{"gene":"ALKBH5","run_date":"2026-06-09T22:02:43","timeline":{"discoveries":[{"year":2012,"finding":"ALKBH5 is a mammalian RNA demethylase that oxidatively reverses N6-methyladenosine (m6A) in mRNA in vitro and in vivo. Its demethylation activity affects mRNA export and RNA metabolism, as well as assembly of mRNA processing factors in nuclear speckles. Alkbh5-deficient male mice show increased m6A in mRNA and impaired fertility due to apoptosis of meiotic metaphase-stage spermatocytes.","method":"In vitro demethylation assay, Alkbh5-knockout mice, mRNA export and nuclear speckle assembly assays, transcriptome profiling of testes","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzymatic assay plus in vivo knockout model with multiple orthogonal readouts; foundational paper replicated across many subsequent studies","pmids":["23177736"],"is_preprint":false},{"year":2022,"finding":"Crystal structures of ALKBH5 in complex with m6A-containing single-stranded RNA 8-mer revealed that the RNA substrate binds in a 5'-3' orientation opposite to that of DNA substrates in other AlkB members. The structures defined the (A/G)m6AC consensus sequence preference and a proton shuttle network involving Lys132 and Tyr139 that enables efficient hemiaminal intermediate demethylation to produce formaldehyde.","method":"X-ray crystallography (three crystal structures), biochemical demethylation assays, mutagenesis of active-site residues","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple crystal structures with biochemical validation and mutagenesis in a single rigorous study","pmids":["35333330"],"is_preprint":false},{"year":2017,"finding":"ALKBH5 demethylates FOXM1 nascent transcripts in glioblastoma stem-like cells (GSCs), leading to enhanced FOXM1 mRNA stability and expression. A long non-coding RNA antisense to FOXM1 (FOXM1-AS) promotes the interaction of ALKBH5 with FOXM1 nascent transcripts. ALKBH5 silencing suppresses GSC proliferation and tumorigenesis through this FOXM1 axis.","method":"Integrated transcriptome and m6A-seq, RNA immunoprecipitation, ALKBH5 knockdown in patient-derived GSCs, in vivo tumor models","journal":"Cancer cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal RIP, m6A-seq, in vivo models, replicated concept across multiple GSC lines","pmids":["28344040"],"is_preprint":false},{"year":2021,"finding":"ROS induces ALKBH5 SUMOylation via ERK/JNK signaling, which inhibits ALKBH5 m6A demethylase activity by blocking substrate accessibility, thereby globally increasing mRNA m6A levels and inducing DNA damage response genes. This ERK/JNK/ALKBH5-PTMs/m6A axis is activated in hematopoietic stem/progenitor cells in vivo.","method":"SUMOylation assays, ERK/JNK inhibitor treatments, ROS induction in cell lines and mouse HSPCs, m6A quantification, mutagenesis of SUMOylation sites","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple PTM assays, in vitro enzymatic activity, in vivo HSPC validation, and mutagenesis in one study","pmids":["34048572"],"is_preprint":false},{"year":2023,"finding":"RBM33 (RNA-binding motif protein 33) forms a complex with ALKBH5 and acts as a substrate-recruiting co-factor that (1) recruits ALKBH5 to specific m6A-marked mRNA targets and (2) activates ALKBH5 demethylase activity by removing its SUMOylation. This interaction selectively directs ALKBH5 to demethylate DDIT4 mRNA to promote autophagy in HNSCC.","method":"Co-immunoprecipitation, in vitro demethylase activity assays, SUMOylation assays, m6A-seq, RBM33 knockdown in cancer cells","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — reciprocal Co-IP, in vitro enzymatic validation, and m6A-seq in a single rigorous study","pmids":["37257451"],"is_preprint":false},{"year":2023,"finding":"EGFR signaling retains ALKBH5 in the nucleus of glioblastoma stem cells by activating SRC kinase, which phosphorylates ALKBH5 and inhibits CRM1-mediated nuclear export. Nuclear ALKBH5 demethylates GCLM mRNA; reduced m6A on GCLM mRNA stabilizes it (preventing YTHDF2-mediated decay), thereby protecting against ferroptosis.","method":"EGFR/SRC inhibition, ALKBH5 phosphorylation assays, nuclear fractionation, YTHDF2-dependent GCLM mRNA decay assays, m6A-seq, GSC in vivo models","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — phosphorylation mechanism, nuclear localization, mRNA decay, and in vivo data with multiple orthogonal methods","pmids":["37979586"],"is_preprint":false},{"year":2023,"finding":"USP36 deubiquitinase directly binds ALKBH5, removes its polyubiquitin chain, and stabilizes the ALKBH5 protein in glioblastoma. USP36 depletion reduces ALKBH5 levels, impairs glioblastoma stem cell self-renewal, and inhibits in vivo tumor growth.","method":"Mass spectrometry, co-immunoprecipitation, in vivo and in vitro ubiquitination assays, USP36 knockdown in GSCs, intracranial tumor xenografts","journal":"Neuro-oncology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — direct binding by MS and Co-IP, in vitro and in vivo ubiquitination assays, in vivo tumor model","pmids":["36239338"],"is_preprint":false},{"year":2023,"finding":"USP9X deubiquitinase directly binds ALKBH5, removes K48-linked polyubiquitin at K57, and stabilizes the ALKBH5 protein in AML cells. USP9X depletion reduces ALKBH5 levels and promotes AML cell apoptosis; ectopic ALKBH5 expression partially rescues USP9X knockdown effects.","method":"Mass spectrometry, co-immunoprecipitation, K48-linked ubiquitination assays, site-specific mutagenesis (K57), genetic knockdown, murine AML model","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — MS identification, Co-IP, site-specific ubiquitination mutagenesis, and rescue experiments","pmids":["37454738"],"is_preprint":false},{"year":2020,"finding":"ALKBH5 is required for AML development and leukemia stem cell self-renewal but is dispensable for normal hematopoiesis. Mechanistically, ALKBH5 demethylates TACC3 mRNA, stabilizing it and promoting TACC3 protein expression, which drives leukemogenesis.","method":"Alkbh5 conditional knockout mice, human AML cell lines, m6A-seq, RNA-seq, MeRIP-qPCR, in vivo AML models","journal":"Cell stem cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO mouse model, m6A-seq/RNA-seq, and in vivo leukemia models demonstrating pathway specificity","pmids":["32402250"],"is_preprint":false},{"year":2020,"finding":"KDM4C histone demethylase regulates ALKBH5 expression in AML by reducing H3K9me3 at the ALKBH5 locus, increasing chromatin accessibility, and promoting MYB/Pol II recruitment. ALKBH5 in turn affects AXL mRNA stability in an m6A-dependent manner to maintain leukemia stem cell function.","method":"ChIP-seq, ATAC-seq, chromatin accessibility assays, KDM4C knockdown, MeRIP-seq, AML mouse models","journal":"Cell stem cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP-seq, ATAC-seq, m6A-seq, and in vivo AML model with multiple orthogonal methods","pmids":["32402251"],"is_preprint":false},{"year":2019,"finding":"METTL3 and ALKBH5 oppositely regulate m6A modification of TFEB mRNA in cardiomyocytes. METTL3 methylates TFEB 3'-UTR at two m6A residues, promoting HNRNPD binding and decreasing TFEB expression. ALKBH5 demethylates these sites to reverse the effect. TFEB reciprocally binds the ALKBH5 promoter to induce its transcription, establishing a feedback loop.","method":"m6A-seq, RIP, ChIP, METTL3/ALKBH5 knockdown and overexpression in cardiomyocytes, promoter luciferase assay, murine I/R model","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal functional experiments, m6A-seq, RIP, ChIP, and in vivo cardiac model with multiple orthogonal validations","pmids":["30870073"],"is_preprint":false},{"year":2024,"finding":"ALKBH5 undergoes lactylation catalyzed by acetyltransferase ESCO2 (increased during viral infection) and is de-lactylated by SIRT6. Lactylated ALKBH5 binds IFN-β mRNA and demethylates its m6A modifications, promoting IFN-β mRNA biogenesis and antiviral innate immune responses against HSV-1, KSHV, and mpox virus.","method":"Lactylation mass spectrometry, ESCO2/SIRT6 co-immunoprecipitation, RIP of IFN-β mRNA, m6A quantification, viral infection assays, ESCO2 overexpression/SIRT6 depletion","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — PTM identification by MS, writer/eraser identification by Co-IP, direct RNA-binding by RIP, and functional antiviral assays","pmids":["39413129"],"is_preprint":false},{"year":2025,"finding":"KRAS mutants activate ERK/JNK signaling in NSCLC, which promotes ALKBH5 SUMOylation (inhibiting its demethylase activity), resulting in increased m6A methylation on DDB2 and XPC mRNAs. Stabilization of these DNA repair transcripts enhances nucleotide excision repair, conferring platinum resistance. A SUMOylation-deficient ALKBH5 mutant restores platinum sensitivity.","method":"SUMOylation assays, m6A-seq, RNA stability assays, NER functional assays, ALKBH5 mutagenesis, in vivo xenograft models with KRAS mutant NSCLC","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — ALKBH5 mutagenesis, m6A-seq, NER assays, and in vivo model; mechanistically connects KRAS→ERK/JNK→ALKBH5 SUMOylation→m6A→DNA repair","pmids":["39960727"],"is_preprint":false},{"year":2025,"finding":"Protein kinase A (PKA) phosphorylates ALKBH5, promoting its degradation. Loss of ALKBH5 maintains m6A modification on GPX4 mRNA, stabilizing GPX4 and thereby suppressing ferroptosis. PKA thus acts as a regulator of ferroptosis through the ALKBH5-GPX4 m6A axis.","method":"PKA phosphorylation assays, ALKBH5 deletion/reconstitution, m6A quantification of GPX4 mRNA, ferroptosis assays, in vivo tumor models","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — PKA-ALKBH5 phosphorylation and GPX4 m6A regulation demonstrated in single lab with multiple functional readouts; no mutagenesis of specific phospho-site reported in abstract","pmids":["39901038"],"is_preprint":false},{"year":2021,"finding":"ALKBH5 demethylates FOXM1 mRNA in uveal melanoma, increasing its stability and expression, promoting tumor growth and metastasis via EMT. EP300-mediated H3K27 acetylation at the ALKBH5 locus activates ALKBH5 transcription.","method":"MeRIP-qPCR for m6A on FOXM1 mRNA, ALKBH5 knockdown in UM cell lines, in vivo xenograft model, ChIP for H3K27ac","journal":"Aging","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MeRIP, in vivo model, ChIP, single lab; extends FOXM1 m6A finding from GBM to UM","pmids":["33428593"],"is_preprint":false},{"year":2022,"finding":"ALKBH5 mediates m6A demethylation of NEAT1 lncRNA under hypoxia, stabilizing NEAT1 and facilitating paraspeckle assembly. This causes relocalization of transcriptional repressor SFPQ from the CXCL8 promoter to paraspeckles, upregulating CXCL8/IL8 secretion and promoting tumor-associated macrophage recruitment in GBM.","method":"m6A-seq on hypoxic GBM cells, NEAT1 stability assays, SFPQ chromatin immunoprecipitation, ALKBH5 depletion/inactivation, allograft tumor models","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — m6A-seq, ChIP, RIP, in vivo allograft with multiple orthogonal mechanistic steps validated","pmids":["34670781"],"is_preprint":false},{"year":2023,"finding":"The disordered C-terminal intrinsically disordered region (cIDR) of ALKBH5 promotes liquid-liquid phase separation and incorporation into paraspeckles. Under hypoxia, rapid ALKBH5 condensation in paraspeckles induces m6A demethylation of NEAT1, further facilitating paraspeckle assembly. ALKBH5 lacking cIDR fails to support paraspeckle formation and NEAT1 stabilization.","method":"Phase separation assays, deletion mutants of ALKBH5-cIDR, live-cell imaging of ALKBH5 condensates, m6A demethylation of NEAT1, hypoxia-induced invasion assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phase separation assays and functional cIDR deletions in single lab; mechanistically extends paraspeckle/NEAT1 finding","pmids":["37474102"],"is_preprint":false},{"year":2023,"finding":"LATS2 kinase phosphorylates ALKBH5, preventing its nuclear export and enhancing its protein stability. Phosphorylated ALKBH5 reciprocally erases m6A from LATS2 mRNA, stabilizing this transcript and establishing a positive feedback loop that promotes glioblastoma stem cell self-renewal.","method":"Kinase assay, nuclear fractionation, ALKBH5/LATS2 Co-IP, m6A-seq on LATS2 mRNA, ALKBH5 phosphorylation-site mutants, GBM xenograft models","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — kinase assay, Co-IP, m6A-seq, phospho-mutants, and in vivo data from single lab","pmids":["38568805"],"is_preprint":false},{"year":2025,"finding":"USP14 deubiquitinase stabilizes ALKBH5 by removing K48-linked ubiquitination through HECW2. MST4 kinase phosphorylates ALKBH5 at S64 and S69, increasing its interaction with USP14 and promoting ALKBH5 deubiquitylation. ALKBH5 also binds and stabilizes USP14 mRNA via YTHDF2-dependent mechanism, creating a positive feedback loop. This MST4-USP14-ALKBH5 axis promotes GSC radioresistance.","method":"Mass spectrometry, co-immunoprecipitation, ubiquitination assays, phospho-site mutagenesis (S64/S69), m6A-seq, transcriptome analysis, GSC radioresistance assays, xenograft models","journal":"Theranostics","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — MS identification, kinase assay, ubiquitination assays, phospho-site mutagenesis, m6A-seq, and in vivo xenograft in a single rigorous study","pmids":["39990235"],"is_preprint":false},{"year":2021,"finding":"ALKBH5 demethylates TRAF1 mRNA 3'-UTR, decreasing m6A abundance and enhancing TRAF1 mRNA stability, which promotes TRAF1 protein expression and activates NF-κB and MAPK signaling to drive multiple myeloma cell growth and survival.","method":"MeRIP-qPCR, mRNA stability assays, ALKBH5 knockdown in MM cells, in vivo xenograft models, NF-κB/MAPK pathway readouts","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MeRIP, mRNA stability, in vivo model; single lab with multiple functional readouts","pmids":["34759347"],"is_preprint":false},{"year":2023,"finding":"ALKBH5 binds and demethylates AXIN2 mRNA, causing AXIN2 mRNA dissociation from IGF2BP1 and subsequent mRNA degradation, resulting in hyperactivated Wnt/β-catenin signaling and induction of DKK1, which recruits myeloid-derived suppressor cells to drive immunosuppression in colorectal cancer.","method":"MeRIP-seq, RNA-seq, RIP for IGF2BP1/AXIN2 interaction, ALKBH5 knockin mice (intestine-specific), CD34+ humanized mice, allografts, vesicle-nanoparticle siRNA delivery","journal":"Gastroenterology","confidence":"High","confidence_rationale":"Tier 2 / Strong — MeRIP-seq, multiple in vivo models (knockin mice, humanized mice, allografts), RIP validation, with mechanistic pathway dissection","pmids":["37169182"],"is_preprint":false},{"year":2024,"finding":"ALKBH5 in macrophages demethylates IL-11 mRNA, increasing its stability and IL-11 protein levels, which drives macrophage-to-myofibroblast transition (MMT) under angiotensin II-induced hypertension. Macrophage-specific ALKBH5 knockout inhibits MMT and ameliorates cardiac fibrosis.","method":"RIP-seq (identified IL-11 mRNA as target), single-cell transcriptomics, lineage tracing, parabiosis, macrophage-specific ALKBH5 knockout mice, Ang II infusion model, IL-11 overexpression rescue","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — RIP-seq, cell-type-specific KO, lineage tracing, parabiosis, and in vivo rescue experiments","pmids":["38443404"],"is_preprint":false},{"year":2022,"finding":"ALKBH5 demethylates PKMYT1 mRNA; loss of ALKBH5 increases m6A on PKMYT1 mRNA, and the m6A reader IGF2BP3 stabilizes PKMYT1 mRNA, upregulating PKMYT1 expression and promoting gastric cancer invasion and metastasis.","method":"MeRIP-seq, RNA pulldown, mass spectrometry, RIP, ALKBH5 demethylase activity mutant, in vivo lung metastasis model","journal":"Molecular cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MeRIP-seq, RNA pulldown, demethylase mutant, and in vivo metastasis; single lab","pmids":["35114989"],"is_preprint":false},{"year":2021,"finding":"ALKBH5 deficiency in PD-L1 mRNA 3'-UTR enriches m6A modification, promoting YTHDF2-dependent PD-L1 mRNA degradation and reducing tumor PD-L1 expression. ALKBH5 thus sustains PD-L1 expression to promote immune evasion in intrahepatic cholangiocarcinoma.","method":"m6A methylome sequencing, ALKBH5-PD-L1 mRNA RIP, YTHDF2 knockdown rescue, in vitro and in vivo ICC tumor models","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — m6A-seq, RIP, YTHDF2 rescue experiments, in vivo ICC model; single lab","pmids":["34301762"],"is_preprint":false},{"year":2020,"finding":"ALKBH5 deletion in tumor cells modulates Mct4/Slc16a3 mRNA m6A levels and reduces lactate content in the tumor microenvironment, altering infiltration of Treg and myeloid-derived suppressor cells to sensitize tumors to anti-PD-1 immunotherapy.","method":"Alkbh5 deletion in tumor cell lines, m6A density analysis, splicing analysis, lactate measurement, flow cytometry of tumor-infiltrating immune cells, small-molecule ALKBH5 inhibitor treatment, in vivo tumor models","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo deletion model with m6A-seq, splicing analysis, and immune cell profiling; single lab","pmids":["32747553"],"is_preprint":false},{"year":2022,"finding":"ALKBH5 mediates m6A demethylation of ITGB1 mRNA in ovarian cancer, suppressing YTHDF2-mediated ITGB1 mRNA degradation and increasing ITGB1 expression, which activates FAK/Src phosphorylation and promotes tumor-associated lymphangiogenesis and lymph node metastasis. Hypoxia induces HIF1α-dependent ALKBH5 upregulation that feeds this pathway.","method":"RNA pulldown, RIP-qPCR, Co-IP, MeRIP-qPCR, luciferase reporter assay, in vitro and in vivo lymphangiogenesis models","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNA pulldown, RIP, MeRIP, Co-IP, and in vivo models; single lab","pmids":["36632222"],"is_preprint":false},{"year":2022,"finding":"ALKBH5 mediates m6A demethylation of Drp1 mRNA 3'-UTR. Under low ALKBH5 expression in hepatic stellate cells (HSCs), increased m6A on Drp1 mRNA promotes YTHDF1-mediated translation of DRP1, enhancing mitochondrial fission and HSC proliferation/migration to drive liver fibrosis.","method":"MeRIP-qPCR, mRNA stability assays, polysome fractionation, YTHDF1 co-immunoprecipitation, mitochondrial morphology assays, TGF-β1-induced HSC activation, in vivo fibrosis models","journal":"Pharmacological research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MeRIP, polysome fractionation, Co-IP, and in vivo fibrosis models; single lab","pmids":["36566000"],"is_preprint":false},{"year":2022,"finding":"ALKBH5 demethylates pri-miR-320a-3p, blocking the microprocessor protein DGCR8 from binding and preventing maturation of miR-320a-3p. Reduced mature miR-320a-3p de-represses FOXM1 mRNA (its target), promoting fibroblast activation. ALKBH5 also directly demethylates FOXM1 mRNA in an m6A-dependent manner.","method":"RIP assay for DGCR8/pri-miR-320a-3p interaction, MeRIP, miRNA processing assays, ALKBH5 knockdown in fibroblasts, silica-induced pulmonary fibrosis mouse model","journal":"Cellular & molecular biology letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RIP, MeRIP, microprocessor interaction assay, in vivo fibrosis model; single lab","pmids":["35279083"],"is_preprint":false},{"year":2023,"finding":"Loss of ALKBH5 increases m6A on OGDH (oxoglutarate dehydrogenase) mRNA, destabilizing it and reducing OGDH protein. Limited OGDH slows the TCA cycle, causing α-KG accumulation and conversion to L-2-HG, which inhibits mitochondrial energy production in hematopoietic stem/progenitor cells and impairs HSPC fitness.","method":"Alkbh5 KO mice, m6A-seq, RNA stability assays, metabolomics (α-KG/L-2-HG measurement), HSPC competitive transplantation, human hematopoietic cell in vitro assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — m6A-seq, metabolomics, in vivo competitive transplantation, and human cell validation; multiple orthogonal methods","pmids":["37742191"],"is_preprint":false},{"year":2023,"finding":"ALKBH5 erases m6A on CSF3R mRNA (encoding G-CSFR), stabilizing it and increasing G-CSFR surface expression and downstream STAT3 signaling in neutrophils. This drives emergency granulopoiesis and neutrophil mobilization during bacterial sepsis. ALKBH5 direct binding to CSF3R mRNA was confirmed by RIP-qPCR.","method":"Alkbh5-deficient mice in CLP sepsis model, RIP-qPCR, m6A quantification of CSF3R mRNA, mRNA stability assays, surface G-CSFR expression, STAT3 signaling readouts","journal":"Cellular & molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RIP-qPCR, m6A assays, mRNA stability, in vivo sepsis model; single lab","pmids":["38114747"],"is_preprint":false},{"year":2022,"finding":"ALKBH5-mediated m6A demethylation of neutrophil migration-related mRNA targets (including CXCR2, NLRP12, PTGER4, TNC, WNK1) imprints a migration-promoting transcriptome in neutrophils. Loss of ALKBH5 reduces CXCR2 expression and impairs neutrophil migration toward CXCL2, increasing mortality in sepsis.","method":"Alkbh5-deficient mice in CLP model, CXCR2 surface expression, m6A RNA decay assays, mRNA stability analysis, neutrophil migration assays","journal":"Signal transduction and targeted therapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo sepsis model, m6A RNA decay assays, mRNA stability assays; single lab","pmids":["35764614"],"is_preprint":false},{"year":2022,"finding":"Loss of ALKBH5 in lymphoid cells increases m6A on Jagged1 and Notch2 mRNAs, reducing their expression and impairing Jagged1/Notch2 signaling. This favors γδ T cell precursor expansion and differentiation, expanding the mature γδ T cell repertoire and enhancing protection against Salmonella infection.","method":"Alkbh5 conditional KO in lymphocytes, m6A-seq in thymocytes, flow cytometry of γδ T cell populations, Jagged1/Notch2 expression analysis, Salmonella infection challenge","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — lymphoid-specific KO, m6A-seq, flow cytometry, in vivo infection model; single lab","pmids":["35939687"],"is_preprint":false},{"year":2023,"finding":"ALKBH5-mediated m6A demethylation ensures timely degradation of maternal RNAs during oocyte meiosis. In Alkbh5-/- oocytes, certain maternal transcripts accumulate with persistent m6A peaks and are recognized by IGF2BP2, stabilizing them and impairing mRNA clearance. Reducing IGF2BP2 in Alkbh5-/- oocytes partially rescues meiotic defects.","method":"Alkbh5 knockout female mice, temporal maternal transcriptomics, m6A dynamics profiling, m6A-seq, IGF2BP2 knockdown rescue in Alkbh5-/- oocytes","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse model, temporal m6A-seq, and IGF2BP2 rescue experiments with multiple orthogonal readouts","pmids":["37848452"],"is_preprint":false},{"year":2022,"finding":"ALKBH5 in Sertoli cells regulates m6A modification on Cdh2 mRNA (encoding N-cadherin). Removal of m6A by ALKBH5 promotes Cdh2 mRNA translation via IGF2BP1/2/3 and YTHDF1 complexes, maintaining N-cadherin levels and blood-testis barrier integrity. Alkbh5 knockout mice show disordered basal endoplasmic specialization.","method":"m6A-seq, MeRIP-qPCR, RIP-qPCR, Co-IP, polysome fractionation-qPCR, BTB integrity assay, transmission electron microscopy of Alkbh5-KO testes","journal":"Cellular & molecular biology letters","confidence":"High","confidence_rationale":"Tier 2 / Strong — m6A-seq, polysome fractionation, Co-IP, electron microscopy, and KO mouse model; multiple orthogonal methods","pmids":["36418936"],"is_preprint":false},{"year":2022,"finding":"Hypoxia upregulates ALKBH5 in trophoblast cells, which translocates from nucleus to cytoplasm and demethylates SMAD1/SMAD5 mRNAs, enhancing their translation and promoting MMP9 and ITGA1 production to support trophoblast invasion. ALKBH5 knockdown in mouse placenta suppresses trophoblast invasion and causes fetal abortion.","method":"m6A-seq in hypoxia-treated trophoblast, nuclear/cytoplasmic fractionation, MeRIP-qPCR, mRNA translation assays for SMAD1/5, trophoblast-specific ALKBH5 knockdown in vivo","journal":"Biochimica et biophysica acta. Molecular cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — m6A-seq, nuclear/cytoplasmic fractionation, in vivo knockdown; single lab","pmids":["35724807"],"is_preprint":false},{"year":2025,"finding":"ALKBH5 is phosphorylated by protein kinase A (PKA), causing its translocation from nucleus to cytosol in hepatocytes during obesity. Hepatocyte-specific deletion of Alkbh5 reduces glucose and lipids by inhibiting GCGR and mTORC1 signaling pathways. Targeted knockdown of hepatic Alkbh5 reverses T2DM and MAFLD in diabetic mice.","method":"PKA phosphorylation assays of ALKBH5, nuclear/cytoplasmic fractionation, hepatocyte-specific Alkbh5 knockout mice, GCGR and mTORC1 pathway analyses, diabetic mouse models","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 2 / Strong — PKA phosphorylation, subcellular fractionation, cell-type-specific KO, and in vivo metabolic phenotyping with multiple readouts","pmids":["40014709"],"is_preprint":false},{"year":2024,"finding":"PRMT5 directly catalyzes symmetric dimethylation of ALKBH5 at R316. This modification enhances TRIM28-mediated ubiquitination and degradation of ALKBH5, reducing its demethylase activity and increasing m6A on CD276 mRNA, thereby stabilizing CD276 expression and promoting CRC immune evasion.","method":"Co-IP, mass spectrometry for meR316, in vitro PRMT5 methylation assay, TRIM28 ubiquitination assay, MeRIP for CD276 mRNA, CRC in vivo models","journal":"Research (Washington, D.C.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro methylation assay, ubiquitination assay, MeRIP, in vivo model; single lab","pmids":["39781264"],"is_preprint":false},{"year":2023,"finding":"ALKBH5-mediated m6A demethylation of JARID2 mRNA stabilizes JARID2 transcripts in cooperation with IGF2BP3, promoting proliferation, migration, and invasion of rheumatoid arthritis fibroblast-like synoviocytes. ALKBH5 knockdown attenuates arthritis severity in CIA and DTHA mouse models.","method":"m6A-seq, RNA-seq, RIP, RNA pulldown, ALKBH5 knockdown/overexpression in RA FLSs, CIA/DTHA mouse models with ALKBH5 KO or shRNA injection","journal":"Arthritis & rheumatology (Hoboken, N.J.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — m6A-seq, RIP, RNA pulldown, and in vivo arthritis models; single lab","pmids":["37584615"],"is_preprint":false},{"year":2023,"finding":"ALKBH5 promotes axonal regeneration in dorsal root ganglion neurons by stabilizing Lpin2 mRNA (via m6A demethylation), thereby limiting regenerative lipid metabolism. Knockdown of ALKBH5 enhances sensory axonal regeneration in PNS, and overexpression impairs it in an m6A-dependent manner. In CNS, ALKBH5 knockdown enhances retinal ganglion cell survival and axon regeneration after optic nerve injury.","method":"Systematic m6A enzyme screen in axon regeneration, ALKBH5 knockdown and overexpression in rodent DRG neurons, Lpin2 mRNA stability assay, ALKBH5 demethylase-inactive mutant, optic nerve crush model","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — demethylase-inactive mutant, mRNA stability, and in vivo PNS/CNS regeneration models; single lab","pmids":["37535403"],"is_preprint":false},{"year":2021,"finding":"ALKBH5 demethylates TFEB mRNA to promote TFEB expression. TFEB in turn transcriptionally activates the ALKBH5 promoter (binding directly to it), while inhibiting METTL3 via mRNA stability downregulation, establishing a positive feedback loop relevant to autophagy regulation. (This is the TFEB→ALKBH5 transcriptional regulation component of PMID 30870073.)","method":"ChIP of TFEB at ALKBH5 promoter, luciferase promoter assay, TFEB knockdown/overexpression, mRNA stability of METTL3","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP, promoter assay, and reciprocal regulation demonstrated; already noted in PMID 30870073 entry above","pmids":["30870073"],"is_preprint":false},{"year":2022,"finding":"ALKBH5 demethylates CYP1B1 mRNA. In aging MSCs where ALKBH5 is decreased, increased m6A on CYP1B1 mRNA is recognized by IGF2BP1, stabilizing CYP1B1 mRNA, inducing mitochondrial dysfunction and cellular senescence. Alkbh5 knockout in MSCs aggravates spontaneous osteoarthritis.","method":"m6A quantification, MeRIP-qPCR for CYP1B1, IGF2BP1 RIP, mitochondrial function assays, Alkbh5 KO mouse spontaneous OA model, MSC senescence assays","journal":"Experimental & molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MeRIP, RIP, in vivo KO model, mitochondrial assays; single lab","pmids":["37524872"],"is_preprint":false},{"year":2023,"finding":"ALKBH5 knockdown in NSCLC cells reduces nuclear ALKBH5 distribution via interaction with circEML4 (delivered by extracellular vesicles from M2 macrophages), elevating m6A modifications on SOCS2 mRNA (identified by m6A-seq/RNA-seq), activating the JAK-STAT pathway to promote NSCLC malignancy.","method":"circEML4-ALKBH5 interaction assay (Co-IP/RIP), m6A-seq, RNA-seq for SOCS2, nuclear fractionation, EVs transfer assay, in vivo NSCLC xenograft","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — m6A-seq, Co-IP, nuclear fractionation, in vivo models; single lab","pmids":["37246269"],"is_preprint":false},{"year":2024,"finding":"Astrocytic ALKBH5 demethylates GLT-1 (glutamate transporter-1, SLC1A2) mRNA, increasing GLT-1 expression in astrocytes. Selective deletion of ALKBH5 in astrocytes (but not neurons or endothelial cells) produces antidepressant-like behaviors and preserves stress-induced disruption of glutamatergic synaptic transmission and neuronal integrity in the mPFC.","method":"Astrocyte-specific, neuron-specific, and endothelial cell-specific Alkbh5 conditional KO mice, GLT-1 mRNA m6A assay, depression behavioral tests, Ca2+ imaging, synaptic transmission recordings","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — cell-type-specific KO comparison, m6A assay for GLT-1 mRNA, electrophysiology, and Ca2+ imaging; multiple orthogonal methods in vivo","pmids":["38773146"],"is_preprint":false},{"year":2021,"finding":"ALKBH5 demethylates USP1 mRNA, reducing m6A levels and enhancing USP1 mRNA stability, thereby increasing USP1 expression. USP1 confers glucocorticoid resistance in T-ALL by deubiquitinating Aurora B. ALKBH5 knockdown reduces USP1 and Aurora B, sensitizing cells to dexamethasone.","method":"MeRIP-qPCR for USP1 m6A, mRNA stability assays, ALKBH5/USP1 knockdown in T-ALL cells, USP1 rescue experiments, in vivo T-ALL xenograft","journal":"Molecular carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MeRIP, stability assay, rescue experiments, in vivo model; single lab","pmids":["34169564"],"is_preprint":false},{"year":2020,"finding":"ALKBH5 demethylates IGF1R mRNA, enhancing IGF1R mRNA stability and translation, consequently activating IGF1R signaling and promoting endometrial cancer cell proliferation and invasion.","method":"MeRIP-qPCR, mRNA stability assays, ALKBH5 knockdown in endometrial cancer cells, IGF1R signaling pathway readouts","journal":"Journal of Cancer","confidence":"Low","confidence_rationale":"Tier 3 / Weak — MeRIP-qPCR and mRNA stability in single lab with limited mechanistic depth reported in abstract","pmids":["32913456"],"is_preprint":false},{"year":2025,"finding":"Psychological stress activates sympathetic nerves to release noradrenaline, which downregulates ALKBH5 in pancreatic cancer cells. ALKBH5 deficiency causes aberrant m6A modification of RNAs, which are packed into extracellular vesicles and delivered to nerves in the tumor microenvironment, enhancing hyperinnervation and PDAC progression.","method":"Mouse stress model, ALKBH5 knockdown in PDAC cells, EV m6A RNA profiling, in vivo nerve innervation assays, fisetin treatment to block EV uptake by neurons","journal":"Nature cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo stress model, EV m6A profiling, and pharmacological rescue; single lab","pmids":["40419796"],"is_preprint":false},{"year":2020,"finding":"In cerebral ischemia-reperfusion, Alkbh5 expression increases and, together with FTO, selectively demethylates Bcl2 mRNA, preventing its degradation and enhancing Bcl2 protein expression. Knockdown of Alkbh5 aggravates neuronal damage in this context.","method":"Rat MCAO model, primary neuron oxygen deprivation/reoxygenation, Alkbh5 shRNA, m6A quantification, Bcl2 mRNA stability analysis","journal":"Therapeutic advances in chronic disease","confidence":"Low","confidence_rationale":"Tier 3 / Weak — mRNA stability assay and in vivo knockdown; limited biochemical detail on direct demethylation of Bcl2 by ALKBH5 vs FTO","pmids":["32426101"],"is_preprint":false},{"year":2021,"finding":"ALKBH5 demethylates Runx2 mRNA, increasing its stability and promoting osteoblast differentiation. Expression of catalytically inactive ALKBH5 (active-site mutant) fails to rescue osteogenesis inhibition caused by ALKBH5 knockdown.","method":"ALKBH5 knockdown and overexpression during osteoblast differentiation, catalytic mutant rescue experiment, mRNA stability assay for Runx2","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — catalytic mutant rescue, mRNA stability, and differentiation assay; single lab, limited mechanistic depth","pmids":["34105773"],"is_preprint":false},{"year":2020,"finding":"TLR4 signaling in the tumor microenvironment activates NF-κB, which upregulates ALKBH5 expression in ovarian cancer cells co-cultured with M2 macrophages. ALKBH5-mediated m6A demethylation increases NANOG mRNA expression, enhancing ovarian cancer aggressiveness.","method":"Macrophage-cancer cell co-culture, TLR4/NF-κB pathway inhibitors, transcriptome sequencing, m6A-seq, MeRIP for NANOG mRNA","journal":"Journal of cellular and molecular medicine","confidence":"Low","confidence_rationale":"Tier 3 / Weak — co-culture model, MeRIP, transcriptome; limited mechanistic detail on how NF-κB directly activates ALKBH5 transcription","pmids":["32329191"],"is_preprint":false},{"year":2023,"finding":"ALKBH5 demethylates and stabilizes DDIT4 mRNA in HNSCC via the RBM33 co-factor (see RBM33 discovery), promoting autophagy and oncogenic function. (This is the downstream functional consequence of ALKBH5-RBM33 complex described in PMID 37257451.)","method":"ALKBH5 and RBM33 knockdown in HNSCC, MeRIP for DDIT4, autophagy assays","journal":"Molecular cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — part of the same rigorous study as RBM33/ALKBH5 interaction above; DDIT4 is the validated downstream target","pmids":["37257451"],"is_preprint":false},{"year":2022,"finding":"ALKBH5 demethylates MAP3K8 mRNA in an m6A-dependent manner, promoting MAP3K8 expression, JNK/ERK pathway activation, IL-8 secretion, and macrophage recruitment in hepatocellular carcinoma.","method":"MeRIP-qPCR for MAP3K8 m6A, ALKBH5 knockdown in HCC cells, JNK/ERK pathway readouts, macrophage recruitment assays, single-cell sequencing GSVA","journal":"International journal of biological sciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — MeRIP-qPCR and pathway readouts; single lab, limited direct mechanistic validation of demethylation step","pmids":["35982895"],"is_preprint":false},{"year":2020,"finding":"Alkbh5 in mouse neurons is primarily localized to the nucleus, based on immunofluorescence co-localization with NeuN and nuclear markers in adult mouse brain, with expression decreasing dramatically during brain development.","method":"Immunofluorescence in adult mouse brain sections and cell lines, co-localization with NeuN and nuclear markers, developmental Western blot","journal":"Brain research bulletin","confidence":"Low","confidence_rationale":"Tier 3 / Weak — localization by immunofluorescence only, no direct functional consequence linked to nuclear localization in this study","pmids":["32717204"],"is_preprint":false},{"year":2022,"finding":"ALKBH5 inhibits SAV1 mRNA m6A modification; in ALKBH5-knockdown multiple myeloma cells, increased m6A on SAV1 mRNA decreases SAV1 stability and expression, suppressing HIPPO-pathway signaling and activating YAP, thus exerting an anti-myeloma effect. ALKBH5 also maintains MM stem cell pluripotency.","method":"MeRIP-seq for SAV1 m6A, mRNA stability assays, ALKBH5 knockdown in MM cells, HIPPO/YAP pathway readouts, in vivo and in vitro MM models","journal":"International journal of biological sciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — MeRIP-seq and mRNA stability; single lab, limited validation of direct ALKBH5-SAV1 interaction","pmids":["35414790"],"is_preprint":false}],"current_model":"ALKBH5 is a ferrous iron- and 2-oxoglutarate-dependent RNA demethylase (the second identified mammalian m6A eraser) that binds m6A-containing single-stranded RNA in a 5'-3' orientation, uses an active-site proton shuttle (Lys132/Tyr139) to generate a hemiaminal intermediate, and releases formaldehyde to regenerate unmodified adenosine; its substrate selectivity, activity, and nuclear retention are regulated by multiple post-translational modifications—including SUMOylation (activated by ERK/JNK; inhibitory), phosphorylation (by PKA, SRC, LATS2, MST4; effects on nuclear export or stability), lactylation (by ESCO2; activating for IFN-β demethylation), arginine methylation (by PRMT5; promotes ubiquitination/degradation), and deubiquitination (by USP9X, USP14, or USP36; stabilizing)—as well as by the RNA-binding co-factor RBM33 that removes inhibitory SUMOylation and directs it to specific transcripts; in the nucleus ALKBH5 demethylates m6A from diverse target mRNAs (FOXM1, TACC3, TFEB, TRAF1, AXL, AXIN2, IL-11, CSF3R, GPX4, Cdh2, OGDH, SMAD1/5, etc.) to regulate their stability or translation, and also demethylates non-coding RNAs such as NEAT1 (facilitating paraspeckle assembly via phase separation through its C-terminal disordered region); at the organismal level its demethylase activity is essential for male spermatogenesis, female oocyte meiotic RNA clearance, blood-testis barrier integrity, hematopoietic stem cell metabolism, neutrophil migration, γδ T cell development, astrocyte-mediated glutamate homeostasis, hepatic glucose/lipid metabolism, and cardiac fibrosis, while its dysregulation drives tumorigenesis in glioblastoma, AML, multiple myeloma, colorectal, gastric, and other cancers through context-dependent stabilization or destabilization of oncogenic or tumor-suppressive transcripts."},"narrative":{"mechanistic_narrative":"ALKBH5 is a mammalian RNA demethylase that oxidatively reverses N6-methyladenosine (m6A) in mRNA in vitro and in vivo, the second identified mammalian m6A eraser, controlling mRNA export, nuclear speckle assembly, and RNA metabolism [PMID:23177736]. Crystal structures of ALKBH5 bound to m6A-containing single-stranded RNA show the substrate engaged in a 5'-3' orientation, define an (A/G)m6AC consensus preference, and identify a Lys132/Tyr139 proton-shuttle network that drives hemiaminal-intermediate demethylation and formaldehyde release [PMID:35333330]. Substrate selection is not intrinsic to the enzyme alone: the RNA-binding co-factor RBM33 forms a complex with ALKBH5, removes its inhibitory SUMOylation, and recruits it to specific transcripts such as DDIT4 [PMID:37257451]. ALKBH5 activity and localization are tuned by a layered post-translational code—ROS/ERK-JNK-driven SUMOylation that blocks substrate access [PMID:34048572], kinase phosphorylation (SRC, LATS2, MST4, PKA) that controls nuclear retention, export, or stability [PMID:37979586, PMID:38568805, PMID:39990235, PMID:40014709], PRMT5-catalyzed arginine methylation that promotes TRIM28-dependent degradation [PMID:39781264], lactylation that licenses IFN-β mRNA demethylation [PMID:39413129], and deubiquitination by USP9X, USP36, and USP14 that stabilizes the protein [PMID:36239338, PMID:37454738, PMID:39990235]. Through demethylation of defined mRNA targets, ALKBH5 alters transcript stability or translation—generally protecting transcripts from m6A-reader-mediated decay (e.g. YTHDF2) or shifting reader engagement (IGF2BP, YTHDF1)—to regulate processes including spermatogenesis [PMID:23177736], oocyte maternal-RNA clearance via IGF2BP2 [PMID:37848452], blood-testis barrier integrity through Cdh2 [PMID:36418936], hematopoietic stem cell metabolism via OGDH [PMID:37742191], neutrophil mobilization and migration [PMID:38114747, PMID:35764614], astrocytic glutamate homeostasis via GLT-1 [PMID:38773146], and hepatic glucose/lipid metabolism [PMID:40014709]. ALKBH5 also acts on non-coding RNA: under hypoxia it condenses into paraspeckles through its C-terminal disordered region and demethylates NEAT1 to drive paraspeckle assembly and CXCL8-mediated macrophage recruitment [PMID:34670781, PMID:37474102]. Dysregulated ALKBH5 drives tumorigenesis in glioblastoma, AML, multiple myeloma, colorectal, gastric, and other cancers through context-dependent stabilization or destabilization of oncogenic transcripts including FOXM1, TACC3, and AXL [PMID:28344040, PMID:32402250, PMID:37169182].","teleology":[{"year":2012,"claim":"Established that mammals possess a second, dedicated m6A eraser, defining ALKBH5 as an RNA demethylase with a physiological role in fertility.","evidence":"In vitro demethylation assay plus Alkbh5-knockout mice with mRNA export and nuclear speckle readouts and testis transcriptomics","pmids":["23177736"],"confidence":"High","gaps":["No structural basis for substrate recognition","Direct mRNA targets in spermatocytes not individually defined"]},{"year":2022,"claim":"Resolved how ALKBH5 recognizes and chemically processes its substrate, explaining sequence preference and the catalytic chemistry distinguishing it from DNA-acting AlkB members.","evidence":"Three X-ray crystal structures with m6A ssRNA, biochemical demethylation assays, and active-site mutagenesis","pmids":["35333330"],"confidence":"High","gaps":["Structures do not address how cellular co-factors redirect substrate choice","No structure of post-translationally modified ALKBH5"]},{"year":2017,"claim":"Showed that ALKBH5 acts on specific nascent transcripts in disease, demethylating FOXM1 to stabilize it and sustain glioblastoma stem cell proliferation, guided by an antisense lncRNA.","evidence":"m6A-seq, reciprocal RIP, ALKBH5 knockdown in patient-derived GSCs, and in vivo tumor models","pmids":["28344040"],"confidence":"High","gaps":["Mechanism by which FOXM1-AS tethers ALKBH5 to nascent RNA not structurally defined","Generalizability of antisense-RNA targeting to other transcripts unknown"]},{"year":2020,"claim":"Demonstrated ALKBH5 as a selective dependency of leukemia stem cells (dispensable for normal hematopoiesis) acting through TACC3 and AXL, and showed its expression is set epigenetically via KDM4C/H3K9me3.","evidence":"Conditional KO mice, human AML lines, m6A-seq/RNA-seq, ChIP-seq/ATAC-seq, and in vivo leukemia models","pmids":["32402250","32402251"],"confidence":"High","gaps":["Why LSCs but not normal HSCs depend on ALKBH5 not fully explained","Reader proteins decoding the retained m6A marks not defined in these studies"]},{"year":2021,"claim":"Revealed that ALKBH5 enzymatic activity is gated by SUMOylation downstream of ROS/ERK-JNK signaling, providing a signal-responsive switch over global m6A.","evidence":"SUMOylation assays, ERK/JNK inhibition, ROS induction in cells and mouse HSPCs, and SUMO-site mutagenesis","pmids":["34048572"],"confidence":"High","gaps":["SUMO ligase responsible not identified","How SUMOylation physically blocks substrate access not structurally resolved"]},{"year":2021,"claim":"Extended the demethylation-stabilization logic across diverse cancers and tissues, showing ALKBH5 acts on target-specific transcripts (TRAF1, FOXM1, USP1, NANOG, IGF1R) to drive distinct oncogenic programs.","evidence":"MeRIP-qPCR, mRNA stability assays, knockdown/rescue, and in vivo tumor models across myeloma, melanoma, T-ALL, ovarian, and endometrial cancers","pmids":["34759347","33428593","34169564","32329191","32913456"],"confidence":"Medium","gaps":["Several rely on single-lab MeRIP without reciprocal validation","Direct ALKBH5-transcript binding not always demonstrated"]},{"year":2022,"claim":"Showed ALKBH5 also regulates non-coding RNA and organizes membraneless compartments, condensing via its disordered C-terminus into paraspeckles to demethylate and stabilize NEAT1 under hypoxia.","evidence":"m6A-seq, NEAT1 stability and SFPQ ChIP assays, phase-separation assays, and cIDR deletion mutants","pmids":["34670781","37474102"],"confidence":"Medium","gaps":["Whether condensation is required for catalysis on mRNA targets is unresolved","Triggers of hypoxia-induced condensation not fully mapped"]},{"year":2022,"claim":"Established ALKBH5 as a tissue-level regulator through cell-type-specific knockouts, governing blood-testis barrier integrity, hematopoietic metabolism, oocyte RNA clearance, neutrophil function, and γδ T cell development via discrete mRNA targets.","evidence":"Cell-type-specific Alkbh5 KO mice, m6A-seq, polysome fractionation, metabolomics, and in vivo infection/transplantation models","pmids":["36418936","37742191","37848452","38114747","35764614","35939687"],"confidence":"High","gaps":["Reader partners (IGF2BP1/2/3, YTHDF1/2) coupling differs by target and is incompletely mapped","Whether single targets account for full phenotypes is uncertain"]},{"year":2023,"claim":"Identified RBM33 as the substrate-recruiting co-factor that both de-SUMOylates and activates ALKBH5, solving how the enzyme achieves transcript selectivity.","evidence":"Reciprocal Co-IP, in vitro demethylase assays, SUMOylation assays, and m6A-seq in HNSCC targeting DDIT4","pmids":["37257451"],"confidence":"High","gaps":["Whether RBM33 directs ALKBH5 to its many other reported targets is untested","Other adaptor co-factors not excluded"]},{"year":2023,"claim":"Defined an extensive deubiquitinase/kinase network (USP9X, USP36, USP14, SRC, LATS2, MST4) controlling ALKBH5 protein abundance and nuclear retention, frequently within positive feedback loops sustaining glioblastoma and AML stem cells.","evidence":"Mass spectrometry, Co-IP, site-specific ubiquitination and phospho-mutagenesis, m6A-seq, and xenograft models","pmids":["36239338","37454738","37979586","38568805","39990235"],"confidence":"High","gaps":["The E3 ligases opposing these DUBs are largely undefined","How multiple PTMs are integrated on the same molecule is unknown"]},{"year":2024,"claim":"Expanded the PTM repertoire to lactylation (ESCO2/SIRT6) and arginine methylation (PRMT5/TRIM28), linking ALKBH5 regulation to antiviral innate immunity and to degradation-driven cancer immune evasion.","evidence":"Lactylation/methylation mass spectrometry, writer-eraser Co-IP, in vitro PRMT5 methylation and TRIM28 ubiquitination assays, RIP, and functional immune/tumor assays","pmids":["39413129","39781264"],"confidence":"Medium","gaps":["Crosstalk between lactylation, SUMOylation, and methylation on the same residues unresolved","PRMT5/TRIM28 axis shown in single lab"]},{"year":2025,"claim":"Connected ALKBH5 regulation to systemic physiology and therapy resistance, showing PKA-driven phosphorylation/relocalization governs hepatic glucose-lipid metabolism and ferroptosis, and KRAS-ERK/JNK SUMOylation confers platinum resistance via DNA-repair transcripts.","evidence":"PKA phosphorylation assays, hepatocyte-specific KO, SUMO-deficient mutants, NER and ferroptosis assays, and diabetic/NSCLC in vivo models","pmids":["40014709","39960727","39901038"],"confidence":"High","gaps":["Phospho-sites for the PKA-ferroptosis axis not all mapped","Whether hepatic and tumor PKA effects share the same mechanism is unclear"]},{"year":null,"claim":"How the combinatorial post-translational code, co-factor recruitment, and phase separation are integrated to select among the many reported ALKBH5 targets in a given cell remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking PTM state to target choice","Reader-protein coupling that dictates stabilization vs destabilization not systematically defined","E3 ligases and SUMO ligases acting on ALKBH5 largely unidentified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[0,1,2,8,32]},{"term_id":"GO:0016491","term_label":"oxidoreductase activity","supporting_discovery_ids":[0,1]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[1,2,11,29]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[1]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,5,17,51]},{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[0,16]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[34,35]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,1,2,8,32]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[2,8,20,12]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[11,29,30,31]}],"complexes":["paraspeckle"],"partners":["RBM33","USP9X","USP36","USP14","PRMT5","SRC","LATS2","MST4"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q6P6C2","full_name":"RNA demethylase ALKBH5","aliases":["Alkylated DNA repair protein alkB homolog 5","Alpha-ketoglutarate-dependent dioxygenase alkB homolog 5"],"length_aa":394,"mass_kda":44.3,"function":"Dioxygenase that specifically demethylates N(6)-methyladenosine (m6A) RNA, the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes (PubMed:23177736, PubMed:24489119, PubMed:24616105, PubMed:24778178, PubMed:34048572, PubMed:36944332, PubMed:37257451, PubMed:37369679, PubMed:40793791). Demethylates RNA by oxidative demethylation, which requires molecular oxygen, alpha-ketoglutarate and iron (PubMed:21264265, PubMed:23177736, PubMed:24489119, PubMed:24616105, PubMed:24778178). Demethylation of m6A mRNA affects mRNA processing, translation and export (PubMed:23177736, PubMed:34048572, PubMed:36944332, PubMed:37257451). Can also demethylate N(6)-methyladenosine in single-stranded DNA (in vitro) (PubMed:24616105). Required for the late meiotic and haploid phases of spermatogenesis by mediating m6A demethylation in spermatocytes and round spermatids: m6A demethylation of target transcripts is required for correct splicing and the production of longer 3'-UTR mRNAs in male germ cells (By similarity). Involved in paraspeckle assembly, a nuclear membraneless organelle, by undergoing liquid-liquid phase separation (PubMed:37369679, PubMed:37474102). Paraspeckle assembly is coupled with m6A demethylation of RNAs, such as NEAT1 non-coding RNA (PubMed:37474102). Also acts as a negative regulator of T-cell development: inhibits gamma-delta T-cell proliferation via demethylation of JAG1 and NOTCH2 transcripts (By similarity). Inhibits regulatory T-cell (Treg) recruitment by mediating demethylation and destabilization of CCL28 mRNAs (By similarity)","subcellular_location":"Nucleus speckle; Nucleus, nucleoplasm","url":"https://www.uniprot.org/uniprotkb/Q6P6C2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ALKBH5","classification":"Not Classified","n_dependent_lines":20,"n_total_lines":1208,"dependency_fraction":0.016556291390728478},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"KPNA6","stoichiometry":4.0},{"gene":"MINK1","stoichiometry":4.0},{"gene":"DDX39B","stoichiometry":0.2},{"gene":"KPNA1","stoichiometry":0.2},{"gene":"PRPF4B","stoichiometry":0.2},{"gene":"RBM8A","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/ALKBH5","total_profiled":1310},"omim":[{"mim_id":"620833","title":"RNA-BINDING MOTIF PROTEIN 33; RBM33","url":"https://www.omim.org/entry/620833"},{"mim_id":"613303","title":"AlkB HOMOLOG 5, RNA DEMETHYLASE; ALKBH5","url":"https://www.omim.org/entry/613303"},{"mim_id":"613022","title":"OXOGLUTARATE DEHYDROGENASE; OGDH","url":"https://www.omim.org/entry/613022"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"},{"location":"Golgi apparatus","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"skeletal muscle","ntpm":221.6}],"url":"https://www.proteinatlas.org/search/ALKBH5"},"hgnc":{"alias_symbol":["FLJ20308"],"prev_symbol":["OFOXD1"]},"alphafold":{"accession":"Q6P6C2","domains":[{"cath_id":"2.60.120.590","chopping":"78-284","consensus_level":"high","plddt":93.354,"start":78,"end":284}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6P6C2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q6P6C2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q6P6C2-F1-predicted_aligned_error_v6.png","plddt_mean":72.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ALKBH5","jax_strain_url":"https://www.jax.org/strain/search?query=ALKBH5"},"sequence":{"accession":"Q6P6C2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q6P6C2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q6P6C2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6P6C2"}},"corpus_meta":[{"pmid":"23177736","id":"PMC_23177736","title":"ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility.","date":"2012","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/23177736","citation_count":2889,"is_preprint":false},{"pmid":"28344040","id":"PMC_28344040","title":"m6A Demethylase ALKBH5 Maintains Tumorigenicity of Glioblastoma Stem-like Cells by Sustaining FOXM1 Expression and Cell Proliferation Program.","date":"2017","source":"Cancer cell","url":"https://pubmed.ncbi.nlm.nih.gov/28344040","citation_count":1217,"is_preprint":false},{"pmid":"32747553","id":"PMC_32747553","title":"ALKBH5 regulates anti-PD-1 therapy response by modulating lactate and suppressive immune cell accumulation in tumor microenvironment.","date":"2020","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/32747553","citation_count":469,"is_preprint":false},{"pmid":"30870073","id":"PMC_30870073","title":"METTL3 and ALKBH5 oppositely regulate m6A modification of 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research","url":"https://pubmed.ncbi.nlm.nih.gov/39060657","citation_count":17,"is_preprint":false},{"pmid":"40001461","id":"PMC_40001461","title":"M6A Demethylase ALKBH5 in Human Diseases: From Structure to Mechanisms.","date":"2025","source":"Biomolecules","url":"https://pubmed.ncbi.nlm.nih.gov/40001461","citation_count":17,"is_preprint":false},{"pmid":"37454738","id":"PMC_37454738","title":"Deubiquitinase USP9X stabilizes RNA m6A demethylase ALKBH5 and promotes acute myeloid leukemia cell survival.","date":"2023","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/37454738","citation_count":16,"is_preprint":false},{"pmid":"35182329","id":"PMC_35182329","title":"ALKBH5 promotes the progression of infantile hemangioma through regulating the NEAT1/miR-378b/FOSL1 axis.","date":"2022","source":"Molecular and cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/35182329","citation_count":15,"is_preprint":false},{"pmid":"39976119","id":"PMC_39976119","title":"Discovery of Covalent and Cell-Active ALKBH5 Inhibitors with Potent Antileukemia Effects In Vivo.","date":"2025","source":"Angewandte Chemie (International ed. in English)","url":"https://pubmed.ncbi.nlm.nih.gov/39976119","citation_count":14,"is_preprint":false},{"pmid":"37474102","id":"PMC_37474102","title":"The disordered C terminus of ALKBH5 promotes phase separation and paraspeckles assembly.","date":"2023","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/37474102","citation_count":14,"is_preprint":false},{"pmid":"38156523","id":"PMC_38156523","title":"Aging-Associated ALKBH5-m6A Modification Exacerbates Doxorubicin-Induced Cardiomyocyte Apoptosis Via AT-Rich Interaction Domain 2.","date":"2023","source":"Journal of the American Heart Association","url":"https://pubmed.ncbi.nlm.nih.gov/38156523","citation_count":14,"is_preprint":false},{"pmid":"38227578","id":"PMC_38227578","title":"The N6-methyladenosine demethylase ALKBH5 regulates the hypoxic HBV transcriptome.","date":"2024","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/38227578","citation_count":14,"is_preprint":false},{"pmid":"38568805","id":"PMC_38568805","title":"A LATS2 and ALKBH5 positive feedback loop supports their oncogenic roles.","date":"2024","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/38568805","citation_count":13,"is_preprint":false},{"pmid":"33790968","id":"PMC_33790968","title":"ALKBH5 Gene Polymorphisms and Hepatoblastoma Susceptibility in Chinese Children.","date":"2021","source":"Journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/33790968","citation_count":13,"is_preprint":false},{"pmid":"37818080","id":"PMC_37818080","title":"Hypoxia-induced m6A demethylase ALKBH5 promotes ovarian cancer tumorigenicity by decreasing methylation of the lncRNA RMRP.","date":"2023","source":"American journal of cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/37818080","citation_count":13,"is_preprint":false},{"pmid":"38946930","id":"PMC_38946930","title":"ALKBH5 gene polymorphisms and risk of neuroblastoma in Chinese children from Jiangsu Province.","date":"2023","source":"Cancer innovation","url":"https://pubmed.ncbi.nlm.nih.gov/38946930","citation_count":13,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":53991,"output_tokens":14464,"usd":0.189467,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":26455,"output_tokens":4866,"usd":0.126963,"stage2_stop_reason":"end_turn"},"total_usd":0.31643,"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\": 2012,\n      \"finding\": \"ALKBH5 is a mammalian RNA demethylase that oxidatively reverses N6-methyladenosine (m6A) in mRNA in vitro and in vivo. Its demethylation activity affects mRNA export and RNA metabolism, as well as assembly of mRNA processing factors in nuclear speckles. Alkbh5-deficient male mice show increased m6A in mRNA and impaired fertility due to apoptosis of meiotic metaphase-stage spermatocytes.\",\n      \"method\": \"In vitro demethylation assay, Alkbh5-knockout mice, mRNA export and nuclear speckle assembly assays, transcriptome profiling of testes\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzymatic assay plus in vivo knockout model with multiple orthogonal readouts; foundational paper replicated across many subsequent studies\",\n      \"pmids\": [\"23177736\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Crystal structures of ALKBH5 in complex with m6A-containing single-stranded RNA 8-mer revealed that the RNA substrate binds in a 5'-3' orientation opposite to that of DNA substrates in other AlkB members. The structures defined the (A/G)m6AC consensus sequence preference and a proton shuttle network involving Lys132 and Tyr139 that enables efficient hemiaminal intermediate demethylation to produce formaldehyde.\",\n      \"method\": \"X-ray crystallography (three crystal structures), biochemical demethylation assays, mutagenesis of active-site residues\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple crystal structures with biochemical validation and mutagenesis in a single rigorous study\",\n      \"pmids\": [\"35333330\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ALKBH5 demethylates FOXM1 nascent transcripts in glioblastoma stem-like cells (GSCs), leading to enhanced FOXM1 mRNA stability and expression. A long non-coding RNA antisense to FOXM1 (FOXM1-AS) promotes the interaction of ALKBH5 with FOXM1 nascent transcripts. ALKBH5 silencing suppresses GSC proliferation and tumorigenesis through this FOXM1 axis.\",\n      \"method\": \"Integrated transcriptome and m6A-seq, RNA immunoprecipitation, ALKBH5 knockdown in patient-derived GSCs, in vivo tumor models\",\n      \"journal\": \"Cancer cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal RIP, m6A-seq, in vivo models, replicated concept across multiple GSC lines\",\n      \"pmids\": [\"28344040\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ROS induces ALKBH5 SUMOylation via ERK/JNK signaling, which inhibits ALKBH5 m6A demethylase activity by blocking substrate accessibility, thereby globally increasing mRNA m6A levels and inducing DNA damage response genes. This ERK/JNK/ALKBH5-PTMs/m6A axis is activated in hematopoietic stem/progenitor cells in vivo.\",\n      \"method\": \"SUMOylation assays, ERK/JNK inhibitor treatments, ROS induction in cell lines and mouse HSPCs, m6A quantification, mutagenesis of SUMOylation sites\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple PTM assays, in vitro enzymatic activity, in vivo HSPC validation, and mutagenesis in one study\",\n      \"pmids\": [\"34048572\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RBM33 (RNA-binding motif protein 33) forms a complex with ALKBH5 and acts as a substrate-recruiting co-factor that (1) recruits ALKBH5 to specific m6A-marked mRNA targets and (2) activates ALKBH5 demethylase activity by removing its SUMOylation. This interaction selectively directs ALKBH5 to demethylate DDIT4 mRNA to promote autophagy in HNSCC.\",\n      \"method\": \"Co-immunoprecipitation, in vitro demethylase activity assays, SUMOylation assays, m6A-seq, RBM33 knockdown in cancer cells\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — reciprocal Co-IP, in vitro enzymatic validation, and m6A-seq in a single rigorous study\",\n      \"pmids\": [\"37257451\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EGFR signaling retains ALKBH5 in the nucleus of glioblastoma stem cells by activating SRC kinase, which phosphorylates ALKBH5 and inhibits CRM1-mediated nuclear export. Nuclear ALKBH5 demethylates GCLM mRNA; reduced m6A on GCLM mRNA stabilizes it (preventing YTHDF2-mediated decay), thereby protecting against ferroptosis.\",\n      \"method\": \"EGFR/SRC inhibition, ALKBH5 phosphorylation assays, nuclear fractionation, YTHDF2-dependent GCLM mRNA decay assays, m6A-seq, GSC in vivo models\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — phosphorylation mechanism, nuclear localization, mRNA decay, and in vivo data with multiple orthogonal methods\",\n      \"pmids\": [\"37979586\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP36 deubiquitinase directly binds ALKBH5, removes its polyubiquitin chain, and stabilizes the ALKBH5 protein in glioblastoma. USP36 depletion reduces ALKBH5 levels, impairs glioblastoma stem cell self-renewal, and inhibits in vivo tumor growth.\",\n      \"method\": \"Mass spectrometry, co-immunoprecipitation, in vivo and in vitro ubiquitination assays, USP36 knockdown in GSCs, intracranial tumor xenografts\",\n      \"journal\": \"Neuro-oncology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — direct binding by MS and Co-IP, in vitro and in vivo ubiquitination assays, in vivo tumor model\",\n      \"pmids\": [\"36239338\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"USP9X deubiquitinase directly binds ALKBH5, removes K48-linked polyubiquitin at K57, and stabilizes the ALKBH5 protein in AML cells. USP9X depletion reduces ALKBH5 levels and promotes AML cell apoptosis; ectopic ALKBH5 expression partially rescues USP9X knockdown effects.\",\n      \"method\": \"Mass spectrometry, co-immunoprecipitation, K48-linked ubiquitination assays, site-specific mutagenesis (K57), genetic knockdown, murine AML model\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — MS identification, Co-IP, site-specific ubiquitination mutagenesis, and rescue experiments\",\n      \"pmids\": [\"37454738\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ALKBH5 is required for AML development and leukemia stem cell self-renewal but is dispensable for normal hematopoiesis. Mechanistically, ALKBH5 demethylates TACC3 mRNA, stabilizing it and promoting TACC3 protein expression, which drives leukemogenesis.\",\n      \"method\": \"Alkbh5 conditional knockout mice, human AML cell lines, m6A-seq, RNA-seq, MeRIP-qPCR, in vivo AML models\",\n      \"journal\": \"Cell stem cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO mouse model, m6A-seq/RNA-seq, and in vivo leukemia models demonstrating pathway specificity\",\n      \"pmids\": [\"32402250\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"KDM4C histone demethylase regulates ALKBH5 expression in AML by reducing H3K9me3 at the ALKBH5 locus, increasing chromatin accessibility, and promoting MYB/Pol II recruitment. ALKBH5 in turn affects AXL mRNA stability in an m6A-dependent manner to maintain leukemia stem cell function.\",\n      \"method\": \"ChIP-seq, ATAC-seq, chromatin accessibility assays, KDM4C knockdown, MeRIP-seq, AML mouse models\",\n      \"journal\": \"Cell stem cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP-seq, ATAC-seq, m6A-seq, and in vivo AML model with multiple orthogonal methods\",\n      \"pmids\": [\"32402251\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"METTL3 and ALKBH5 oppositely regulate m6A modification of TFEB mRNA in cardiomyocytes. METTL3 methylates TFEB 3'-UTR at two m6A residues, promoting HNRNPD binding and decreasing TFEB expression. ALKBH5 demethylates these sites to reverse the effect. TFEB reciprocally binds the ALKBH5 promoter to induce its transcription, establishing a feedback loop.\",\n      \"method\": \"m6A-seq, RIP, ChIP, METTL3/ALKBH5 knockdown and overexpression in cardiomyocytes, promoter luciferase assay, murine I/R model\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal functional experiments, m6A-seq, RIP, ChIP, and in vivo cardiac model with multiple orthogonal validations\",\n      \"pmids\": [\"30870073\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ALKBH5 undergoes lactylation catalyzed by acetyltransferase ESCO2 (increased during viral infection) and is de-lactylated by SIRT6. Lactylated ALKBH5 binds IFN-β mRNA and demethylates its m6A modifications, promoting IFN-β mRNA biogenesis and antiviral innate immune responses against HSV-1, KSHV, and mpox virus.\",\n      \"method\": \"Lactylation mass spectrometry, ESCO2/SIRT6 co-immunoprecipitation, RIP of IFN-β mRNA, m6A quantification, viral infection assays, ESCO2 overexpression/SIRT6 depletion\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — PTM identification by MS, writer/eraser identification by Co-IP, direct RNA-binding by RIP, and functional antiviral assays\",\n      \"pmids\": [\"39413129\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"KRAS mutants activate ERK/JNK signaling in NSCLC, which promotes ALKBH5 SUMOylation (inhibiting its demethylase activity), resulting in increased m6A methylation on DDB2 and XPC mRNAs. Stabilization of these DNA repair transcripts enhances nucleotide excision repair, conferring platinum resistance. A SUMOylation-deficient ALKBH5 mutant restores platinum sensitivity.\",\n      \"method\": \"SUMOylation assays, m6A-seq, RNA stability assays, NER functional assays, ALKBH5 mutagenesis, in vivo xenograft models with KRAS mutant NSCLC\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — ALKBH5 mutagenesis, m6A-seq, NER assays, and in vivo model; mechanistically connects KRAS→ERK/JNK→ALKBH5 SUMOylation→m6A→DNA repair\",\n      \"pmids\": [\"39960727\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Protein kinase A (PKA) phosphorylates ALKBH5, promoting its degradation. Loss of ALKBH5 maintains m6A modification on GPX4 mRNA, stabilizing GPX4 and thereby suppressing ferroptosis. PKA thus acts as a regulator of ferroptosis through the ALKBH5-GPX4 m6A axis.\",\n      \"method\": \"PKA phosphorylation assays, ALKBH5 deletion/reconstitution, m6A quantification of GPX4 mRNA, ferroptosis assays, in vivo tumor models\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — PKA-ALKBH5 phosphorylation and GPX4 m6A regulation demonstrated in single lab with multiple functional readouts; no mutagenesis of specific phospho-site reported in abstract\",\n      \"pmids\": [\"39901038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ALKBH5 demethylates FOXM1 mRNA in uveal melanoma, increasing its stability and expression, promoting tumor growth and metastasis via EMT. EP300-mediated H3K27 acetylation at the ALKBH5 locus activates ALKBH5 transcription.\",\n      \"method\": \"MeRIP-qPCR for m6A on FOXM1 mRNA, ALKBH5 knockdown in UM cell lines, in vivo xenograft model, ChIP for H3K27ac\",\n      \"journal\": \"Aging\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MeRIP, in vivo model, ChIP, single lab; extends FOXM1 m6A finding from GBM to UM\",\n      \"pmids\": [\"33428593\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ALKBH5 mediates m6A demethylation of NEAT1 lncRNA under hypoxia, stabilizing NEAT1 and facilitating paraspeckle assembly. This causes relocalization of transcriptional repressor SFPQ from the CXCL8 promoter to paraspeckles, upregulating CXCL8/IL8 secretion and promoting tumor-associated macrophage recruitment in GBM.\",\n      \"method\": \"m6A-seq on hypoxic GBM cells, NEAT1 stability assays, SFPQ chromatin immunoprecipitation, ALKBH5 depletion/inactivation, allograft tumor models\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — m6A-seq, ChIP, RIP, in vivo allograft with multiple orthogonal mechanistic steps validated\",\n      \"pmids\": [\"34670781\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The disordered C-terminal intrinsically disordered region (cIDR) of ALKBH5 promotes liquid-liquid phase separation and incorporation into paraspeckles. Under hypoxia, rapid ALKBH5 condensation in paraspeckles induces m6A demethylation of NEAT1, further facilitating paraspeckle assembly. ALKBH5 lacking cIDR fails to support paraspeckle formation and NEAT1 stabilization.\",\n      \"method\": \"Phase separation assays, deletion mutants of ALKBH5-cIDR, live-cell imaging of ALKBH5 condensates, m6A demethylation of NEAT1, hypoxia-induced invasion assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phase separation assays and functional cIDR deletions in single lab; mechanistically extends paraspeckle/NEAT1 finding\",\n      \"pmids\": [\"37474102\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"LATS2 kinase phosphorylates ALKBH5, preventing its nuclear export and enhancing its protein stability. Phosphorylated ALKBH5 reciprocally erases m6A from LATS2 mRNA, stabilizing this transcript and establishing a positive feedback loop that promotes glioblastoma stem cell self-renewal.\",\n      \"method\": \"Kinase assay, nuclear fractionation, ALKBH5/LATS2 Co-IP, m6A-seq on LATS2 mRNA, ALKBH5 phosphorylation-site mutants, GBM xenograft models\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — kinase assay, Co-IP, m6A-seq, phospho-mutants, and in vivo data from single lab\",\n      \"pmids\": [\"38568805\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP14 deubiquitinase stabilizes ALKBH5 by removing K48-linked ubiquitination through HECW2. MST4 kinase phosphorylates ALKBH5 at S64 and S69, increasing its interaction with USP14 and promoting ALKBH5 deubiquitylation. ALKBH5 also binds and stabilizes USP14 mRNA via YTHDF2-dependent mechanism, creating a positive feedback loop. This MST4-USP14-ALKBH5 axis promotes GSC radioresistance.\",\n      \"method\": \"Mass spectrometry, co-immunoprecipitation, ubiquitination assays, phospho-site mutagenesis (S64/S69), m6A-seq, transcriptome analysis, GSC radioresistance assays, xenograft models\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — MS identification, kinase assay, ubiquitination assays, phospho-site mutagenesis, m6A-seq, and in vivo xenograft in a single rigorous study\",\n      \"pmids\": [\"39990235\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ALKBH5 demethylates TRAF1 mRNA 3'-UTR, decreasing m6A abundance and enhancing TRAF1 mRNA stability, which promotes TRAF1 protein expression and activates NF-κB and MAPK signaling to drive multiple myeloma cell growth and survival.\",\n      \"method\": \"MeRIP-qPCR, mRNA stability assays, ALKBH5 knockdown in MM cells, in vivo xenograft models, NF-κB/MAPK pathway readouts\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MeRIP, mRNA stability, in vivo model; single lab with multiple functional readouts\",\n      \"pmids\": [\"34759347\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ALKBH5 binds and demethylates AXIN2 mRNA, causing AXIN2 mRNA dissociation from IGF2BP1 and subsequent mRNA degradation, resulting in hyperactivated Wnt/β-catenin signaling and induction of DKK1, which recruits myeloid-derived suppressor cells to drive immunosuppression in colorectal cancer.\",\n      \"method\": \"MeRIP-seq, RNA-seq, RIP for IGF2BP1/AXIN2 interaction, ALKBH5 knockin mice (intestine-specific), CD34+ humanized mice, allografts, vesicle-nanoparticle siRNA delivery\",\n      \"journal\": \"Gastroenterology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — MeRIP-seq, multiple in vivo models (knockin mice, humanized mice, allografts), RIP validation, with mechanistic pathway dissection\",\n      \"pmids\": [\"37169182\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ALKBH5 in macrophages demethylates IL-11 mRNA, increasing its stability and IL-11 protein levels, which drives macrophage-to-myofibroblast transition (MMT) under angiotensin II-induced hypertension. Macrophage-specific ALKBH5 knockout inhibits MMT and ameliorates cardiac fibrosis.\",\n      \"method\": \"RIP-seq (identified IL-11 mRNA as target), single-cell transcriptomics, lineage tracing, parabiosis, macrophage-specific ALKBH5 knockout mice, Ang II infusion model, IL-11 overexpression rescue\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — RIP-seq, cell-type-specific KO, lineage tracing, parabiosis, and in vivo rescue experiments\",\n      \"pmids\": [\"38443404\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ALKBH5 demethylates PKMYT1 mRNA; loss of ALKBH5 increases m6A on PKMYT1 mRNA, and the m6A reader IGF2BP3 stabilizes PKMYT1 mRNA, upregulating PKMYT1 expression and promoting gastric cancer invasion and metastasis.\",\n      \"method\": \"MeRIP-seq, RNA pulldown, mass spectrometry, RIP, ALKBH5 demethylase activity mutant, in vivo lung metastasis model\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MeRIP-seq, RNA pulldown, demethylase mutant, and in vivo metastasis; single lab\",\n      \"pmids\": [\"35114989\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ALKBH5 deficiency in PD-L1 mRNA 3'-UTR enriches m6A modification, promoting YTHDF2-dependent PD-L1 mRNA degradation and reducing tumor PD-L1 expression. ALKBH5 thus sustains PD-L1 expression to promote immune evasion in intrahepatic cholangiocarcinoma.\",\n      \"method\": \"m6A methylome sequencing, ALKBH5-PD-L1 mRNA RIP, YTHDF2 knockdown rescue, in vitro and in vivo ICC tumor models\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — m6A-seq, RIP, YTHDF2 rescue experiments, in vivo ICC model; single lab\",\n      \"pmids\": [\"34301762\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ALKBH5 deletion in tumor cells modulates Mct4/Slc16a3 mRNA m6A levels and reduces lactate content in the tumor microenvironment, altering infiltration of Treg and myeloid-derived suppressor cells to sensitize tumors to anti-PD-1 immunotherapy.\",\n      \"method\": \"Alkbh5 deletion in tumor cell lines, m6A density analysis, splicing analysis, lactate measurement, flow cytometry of tumor-infiltrating immune cells, small-molecule ALKBH5 inhibitor treatment, in vivo tumor models\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo deletion model with m6A-seq, splicing analysis, and immune cell profiling; single lab\",\n      \"pmids\": [\"32747553\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ALKBH5 mediates m6A demethylation of ITGB1 mRNA in ovarian cancer, suppressing YTHDF2-mediated ITGB1 mRNA degradation and increasing ITGB1 expression, which activates FAK/Src phosphorylation and promotes tumor-associated lymphangiogenesis and lymph node metastasis. Hypoxia induces HIF1α-dependent ALKBH5 upregulation that feeds this pathway.\",\n      \"method\": \"RNA pulldown, RIP-qPCR, Co-IP, MeRIP-qPCR, luciferase reporter assay, in vitro and in vivo lymphangiogenesis models\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNA pulldown, RIP, MeRIP, Co-IP, and in vivo models; single lab\",\n      \"pmids\": [\"36632222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ALKBH5 mediates m6A demethylation of Drp1 mRNA 3'-UTR. Under low ALKBH5 expression in hepatic stellate cells (HSCs), increased m6A on Drp1 mRNA promotes YTHDF1-mediated translation of DRP1, enhancing mitochondrial fission and HSC proliferation/migration to drive liver fibrosis.\",\n      \"method\": \"MeRIP-qPCR, mRNA stability assays, polysome fractionation, YTHDF1 co-immunoprecipitation, mitochondrial morphology assays, TGF-β1-induced HSC activation, in vivo fibrosis models\",\n      \"journal\": \"Pharmacological research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MeRIP, polysome fractionation, Co-IP, and in vivo fibrosis models; single lab\",\n      \"pmids\": [\"36566000\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ALKBH5 demethylates pri-miR-320a-3p, blocking the microprocessor protein DGCR8 from binding and preventing maturation of miR-320a-3p. Reduced mature miR-320a-3p de-represses FOXM1 mRNA (its target), promoting fibroblast activation. ALKBH5 also directly demethylates FOXM1 mRNA in an m6A-dependent manner.\",\n      \"method\": \"RIP assay for DGCR8/pri-miR-320a-3p interaction, MeRIP, miRNA processing assays, ALKBH5 knockdown in fibroblasts, silica-induced pulmonary fibrosis mouse model\",\n      \"journal\": \"Cellular & molecular biology letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RIP, MeRIP, microprocessor interaction assay, in vivo fibrosis model; single lab\",\n      \"pmids\": [\"35279083\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Loss of ALKBH5 increases m6A on OGDH (oxoglutarate dehydrogenase) mRNA, destabilizing it and reducing OGDH protein. Limited OGDH slows the TCA cycle, causing α-KG accumulation and conversion to L-2-HG, which inhibits mitochondrial energy production in hematopoietic stem/progenitor cells and impairs HSPC fitness.\",\n      \"method\": \"Alkbh5 KO mice, m6A-seq, RNA stability assays, metabolomics (α-KG/L-2-HG measurement), HSPC competitive transplantation, human hematopoietic cell in vitro assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — m6A-seq, metabolomics, in vivo competitive transplantation, and human cell validation; multiple orthogonal methods\",\n      \"pmids\": [\"37742191\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ALKBH5 erases m6A on CSF3R mRNA (encoding G-CSFR), stabilizing it and increasing G-CSFR surface expression and downstream STAT3 signaling in neutrophils. This drives emergency granulopoiesis and neutrophil mobilization during bacterial sepsis. ALKBH5 direct binding to CSF3R mRNA was confirmed by RIP-qPCR.\",\n      \"method\": \"Alkbh5-deficient mice in CLP sepsis model, RIP-qPCR, m6A quantification of CSF3R mRNA, mRNA stability assays, surface G-CSFR expression, STAT3 signaling readouts\",\n      \"journal\": \"Cellular & molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RIP-qPCR, m6A assays, mRNA stability, in vivo sepsis model; single lab\",\n      \"pmids\": [\"38114747\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ALKBH5-mediated m6A demethylation of neutrophil migration-related mRNA targets (including CXCR2, NLRP12, PTGER4, TNC, WNK1) imprints a migration-promoting transcriptome in neutrophils. Loss of ALKBH5 reduces CXCR2 expression and impairs neutrophil migration toward CXCL2, increasing mortality in sepsis.\",\n      \"method\": \"Alkbh5-deficient mice in CLP model, CXCR2 surface expression, m6A RNA decay assays, mRNA stability analysis, neutrophil migration assays\",\n      \"journal\": \"Signal transduction and targeted therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo sepsis model, m6A RNA decay assays, mRNA stability assays; single lab\",\n      \"pmids\": [\"35764614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Loss of ALKBH5 in lymphoid cells increases m6A on Jagged1 and Notch2 mRNAs, reducing their expression and impairing Jagged1/Notch2 signaling. This favors γδ T cell precursor expansion and differentiation, expanding the mature γδ T cell repertoire and enhancing protection against Salmonella infection.\",\n      \"method\": \"Alkbh5 conditional KO in lymphocytes, m6A-seq in thymocytes, flow cytometry of γδ T cell populations, Jagged1/Notch2 expression analysis, Salmonella infection challenge\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — lymphoid-specific KO, m6A-seq, flow cytometry, in vivo infection model; single lab\",\n      \"pmids\": [\"35939687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ALKBH5-mediated m6A demethylation ensures timely degradation of maternal RNAs during oocyte meiosis. In Alkbh5-/- oocytes, certain maternal transcripts accumulate with persistent m6A peaks and are recognized by IGF2BP2, stabilizing them and impairing mRNA clearance. Reducing IGF2BP2 in Alkbh5-/- oocytes partially rescues meiotic defects.\",\n      \"method\": \"Alkbh5 knockout female mice, temporal maternal transcriptomics, m6A dynamics profiling, m6A-seq, IGF2BP2 knockdown rescue in Alkbh5-/- oocytes\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse model, temporal m6A-seq, and IGF2BP2 rescue experiments with multiple orthogonal readouts\",\n      \"pmids\": [\"37848452\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ALKBH5 in Sertoli cells regulates m6A modification on Cdh2 mRNA (encoding N-cadherin). Removal of m6A by ALKBH5 promotes Cdh2 mRNA translation via IGF2BP1/2/3 and YTHDF1 complexes, maintaining N-cadherin levels and blood-testis barrier integrity. Alkbh5 knockout mice show disordered basal endoplasmic specialization.\",\n      \"method\": \"m6A-seq, MeRIP-qPCR, RIP-qPCR, Co-IP, polysome fractionation-qPCR, BTB integrity assay, transmission electron microscopy of Alkbh5-KO testes\",\n      \"journal\": \"Cellular & molecular biology letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — m6A-seq, polysome fractionation, Co-IP, electron microscopy, and KO mouse model; multiple orthogonal methods\",\n      \"pmids\": [\"36418936\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Hypoxia upregulates ALKBH5 in trophoblast cells, which translocates from nucleus to cytoplasm and demethylates SMAD1/SMAD5 mRNAs, enhancing their translation and promoting MMP9 and ITGA1 production to support trophoblast invasion. ALKBH5 knockdown in mouse placenta suppresses trophoblast invasion and causes fetal abortion.\",\n      \"method\": \"m6A-seq in hypoxia-treated trophoblast, nuclear/cytoplasmic fractionation, MeRIP-qPCR, mRNA translation assays for SMAD1/5, trophoblast-specific ALKBH5 knockdown in vivo\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — m6A-seq, nuclear/cytoplasmic fractionation, in vivo knockdown; single lab\",\n      \"pmids\": [\"35724807\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ALKBH5 is phosphorylated by protein kinase A (PKA), causing its translocation from nucleus to cytosol in hepatocytes during obesity. Hepatocyte-specific deletion of Alkbh5 reduces glucose and lipids by inhibiting GCGR and mTORC1 signaling pathways. Targeted knockdown of hepatic Alkbh5 reverses T2DM and MAFLD in diabetic mice.\",\n      \"method\": \"PKA phosphorylation assays of ALKBH5, nuclear/cytoplasmic fractionation, hepatocyte-specific Alkbh5 knockout mice, GCGR and mTORC1 pathway analyses, diabetic mouse models\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — PKA phosphorylation, subcellular fractionation, cell-type-specific KO, and in vivo metabolic phenotyping with multiple readouts\",\n      \"pmids\": [\"40014709\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PRMT5 directly catalyzes symmetric dimethylation of ALKBH5 at R316. This modification enhances TRIM28-mediated ubiquitination and degradation of ALKBH5, reducing its demethylase activity and increasing m6A on CD276 mRNA, thereby stabilizing CD276 expression and promoting CRC immune evasion.\",\n      \"method\": \"Co-IP, mass spectrometry for meR316, in vitro PRMT5 methylation assay, TRIM28 ubiquitination assay, MeRIP for CD276 mRNA, CRC in vivo models\",\n      \"journal\": \"Research (Washington, D.C.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro methylation assay, ubiquitination assay, MeRIP, in vivo model; single lab\",\n      \"pmids\": [\"39781264\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ALKBH5-mediated m6A demethylation of JARID2 mRNA stabilizes JARID2 transcripts in cooperation with IGF2BP3, promoting proliferation, migration, and invasion of rheumatoid arthritis fibroblast-like synoviocytes. ALKBH5 knockdown attenuates arthritis severity in CIA and DTHA mouse models.\",\n      \"method\": \"m6A-seq, RNA-seq, RIP, RNA pulldown, ALKBH5 knockdown/overexpression in RA FLSs, CIA/DTHA mouse models with ALKBH5 KO or shRNA injection\",\n      \"journal\": \"Arthritis & rheumatology (Hoboken, N.J.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — m6A-seq, RIP, RNA pulldown, and in vivo arthritis models; single lab\",\n      \"pmids\": [\"37584615\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ALKBH5 promotes axonal regeneration in dorsal root ganglion neurons by stabilizing Lpin2 mRNA (via m6A demethylation), thereby limiting regenerative lipid metabolism. Knockdown of ALKBH5 enhances sensory axonal regeneration in PNS, and overexpression impairs it in an m6A-dependent manner. In CNS, ALKBH5 knockdown enhances retinal ganglion cell survival and axon regeneration after optic nerve injury.\",\n      \"method\": \"Systematic m6A enzyme screen in axon regeneration, ALKBH5 knockdown and overexpression in rodent DRG neurons, Lpin2 mRNA stability assay, ALKBH5 demethylase-inactive mutant, optic nerve crush model\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — demethylase-inactive mutant, mRNA stability, and in vivo PNS/CNS regeneration models; single lab\",\n      \"pmids\": [\"37535403\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ALKBH5 demethylates TFEB mRNA to promote TFEB expression. TFEB in turn transcriptionally activates the ALKBH5 promoter (binding directly to it), while inhibiting METTL3 via mRNA stability downregulation, establishing a positive feedback loop relevant to autophagy regulation. (This is the TFEB→ALKBH5 transcriptional regulation component of PMID 30870073.)\",\n      \"method\": \"ChIP of TFEB at ALKBH5 promoter, luciferase promoter assay, TFEB knockdown/overexpression, mRNA stability of METTL3\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP, promoter assay, and reciprocal regulation demonstrated; already noted in PMID 30870073 entry above\",\n      \"pmids\": [\"30870073\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ALKBH5 demethylates CYP1B1 mRNA. In aging MSCs where ALKBH5 is decreased, increased m6A on CYP1B1 mRNA is recognized by IGF2BP1, stabilizing CYP1B1 mRNA, inducing mitochondrial dysfunction and cellular senescence. Alkbh5 knockout in MSCs aggravates spontaneous osteoarthritis.\",\n      \"method\": \"m6A quantification, MeRIP-qPCR for CYP1B1, IGF2BP1 RIP, mitochondrial function assays, Alkbh5 KO mouse spontaneous OA model, MSC senescence assays\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MeRIP, RIP, in vivo KO model, mitochondrial assays; single lab\",\n      \"pmids\": [\"37524872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ALKBH5 knockdown in NSCLC cells reduces nuclear ALKBH5 distribution via interaction with circEML4 (delivered by extracellular vesicles from M2 macrophages), elevating m6A modifications on SOCS2 mRNA (identified by m6A-seq/RNA-seq), activating the JAK-STAT pathway to promote NSCLC malignancy.\",\n      \"method\": \"circEML4-ALKBH5 interaction assay (Co-IP/RIP), m6A-seq, RNA-seq for SOCS2, nuclear fractionation, EVs transfer assay, in vivo NSCLC xenograft\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — m6A-seq, Co-IP, nuclear fractionation, in vivo models; single lab\",\n      \"pmids\": [\"37246269\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Astrocytic ALKBH5 demethylates GLT-1 (glutamate transporter-1, SLC1A2) mRNA, increasing GLT-1 expression in astrocytes. Selective deletion of ALKBH5 in astrocytes (but not neurons or endothelial cells) produces antidepressant-like behaviors and preserves stress-induced disruption of glutamatergic synaptic transmission and neuronal integrity in the mPFC.\",\n      \"method\": \"Astrocyte-specific, neuron-specific, and endothelial cell-specific Alkbh5 conditional KO mice, GLT-1 mRNA m6A assay, depression behavioral tests, Ca2+ imaging, synaptic transmission recordings\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cell-type-specific KO comparison, m6A assay for GLT-1 mRNA, electrophysiology, and Ca2+ imaging; multiple orthogonal methods in vivo\",\n      \"pmids\": [\"38773146\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ALKBH5 demethylates USP1 mRNA, reducing m6A levels and enhancing USP1 mRNA stability, thereby increasing USP1 expression. USP1 confers glucocorticoid resistance in T-ALL by deubiquitinating Aurora B. ALKBH5 knockdown reduces USP1 and Aurora B, sensitizing cells to dexamethasone.\",\n      \"method\": \"MeRIP-qPCR for USP1 m6A, mRNA stability assays, ALKBH5/USP1 knockdown in T-ALL cells, USP1 rescue experiments, in vivo T-ALL xenograft\",\n      \"journal\": \"Molecular carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MeRIP, stability assay, rescue experiments, in vivo model; single lab\",\n      \"pmids\": [\"34169564\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ALKBH5 demethylates IGF1R mRNA, enhancing IGF1R mRNA stability and translation, consequently activating IGF1R signaling and promoting endometrial cancer cell proliferation and invasion.\",\n      \"method\": \"MeRIP-qPCR, mRNA stability assays, ALKBH5 knockdown in endometrial cancer cells, IGF1R signaling pathway readouts\",\n      \"journal\": \"Journal of Cancer\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — MeRIP-qPCR and mRNA stability in single lab with limited mechanistic depth reported in abstract\",\n      \"pmids\": [\"32913456\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Psychological stress activates sympathetic nerves to release noradrenaline, which downregulates ALKBH5 in pancreatic cancer cells. ALKBH5 deficiency causes aberrant m6A modification of RNAs, which are packed into extracellular vesicles and delivered to nerves in the tumor microenvironment, enhancing hyperinnervation and PDAC progression.\",\n      \"method\": \"Mouse stress model, ALKBH5 knockdown in PDAC cells, EV m6A RNA profiling, in vivo nerve innervation assays, fisetin treatment to block EV uptake by neurons\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo stress model, EV m6A profiling, and pharmacological rescue; single lab\",\n      \"pmids\": [\"40419796\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In cerebral ischemia-reperfusion, Alkbh5 expression increases and, together with FTO, selectively demethylates Bcl2 mRNA, preventing its degradation and enhancing Bcl2 protein expression. Knockdown of Alkbh5 aggravates neuronal damage in this context.\",\n      \"method\": \"Rat MCAO model, primary neuron oxygen deprivation/reoxygenation, Alkbh5 shRNA, m6A quantification, Bcl2 mRNA stability analysis\",\n      \"journal\": \"Therapeutic advances in chronic disease\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — mRNA stability assay and in vivo knockdown; limited biochemical detail on direct demethylation of Bcl2 by ALKBH5 vs FTO\",\n      \"pmids\": [\"32426101\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ALKBH5 demethylates Runx2 mRNA, increasing its stability and promoting osteoblast differentiation. Expression of catalytically inactive ALKBH5 (active-site mutant) fails to rescue osteogenesis inhibition caused by ALKBH5 knockdown.\",\n      \"method\": \"ALKBH5 knockdown and overexpression during osteoblast differentiation, catalytic mutant rescue experiment, mRNA stability assay for Runx2\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — catalytic mutant rescue, mRNA stability, and differentiation assay; single lab, limited mechanistic depth\",\n      \"pmids\": [\"34105773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TLR4 signaling in the tumor microenvironment activates NF-κB, which upregulates ALKBH5 expression in ovarian cancer cells co-cultured with M2 macrophages. ALKBH5-mediated m6A demethylation increases NANOG mRNA expression, enhancing ovarian cancer aggressiveness.\",\n      \"method\": \"Macrophage-cancer cell co-culture, TLR4/NF-κB pathway inhibitors, transcriptome sequencing, m6A-seq, MeRIP for NANOG mRNA\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — co-culture model, MeRIP, transcriptome; limited mechanistic detail on how NF-κB directly activates ALKBH5 transcription\",\n      \"pmids\": [\"32329191\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ALKBH5 demethylates and stabilizes DDIT4 mRNA in HNSCC via the RBM33 co-factor (see RBM33 discovery), promoting autophagy and oncogenic function. (This is the downstream functional consequence of ALKBH5-RBM33 complex described in PMID 37257451.)\",\n      \"method\": \"ALKBH5 and RBM33 knockdown in HNSCC, MeRIP for DDIT4, autophagy assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — part of the same rigorous study as RBM33/ALKBH5 interaction above; DDIT4 is the validated downstream target\",\n      \"pmids\": [\"37257451\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ALKBH5 demethylates MAP3K8 mRNA in an m6A-dependent manner, promoting MAP3K8 expression, JNK/ERK pathway activation, IL-8 secretion, and macrophage recruitment in hepatocellular carcinoma.\",\n      \"method\": \"MeRIP-qPCR for MAP3K8 m6A, ALKBH5 knockdown in HCC cells, JNK/ERK pathway readouts, macrophage recruitment assays, single-cell sequencing GSVA\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — MeRIP-qPCR and pathway readouts; single lab, limited direct mechanistic validation of demethylation step\",\n      \"pmids\": [\"35982895\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Alkbh5 in mouse neurons is primarily localized to the nucleus, based on immunofluorescence co-localization with NeuN and nuclear markers in adult mouse brain, with expression decreasing dramatically during brain development.\",\n      \"method\": \"Immunofluorescence in adult mouse brain sections and cell lines, co-localization with NeuN and nuclear markers, developmental Western blot\",\n      \"journal\": \"Brain research bulletin\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — localization by immunofluorescence only, no direct functional consequence linked to nuclear localization in this study\",\n      \"pmids\": [\"32717204\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ALKBH5 inhibits SAV1 mRNA m6A modification; in ALKBH5-knockdown multiple myeloma cells, increased m6A on SAV1 mRNA decreases SAV1 stability and expression, suppressing HIPPO-pathway signaling and activating YAP, thus exerting an anti-myeloma effect. ALKBH5 also maintains MM stem cell pluripotency.\",\n      \"method\": \"MeRIP-seq for SAV1 m6A, mRNA stability assays, ALKBH5 knockdown in MM cells, HIPPO/YAP pathway readouts, in vivo and in vitro MM models\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — MeRIP-seq and mRNA stability; single lab, limited validation of direct ALKBH5-SAV1 interaction\",\n      \"pmids\": [\"35414790\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ALKBH5 is a ferrous iron- and 2-oxoglutarate-dependent RNA demethylase (the second identified mammalian m6A eraser) that binds m6A-containing single-stranded RNA in a 5'-3' orientation, uses an active-site proton shuttle (Lys132/Tyr139) to generate a hemiaminal intermediate, and releases formaldehyde to regenerate unmodified adenosine; its substrate selectivity, activity, and nuclear retention are regulated by multiple post-translational modifications—including SUMOylation (activated by ERK/JNK; inhibitory), phosphorylation (by PKA, SRC, LATS2, MST4; effects on nuclear export or stability), lactylation (by ESCO2; activating for IFN-β demethylation), arginine methylation (by PRMT5; promotes ubiquitination/degradation), and deubiquitination (by USP9X, USP14, or USP36; stabilizing)—as well as by the RNA-binding co-factor RBM33 that removes inhibitory SUMOylation and directs it to specific transcripts; in the nucleus ALKBH5 demethylates m6A from diverse target mRNAs (FOXM1, TACC3, TFEB, TRAF1, AXL, AXIN2, IL-11, CSF3R, GPX4, Cdh2, OGDH, SMAD1/5, etc.) to regulate their stability or translation, and also demethylates non-coding RNAs such as NEAT1 (facilitating paraspeckle assembly via phase separation through its C-terminal disordered region); at the organismal level its demethylase activity is essential for male spermatogenesis, female oocyte meiotic RNA clearance, blood-testis barrier integrity, hematopoietic stem cell metabolism, neutrophil migration, γδ T cell development, astrocyte-mediated glutamate homeostasis, hepatic glucose/lipid metabolism, and cardiac fibrosis, while its dysregulation drives tumorigenesis in glioblastoma, AML, multiple myeloma, colorectal, gastric, and other cancers through context-dependent stabilization or destabilization of oncogenic or tumor-suppressive transcripts.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ALKBH5 is a mammalian RNA demethylase that oxidatively reverses N6-methyladenosine (m6A) in mRNA in vitro and in vivo, the second identified mammalian m6A eraser, controlling mRNA export, nuclear speckle assembly, and RNA metabolism [#0]. Crystal structures of ALKBH5 bound to m6A-containing single-stranded RNA show the substrate engaged in a 5'-3' orientation, define an (A/G)m6AC consensus preference, and identify a Lys132/Tyr139 proton-shuttle network that drives hemiaminal-intermediate demethylation and formaldehyde release [#1]. Substrate selection is not intrinsic to the enzyme alone: the RNA-binding co-factor RBM33 forms a complex with ALKBH5, removes its inhibitory SUMOylation, and recruits it to specific transcripts such as DDIT4 [#4, #49]. ALKBH5 activity and localization are tuned by a layered post-translational code—ROS/ERK-JNK-driven SUMOylation that blocks substrate access [#3], kinase phosphorylation (SRC, LATS2, MST4, PKA) that controls nuclear retention, export, or stability [#5, #17, #18, #35], PRMT5-catalyzed arginine methylation that promotes TRIM28-dependent degradation [#36], lactylation that licenses IFN-\\u03b2 mRNA demethylation [#11], and deubiquitination by USP9X, USP36, and USP14 that stabilizes the protein [#6, #7, #18]. Through demethylation of defined mRNA targets, ALKBH5 alters transcript stability or translation—generally protecting transcripts from m6A-reader-mediated decay (e.g. YTHDF2) or shifting reader engagement (IGF2BP, YTHDF1)—to regulate processes including spermatogenesis [#0], oocyte maternal-RNA clearance via IGF2BP2 [#32], blood-testis barrier integrity through Cdh2 [#33], hematopoietic stem cell metabolism via OGDH [#28], neutrophil mobilization and migration [#29, #30], astrocytic glutamate homeostasis via GLT-1 [#42], and hepatic glucose/lipid metabolism [#35]. ALKBH5 also acts on non-coding RNA: under hypoxia it condenses into paraspeckles through its C-terminal disordered region and demethylates NEAT1 to drive paraspeckle assembly and CXCL8-mediated macrophage recruitment [#15, #16]. Dysregulated ALKBH5 drives tumorigenesis in glioblastoma, AML, multiple myeloma, colorectal, gastric, and other cancers through context-dependent stabilization or destabilization of oncogenic transcripts including FOXM1, TACC3, and AXL [#2, #8, #20].\",\n  \"teleology\": [\n    {\n      \"year\": 2012,\n      \"claim\": \"Established that mammals possess a second, dedicated m6A eraser, defining ALKBH5 as an RNA demethylase with a physiological role in fertility.\",\n      \"evidence\": \"In vitro demethylation assay plus Alkbh5-knockout mice with mRNA export and nuclear speckle readouts and testis transcriptomics\",\n      \"pmids\": [\"23177736\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural basis for substrate recognition\", \"Direct mRNA targets in spermatocytes not individually defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Resolved how ALKBH5 recognizes and chemically processes its substrate, explaining sequence preference and the catalytic chemistry distinguishing it from DNA-acting AlkB members.\",\n      \"evidence\": \"Three X-ray crystal structures with m6A ssRNA, biochemical demethylation assays, and active-site mutagenesis\",\n      \"pmids\": [\"35333330\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structures do not address how cellular co-factors redirect substrate choice\", \"No structure of post-translationally modified ALKBH5\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed that ALKBH5 acts on specific nascent transcripts in disease, demethylating FOXM1 to stabilize it and sustain glioblastoma stem cell proliferation, guided by an antisense lncRNA.\",\n      \"evidence\": \"m6A-seq, reciprocal RIP, ALKBH5 knockdown in patient-derived GSCs, and in vivo tumor models\",\n      \"pmids\": [\"28344040\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which FOXM1-AS tethers ALKBH5 to nascent RNA not structurally defined\", \"Generalizability of antisense-RNA targeting to other transcripts unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrated ALKBH5 as a selective dependency of leukemia stem cells (dispensable for normal hematopoiesis) acting through TACC3 and AXL, and showed its expression is set epigenetically via KDM4C/H3K9me3.\",\n      \"evidence\": \"Conditional KO mice, human AML lines, m6A-seq/RNA-seq, ChIP-seq/ATAC-seq, and in vivo leukemia models\",\n      \"pmids\": [\"32402250\", \"32402251\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why LSCs but not normal HSCs depend on ALKBH5 not fully explained\", \"Reader proteins decoding the retained m6A marks not defined in these studies\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Revealed that ALKBH5 enzymatic activity is gated by SUMOylation downstream of ROS/ERK-JNK signaling, providing a signal-responsive switch over global m6A.\",\n      \"evidence\": \"SUMOylation assays, ERK/JNK inhibition, ROS induction in cells and mouse HSPCs, and SUMO-site mutagenesis\",\n      \"pmids\": [\"34048572\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"SUMO ligase responsible not identified\", \"How SUMOylation physically blocks substrate access not structurally resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Extended the demethylation-stabilization logic across diverse cancers and tissues, showing ALKBH5 acts on target-specific transcripts (TRAF1, FOXM1, USP1, NANOG, IGF1R) to drive distinct oncogenic programs.\",\n      \"evidence\": \"MeRIP-qPCR, mRNA stability assays, knockdown/rescue, and in vivo tumor models across myeloma, melanoma, T-ALL, ovarian, and endometrial cancers\",\n      \"pmids\": [\"34759347\", \"33428593\", \"34169564\", \"32329191\", \"32913456\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Several rely on single-lab MeRIP without reciprocal validation\", \"Direct ALKBH5-transcript binding not always demonstrated\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showed ALKBH5 also regulates non-coding RNA and organizes membraneless compartments, condensing via its disordered C-terminus into paraspeckles to demethylate and stabilize NEAT1 under hypoxia.\",\n      \"evidence\": \"m6A-seq, NEAT1 stability and SFPQ ChIP assays, phase-separation assays, and cIDR deletion mutants\",\n      \"pmids\": [\"34670781\", \"37474102\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether condensation is required for catalysis on mRNA targets is unresolved\", \"Triggers of hypoxia-induced condensation not fully mapped\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Established ALKBH5 as a tissue-level regulator through cell-type-specific knockouts, governing blood-testis barrier integrity, hematopoietic metabolism, oocyte RNA clearance, neutrophil function, and \\u03b3\\u03b4 T cell development via discrete mRNA targets.\",\n      \"evidence\": \"Cell-type-specific Alkbh5 KO mice, m6A-seq, polysome fractionation, metabolomics, and in vivo infection/transplantation models\",\n      \"pmids\": [\"36418936\", \"37742191\", \"37848452\", \"38114747\", \"35764614\", \"35939687\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reader partners (IGF2BP1/2/3, YTHDF1/2) coupling differs by target and is incompletely mapped\", \"Whether single targets account for full phenotypes is uncertain\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified RBM33 as the substrate-recruiting co-factor that both de-SUMOylates and activates ALKBH5, solving how the enzyme achieves transcript selectivity.\",\n      \"evidence\": \"Reciprocal Co-IP, in vitro demethylase assays, SUMOylation assays, and m6A-seq in HNSCC targeting DDIT4\",\n      \"pmids\": [\"37257451\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether RBM33 directs ALKBH5 to its many other reported targets is untested\", \"Other adaptor co-factors not excluded\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined an extensive deubiquitinase/kinase network (USP9X, USP36, USP14, SRC, LATS2, MST4) controlling ALKBH5 protein abundance and nuclear retention, frequently within positive feedback loops sustaining glioblastoma and AML stem cells.\",\n      \"evidence\": \"Mass spectrometry, Co-IP, site-specific ubiquitination and phospho-mutagenesis, m6A-seq, and xenograft models\",\n      \"pmids\": [\"36239338\", \"37454738\", \"37979586\", \"38568805\", \"39990235\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The E3 ligases opposing these DUBs are largely undefined\", \"How multiple PTMs are integrated on the same molecule is unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Expanded the PTM repertoire to lactylation (ESCO2/SIRT6) and arginine methylation (PRMT5/TRIM28), linking ALKBH5 regulation to antiviral innate immunity and to degradation-driven cancer immune evasion.\",\n      \"evidence\": \"Lactylation/methylation mass spectrometry, writer-eraser Co-IP, in vitro PRMT5 methylation and TRIM28 ubiquitination assays, RIP, and functional immune/tumor assays\",\n      \"pmids\": [\"39413129\", \"39781264\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Crosstalk between lactylation, SUMOylation, and methylation on the same residues unresolved\", \"PRMT5/TRIM28 axis shown in single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Connected ALKBH5 regulation to systemic physiology and therapy resistance, showing PKA-driven phosphorylation/relocalization governs hepatic glucose-lipid metabolism and ferroptosis, and KRAS-ERK/JNK SUMOylation confers platinum resistance via DNA-repair transcripts.\",\n      \"evidence\": \"PKA phosphorylation assays, hepatocyte-specific KO, SUMO-deficient mutants, NER and ferroptosis assays, and diabetic/NSCLC in vivo models\",\n      \"pmids\": [\"40014709\", \"39960727\", \"39901038\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phospho-sites for the PKA-ferroptosis axis not all mapped\", \"Whether hepatic and tumor PKA effects share the same mechanism is unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the combinatorial post-translational code, co-factor recruitment, and phase separation are integrated to select among the many reported ALKBH5 targets in a given cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model linking PTM state to target choice\", \"Reader-protein coupling that dictates stabilization vs destabilization not systematically defined\", \"E3 ligases and SUMO ligases acting on ALKBH5 largely unidentified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [0, 1, 2, 8, 32]},\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [1, 2, 11, 29]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 5, 17, 51]},\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [0, 16]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [34, 35]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 1, 2, 8, 32]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [2, 8, 20, 12]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [11, 29, 30, 31]}\n    ],\n    \"complexes\": [\"paraspeckle\"],\n    \"partners\": [\"RBM33\", \"USP9X\", \"USP36\", \"USP14\", \"PRMT5\", \"SRC\", \"LATS2\", \"MST4\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}