Fe(II)/2-oxoglutarate-dependent dioxygenase of the AlkB family, encoded by a nuclear gene and imported into the mitochondrial matrix, with a smaller nuclear and cytoplasmic pool. Its best-established activity is on transfer RNA: it performs the second, oxidative step of wobble-base maturation in mitochondrial tRNA-Met, converting NSUN3-generated 5-methylcytidine at position 34 first to 5-hydroxymethylcytidine and then to 5-formylcytidine (f5C34). That modification allows the single mitochondrial tRNA-Met to decode AUA and AUU as well as AUG under the non-universal mitochondrial genetic code, so loss of ALKBH1 impairs mitochondrial translation and respiratory complex activity. ALKBH1 makes analogous 2'-O-methylated hydroxymethyl- and formyl-cytidines at position 34 of cytoplasmic tRNA-Leu, and it demethylates N1-methyladenosine in cytoplasmic and mitochondrial tRNAs, which modulates tRNA usage and the rate of translation initiation. Additional activities demonstrated in vitro include oxidative demethylation of 3-methylcytosine in single-stranded DNA and in RNA, and an AP (abasic site) lyase activity that arises from an active site distinct from the dioxygenase centre. A widely cited role as the eraser of N6-methyladenine (6mA) in genomic DNA is disputed: ALKBH1 demethylates 6mA efficiently in single-stranded or bubbled DNA but poorly in duplex DNA, and several independent methodological audits attribute most reported mammalian genomic 6mA to bacterial and RNA contamination, antibody cross-reactivity and misincorporation of N6-methyladenosine from the nucleotide salvage pool, while comparative genomics places robust eukaryotic 6mA only in lineages retaining the AMT1 adenine methyltransferase, which animals have lost. In mouse, loss of Alkbh1 causes embryonic lethality with defects in placental trophoblast and neural differentiation.
Definition: Catalysis of the Fe(II)- and 2-oxoglutarate-dependent oxidation of a methylated residue in histone H2A.
Justification: ALKBH1 was reported to act as a dioxygenase specifically on histone H2A, with histone H2A from Alkbh1-null mice showing altered methylation. GOA carries a nucleus annotation from this study but no molecular function capturing the reported histone substrate. The activity has not been independently replicated, so this is offered as a candidate term rather than as a recommended annotation.
Parent term: dioxygenase activity
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005634 nucleus | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic assertion that AlkB-family members of this clade act in the nucleus. A nuclear pool of ALKBH1 is independently documented in human cells. Reason: Correct. ALKBH1 is dual-localised; the nuclear pool is seen by fluorescent fusion protein imaging, by HPA immunofluorescence, and in chromatin work on histone H2A and on euchromatin. |
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic assertion of cytoplasmic activity, consistent with ALKBH1 acting on cytoplasmic tRNA-Leu and cytoplasmic tRNA m1A. Reason: Correct, though general. The mitochondrion (GO:0005739) and mitochondrial matrix (GO:0005759) annotations carried by this gene are far more informative about where the bulk of the protein acts; cytoplasm is retained because a genuine extramitochondrial pool modifies cytoplasmic tRNA-Leu. |
| GO:0035513 oxidative RNA demethylation | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic assertion of oxidative RNA demethylation, matching the experimentally demonstrated m1A demethylation of tRNAs by human ALKBH1. Reason: Consistent with direct human experimental evidence. The gene's own experimental annotation appears in the WITH/FROM field, which is the expected marker that the PAINT node was seeded in part by experimental data on this very gene, not a sign of circularity. Supporting Evidence: PMID:27745969 Here, we show that mammalian ALKBH1 is a tRNA demethylase. It mediates the demethylation of N1-methyladenosine (m1A) in tRNAs. |
| GO:0035516 broad specificity oxidative DNA demethylase activity | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Phylogenetic transfer of the ancestral AlkB DNA-demethylase function, seeded from E. coli AlkB (UniProtKB:P05050) and mouse Alkbh1. Reason: The ancestral AlkB activity is real and human ALKBH1 does demethylate 3-methylcytosine in vitro, but only in single-stranded DNA, and two independent early studies failed to detect any repair activity at all. In cells the demonstrated substrates of ALKBH1 are tRNAs, not DNA. Retained as a family-level activity but demoted from core; the node placement itself is not disputed, so no propagation_review is asserted. Supporting Evidence: PMID:18603530 Employing three different methods we demonstrate that hABH1 demethylates 3-methylcytosine in single-stranded DNA and RNA in vitro. PMID:18603530 Surprisingly, hABH1, which displays the strongest homology to AlkB, failed to show repair activity in two independent studies. |
| GO:0008198 ferrous iron binding | IBA GO_REF:0000033 | ACCEPT | Summary: Fe(II) binding at the conserved 2-His-1-carboxylate facial triad, required for every dioxygenase activity of this protein. Reason: Core cofactor-binding function; directly confirmed for the human protein by site-directed mutagenesis of the predicted Fe(II)- and 2-oxoglutarate-binding residues. Supporting Evidence: PMID:18603530 Site-specific mutagenesis confirmed that the putative Fe(II) and 2OG binding residues are essential for activity. |
| GO:0035515 oxidative RNA demethylase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic assertion of oxidative RNA demethylase activity, matching the human tRNA m1A demethylase data. Reason: Well supported by direct human evidence in two independent labs. Supporting Evidence: PMID:27745969 Here, we show that mammalian ALKBH1 is a tRNA demethylase. It mediates the demethylation of N1-methyladenosine (m1A) in tRNAs. |
| GO:0005634 nucleus | IEA GO_REF:0000120 | ACCEPT | Summary: Automated subcellular-location mapping to the nucleus. Reason: Correct; a nuclear pool is experimentally documented. |
| GO:0005739 mitochondrion | IEA GO_REF:0000044 | ACCEPT | Summary: Automated transfer from the UniProt subcellular-location vocabulary. Reason: Correct and central. The mitochondrion is where ALKBH1's best-supported substrate (mt-tRNA-Met) resides. |
| GO:0035516 broad specificity oxidative DNA demethylase activity | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Automated assignment of the AlkB-family DNA demethylase activity via ARBA, orthology and the RHEA/EC 1.14.11.33 mapping. Reason: The ancestral AlkB activity is real and human ALKBH1 does demethylate 3-methylcytosine in vitro, but only in single-stranded DNA, and two independent early studies failed to detect any repair activity at all. In cells the demonstrated substrates of ALKBH1 are tRNAs, not DNA. Retained as a family-level activity but demoted from core. |
| GO:0050918 positive chemotaxis | IEA GO_REF:0000108 | REMOVE | Summary: Logically inferred from the GO:0042056 chemoattractant activity annotation, which is itself an erroneous propagation. Reason: This annotation exists only as an inter-ontology inference from GO:0042056 chemoattractant activity. That parent annotation traces, through Ensembl Compara transfer from mouse Alkbh1 (UniProtKB:P0CB42), to an MGI IDA on PMID:16860792 - a paper about NRP (neural regeneration protein), a secreted SDF-1-like neurotrophic factor encoded in a different reading frame of the Alkbh1 locus, not the ALKBH1 dioxygenase polypeptide. Removing the unsound parent removes the inference. No experimental annotation is affected. Supporting Evidence: PMID:16860792 The Nrp gene is encoded as a forward frameshift to the hypothetical alkylated DNA repair protein AlkB. |
| GO:0140078 class I DNA-(apurinic or apyrimidinic site) endonuclease activity | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Automated assignment from the RHEA:66592 / EC 4.2.99.18 mapping, i.e. the AP lyase activity. Reason: The AP lyase activity is genuine and was measured directly for the human protein, but it arises from an active site distinct from the dioxygenase centre, is metal- and 2-oxoglutarate-independent, and its physiological significance is explicitly hedged by the authors who found it. Secondary to the tRNA-modifying role. |
| GO:0141131 DNA N6-methyladenine demethylase activity | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Automated assignment from the RHEA:49524 / EC 1.14.11.51 mapping for 6mA demethylation. Reason: The enzymology is real but the physiological substrate is disputed. ALKBH1 demethylates 6mA efficiently only in single-stranded or bubbled/bulged DNA and poorly in duplex DNA, as conceded by the proponents of the pathway themselves (PMID:40715766). More seriously, the existence of regulated genomic 6mA in mammals is contested by three orthogonal methodological audits: antibody non-specificity plus bacterial and RNA contamination (PMID:32206710), misincorporation of ribo-N6-methyladenosine from the nucleotide-salvage pool by DNA polymerases (PMID:32203414), and quantitative metagenomic deconvolution finding no high-abundance 6mA in humans (PMID:35113693). A 2025 comparative-genomics survey places robust eukaryotic 6mA only in AMT1-encoding lineages and reports that animals lost the AMT1-6mA pathway (PMID:41254163). Retained because the in vitro reaction is reproducible - a dedicated eraser can exist without a regulated mark - but firmly non-core. Supporting Evidence: PMID:40715766 ALKBH1 preferentially exhibits 6mA demethylase activity for single-stranded DNA (ssDNA) or bubbled/bulged DNA, but not for double-stranded DNA (dsDNA). PMID:32203414 However, the bulk of genomic m6dA originates from ribo-N6-methyladenosine, which is processed via the nucleotide-salvage pathway and misincorporated by DNA polymerases. |
| GO:0160290 2-oxoglutarate-dependent tRNA 5-methylcytidine formyltransferase activity | IEA GO_REF:0000116 | ACCEPT | Summary: Automated assignment from the RHEA:54144 reaction mapping for the m5C34 to f5C34 conversion in mt-tRNA-Met. Reason: This is ALKBH1's best-supported molecular function and the electronic assignment agrees with the direct experimental evidence. Supporting Evidence: PMID:27497299 We further identify ALKBH1/ABH1 as the dioxygenase responsible for oxidising m5C34 of mt-tRNAMet to generate an f5C34 modification. |
| GO:0000791 euchromatin | IEA GO_REF:0000107 | KEEP AS NON CORE | Summary: Ensembl Compara transfer from a mouse IDA showing Alkbh1 localising to nuclear euchromatin. Reason: The mouse source (PMID:18163532) is a genuine observation on the Alkbh1 protein itself, unlike the chemoattractant transfer from the same orthology pipeline. Retained as a sub-nuclear location, but peripheral to the mitochondrial tRNA-modifying core function. Supporting Evidence: PMID:18163532 Alkbh1 localizes to nuclear euchromatin, and interacts strongly with Mrj, an essential placental gene that mediates gene repression by recruitment of class II histone deacetylases (HDACs). |
| GO:0042056 chemoattractant activity | IEA GO_REF:0000107 | REMOVE | Summary: Ensembl Compara transfer of a mouse chemoattractant-activity annotation onto human ALKBH1. The mouse annotation describes a different protein product of the locus. Reason: The mouse source annotation (UniProtKB:P0CB42 enables GO:0042056, IDA, PMID:16860792, assigned by MGI) is based on NRP, "neural regeneration protein", which that paper states is encoded as a forward frameshift relative to the AlkB-homologous reading frame. NRP is described as a secreted SDF-1-like factor with trefoil (TFF-1) and survival-promoting-peptide homology - a different polypeptide from the same locus, not the ALKBH1 dioxygenase. Orthology transfer of a secreted chemokine activity onto Q13686, a matrix-targeted mitochondrial 2-oxoglutarate/Fe(II) oxygenase with no signal peptide, is biologically incoherent. This is an electronic over-propagation that can be argued against on biological grounds; no experimental annotation on this gene is touched. Supporting Evidence: PMID:16860792 The Nrp gene is encoded as a forward frameshift to the hypothetical alkylated DNA repair protein AlkB. |
| GO:0141137 positive regulation of gene expression, epigenetic | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Automated/orthology assignment of an epigenetic gene-activation role, derived from the genomic 6mA demethylation model. Reason: This process annotation is entirely downstream of the contested genomic-6mA premise - ALKBH1 is held to activate transcription by erasing a repressive 6mA mark. Since the existence of regulated mammalian genomic 6mA is disputed (PMID:32206710, PMID:32203414, PMID:35113693, PMID:41254163), the process cannot be treated as core. Retained rather than removed because the underlying knockdown and overexpression phenotypes are real observations whose mechanistic interpretation, not whose existence, is in question. |
| GO:1990983 regulation of translational initiation by tRNA modification | IEA GO_REF:0000120 | ACCEPT | Summary: Automated/orthology assignment of the tRNA-modification-dependent control of translation initiation. Reason: Agrees with direct human experimental evidence that ALKBH1-mediated tRNA m1A demethylation attenuates translation initiation. Supporting Evidence: PMID:27745969 The ALKBH1-catalyzed demethylation of the target tRNAs results in attenuated translation initiation and decreased usage of tRNAs in protein synthesis. |
| GO:0005654 nucleoplasm | IDA GO_REF:0000052 | ACCEPT | Summary: HPA immunofluorescence localising ALKBH1 to the nucleoplasm. Reason: Consistent with the documented nuclear pool of the protein. |
| GO:0160290 2-oxoglutarate-dependent tRNA 5-methylcytidine formyltransferase activity | IDA PMID:27497299 NSUN3 and ABH1 modify the wobble position of mt-tRNAMet to e... | ACCEPT | Summary: Direct demonstration that ALKBH1/ABH1 is the dioxygenase that oxidises NSUN3-generated m5C34 of mt-tRNA-Met to f5C34. Reason: Core molecular function. Independently reproduced and mechanistically resolved into two successive oxidation steps by a second lab, and re-endorsed in 2025 by a study designed specifically to eliminate f5C detection artifacts. Supporting Evidence: PMID:27497299 We further identify ALKBH1/ABH1 as the dioxygenase responsible for oxidising m5C34 of mt-tRNAMet to generate an f5C34 modification. PMID:41219180 Instead, the bulk of mammalian f5C resides in the well-established mitochondrial tRNA Methionine (mt-tRNAMet) and is mediated by ALKBH1. |
| GO:0002101 tRNA wobble cytosine modification | IMP PMID:28472312 ALKBH1 is an RNA dioxygenase responsible for cytoplasmic and... | ACCEPT | Summary: ALKBH1 knockout abolishes f5C34 of mt-tRNA-Met and the hm5Cm/f5Cm modifications at position 34 of cytoplasmic tRNA-Leu. Reason: Core biological process, directly downstream of the formyltransferase/oxidase activity. Supporting Evidence: PMID:28472312 found that ALKBH1 first hydroxylated m5C34 to form hm5C34, and then oxidized hm5C34 to form f5C34. |
| GO:0005759 mitochondrial matrix | TAS PMID:28472312 ALKBH1 is an RNA dioxygenase responsible for cytoplasmic and... | ACCEPT | Summary: ALKBH1 is active in the mitochondrial matrix, where mt-tRNA-Met is transcribed, matured and used. Reason: The most informative location term for the core function; specific and correct. Supporting Evidence: PMID:28472312 ALKBH1-knockout cells exhibited a strong reduction in mitochondrial translation and reduced respiratory complex activities, indicating that f5C34 formation mediated by ALKBH1 is required for efficient mitochondrial functions. |
| GO:0160290 2-oxoglutarate-dependent tRNA 5-methylcytidine formyltransferase activity | IMP PMID:28472312 ALKBH1 is an RNA dioxygenase responsible for cytoplasmic and... | ACCEPT | Summary: Loss-of-function evidence that ALKBH1 generates f5C34 on mt-tRNA-Met, with in vitro reconstitution of both oxidation steps. Reason: Core molecular function, supported by both knockout and reconstitution in the same study. Supporting Evidence: PMID:28472312 ALKBH1-knockout cells exhibited a strong reduction in mitochondrial translation and reduced respiratory complex activities, indicating that f5C34 formation mediated by ALKBH1 is required for efficient mitochondrial functions. |
| GO:0005634 nucleus | EXP PMID:22961808 ALKBH1 is a histone H2A dioxygenase involved in neural diffe... | ACCEPT | Summary: Nuclear localisation in the context of ALKBH1 acting on histone H2A and regulating neural differentiation genes by chromatin immunoprecipitation. Reason: A genuine nuclear pool exists alongside the dominant mitochondrial pool. Supporting Evidence: PMID:22961808 Whole-genome expression analysis and chromatin immunoprecipitation revealed that ALKBH1 regulates both directly and indirectly, a subset of genes required for neural development. |
| GO:0005739 mitochondrion | EXP PMID:17979886 Expression and sub-cellular localization of human ABH family... | ACCEPT | Summary: Subcellular localisation survey of the human AlkB homologue family. Reason: Mitochondrial localisation of ALKBH1 is abundantly and independently supported (IDA in PMID:18603530 and PMID:27497299, high-throughput mitochondrial proteomics in PMID:34800366, UniProt subcellular mapping). Caveat recorded for transparency: the cached abstract of this particular reference reports hABH1 in cytoplasm and nuclei and does not itself foreground mitochondria. The full text was not available to this review, so the curator's reading is deferred to, and the conclusion is unchanged because the location is established elsewhere. Supporting Evidence: PMID:18603530 Here, we show that hABH1 is a mitochondrial protein, as demonstrated using fluorescent fusion protein expression, immunocytochemistry, and Western blot analysis. |
| GO:0141131 DNA N6-methyladenine demethylase activity | EXP PMID:30017583 N(6)-Methyladenine DNA Modification in the Human Genome. | KEEP AS NON CORE | Summary: Reported identification of ALKBH1 as the demethylase for 6mA in human genomic DNA, paired with N6AMT1 as the writer. Reason: The enzymology is real but the physiological substrate is disputed. ALKBH1 demethylates 6mA efficiently only in single-stranded or bubbled/bulged DNA and poorly in duplex DNA, as conceded by the proponents of the pathway themselves (PMID:40715766). More seriously, the existence of regulated genomic 6mA in mammals is contested by three orthogonal methodological audits: antibody non-specificity plus bacterial and RNA contamination (PMID:32206710), misincorporation of ribo-N6-methyladenosine from the nucleotide-salvage pool by DNA polymerases (PMID:32203414), and quantitative metagenomic deconvolution finding no high-abundance 6mA in humans (PMID:35113693). A 2025 comparative-genomics survey places robust eukaryotic 6mA only in AMT1-encoding lineages and reports that animals lost the AMT1-6mA pathway (PMID:41254163). Not removed, because this is an experimental annotation whose full text was unavailable here and the in vitro demethylation chemistry is reproducible. Demoted to non-core pending resolution of whether the mark exists in mammalian duplex genomic DNA. Supporting Evidence: PMID:30017583 DNA 6mA and N6-demethyladenine modification in the human genome were mediated by methyltransferase N6AMT1 and demethylase ALKBH1, respectively. PMID:32206710 Together, our results strongly imply that the evidence published to date is not sufficient to support the presence of 6mdA in mammals. |
| GO:0141131 DNA N6-methyladenine demethylase activity | EXP PMID:30392959 N(6)-methyladenine DNA Modification in Glioblastoma. | KEEP AS NON CORE | Summary: ALKBH1 reported as the demethylase regulating N6-mA levels in glioblastoma stem cells, with in vitro demethylation of 6mA-containing oligonucleotides by dot blot and UHPLC-MS. Reason: The enzymology is real but the physiological substrate is disputed. ALKBH1 demethylates 6mA efficiently only in single-stranded or bubbled/bulged DNA and poorly in duplex DNA, as conceded by the proponents of the pathway themselves (PMID:40715766). More seriously, the existence of regulated genomic 6mA in mammals is contested by three orthogonal methodological audits: antibody non-specificity plus bacterial and RNA contamination (PMID:32206710), misincorporation of ribo-N6-methyladenosine from the nucleotide-salvage pool by DNA polymerases (PMID:32203414), and quantitative metagenomic deconvolution finding no high-abundance 6mA in humans (PMID:35113693). A 2025 comparative-genomics survey places robust eukaryotic 6mA only in AMT1-encoding lineages and reports that animals lost the AMT1-6mA pathway (PMID:41254163). Not removed, because this is an experimental annotation and the in vitro demethylation chemistry is reproducible. Demoted to non-core pending resolution of whether the mark exists in mammalian duplex genomic DNA. Supporting Evidence: PMID:30392959 N6-mA levels were dynamically regulated by the DNA demethylase ALKBH1, depletion of which led to transcriptional silencing of oncogenic pathways through decreasing chromatin accessibility. PMID:41254163 Intriguingly, 5mC is predominantly retained in major multicellular eukaryotic lineages (plants, animals and Dikarya fungi), whereas the AMT1–6mA pathway was lost in these lineages. |
| GO:0005739 mitochondrion | HTP PMID:34800366 Quantitative high-confidence human mitochondrial proteome an... | ACCEPT | Summary: ALKBH1 detected in a quantitative high-confidence human mitochondrial proteome. Reason: Orthogonal, unbiased support for the mitochondrial localisation. Supporting Evidence: PMID:34800366 We classified >8,000 proteins in mitochondrial preparations of human cells and defined a mitochondrial high-confidence proteome of >1,100 proteins (MitoCoP). |
| GO:0005739 mitochondrion | IDA PMID:18603530 Human AlkB homolog 1 is a mitochondrial protein that demethy... | ACCEPT | Summary: Direct demonstration by fluorescent fusion protein expression, immunocytochemistry and Western blotting that hABH1 is a mitochondrial protein. Reason: Primary and best-characterised location. Supporting Evidence: PMID:18603530 Here, we show that hABH1 is a mitochondrial protein, as demonstrated using fluorescent fusion protein expression, immunocytochemistry, and Western blot analysis. PMID:18603530 A fraction is apparently nuclear and this fraction increases strongly if the fluorescent tag is placed at the N-terminal end of the protein, thus interfering with mitochondrial targeting. |
| GO:0141137 positive regulation of gene expression, epigenetic | IDA PMID:30017583 N(6)-Methyladenine DNA Modification in the Human Genome. | KEEP AS NON CORE | Summary: Transcriptional activation attributed to ALKBH1-mediated removal of genomic 6mA. Reason: This process annotation is entirely downstream of the contested genomic-6mA premise - ALKBH1 is held to activate transcription by erasing a repressive 6mA mark. Since the existence of regulated mammalian genomic 6mA is disputed (PMID:32206710, PMID:32203414, PMID:35113693, PMID:41254163), the process cannot be treated as core. Retained rather than removed because the underlying expression phenotypes are real observations whose mechanistic interpretation, not whose existence, is in question. Supporting Evidence: PMID:30017583 6mA sites were enriched in the coding regions and mark actively transcribed genes in human cells. |
| GO:0141137 positive regulation of gene expression, epigenetic | IDA PMID:30392959 N(6)-methyladenine DNA Modification in Glioblastoma. | KEEP AS NON CORE | Summary: ALKBH1 depletion in glioblastoma stem cells silenced oncogenic transcriptional programmes and decreased chromatin accessibility. Reason: This process annotation is entirely downstream of the contested genomic-6mA premise - ALKBH1 is held to activate transcription by erasing a repressive 6mA mark. Since the existence of regulated mammalian genomic 6mA is disputed (PMID:32206710, PMID:32203414, PMID:35113693, PMID:41254163), the process cannot be treated as core. Retained rather than removed because the underlying expression phenotypes are real observations whose mechanistic interpretation, not whose existence, is in question. Supporting Evidence: PMID:30392959 N6-mA levels were dynamically regulated by the DNA demethylase ALKBH1, depletion of which led to transcriptional silencing of oncogenic pathways through decreasing chromatin accessibility. |
| GO:0035515 oxidative RNA demethylase activity | IDA PMID:18603530 Human AlkB homolog 1 is a mitochondrial protein that demethy... | ACCEPT | Summary: Direct in vitro demonstration that hABH1 demethylates 3-methylcytosine in RNA as well as in single-stranded DNA. Reason: RNA is the substrate class for which ALKBH1 has consistent in-cell support, and the oxidative RNA demethylase activity is confirmed independently for tRNA m1A. Supporting Evidence: PMID:18603530 Employing three different methods we demonstrate that hABH1 demethylates 3-methylcytosine in single-stranded DNA and RNA in vitro. |
| GO:0035516 broad specificity oxidative DNA demethylase activity | IDA PMID:18603530 Human AlkB homolog 1 is a mitochondrial protein that demethy... | KEEP AS NON CORE | Summary: In vitro demethylation of 3-methylcytosine in single-stranded DNA. Reason: A real in vitro activity, but restricted to single-stranded DNA and never shown to operate on chromatin in cells; the same study notes that two earlier independent efforts detected no repair activity for this protein at all. The demonstrated cellular substrates of ALKBH1 are tRNAs. Retained, demoted from core. Supporting Evidence: PMID:18603530 Employing three different methods we demonstrate that hABH1 demethylates 3-methylcytosine in single-stranded DNA and RNA in vitro. |
| GO:0035516 broad specificity oxidative DNA demethylase activity | IDA PMID:30392959 N(6)-methyladenine DNA Modification in Glioblastoma. | KEEP AS NON CORE | Summary: DNA demethylase activity measured on 6mA-containing oligonucleotides by dot blot and UHPLC-MS. Reason: A real in vitro activity, but the physiological relevance rests on the contested premise that regulated 6mA exists in mammalian genomic duplex DNA. Retained, demoted from core, in line with the treatment of the specific GO:0141131 annotation from the same study. |
| GO:0035513 oxidative RNA demethylation | IDA PMID:31188562 AlkB Homologue 1 Demethylates N(3)-Methylcytidine in mRNA of... | ACCEPT | Summary: ALKBH1 overexpression and knockdown decrease and increase, respectively, the level of 3-methylcytidine in mRNA. Reason: Oxidative RNA demethylation by ALKBH1 is well supported overall. Caveat: the specific mRNA-m3C substrate claim depends on METTL8 being an mRNA m3C writer, and METTL8 has since been recharacterised as a mitochondrial tRNA m3C32 methyltransferase, so the mRNA compartment assignment is weaker than the tRNA one. The process term itself is not in doubt. Supporting Evidence: PMID:31188562 Overexpression and knockdown of ALKBH1 in cultured human cells can induce decrease and increase of the level of m3C in mRNA, respectively, revealing the eraser enzyme property of ALKBH1 on m3C in mRNA. |
| GO:0035515 oxidative RNA demethylase activity | IDA PMID:31188562 AlkB Homologue 1 Demethylates N(3)-Methylcytidine in mRNA of... | ACCEPT | Summary: In vitro demethylation of 3-methylcytidine in RNA by recombinant ALKBH1. Reason: Consistent with the well-established oxidative RNA demethylase activity of this protein. Supporting Evidence: PMID:31188562 In the current study, we found that the AlkB homologue 1 (ALKBH1) was capable of demethylating m3C in mRNA of mammalian cells in vitro. |
| GO:0035516 broad specificity oxidative DNA demethylase activity | IDA PMID:30017583 N(6)-Methyladenine DNA Modification in the Human Genome. | KEEP AS NON CORE | Summary: DNA demethylase activity on 6mA-containing substrates reported in the human genomic 6mA study. Reason: A real in vitro activity, but the physiological relevance rests on the contested premise that regulated 6mA exists in mammalian genomic duplex DNA. Retained, demoted from core, in line with the treatment of the specific GO:0141131 annotation from the same study. |
| GO:0140078 class I DNA-(apurinic or apyrimidinic site) endonuclease activity | IDA PMID:19959401 Human AlkB homologue 1 (ABH1) exhibits DNA lyase activity at... | KEEP AS NON CORE | Summary: Direct demonstration that ABH1 cleaves DNA at abasic sites by a lyase mechanism, from an active site distinct from the dioxygenase centre. Reason: A well-controlled in vitro activity (metal- and 2-oxoglutarate-independent, unaffected by mutation of the iron-binding residues), but the authors themselves present its physiological role as speculative, and no cellular phenotype links ALKBH1 to base-excision repair. Retained as a genuine secondary activity. Supporting Evidence: PMID:19959401 Here, we show that ABH1 unexpectedly has a second activity, cleaving DNA at abasic (AP) sites such as those arising spontaneously from alkylation-dependent depurination reactions. PMID:19959401 ABH1 can cleave at closely spaced AP-sites on opposite DNA strands yielding double-strand breaks in vitro and this reaction may relate to the physiological role of this unexpected AP lyase activity. |
| GO:0000049 tRNA binding | IDA PMID:27497299 NSUN3 and ABH1 modify the wobble position of mt-tRNAMet to e... | ACCEPT | Summary: Direct binding of ALKBH1 to mitochondrial tRNA-Met. Reason: Substrate recognition underpinning the core catalytic function; far more informative than a bare binding term because the ligand class is specified. Supporting Evidence: PMID:27497299 We further identify ALKBH1/ABH1 as the dioxygenase responsible for oxidising m5C34 of mt-tRNAMet to generate an f5C34 modification. |
| GO:0002101 tRNA wobble cytosine modification | IDA PMID:27497299 NSUN3 and ABH1 modify the wobble position of mt-tRNAMet to e... | ACCEPT | Summary: ALKBH1 generates f5C at the wobble position (C34) of mt-tRNA-Met. Reason: Core biological process, the direct output of the formyltransferase/oxidase activity. Supporting Evidence: PMID:27497299 We further identify ALKBH1/ABH1 as the dioxygenase responsible for oxidising m5C34 of mt-tRNAMet to generate an f5C34 modification. |
| GO:0005739 mitochondrion | IDA PMID:27497299 NSUN3 and ABH1 modify the wobble position of mt-tRNAMet to e... | ACCEPT | Summary: Mitochondrial localisation shown alongside the demonstration of ALKBH1 acting on mt-tRNA-Met. Reason: Correct and directly tied to the core function. Supporting Evidence: PMID:27497299 We show that the RNA methyltransferase NSUN3 localises to mitochondria and interacts with mt-tRNAMet to methylate cytosine 34 (C34) at the wobble position. |
| GO:0006446 regulation of translational initiation | IMP PMID:27745969 ALKBH1-Mediated tRNA Demethylation Regulates Translation. | MODIFY | Summary: ALKBH1-mediated tRNA m1A demethylation attenuates translation initiation. Reason: Correct but under-specific. The mechanism is explicitly tRNA-modification-dependent, and this gene already carries the more informative child term GO:1990983 for the same finding. Proposed replacements: regulation of translational initiation by tRNA modification Supporting Evidence: PMID:27745969 The ALKBH1-catalyzed demethylation of the target tRNAs results in attenuated translation initiation and decreased usage of tRNAs in protein synthesis. |
| GO:0006448 regulation of translational elongation | IMP PMID:27745969 ALKBH1-Mediated tRNA Demethylation Regulates Translation. | KEEP AS NON CORE | Summary: Altered tRNA usage in protein synthesis upon ALKBH1 manipulation, affecting elongation as well as initiation. Reason: A real downstream consequence of changing the tRNA modification landscape, but a step removed from the molecular activity; initiation is the better-documented effect. |
| GO:0070129 regulation of mitochondrial translation | IMP PMID:27497299 NSUN3 and ABH1 modify the wobble position of mt-tRNAMet to e... | ACCEPT | Summary: Depletion of ALKBH1 strongly reduces mitochondrial translation, via loss of f5C34 on mt-tRNA-Met. Reason: Core biological process, reproduced independently in ALKBH1-knockout cells with an accompanying respiratory-complex defect. Supporting Evidence: PMID:27497299 Depletion of either NSUN3 or ABH1 strongly affects mitochondrial translation in human cells, implying that modifications generated by both enzymes are necessary for mt-tRNAMet function. PMID:28472312 ALKBH1-knockout cells exhibited a strong reduction in mitochondrial translation and reduced respiratory complex activities, indicating that f5C34 formation mediated by ALKBH1 is required for efficient mitochondrial functions. |
| GO:1990983 regulation of translational initiation by tRNA modification | IDA PMID:27745969 ALKBH1-Mediated tRNA Demethylation Regulates Translation. | ACCEPT | Summary: Demethylation of m1A in tRNAs by ALKBH1 changes tRNA usage and attenuates translation initiation, in a glucose-responsive manner. Reason: Precise and mechanistically explicit; the best available process term for the tRNA m1A demethylase activity. Supporting Evidence: PMID:27745969 The ALKBH1-catalyzed demethylation of the target tRNAs results in attenuated translation initiation and decreased usage of tRNAs in protein synthesis. |
| GO:1990984 tRNA demethylase activity | IDA PMID:27745969 ALKBH1-Mediated tRNA Demethylation Regulates Translation. | ACCEPT | Summary: Direct demonstration that mammalian ALKBH1 demethylates N1-methyladenosine in tRNAs. Reason: Core molecular function. Corroborated independently by the increase in m1A in two mitochondrial tRNAs observed in ALKBH1-knockout cells by a different group. Supporting Evidence: PMID:27745969 Here, we show that mammalian ALKBH1 is a tRNA demethylase. It mediates the demethylation of N1-methyladenosine (m1A) in tRNAs. PMID:28472312 we found that the frequency of 1-methyladenosine (m1A) in two mitochondrial tRNAs increased in ALKBH1-knockout cells, indicating that ALKBH1 also has demethylation activity toward m1A in mt-tRNAs. |
| GO:0008198 ferrous iron binding | IDA PMID:18603530 Human AlkB homolog 1 is a mitochondrial protein that demethy... | ACCEPT | Summary: Fe(II) dependence established directly, with site-specific mutagenesis of the predicted iron- and 2-oxoglutarate-binding residues abolishing activity. Reason: Core cofactor binding; every catalytic activity of this protein depends on it. Supporting Evidence: PMID:18603530 Site-specific mutagenesis confirmed that the putative Fe(II) and 2OG binding residues are essential for activity. |
| GO:0006281 DNA repair | IDA PMID:19959401 Human AlkB homologue 1 (ABH1) exhibits DNA lyase activity at... | KEEP AS NON CORE | Summary: DNA repair role inferred from the AP lyase activity measured in vitro. Reason: Follows the same reasoning as the AP lyase molecular function - a genuine biochemical observation whose physiological role the authors explicitly leave open, and which is not supported by any cellular repair phenotype for ALKBH1. Retained as non-core. Supporting Evidence: PMID:19959401 ABH1 can cleave at closely spaced AP-sites on opposite DNA strands yielding double-strand breaks in vitro and this reaction may relate to the physiological role of this unexpected AP lyase activity. |
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Download this section (compressed HTML)Q: Does regulated N6-methyladenine exist in mammalian genomic duplex DNA at all? If nanopore or orthogonal mass-spectrometry profiling with full bacterial, RNA and salvage-pathway controls finds none, what remains of the ALKBH1 genomic-eraser assignment?
Suggested experts: Chromatin and DNA modification, Analytical nucleic acid chemistry
Q: If the bulk of genomic m6dA derives from misincorporated ribo-N6-methyladenosine via the nucleotide salvage pathway, could the apparent effect of ALKBH1 depletion on cellular 6mA levels be an indirect consequence of its RNA demethylase activity altering the m6A ribonucleotide pool, rather than DNA demethylation?
Suggested experts: Nucleotide metabolism, RNA modification
Q: Which mammalian enzyme, if any, writes genomic 6mA? Animals appear to have lost the AMT1 family, so is there a non-AMT1 writer, or is there no writer to pair with the proposed eraser?
Suggested experts: Comparative genomics, Evolution of epigenetic systems
Q: Do the neural-differentiation and placental-trophoblast phenotypes of Alkbh1-null mice arise from impaired mitochondrial translation (loss of f5C34) rather than from changes in DNA or histone methylation?
Suggested experts: Mitochondrial biology, Developmental genetics
Q: Is the histone H2A dioxygenase activity reproducible, and which residue is modified?
Suggested experts: Chromatin biochemistry
Q: Does the AP lyase activity of ALKBH1 operate in cells, and if so in which compartment - is there a role in mitochondrial DNA abasic-site processing?
Suggested experts: DNA repair, Mitochondrial genome maintenance
Experiment: Metabolically label cells with isotopically distinct adenine and N6-methyladenosine precursors, then quantify genomic m6dA by ultrasensitive LC-MS/MS in wild-type, ALKBH1-knockout and ALKBH1-overexpressing cells, distinguishing salvage-derived from in-situ-methylated species by their isotopic signature. A catalytically dead ALKBH1 mutant and an RNA-binding-deficient mutant separate the DNA and RNA hypotheses.
Hypothesis: The reported decrease in cellular genomic 6mA upon ALKBH1 overexpression reflects a change in the m6A ribonucleotide salvage pool rather than direct DNA demethylation.
Type: isotope tracing with mass spectrometry
Experiment: Reconstitute nucleosomes on 6mA-containing duplex templates and assay demethylation by purified ALKBH1 with and without purified YTHDF3, reading out by LC-MS/MS rather than antibody dot blot. Compare against matched single-stranded and bubbled substrates to quantify the conformational preference directly.
Hypothesis: ALKBH1 cannot access 6mA in nucleosomal duplex DNA even in the presence of YTHDF3.
Type: in vitro reconstitution biochemistry
Experiment: Compare an ALKBH1-null background rescued with wild-type ALKBH1, with a mitochondrially excluded (targeting-signal-deleted) ALKBH1, and with a catalytically dead variant, scoring mitochondrial translation, respiratory complex activity, neural progenitor differentiation and trophoblast marker expression. If mitochondrial targeting is necessary and sufficient for rescue, the nuclear and DNA-directed models are not needed to explain the phenotype.
Hypothesis: The organismal phenotypes of ALKBH1 loss are explained by the mitochondrial tRNA function.
Type: genetic complementation
Experiment: Apply nanopore sequencing with 6mA-aware basecalling to human cells grown under strictly axenic conditions, with spike-in controls of known 6mA stoichiometry and parallel quantification of bacterial DNA content, plus RNase treatment to exclude ribonucleotide carryover. Report a detection limit alongside any positive calls.
Hypothesis: Genomic 6mA in human cells is below the level at which any regulated function is possible.
Type: single-molecule sequencing
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