Affinage

ALKBH8

tRNA (carboxymethyluridine(34)-5-O)-methyltransferase ALKBH8 · UniProt Q96BT7

Length
664 aa
Mass
75.2 kDa
Annotated
2026-04-28
20 papers in source corpus 15 papers cited in narrative 15 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

ALKBH8 is a bifunctional tRNA-modifying enzyme that regulates codon-specific translation elongation by installing wobble uridine (U34) modifications on select tRNAs. Its methyltransferase domain, activated by the obligate partner TRM112, catalyzes the conversion of cm5U to mcm5U—a prerequisite for downstream thiolation (mcm5s2U) and 2'-O-ribose methylation—while its AlkB oxygenase domain hydroxylates mcm5U to (S)-mchm5U specifically on tRNA-Gly(UCC) (PMID:20123966, PMID:21285950, PMID:26438534). These modifications promote efficient decoding of adenine-ending codons, thereby controlling translation of codon-biased mRNAs including selenoprotein mRNAs (via tRNASec modification to support UGA recoding) and oncogenes such as KRAS, whose translation depends on ALKBH8 catalytic activity downstream of Wnt/β-catenin transcriptional activation (PMID:20123966, PMID:32303148, PMID:41083459). Loss-of-function mutations in the ALKBH8 methyltransferase domain cause intellectual disability (MRT71) through complete abrogation of ALKBH8-dependent tRNA modifications (PMID:34757492).

Mechanistic history

Synthesis pass · year-by-year structured walk · 10 steps
  1. 2007 High

    Establishing that wobble uridine methylation by the ALKBH8 ortholog Trm9 has a translational output—it selectively enhances expression of codon-biased proteins involved in the DNA damage response—moved the modification from a biochemical curiosity to a gene-expression regulatory mechanism.

    Evidence Yeast trm9Δ deletion with tRNA modification analysis, codon usage computation, and western blot for target proteins

    PMID:18082610

    Open questions at the time
    • Whether the same codon-specific translational control operates in mammalian systems was untested
    • Direct ribosome-level evidence of elongation rate change was lacking
  2. 2010 High

    Generation of Alkbh8-knockout mice demonstrated that ALKBH8 is the mammalian enzyme responsible for the cm5U-to-mcm5U methylation step, that TRM112 is its obligate activating partner, and that loss of this modification disrupts selenoprotein translation by impairing tRNASec modification.

    Evidence Alkbh8−/− mouse, HPLC/MS tRNA modification profiling, in vitro methyltransferase assay, co-immunoprecipitation of ALKBH8–TRM112

    PMID:20123966

    Open questions at the time
    • Function of the AlkB oxygenase domain remained unknown
    • Whether translational fidelity is affected in mammalian cells was not assessed
  3. 2011 High

    Identification of the AlkB domain as an RNA hydroxylase that converts mcm5U to (S)-mchm5U on tRNA-Gly(UCC) revealed that ALKBH8 is a true bifunctional enzyme and expanded the ALKBH family beyond DNA repair functions.

    Evidence In vitro hydroxylation assay with recombinant AlkB domain, mass spectrometry, validation in Alkbh8−/− mouse tRNA

    PMID:21285950

    Open questions at the time
    • Biological consequence of the hydroxylation product (S)-mchm5U was not defined
    • No structural information for the mammalian enzyme
  4. 2012 High

    Demonstrating that loss of Trm9-dependent modifications causes translational infidelity and triggers the unfolded protein response established that these wobble modifications are required for accurate decoding, not merely for efficiency.

    Evidence Translational fidelity reporters and phenotypic assays in trm9Δ yeast

    PMID:22832247

    Open questions at the time
    • Whether translational infidelity occurs in mammalian ALKBH8-null cells was unknown
    • Specific miscoded codons in vivo were not fully mapped
  5. 2015 High

    The crystal structure of the Trm9–Trm112 complex revealed how TRM112 serves as a structurally plastic activating platform shared among multiple methyltransferases, explaining the obligate requirement for this partner.

    Evidence X-ray crystallography with structure-guided mutagenesis

    PMID:26438534

    Open questions at the time
    • No structure of full-length mammalian ALKBH8 including the AlkB domain
    • How substrate tRNA is recognized by the complex was not resolved
  6. 2020 Medium

    Linking ALKBH8-dependent selenoprotein translation to an in vivo oxidative stress phenotype established that wobble uridine modification is physiologically required for antioxidant defense, specifically through thioredoxin reductase expression.

    Evidence Alkbh8−/− mice challenged with naphthalene; western blot for thioredoxin reductase and oxidative stress markers

    PMID:32303148

    Open questions at the time
    • Which specific selenoproteins are most affected was not comprehensively profiled
    • Single-lab study without independent replication
  7. 2021 Medium

    Patient-derived evidence that a missense variant in the ALKBH8 methyltransferase domain abolishes all ALKBH8-dependent tRNA modifications without reducing protein levels established enzymatic loss-of-function as the disease mechanism for MRT71 intellectual disability.

    Evidence Patient cell analysis with mass spectrometry-based tRNA modification profiling and targeted proteomics

    PMID:34757492

    Open questions at the time
    • Only a single variant/family was studied
    • Neuronal-specific consequences of modification loss were not mechanistically dissected
  8. 2022 Medium

    Transcriptome-wide binding studies revealed that ALKBH8 directly binds its known substrate tRNAs in their mature CCA-modified form and also associates with C/D box snoRNAs, suggesting possible non-tRNA functions.

    Evidence HITS-CLIP and RIP-seq in human cells

    PMID:36192131

    Open questions at the time
    • Functional significance of snoRNA interaction is unknown
    • Whether snoRNA binding reflects a catalytic or regulatory role is unresolved
  9. 2024 Medium

    Comprehensive phenotyping of Alkbh8−/− mice revealed tissue-specific compensation of tRNA modification (partial rescue in brain), impaired erythropoiesis, and neural dysfunction with reduced mitochondrial membrane potential, connecting wobble uridine modification to mitochondrial and neurodevelopmental physiology.

    Evidence Alkbh8−/− mouse model with UPLC-MS/MS tRNA modification profiling, proteomics, behavioral testing, and mitochondrial assays

    PMID:38550277 PMID:39280612

    Open questions at the time
    • Mechanism of tissue-specific compensation is unknown
    • Identity of the compensating enzyme(s) in brain is not established
  10. 2025 High

    Identifying ALKBH8 as a direct Wnt/β-catenin target gene whose methyltransferase activity specifically promotes KRAS translation through codon-dependent ribosome elongation provided the first mechanistic link between an epitranscriptomic modifier and oncogenic signaling in colorectal cancer.

    Evidence CRISPR knockout, ribosome profiling showing ribosome pausing at A-ending codons, rescue with catalytically dead mutant, Apcmin/+ and AOM/DSS mouse tumor models

    PMID:41083459

    Open questions at the time
    • Whether other oncogenes with similar codon bias are co-regulated was not systematically addressed
    • Therapeutic targetability of ALKBH8 catalytic activity is untested

Open questions

Synthesis pass · forward-looking unresolved questions
  • Key unresolved questions include the biological function of ALKBH8's hydroxylation product (S)-mchm5U, the mechanism of substrate tRNA recognition by the full-length mammalian enzyme, the functional significance of snoRNA interactions, and the basis for tissue-specific compensation of tRNA modification loss.
  • No structure of full-length mammalian ALKBH8
  • Biological role of (S)-mchm5U hydroxylation uncharacterized
  • snoRNA interaction function unknown
  • Tissue-specific compensation mechanism undefined

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0016740 transferase activity 6 GO:0003723 RNA binding 2 GO:0016491 oxidoreductase activity 2
Localization
GO:0005829 cytosol 1
Pathway
R-HSA-8953854 Metabolism of RNA 5 R-HSA-392499 Metabolism of proteins 4 GO:0003723 RNA binding 1 R-HSA-162582 Signal Transduction 1
Partners
TRM112
Complex memberships
ALKBH8–TRM112 methyltransferase complex

Evidence

Reading pass · 15 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2010 ALKBH8 is a tRNA methyltransferase required for the final step in biogenesis of 5-methoxycarbonylmethyluridine (mcm5U) at the wobble position of tRNA. Interaction with the small accessory protein TRM112 is required to form a functional tRNA methyltransferase complex. Prior ALKBH8-mediated methylation is a prerequisite for subsequent thiolation (mcm5s2U) and 2'-O-ribose methylation (mcm5Um). Loss of these modifications in Alkbh8-/- mice causes aberrant modification of selenocysteine tRNA (tRNASec) and reduced UGA stop codon recoding to selenocysteine for Gpx1. Alkbh8-/- mouse knockout, tRNA modification analysis (HPLC/MS), in vitro methyltransferase assay, co-immunoprecipitation of ALKBH8-TRM112 complex Molecular and cellular biology High 20123966
2011 The AlkB oxygenase domain of ALKBH8 specifically hydroxylates mcm5U to (S)-mchm5U in tRNA-Gly(UCC), generating a novel diastereomeric pair of wobble nucleosides. The mammalian ALKBH8 AlkB domain acts as an RNA hydroxylase rather than a DNA repair enzyme, expanding ALKBH oxygenase function beyond nucleic acid repair. In vitro hydroxylation assay with recombinant ALKBH8 AlkB domain, tRNA modification analysis by mass spectrometry, in vivo analysis using Alkbh8-/- mouse tRNA Nature communications High 21285950
2011 Trm9 (yeast ortholog of ALKBH8) and Trm112 physically interact and function together as a complex for the final methylation step (cm5U → mcm5U) in wobble uridine modification. Co-expression of His-tagged Trm9 with native Trm112 in E. coli yielded a purified complex, and Trm112 dramatically improves Trm9 methyltransferase activity in vitro. Co-expression and co-purification from E. coli, in vitro methyltransferase assay, tRNA modification analysis by HPLC from trm9Δ and trm112Δ yeast strains PloS one High 21687733
2015 Crystal structure of the Trm9-Trm112 complex reveals the structural basis for mcm5U modification of tRNA anticodon wobble position. Trm112 acts as an obligate activating platform, interacting with Trm9 through a structurally plastic interface that is shared across multiple Trm112-methyltransferase complexes despite low sequence identity among partners. X-ray crystallography, structure-function analysis with mutagenesis Nucleic acids research High 26438534
2007 Yeast Trm9 (ALKBH8 ortholog) methylates the wobble uridine of tRNAARG(UCU) and tRNAGLU(UUC), and this modification enhances codon-specific translation elongation, promoting increased protein levels of DNA damage response proteins (Yef3, Rnr1, Rnr3) that are enriched in cognate arginine and glutamic acid codons. Genetic deletion (trm9Δ), tRNA modification analysis, reporter assays, protein level measurement by western blot, computational codon usage analysis Molecular cell High 18082610
2012 Trm9-catalyzed wobble uridine modifications (mcm5U and mcm5s2U) are required for translational fidelity; loss of these two modifications in trm9Δ yeast causes translational errors at specific arginine and glutamic acid codons, leading to protein misfolding and activation of unfolded protein and heat shock responses. Phenotypic assays, translational fidelity reporters, quantitative tRNA modification analysis (HPLC), protein-based fidelity assays in trm9Δ yeast, codon reengineering RNA biology High 22832247
2009 ALKBH8 silencing in bladder cancer cells reduces ROS production via downregulation of NOX-1 and induces apoptosis through activation of JNK and p38, which cause γH2AX phosphorylation. ALKBH8 knockdown also suppresses invasion and angiogenesis in vivo. siRNA knockdown, ROS measurement, western blot for pathway components (JNK, p38, γH2AX, NOX-1), chorioallantoic membrane assay, orthotopic mouse model Cancer research Medium 19293182
2016 ALKBH8 promotes bladder cancer cell survival by maintaining protein expression of survivin (an anti-apoptotic factor); ALKBH8 knockdown induces apoptosis via downregulation of survivin, and ALKBH8 transgenic mice show accelerated bladder tumor growth and invasiveness. siRNA knockdown, western blot for survivin, transgenic mouse model with carcinogen-induced bladder cancer (N-butyl-N-(4-hydroxybutyl)-nitrosamine) Biochemical and biophysical research communications Medium 27329810
2014 Protozoan ALKBH8 proteins display dual activity: DNA repair (dealkylation) and tRNA wobble uridine hydroxylation (mcm5U modification in tRNAGly(UCC)), demonstrating functional duality within the ALKBH8 family. Bacterial ALKBH8 shows DNA repair activity in vitro but does not modify tRNAGly(UCC) wobble uridine in vivo. In vitro DNA repair assay, in vitro tRNA modification assay, in vivo tRNA modification analysis in Agrobacterium ALKBH8 mutant PloS one Medium 24914785
2021 A missense variant in the ALKBH8 methyltransferase domain causes complete absence of ALKBH8-dependent tRNA modifications in patient cells (confirmed by targeted proteomics), establishing that loss of methyltransferase activity is the disease mechanism for MRT71 intellectual disability, without loss of ALKBH8 protein expression. Patient cell analysis, mass spectrometry-based detection of tRNA modifications, targeted proteomics of ALKBH8 protein levels Human genetics Medium 34757492
2022 HITS-CLIP and RIP-seq analysis demonstrates that ALKBH8 binds fully processed, CCA-modified substrate tRNAs (the known wobble U-containing tRNAs) and also interacts with several noncoding RNAs, particularly C/D box snoRNAs, in human cells. HITS-CLIP, RIP-seq RNA (New York, N.Y.) Medium 36192131
2020 ALKBH8 modifies the wobble uridine of selenocysteine tRNA to promote selenoprotein translation; Alkbh8-deficient mice show increased oxidative stress markers and decreased thioredoxin reductase protein levels under basal conditions, and fail to develop naphthalene tolerance, establishing ALKBH8 as protective against oxidative lung damage via selenoprotein-dependent antioxidant mechanisms. Alkbh8-/- mouse model, western blot for oxidative stress markers and thioredoxin reductase, naphthalene exposure challenge experiments Epigenetics Medium 32303148
2024 In Alkbh8-/- mice, mcm5U modification is reduced in most tissues but partially compensated in the brain. Loss of ALKBH8 reduces tRNA protein translation efficiency, impairs red blood cell differentiation and embryogenesis (shown by proteome analysis linking downregulated factors to red blood cell and protoporphyrin metabolism), and causes neural dysfunction with oxidative stress and reduced mitochondrial membrane potential in neurons and glial cells. Alkbh8-/- mouse model, UPLC-MS/MS for tRNA modification quantification, proteomics, behavioral tests (novel object recognition, rotarod, forced swim), brain histopathology, mitochondrial membrane potential assay iScience / PNAS nexus Medium 38550277 39280612
2025 ALKBH8 is a direct transcriptional target of Wnt/β-catenin signaling. Its methyltransferase activity promotes translation elongation at adenine-ending codons by modifying U34 tRNA, specifically regulating KRAS translation in a codon-dependent manner. Rescue experiments with methyltransferase-dead ALKBH8 fail to restore KRAS translation, confirming catalytic activity requirement. Loss of ALKBH8 induces ribosome pausing at adenine-ending codons. CRISPR knockout, ribosome profiling (ribosome pausing analysis), rescue with wildtype vs. catalytically-dead ALKBH8 mutant, Apcmin/+ and AOM/DSS mouse tumor models, xenograft, reporter assays Nature communications High 41083459
2025 During ZIKV infection, ALKBH8 is required for mcm5s2U34 tRNA modification; CRISPR/shRNA-mediated knockdown of ALKBH8 significantly reduces ZIKV replication, indicating that ALKBH8-mediated U34 tRNA modification is exploited by the virus to optimize translation of A-ending codon-biased viral proteins. CRISPR/Cas9 and shRNA knockdown of ALKBH8, mass spectrometry for tRNA modification quantification, codon-biased GFP reporters, viral replication assays bioRxiv (preprint)preprint Medium

Source papers

Stage 0 corpus · 20 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2007 Trm9-catalyzed tRNA modifications link translation to the DNA damage response. Molecular cell 259 18082610
2010 Mammalian ALKBH8 possesses tRNA methyltransferase activity required for the biogenesis of multiple wobble uridine modifications implicated in translational decoding. Molecular and cellular biology 199 20123966
2011 ALKBH8-mediated formation of a novel diastereomeric pair of wobble nucleosides in mammalian tRNA. Nature communications 138 21285950
2009 A novel human AlkB homologue, ALKBH8, contributes to human bladder cancer progression. Cancer research 130 19293182
2006 tRNAGlu wobble uridine methylation by Trm9 identifies Elongator's key role for zymocin-induced cell death in yeast. Molecular microbiology 88 16390459
2012 Translational infidelity-induced protein stress results from a deficiency in Trm9-catalyzed tRNA modifications. RNA biology 83 22832247
2011 Unexpected accumulation of ncm(5)U and ncm(5)S(2) (U) in a trm9 mutant suggests an additional step in the synthesis of mcm(5)U and mcm(5)S(2)U. PloS one 59 21687733
2011 Roles of Trm9- and ALKBH8-like proteins in the formation of modified wobble uridines in Arabidopsis tRNA. Nucleic acids research 45 21653555
2015 Insights into molecular plasticity in protein complexes from Trm9-Trm112 tRNA modifying enzyme crystal structure. Nucleic acids research 35 26438534
2016 ALKBH8 promotes bladder cancer growth and progression through regulating the expression of survivin. Biochemical and biophysical research communications 34 27329810
2014 Protozoan ALKBH8 oxygenases display both DNA repair and tRNA modification activities. PloS one 27 24914785
2021 Neurodevelopmental disorder in an Egyptian family with a biallelic ALKBH8 variant. American journal of medical genetics. Part A 24 33544954
2020 The epitranscriptomic writer ALKBH8 drives tolerance and protects mouse lungs from the environmental pollutant naphthalene. Epigenetics 18 32303148
2021 Insight into ALKBH8-related intellectual developmental disability based on the first pathogenic missense variant. Human genetics 15 34757492
2024 ALKBH8 contributes to neurological function through oxidative stress regulation. PNAS nexus 11 38550277
2023 ALKBH8 as a potential N6 -methyladenosine (m6 A) eraser in insects. Insect molecular biology 10 37119026
2024 RNA-modifying enzyme Alkbh8 is involved in mouse embryonic development. iScience 5 39280612
2022 HITS-CLIP analysis of human ALKBH8 reveals interactions with fully processed substrate tRNAs and with specific noncoding RNAs. RNA (New York, N.Y.) 5 36192131
2024 The first Turkish family with a novel biallelic missense variant of the ALKBH8 gene: A study on the clinical and variant spectrum of ALKBH8-related intellectual developmental disorders. American journal of medical genetics. Part A 4 38189198
2025 ALKBH8-mediated codon-specific translation promotes colorectal tumorigenesis. Nature communications 0 41083459