruits the complex to damage sites via K63-polyubiquitin recognition), interacts with RAD51C to facilitate alkylation repair at 3'-tailed DNA, and regulates diverse cellular processes—including ciliogenesis, neurogenesis, mitophagy, glycolysis, and cell survival—by stabilizing or destabilizing specific mRNAs through m1A demethylation, with substrate specificity dictated by key active-site residues Thr133 and Asp194 as revealed by crystal structures of substrate-crosslinked complexes."},"narrative":{"mechanistic_narrative":"ALKBH3 is a single-stranded nucleic acid demethylase that reverses N1-methyladenine/N1-methyladenosine (m1A) and N3-methylcytosine/N3-methylcytidine (m3C) lesions in both DNA and RNA, functioning both as an alkylation-damage repair enzyme and as an epitranscriptomic eraser that controls mRNA fate [PMID:22055184, PMID:30541109, PMID:26967262]. In its DNA-repair role, ALKBH3 acts within the ASCC complex: the ASCC3 3'-5' helicase generates the single-stranded substrate ALKBH3 prefers, and loss of either protein elevates 3-methylcytosine and triggers DNA-damage signaling and reduced proliferation [PMID:22055184]. The complex is targeted to nuclear alkylation sites through the ASCC2 CUE domain, which reads K63-linked polyubiquitin chains [PMID:34971705], and ALKBH3 repair of methyl adducts on 3'-tailed DNA is further stimulated by direct interaction with the recombination factor RAD51C [PMID:31642493]. Crystal structures of substrate-crosslinked ALKBH3 show that the enzyme grips single-stranded substrate via two beta-hairpins and an alpha2 helix and everts the methylated base, with active-site residue Thr133 dictating m1A/m3C selectivity (its substitution by the corresponding FTO/ALKBH5 residue switches selectivity toward m6A) and Asp194 and Tyr143 contributing to substrate recognition and base eversion [PMID:38158383, PMID:38256217]. As an RNA demethylase, ALKBH3 strips m1A from numerous target mRNAs to govern their stability and translation: by erasing m1A it either prevents reader-driven decay—removing marks recognized by YTHDF2/PAN2-PAN3 or, conversely, by the stabilizing reader YTHDF1—or otherwise alters transcript half-life, thereby controlling targets such as CSF-1, Aurora A, METTL3, ALDOA, HK2, VEGFA, PINK1, ZBED6, and Mmp15 [PMID:30342176, PMID:35277482, PMID:38118002, PMID:40019372, PMID:40654364, PMID:40493193, PMID:41816968, PMID:41816893, PMID:39004750]. Through this mRNA-regulatory activity ALKBH3 influences a broad range of processes including cancer cell invasion and glycolysis, ciliogenesis, hippocampal neurogenesis, mitophagy, neovascularization, and cell-survival decisions [PMID:30342176, PMID:35277482, PMID:39004750, PMID:40493193, PMID:41816968]. ALKBH3 also demethylates tRNA, sensitizing it to angiogenin cleavage and generating tRNA-derived small RNAs that support ribosome assembly and suppress apoptosis [PMID:30541109].","teleology":[{"year":2011,"claim":"Established that ALKBH3 is not a stand-alone repair enzyme but operates within the ASCC complex, where the ASCC3 helicase supplies the single-stranded DNA substrate it acts upon.","evidence":"Complex purification, in vitro helicase assay, and siRNA epistasis with DNA-damage readouts in human cells","pmids":["22055184"],"confidence":"High","gaps":["Did not resolve how the complex is recruited to damage sites","Substrate generation linked to ssDNA but RNA substrates not addressed"]},{"year":2015,"claim":"Showed ALKBH3 occupies transcription-associated chromatin (paused Pol II, enhancers, active promoters), raising the possibility of a chromatin/transcription-linked role beyond bulk DNA repair.","evidence":"Endogenous ALKBH3 ChIP-seq with expression microarray after depletion in prostate cancer cells","pmids":["26221185"],"confidence":"Medium","gaps":["Depletion did not alter transcription of bound genes, leaving the functional consequence of binding unresolved","Mechanism linking ALKBH3 chromatin occupancy to inflammatory gene upregulation unknown"]},{"year":2016,"claim":"Provided quantitative kinetic tools and lesion-specificity data, confirming ALKBH3 acts on m1A with measurable kinetics and on specific N3-alkyl thymidine lesions while sparing others.","evidence":"Fluorogenic m1A probe with Km/kcat and live-cell imaging; transcription-bypass lesion-repair assays for N3-alkyl lesions","pmids":["26967262","26930515"],"confidence":"High","gaps":["In vitro reconstitution for ALKBH3 on N3-EtdT specifically not performed","Cellular substrate scope incompletely defined"]},{"year":2018,"claim":"Opened the epitranscriptomic role by showing ALKBH3 m1A-demethylates a specific mRNA (CSF-1) to extend its half-life and drive cancer invasiveness, establishing mRNA stability as an ALKBH3 output.","evidence":"ALKBH3 overexpression/knockdown, m1A mapping, mRNA half-life and invasion assays in breast/ovarian cancer cells","pmids":["30342176"],"confidence":"Medium","gaps":["The reader interpreting m1A on CSF-1 mRNA was not identified (YTHDF2 excluded)","No in vitro reconstitution of the demethylation event"]},{"year":2019,"claim":"Defined ALKBH3 as an m1A/m3C eraser on tRNA and linked its activity to tRNA-derived small RNA production, ribosome assembly, and apoptosis resistance, expanding substrate scope beyond DNA and mRNA.","evidence":"In vitro demethylation, tDR profiling, ribosome assembly and apoptosis assays, xenograft","pmids":["30541109"],"confidence":"High","gaps":["Specific tRNA species and sites incompletely defined","Coupling between demethylation and angiogenin cleavage not structurally resolved"]},{"year":2019,"claim":"Identified RAD51C as a direct ALKBH3 partner that stimulates repair of methyl adducts on 3'-tailed DNA, connecting ALKBH3 alkylation repair to recombination machinery.","evidence":"Reciprocal co-IP/pulldown, in vitro demethylation on 3'-tailed substrates, cellular alkylation-resistance assay","pmids":["31642493"],"confidence":"Medium","gaps":["Single lab; structural basis of interaction unknown","Relationship to the ASCC complex not addressed"]},{"year":2019,"claim":"Demonstrated that ALKBH3 (with ALKBH2) can oxidize carbon-bound 5-methylcytosine to 5hmC/5fC/5caC in vitro, broadening its potential chemistry beyond N-demethylation.","evidence":"In vitro enzymatic assay with purified protein, mass spectrometry, computational docking","pmids":["31114894"],"confidence":"Medium","gaps":["No cellular validation that this activity occurs in vivo","Biological significance of 5mC oxidation by ALKBH3 unknown"]},{"year":2021,"claim":"Resolved how the ASCC-ALKBH3 complex is recruited to damage and how it accesses mRNA substrates, linking K63-ubiquitin recognition and ribosome disassembly to demethylation.","evidence":"ASCC2 CUE-domain structure/mutagenesis with nuclear recruitment assay; ASCC3 KO cells with MS quantitation of mRNA m1A/m3C and P-body imaging","pmids":["34971705","34217309"],"confidence":"Medium","gaps":["Direct demonstration that ASCC3-driven ribosome disassembly is required for ALKBH3 catalysis not shown","Single labs for each mechanism"]},{"year":2022,"claim":"Provided in vivo proof that ALKBH3 catalytic m1A demethylation of a target mRNA (Aurora A) controls a developmental process (ciliogenesis), as a catalytically dead mutant failed to rescue zebrafish defects.","evidence":"mRNA decay/translation assays, catalytic mutant rescue, zebrafish morpholino phenotype rescue","pmids":["35277482"],"confidence":"High","gaps":["Reader machinery for Aurora A m1A not defined","Generality across cilia-related transcripts not established"]},{"year":2024,"claim":"Delivered the structural and kinetic basis of ALKBH3 substrate selectivity, showing Thr133 governs m1A/m3C versus m6A choice and identifying residues for ssDNA binding and base eversion.","evidence":"X-ray crystallography of substrate-crosslinked complexes plus mutagenesis; stopped-flow/CD kinetics defining binding, eversion, anchoring steps","pmids":["38158383","38256217"],"confidence":"High","gaps":["Structures used DNA oligo substrates; RNA-bound conformation not solved","How active-site selectivity maps onto tRNA/mRNA targets in vivo unresolved"]},{"year":2024,"claim":"Showed ALKBH3 m1A erasure tunes mRNA fate through specific readers — preventing YTHDF1-mediated stabilization (SP100A) — coupling the eraser to defined reader-decay logic, and identified a regulatory input via PUS7 pseudouridylation of ALKBH3 mRNA controlling its own translation.","evidence":"Multiomics with rescue and reader (YTHDF1) interaction assays; locus-specific pseudouridine mapping with polysome profiling and xenograft","pmids":["38118002","39175405"],"confidence":"Medium","gaps":["Reader assignment varies by transcript; rules for reader choice unclear","Upstream control of ALKBH3 expression in physiological contexts incompletely mapped"]},{"year":2025,"claim":"Expanded the m1A-eraser regulon to metabolic and signaling targets — METTL3, HK2/VEGFA, ALDOA — establishing ALKBH3 as a driver of glycolysis, neovascularization, fibrosis, and chemoresistance via YTHDF2/PAN2-PAN3-dependent decay and a lactylation feedback loop.","evidence":"Site-specific m1A editing (dm1ACRISPR), knockdown/overexpression with reader/degradation-complex interaction assays, ChIP for histone lactylation, inhibitor and in vivo models","pmids":["40019372","40493193","40654364"],"confidence":"Medium","gaps":["Each pathway from a single lab","Whether one demethylase coordinates these target sets simultaneously not addressed"]},{"year":2026,"claim":"Extended ALKBH3 target regulation to neuronal and cardiac homeostasis — PINK1-dependent mitophagy in Alzheimer's models and a ZBED6-STAT1-AIM2 axis controlling cardiomyocyte PANoptosis — implicating it in degenerative and ischemic disease.","evidence":"m1A epitranscriptomics, in vivo 5xFAD and I/R models, co-IP, ChIP, luciferase reporter, stability/translation assays","pmids":["41816968","41816893"],"confidence":"Medium","gaps":["Direct mapping of m1A demethylation events partly inferential","Single-lab disease models awaiting independent confirmation"]},{"year":null,"claim":"It remains unresolved how ALKBH3 partitions among its DNA-repair, tRNA, and mRNA substrate pools in a given cell, and what determines which reader and outcome a demethylated transcript receives.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model of substrate selection across DNA vs RNA in vivo","Rules dictating reader assignment (YTHDF1 vs YTHDF2/PAN2-PAN3) per transcript unknown","Regulation balancing nuclear repair vs cytoplasmic mRNA roles undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[1,2,4,8,9,13,15,17]},{"term_id":"GO:0140097","term_label":"catalytic activity, acting on DNA","supporting_discovery_ids":[0,5,9,12]},{"term_id":"GO:0016491","term_label":"oxidoreductase activity","supporting_discovery_ids":[3,8,9]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[1,2,4]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[0,5,9]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,7,11]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[6]}],"pathway":[{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[0,5,11,12]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[1,2,4,6,13]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[7]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[16,18,19]}],"complexes":["ASCC complex"],"partners":["ASCC3","ASCC2","RAD51C"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96Q83","full_name":"Alpha-ketoglutarate-dependent dioxygenase alkB homolog 3","aliases":["Alkylated DNA repair protein alkB homolog 3","hABH3","DEPC-1","Prostate cancer antigen 1"],"length_aa":286,"mass_kda":33.4,"function":"Dioxygenase that mediates demethylation of DNA and RNA containing 1-methyladenosine (m1A) (PubMed:12486230, PubMed:12594517, PubMed:16174769, PubMed:26863196, PubMed:26863410). Repairs alkylated DNA containing 1-methyladenosine (m1A) and 3-methylcytosine (m3C) by oxidative demethylation (PubMed:12486230, PubMed:12594517, PubMed:16174769, PubMed:25944111). Has a strong preference for single-stranded DNA (PubMed:12486230, PubMed:12594517, PubMed:16174769, PubMed:20714506). Able to process alkylated m3C within double-stranded regions via its interaction with ASCC3, which promotes DNA unwinding to generate single-stranded substrate needed for ALKBH3 (PubMed:22055184). Can repair exocyclic 3,N4-ethenocytosine adducs in single-stranded DNA (PubMed:25797601). Also acts on RNA (PubMed:12594517, PubMed:16174769, PubMed:16858410, PubMed:26863196, PubMed:26863410). Demethylates N(1)-methyladenosine (m1A) RNA, an epigenetic internal modification of messenger RNAs (mRNAs) highly enriched within 5'-untranslated regions (UTRs) and in the vicinity of start codons (PubMed:26863196, PubMed:26863410). Requires molecular oxygen, alpha-ketoglutarate and iron (PubMed:16858410, PubMed:22055184)","subcellular_location":"Nucleus; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q96Q83/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ALKBH3","classification":"Not Classified","n_dependent_lines":6,"n_total_lines":1208,"dependency_fraction":0.004966887417218543},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ALKBH3","total_profiled":1310},"omim":[{"mim_id":"620886","title":"tRNA METHYLTRANSFERASE 6, NONCATALYTIC SUBUNIT; TRMT6","url":"https://www.omim.org/entry/620886"},{"mim_id":"620885","title":"tRNA METHYLTRANSFERASE 61A; TRMT61A","url":"https://www.omim.org/entry/620885"},{"mim_id":"610603","title":"AlkB HOMOLOG 3, ALPHA-KETOGLUTARATE-DEPENDENT DIOXYGENASE; ALKBH3","url":"https://www.omim.org/entry/610603"},{"mim_id":"610602","title":"AlkB HOMOLOG 2, ALPHA-KETOGLUTARATE-DEPENDENT DIOXYGENASE; ALKBH2","url":"https://www.omim.org/entry/610602"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Mitochondria","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ALKBH3"},"hgnc":{"alias_symbol":["DEPC-1"],"prev_symbol":[]},"alphafold":{"accession":"Q96Q83","domains":[{"cath_id":"2.60.120.590","chopping":"78-274","consensus_level":"high","plddt":94.1139,"start":78,"end":274}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96Q83","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96Q83-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96Q83-F1-predicted_aligned_error_v6.png","plddt_mean":82.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ALKBH3","jax_strain_url":"https://www.jax.org/strain/search?query=ALKBH3"},"sequence":{"accession":"Q96Q83","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96Q83.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96Q83/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96Q83"}},"corpus_meta":[{"pmid":"30541109","id":"PMC_30541109","title":"Transfer RNA demethylase ALKBH3 promotes cancer progression via induction of tRNA-derived small RNAs.","date":"2019","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/30541109","citation_count":302,"is_preprint":false},{"pmid":"22055184","id":"PMC_22055184","title":"DNA unwinding by ASCC3 helicase is coupled to ALKBH3-dependent DNA alkylation repair and cancer cell proliferation.","date":"2011","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/22055184","citation_count":175,"is_preprint":false},{"pmid":"30342176","id":"PMC_30342176","title":"Human ALKBH3-induced m1A demethylation increases the CSF-1 mRNA stability in breast and ovarian cancer cells.","date":"2018","source":"Biochimica et biophysica acta. 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Loss of either ALKBH3 or ASCC3 leads to increased 3-methylcytosine levels, reduced cell proliferation, and pH2A.X/53BP1 foci formation.\",\n      \"method\": \"Protein complex purification, co-immunoprecipitation, helicase activity assay, siRNA knockdown with DNA damage readouts (pH2A.X, 53BP1 foci), cell proliferation assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — complex purified biochemically, helicase activity demonstrated in vitro, functional epistasis confirmed by orthogonal cellular assays with rigorous controls\",\n      \"pmids\": [\"22055184\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ALKBH3 is a 1-methyladenosine (m1A) and 3-methylcytidine (m3C) demethylase of tRNA. ALKBH3-demethylated tRNA is more sensitive to angiogenin (ANG) cleavage, generating tRNA-derived small RNAs (tDRs) around anticodon regions that strengthen ribosome assembly and prevent cytochrome c-triggered apoptosis.\",\n      \"method\": \"In vitro demethylation assay, tDR profiling, ribosome assembly assay, apoptosis assay (cytochrome c), siRNA knockdown, xenograft tumor model\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro enzymatic assay combined with multiple orthogonal cellular and in vivo readouts in a single study\",\n      \"pmids\": [\"30541109\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ALKBH3-mediated m1A demethylation of CSF-1 mRNA increases CSF-1 mRNA stability (half-life) in breast and ovarian cancer cells, promoting cancer cell invasiveness. The m1A site is mapped to the 5'UTR near the translation initiation site, and YTHDF2 (an m6A reader) is not the reader of m1A-containing CSF-1 mRNA.\",\n      \"method\": \"ALKBH3 overexpression/knockdown, mRNA stability assay (half-life measurement), m1A mapping, invasion assay, YTHDF2 interaction test\",\n      \"journal\": \"Biochimica et biophysica acta. Gene regulatory mechanisms\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — single lab with multiple orthogonal methods (stability assay, m1A mapping, functional invasion assay) but no in vitro reconstitution\",\n      \"pmids\": [\"30342176\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ALKBH2 and ALKBH3 (and E. coli AlkB) can oxidize 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine in vitro, demonstrating capacity to oxidize a methyl group attached to carbon rather than nitrogen.\",\n      \"method\": \"In vitro enzymatic assay with purified proteins, mass spectrometry detection of oxidized products, computational docking\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro reconstitution with purified enzymes, single lab, no cellular validation reported\",\n      \"pmids\": [\"31114894\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ALKBH3 removes m1A from Aurora A mRNA, stabilizing it and promoting its translation; depletion of ALKBH3 enhances Aurora A mRNA decay and inhibits its translation, leading to inhibition of ciliogenesis. The catalytically inactive ALKBH3 mutant cannot rescue ciliary defects in alkbh3 morphant zebrafish, confirming the demethylation activity is required.\",\n      \"method\": \"ALKBH3 knockdown/overexpression, Aurora A mRNA stability and translation assays, catalytic mutant rescue, zebrafish alkbh3 morpholino knockdown with phenotypic rescue experiments\",\n      \"journal\": \"Cell discovery\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (mRNA decay, translation, catalytic mutant, in vivo zebrafish rescue) establishing pathway and catalytic requirement\",\n      \"pmids\": [\"35277482\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ALKBH3 directly interacts with human RAD51 paralogue RAD51C via protein-protein interaction, and RAD51C-ALKBH3 interaction stimulates ALKBH3-mediated repair of methyl-adducts within 3'-tailed DNA substrates; disruption of this interaction impairs ALKBH3 function both in vitro and in vivo.\",\n      \"method\": \"Co-immunoprecipitation, pulldown assay, in vitro demethylation assay with 3'-tailed DNA substrates, cellular alkylation damage resistance assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — reciprocal interaction confirmed, in vitro stimulation assay, cellular validation, single lab\",\n      \"pmids\": [\"31642493\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ASCC3, the ALKBH3 binding partner, mediates P-body formation and promotes selective removal of chemically induced m1A and m3C from mRNA; ASCC3-deficient cells show delayed clearance of MMS-induced m1A and m3C from mRNA and impaired P-body formation, consistent with a model where ASCC3-mediated ribosome disassembly allows ALKBH3-dependent mRNA demethylation.\",\n      \"method\": \"Quantitative mass spectrometry of mRNA methylation, SILAC proteomics of mRNA-binding proteins, ASCC3 knockout cells, P-body imaging\",\n      \"journal\": \"Journal of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (MS quantitation, KO cells, imaging) in single lab\",\n      \"pmids\": [\"34217309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ALKBH3 binds to transcription-associated genomic locations including promoter-proximal paused RNA Pol II sites and enhancers in prostate cancer cells; it strongly binds to transcription initiation sites of a small number of highly active promoters characterized by high levels of Mediator, cohesin, and active histone marks. ALKBH3 depletion does not directly alter transcription of its target genes but induces upregulation of ALKBH3-non-bound inflammatory genes.\",\n      \"method\": \"ChIP-seq (endogenous ALKBH3), microarray gene expression after ALKBH3 depletion\",\n      \"journal\": \"Genome medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-seq with expression analysis, single lab, two orthogonal genome-wide methods\",\n      \"pmids\": [\"26221185\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"A fluorogenic probe (MAQ) exploiting fluorescence quenching of 1-methyladenine enables direct measurement of ALKBH3 repair activity in vitro and in cells; the probe is specific for ALKBH3 over ALKBH2 and shows Km and kcat values equivalent to the native substrate. ALKBH3 activity was imaged and quantified in live cells by microscopy and flow cytometry.\",\n      \"method\": \"Fluorogenic substrate assay, enzyme kinetics (Km, kcat), specificity comparison with ALKBH2, live-cell imaging, flow cytometry\",\n      \"journal\": \"Journal of the American Chemical Society\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic reconstitution with quantitative kinetics, cellular imaging validation, specificity controls; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"26967262\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Crystal structures of ALKBH3 crosslinked to oligonucleotide substrates (obtained with a synthetic antibody chaperone) reveal that ALKBH3 uses two β-hairpins (β4-loop-β5 and β'-loop-β'') and an α2 helix for single-stranded substrate binding. Residue Thr133 in the active pocket is required for specific recognition of m1A and m3C; mutation of Thr133 to the corresponding FTO or ALKBH5 residue converts ALKBH3 substrate selectivity from m1A to m6A. Asp194 forms a bubble-like region also critical for substrate recognition.\",\n      \"method\": \"X-ray crystallography of ALKBH3-oligo crosslinked complexes, site-directed mutagenesis (Thr133, Asp194), in vitro demethylation assay with mutants\",\n      \"journal\": \"Angewandte Chemie (International ed. in English)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with mutagenesis and in vitro enzymatic validation in a single study, multiple orthogonal methods\",\n      \"pmids\": [\"38158383\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Biochemical and mutagenesis analysis identifies Tyr143, Leu177, and His191 as key residues for ALKBH3 secondary structure and catalytic activity toward methylated single-stranded DNA. Tyr143 is critical for binding the flipped-out methylated base and stabilizing its everted conformation; Leu177 and His191 are required for secondary structure integrity. Stopped-flow fluorescence spectroscopy revealed a transient kinetic mechanism comprising substrate binding, base eversion, and anchoring steps.\",\n      \"method\": \"Site-directed mutagenesis, stopped-flow fluorescence spectroscopy, CD spectroscopy, in vitro activity assays\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro reconstitution with mutagenesis, single lab, no structural coordinates reported\",\n      \"pmids\": [\"38256217\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The ASCC2 CUE domain selectively binds K63-linked polyubiquitin chains (diubiquitin) by contacting both the distal and proximal ubiquitin, thereby localizing the ASCC-ALKBH3 repair complex to alkylation damage sites in the nucleus. Mutation of residues in the N-terminal portion of the ASCC2 α1 helix that contact the proximal ubiquitin decreases ASCC2 nuclear recruitment in response to DNA alkylation.\",\n      \"method\": \"Co-crystallography/structural analysis, mutagenesis of ASCC2 CUE domain, cellular ASCC2 recruitment assay upon DNA alkylation damage\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — structural and mutagenesis data combined with cellular recruitment assay, single lab\",\n      \"pmids\": [\"34971705\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Alkbh3 (and Alkbh2), but not alkyladenine DNA glycosylase (Aag), can repair N3-ethylthymidine (N3-EtdT) in mammalian cells, as shown by transcription-based lesion bypass assays. Purified human Alkbh2 directly reverses N3-EtdT in vitro. N3-CMdT, O2-EtdT, O4-EtdT, and O4-CMdT are not repaired by Alkbh2 or Alkbh3.\",\n      \"method\": \"Site-specific lesion in non-replicative vectors, transcription bypass assay in mammalian cells, in vitro repair assay with purified Alkbh2\",\n      \"journal\": \"ACS chemical biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — cell-based repair assay and in vitro enzymatic assay (for Alkbh2), Alkbh3 cellular data without in vitro reconstitution for Alkbh3 specifically\",\n      \"pmids\": [\"26930515\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ALKBH3-mediated m1A demethylation of SP100A mRNA prevents its recognition by YTHDF1 (an m1A reader that promotes RNA stability and translation), thereby reducing SP100A protein levels and attenuating formation of tumor-suppressive PML nuclear condensates. YTHDF1 is identified as a reader of m1A-methylated SP100A mRNA.\",\n      \"method\": \"Multiomics (m1A epitranscriptomics + proteomics), ALKBH3 knockdown with SP100A rescue, YTHDF1 interaction with m1A-containing mRNA, in vitro/in vivo functional assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiomics identification with functional rescue and reader identification, single lab\",\n      \"pmids\": [\"38118002\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ALKBH3 demethylates m1A on METTL3 mRNA, preventing YTHDF2-dependent mRNA decay of METTL3 transcript and thereby increasing METTL3 protein levels. Elevated METTL3 then stabilizes COL1A1 and FN1 mRNAs via m6A modification, promoting pathological skin fibrosis (hypertrophic scars).\",\n      \"method\": \"m1A epitranscriptomics, ALKBH3 knockdown/overexpression, METTL3 rescue experiments, in vitro and in vivo fibrosis models, YTHDF2 interaction assay\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic pathway established through multiple functional assays and rescue experiments, single lab\",\n      \"pmids\": [\"40019372\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"m1A demethylase Alkbh3 promotes neurogenesis by demethylating m1A on Mmp15 mRNA, improving its RNA stability and translational efficacy; depletion of Alkbh3 in neural stem cells decreases neuronal differentiation and proliferation while increasing gliogenesis, and reduces hippocampal neurogenesis and spatial memory in adult mice.\",\n      \"method\": \"Alkbh3 knockdown/overexpression in neural stem cells, Mmp15 mRNA stability and translation assay, m1A profiling, in vivo hippocampal neurogenesis and behavioral testing\",\n      \"journal\": \"Cell & bioscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (mRNA stability, translation, in vivo mouse model), single lab\",\n      \"pmids\": [\"39004750\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ALKBH3 demethylates m1A on HK2 mRNA in retinal pigment epithelial cells, activating glycolysis and excess lactate production. This lactate promotes H3K18 histone lactylation, which binds the ALKBH3 promoter to amplify its transcription, establishing a positive feedback loop. ALKBH3 also directly demethylates VEGFA mRNA to promote choroidal neovascularization.\",\n      \"method\": \"dm1ACRISPR system for site-specific m1A demethylation, gene knockout mice, ChIP for histone lactylation at ALKBH3 promoter, m1A epitranscriptomics, ALKBH3 inhibitor (HUHS015), in vivo AMD model\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-specific editing tool, in vivo model, ChIP, inhibitor validation; single lab\",\n      \"pmids\": [\"40493193\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ALKBH3-mediated m1A demethylation of ALDOA mRNA at the 3'UTR stabilizes ALDOA mRNA by preventing recruitment of the YTHDF2/PAN2-PAN3 complex that drives mRNA degradation; this stabilization potentiates glycolysis and doxorubicin resistance in triple-negative breast cancer cells.\",\n      \"method\": \"ALKBH3 knockdown/overexpression, m1A site mapping in ALDOA 3'UTR, mRNA stability assay, YTHDF2/PAN2-PAN3 interaction assay, glycolysis metabolite measurement, in vivo xenograft\",\n      \"journal\": \"Acta pharmaceutica Sinica. B\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — m1A site mapped, reader/degradation complex identified, functional in vitro and in vivo validation; single lab\",\n      \"pmids\": [\"40654364\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"ALKBH3 removes m1A from PINK1 mRNA, promoting its stability and translation; elevated ALKBH3 in Alzheimer's disease models impairs PINK1-dependent mitophagy, leading to mitochondrial dysfunction and neuronal damage. Alkbh3 reduction decreases amyloid-β plaques and restores cognition in 5xFAD mice.\",\n      \"method\": \"m1A epitranscriptomics, Alkbh3 knockout/reduction in 5xFAD mice, PINK1 mRNA stability and translation assays, mitophagy and mitochondrial function assays, behavioral testing\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — m1A on PINK1 mRNA identified, in vivo Alzheimer's model with multiple readouts; single lab\",\n      \"pmids\": [\"41816968\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"ALKBH3 demethylates m1A on ZBED6 mRNA, enhancing ZBED6 translation; ZBED6 then physically interacts with STAT1 (confirmed by co-immunoprecipitation and ChIP) and represses STAT1-driven AIM2 transcription, thereby suppressing PANoptosis (pyroptosis/apoptosis/necroptosis) in cardiomyocytes during ischemia/reperfusion injury.\",\n      \"method\": \"m1A epitranscriptomics, ALKBH3 overexpression/siRNA in cells and in vivo I/R model, co-immunoprecipitation (ZBED6-STAT1), ChIP, dual-luciferase reporter (AIM2 promoter), loss- and gain-of-function for ZBED6, STAT1, AIM2\",\n      \"journal\": \"Clinical and translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pathway established by multiple orthogonal methods including co-IP, ChIP, reporter assay, and in vivo model; single lab\",\n      \"pmids\": [\"41816893\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PUS7-dependent pseudouridylation of ALKBH3 mRNA at position U696 enhances its translation efficiency, thereby increasing ALKBH3 protein levels and suppressing gastric cancer progression.\",\n      \"method\": \"Locus-specific pseudouridine detection assay, polysome profiling, RT-qPCR, Western blotting, 3D colony formation assay, xenograft model\",\n      \"journal\": \"Clinical and translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-specific modification mapped with functional validation via polysome profiling, in vivo xenograft; single lab\",\n      \"pmids\": [\"39175405\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ALKBH3-mediated m1A demethylation of ATF4 mRNA increases ATF4 expression, which inhibits ferroptosis (by upregulating SLC7A11, GPX4, FTH1) and promotes AML cell survival; ALKBH3 knockdown promotes ferroptosis in KG-1 cells.\",\n      \"method\": \"ALKBH3 knockdown, ATF4 knockdown rescue experiments, ferroptosis markers (ROS, MDA, iron, SOD, GSH), flow cytometry, xenograft model\",\n      \"journal\": \"Hematology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — functional assays performed but m1A site on ATF4 mRNA not directly mapped; mechanistic link inferred from knockdown epistasis, single lab\",\n      \"pmids\": [\"39803678\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ALKBH3 is a Fe(II)/α-ketoglutarate-dependent dioxygenase that oxidatively demethylates N1-methyladenosine (m1A) and N3-methylcytidine (m3C) in both single-stranded DNA and RNA (including tRNA, mRNA, and mitochondrial RNA); it operates in a complex with ASCC (particularly the ASCC3 helicase that generates ssDNA substrate and ASCC2 that recruits the complex to damage sites via K63-polyubiquitin recognition), interacts with RAD51C to facilitate alkylation repair at 3'-tailed DNA, and regulates diverse cellular processes—including ciliogenesis, neurogenesis, mitophagy, glycolysis, and cell survival—by stabilizing or destabilizing specific mRNAs through m1A demethylation, with substrate specificity dictated by key active-site residues Thr133 and Asp194 as revealed by crystal structures of substrate-crosslinked complexes.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ALKBH3 is a single-stranded nucleic acid demethylase that reverses N1-methyladenine/N1-methyladenosine (m1A) and N3-methylcytosine/N3-methylcytidine (m3C) lesions in both DNA and RNA, functioning both as an alkylation-damage repair enzyme and as an epitranscriptomic eraser that controls mRNA fate [#0, #1, #8]. In its DNA-repair role, ALKBH3 acts within the ASCC complex: the ASCC3 3'-5' helicase generates the single-stranded substrate ALKBH3 prefers, and loss of either protein elevates 3-methylcytosine and triggers DNA-damage signaling and reduced proliferation [#0]. The complex is targeted to nuclear alkylation sites through the ASCC2 CUE domain, which reads K63-linked polyubiquitin chains [#11], and ALKBH3 repair of methyl adducts on 3'-tailed DNA is further stimulated by direct interaction with the recombination factor RAD51C [#5]. Crystal structures of substrate-crosslinked ALKBH3 show that the enzyme grips single-stranded substrate via two beta-hairpins and an alpha2 helix and everts the methylated base, with active-site residue Thr133 dictating m1A/m3C selectivity (its substitution by the corresponding FTO/ALKBH5 residue switches selectivity toward m6A) and Asp194 and Tyr143 contributing to substrate recognition and base eversion [#9, #10]. As an RNA demethylase, ALKBH3 strips m1A from numerous target mRNAs to govern their stability and translation: by erasing m1A it either prevents reader-driven decay—removing marks recognized by YTHDF2/PAN2-PAN3 or, conversely, by the stabilizing reader YTHDF1—or otherwise alters transcript half-life, thereby controlling targets such as CSF-1, Aurora A, METTL3, ALDOA, HK2, VEGFA, PINK1, ZBED6, and Mmp15 [#2, #4, #13, #14, #17, #16, #18, #19, #15]. Through this mRNA-regulatory activity ALKBH3 influences a broad range of processes including cancer cell invasion and glycolysis, ciliogenesis, hippocampal neurogenesis, mitophagy, neovascularization, and cell-survival decisions [#2, #4, #15, #16, #18]. ALKBH3 also demethylates tRNA, sensitizing it to angiogenin cleavage and generating tRNA-derived small RNAs that support ribosome assembly and suppress apoptosis [#1].\",\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"Established that ALKBH3 is not a stand-alone repair enzyme but operates within the ASCC complex, where the ASCC3 helicase supplies the single-stranded DNA substrate it acts upon.\",\n      \"evidence\": \"Complex purification, in vitro helicase assay, and siRNA epistasis with DNA-damage readouts in human cells\",\n      \"pmids\": [\"22055184\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve how the complex is recruited to damage sites\", \"Substrate generation linked to ssDNA but RNA substrates not addressed\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showed ALKBH3 occupies transcription-associated chromatin (paused Pol II, enhancers, active promoters), raising the possibility of a chromatin/transcription-linked role beyond bulk DNA repair.\",\n      \"evidence\": \"Endogenous ALKBH3 ChIP-seq with expression microarray after depletion in prostate cancer cells\",\n      \"pmids\": [\"26221185\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Depletion did not alter transcription of bound genes, leaving the functional consequence of binding unresolved\", \"Mechanism linking ALKBH3 chromatin occupancy to inflammatory gene upregulation unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Provided quantitative kinetic tools and lesion-specificity data, confirming ALKBH3 acts on m1A with measurable kinetics and on specific N3-alkyl thymidine lesions while sparing others.\",\n      \"evidence\": \"Fluorogenic m1A probe with Km/kcat and live-cell imaging; transcription-bypass lesion-repair assays for N3-alkyl lesions\",\n      \"pmids\": [\"26967262\", \"26930515\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vitro reconstitution for ALKBH3 on N3-EtdT specifically not performed\", \"Cellular substrate scope incompletely defined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Opened the epitranscriptomic role by showing ALKBH3 m1A-demethylates a specific mRNA (CSF-1) to extend its half-life and drive cancer invasiveness, establishing mRNA stability as an ALKBH3 output.\",\n      \"evidence\": \"ALKBH3 overexpression/knockdown, m1A mapping, mRNA half-life and invasion assays in breast/ovarian cancer cells\",\n      \"pmids\": [\"30342176\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The reader interpreting m1A on CSF-1 mRNA was not identified (YTHDF2 excluded)\", \"No in vitro reconstitution of the demethylation event\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined ALKBH3 as an m1A/m3C eraser on tRNA and linked its activity to tRNA-derived small RNA production, ribosome assembly, and apoptosis resistance, expanding substrate scope beyond DNA and mRNA.\",\n      \"evidence\": \"In vitro demethylation, tDR profiling, ribosome assembly and apoptosis assays, xenograft\",\n      \"pmids\": [\"30541109\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific tRNA species and sites incompletely defined\", \"Coupling between demethylation and angiogenin cleavage not structurally resolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified RAD51C as a direct ALKBH3 partner that stimulates repair of methyl adducts on 3'-tailed DNA, connecting ALKBH3 alkylation repair to recombination machinery.\",\n      \"evidence\": \"Reciprocal co-IP/pulldown, in vitro demethylation on 3'-tailed substrates, cellular alkylation-resistance assay\",\n      \"pmids\": [\"31642493\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; structural basis of interaction unknown\", \"Relationship to the ASCC complex not addressed\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrated that ALKBH3 (with ALKBH2) can oxidize carbon-bound 5-methylcytosine to 5hmC/5fC/5caC in vitro, broadening its potential chemistry beyond N-demethylation.\",\n      \"evidence\": \"In vitro enzymatic assay with purified protein, mass spectrometry, computational docking\",\n      \"pmids\": [\"31114894\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No cellular validation that this activity occurs in vivo\", \"Biological significance of 5mC oxidation by ALKBH3 unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Resolved how the ASCC-ALKBH3 complex is recruited to damage and how it accesses mRNA substrates, linking K63-ubiquitin recognition and ribosome disassembly to demethylation.\",\n      \"evidence\": \"ASCC2 CUE-domain structure/mutagenesis with nuclear recruitment assay; ASCC3 KO cells with MS quantitation of mRNA m1A/m3C and P-body imaging\",\n      \"pmids\": [\"34971705\", \"34217309\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct demonstration that ASCC3-driven ribosome disassembly is required for ALKBH3 catalysis not shown\", \"Single labs for each mechanism\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Provided in vivo proof that ALKBH3 catalytic m1A demethylation of a target mRNA (Aurora A) controls a developmental process (ciliogenesis), as a catalytically dead mutant failed to rescue zebrafish defects.\",\n      \"evidence\": \"mRNA decay/translation assays, catalytic mutant rescue, zebrafish morpholino phenotype rescue\",\n      \"pmids\": [\"35277482\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reader machinery for Aurora A m1A not defined\", \"Generality across cilia-related transcripts not established\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Delivered the structural and kinetic basis of ALKBH3 substrate selectivity, showing Thr133 governs m1A/m3C versus m6A choice and identifying residues for ssDNA binding and base eversion.\",\n      \"evidence\": \"X-ray crystallography of substrate-crosslinked complexes plus mutagenesis; stopped-flow/CD kinetics defining binding, eversion, anchoring steps\",\n      \"pmids\": [\"38158383\", \"38256217\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structures used DNA oligo substrates; RNA-bound conformation not solved\", \"How active-site selectivity maps onto tRNA/mRNA targets in vivo unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed ALKBH3 m1A erasure tunes mRNA fate through specific readers — preventing YTHDF1-mediated stabilization (SP100A) — coupling the eraser to defined reader-decay logic, and identified a regulatory input via PUS7 pseudouridylation of ALKBH3 mRNA controlling its own translation.\",\n      \"evidence\": \"Multiomics with rescue and reader (YTHDF1) interaction assays; locus-specific pseudouridine mapping with polysome profiling and xenograft\",\n      \"pmids\": [\"38118002\", \"39175405\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reader assignment varies by transcript; rules for reader choice unclear\", \"Upstream control of ALKBH3 expression in physiological contexts incompletely mapped\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Expanded the m1A-eraser regulon to metabolic and signaling targets — METTL3, HK2/VEGFA, ALDOA — establishing ALKBH3 as a driver of glycolysis, neovascularization, fibrosis, and chemoresistance via YTHDF2/PAN2-PAN3-dependent decay and a lactylation feedback loop.\",\n      \"evidence\": \"Site-specific m1A editing (dm1ACRISPR), knockdown/overexpression with reader/degradation-complex interaction assays, ChIP for histone lactylation, inhibitor and in vivo models\",\n      \"pmids\": [\"40019372\", \"40493193\", \"40654364\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Each pathway from a single lab\", \"Whether one demethylase coordinates these target sets simultaneously not addressed\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Extended ALKBH3 target regulation to neuronal and cardiac homeostasis — PINK1-dependent mitophagy in Alzheimer's models and a ZBED6-STAT1-AIM2 axis controlling cardiomyocyte PANoptosis — implicating it in degenerative and ischemic disease.\",\n      \"evidence\": \"m1A epitranscriptomics, in vivo 5xFAD and I/R models, co-IP, ChIP, luciferase reporter, stability/translation assays\",\n      \"pmids\": [\"41816968\", \"41816893\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct mapping of m1A demethylation events partly inferential\", \"Single-lab disease models awaiting independent confirmation\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how ALKBH3 partitions among its DNA-repair, tRNA, and mRNA substrate pools in a given cell, and what determines which reader and outcome a demethylated transcript receives.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model of substrate selection across DNA vs RNA in vivo\", \"Rules dictating reader assignment (YTHDF1 vs YTHDF2/PAN2-PAN3) per transcript unknown\", \"Regulation balancing nuclear repair vs cytoplasmic mRNA roles undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [1, 2, 4, 8, 9, 13, 15, 17]},\n      {\"term_id\": \"GO:0140097\", \"supporting_discovery_ids\": [0, 5, 9, 12]},\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [3, 8, 9]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [1, 2, 4]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [0, 5, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 7, 11]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [0, 5, 11, 12]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [1, 2, 4, 6, 13]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [16, 18, 19]}\n    ],\n    \"complexes\": [\"ASCC complex\"],\n    \"partners\": [\"ASCC3\", \"ASCC2\", \"RAD51C\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}