Affinage

METTL8

tRNA N(3)-cytidine methyltransferase METTL8, mitochondrial · UniProt Q9H825

Length
291 aa
Mass
33.4 kDa
Annotated
2026-06-10
16 papers in source corpus 14 papers cited in narrative 14 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 7/7 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

METTL8 is an RNA methyltransferase whose principal characterized activity is the installation of 3-methylcytidine (m3C) at position 32 of mitochondrial tRNAThr and tRNASer(UCN), a modification required for efficient mitochondrial translation and respiratory chain function (PMID:34774131, PMID:35247384). The enzyme is expressed as alternatively spliced isoforms: the mitochondria-targeted Iso1, imported via an N-terminal presequence, carries the catalytic activity, whereas the nucleolar Iso4 lacks the N-terminal extension bearing absolutely conserved modification-critical residues and is inactive for m3C (PMID:35357504, PMID:37573249). Target selection is achieved by crosslinking across the anticodon stem-loop of many mt-tRNAs, but methylation is restricted to substrates bearing the U34G35 plus t6A37/(ms2)i6A37 combination found only in mt-tRNAThr and mt-tRNASer(UCN) (PMID:35017528). Beyond catalysis, Iso1 physically interacts with the mitochondrial aminoacyl-tRNA synthetases SARS2 and TARS2 in an RNA-independent manner and stimulates their aminoacylation activity, coupling m3C deposition to charging of its own substrate tRNAs (PMID:35357504, PMID:37573249). Loss of METTL8 produces ribosome stalling on cognate codons and reduced incorporation of complex I subunits, lowering respiration (PMID:34774131, PMID:35017528). This mitochondrial function underlies physiological roles in embryonic cortical neural stem cell maintenance, where conditional deletion impairs mitochondrial translation and respiration and is rescued by pharmacological enhancement of mitochondrial function (PMID:36764294), and in cancer cell states including glioblastoma stem cells, where METTL8 supports the HIF1α–RTK/Akt axis (PMID:38744809). Additional reported activities include m3C-dependent regulation of specific mRNAs to control their translation or stability (ARID1A, Tcf7) and chromatin-associated functions (PMID:34063990, PMID:41891923), and an earlier-described nuclear RNA-binding complex involved in R-loop stabilization (PMID:32199293).

Mechanistic history

Synthesis pass · year-by-year structured walk · 8 steps
  1. 2018 Medium

    Before any catalytic role was known, METTL8 was placed in a transcriptional and translational regulatory circuit, establishing that it can repress translation of a specific mRNA to influence stem cell fate.

    Evidence STAT3 ChIP/reporter, RNA-IP of Mapkbp1 mRNA, and JNK pathway readout in mouse ESC KO/OE cells

    PMID:29706498

    Open questions at the time
    • No demonstration of m3C or methyltransferase activity in this context
    • Mechanism linking METTL8 binding to translational repression of Mapkbp1 not resolved
    • Single-lab study
  2. 2020 Medium

    An initial biochemical characterization assigned METTL8 to a large nuclear RNA-binding complex and linked its methyltransferase activity to R-loop stabilization, raising the question of a chromatin-associated role.

    Evidence Biochemical fractionation, pulldown, and R-loop quantification after knockout

    PMID:32199293

    Open questions at the time
    • Relationship to the later-defined mitochondrial isoform unclear
    • Identity of complex members and direct RNA targets not fully resolved
    • m3C-dependence of R-loop effect not mapped to specific sites
  3. 2021 High

    The core enzymatic function was defined: METTL8 is a mitochondrial enzyme installing m3C32 on mt-tRNAThr and mt-tRNASer(UCN), connecting the modification to respiratory chain translation.

    Evidence KO/OE cell lines, mitoribosome profiling, mass spectrometry of respiratory complexes, m3C detection

    PMID:34774131

    Open questions at the time
    • Substrate recognition determinants not yet defined
    • Isoform basis of mitochondrial targeting not established at this stage
  4. 2022 High

    The isoform logic, substrate-recognition code, and synthetase coupling were resolved, explaining how METTL8 achieves target specificity and is functionally integrated with tRNA charging.

    Evidence Alternative splicing/localization analysis, in vitro methylation with tRNA mutagenesis, crosslinking to mt-tRNA ASLs, native gel and aminoacylation assays, Co-IP with SARS2

    PMID:35017528 PMID:35247384 PMID:35357504

    Open questions at the time
    • Functional consequence of SARS2 interaction is modest and incompletely defined
    • How m3C32 alters tRNA folding mechanistically not fully resolved
    • Reason m3C-deficient tRNAs still aminoacylate and bind ribosomes yet impair translation unclear
  5. 2023 High

    The catalytic determinants and an in vivo physiological role were established: the N-terminal extension carries the modification-critical residues, METTL8 stimulates both SARS2 and TARS2 aminoacylation, and its mitochondrial activity is required for cortical neural stem cell maintenance.

    Evidence In vitro m3C assays with N-extension mutagenesis, aminoacylation assays with SARS2/TARS2, conditional KO mice with respiration and NSC quantification, pharmacological rescue, human organoids

    PMID:36764294 PMID:37573249

    Open questions at the time
    • Mechanism by which reduced mitochondrial translation impairs NSC maintenance not fully delineated
    • Whether non-mitochondrial activities contribute to the phenotype untested
  6. 2024 Medium

    METTL8's mitochondrial activity was tied to oncogenic signaling, placing it upstream of HIF1α and the RTK/Akt axis in glioblastoma stem cells.

    Evidence Subcellular fractionation, m3C detection, knockdown with HIF1α/RTK/Akt analysis, intracranial xenografts, H2AZ chromatin accessibility assay

    PMID:38744809

    Open questions at the time
    • Causal chain from mitochondrial translation to HIF1α protein levels not mechanistically dissected
    • Single-lab finding
  7. 2025 Medium

    Reported substrate scope expanded toward specific mRNAs, with m3C-dependent control of Tcf7 mRNA stability and a Tcf1-interaction-driven chromatin function maintaining T cell stemness, plus a preprint claim of mt-mRNA modification.

    Evidence Murine Mettl8 deletion, m3C modification of Tcf7 mRNA, Co-IP with Tcf1, chromatin looping at Tox; transcriptome-wide m3C mapping of mt-mRNAs (preprint)

    PMID:41891923 PMID:bio_10.1101_2025.01.15.633161

    Open questions at the time
    • mRNA m3C substrate claims rest largely on single labs and a preprint
    • Direct catalysis vs indirect effects on these mRNAs not fully separated from the mitochondrial role
    • mt-mRNA modification not peer-reviewed or replicated
  8. 2026 Medium

    A further signaling link was reported, with METTL8 negatively regulating the Akt/mTOR/4E-BP1 pathway to restrain neural stem cell differentiation.

    Evidence KD/OE in hippocampal NSCs, proliferation and cell-cycle assays, RNA-seq, phospho-Western, rapamycin rescue

    PMID:42178900

    Open questions at the time
    • Molecular connection between METTL8 catalytic activity and mTOR pathway control not defined
    • Whether effect is mitochondrial or mRNA-mediated unresolved

Open questions

Synthesis pass · forward-looking unresolved questions
  • It remains unresolved how METTL8's non-tRNA activities (mRNA m3C, R-loop regulation, chromatin/Tcf1 functions) mechanistically relate to its mitochondrial catalytic role, and whether they represent isoform-specific or moonlighting functions.
  • No unified model integrating mitochondrial vs nuclear/cytoplasmic activities
  • Direct mRNA substrates and their methylation sites not comprehensively validated
  • No human disease linkage established in the corpus

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0016740 transferase activity 5 GO:0003723 RNA binding 4 GO:0140098 catalytic activity, acting on RNA 4
Localization
GO:0005739 mitochondrion 5 GO:0005730 nucleolus 2 GO:0005634 nucleus 1
Pathway
R-HSA-8953854 Metabolism of RNA 4 R-HSA-392499 Metabolism of proteins 3
Partners
SARS2TARS2TCF7

Evidence

Reading pass · 14 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2021 METTL8 is a mitochondrial protein that installs 3-methylcytidine (m3C) at position C32 of mt-tRNASer(UCN) and mt-tRNAThr. METTL8 knockout reduces respiratory chain activity while overexpression increases it. Mitoribosome profiling revealed stalling on mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons in knockout cells, and mass spectrometry showed reduced incorporation of ND6 and ND1 into complex I. Knockout/overexpression cell lines, mitochondrial ribosome profiling, mass spectrometry of respiratory chain complexes, m3C detection Molecular cell High 34774131
2022 METTL8 exists as alternatively spliced isoforms: METTL8-Iso1 is targeted to mitochondria via an N-terminal pre-sequence and catalyzes m3C32 on mt-tRNAThr and mt-tRNASer(UCN), while METTL8-Iso4 localizes to the nucleolus. Substrate specificity of Iso1 for mt-tRNAThr requires G35 but not t6A37, while mt-tRNASer(UCN) modification critically depends on i6A37. METTL8-Iso1 interacts with mitochondrial seryl-tRNA synthetase (SARS2) in an RNA-independent manner, which modestly accelerates m3C modification activity. Alternative splicing analysis, subcellular fractionation/localization, in vitro methylation assays, mutagenesis of tRNA recognition elements, Co-IP Nucleic acids research High 35357504
2022 METTL8 is a mitochondria-associated protein required for m3C formation in human mt-tRNA-Ser-UGA and mt-tRNA-Thr-UGU but not nuclear-encoded tRNAs. METTL8 interacts with mitochondrial seryl-tRNA synthetase and mt-tRNAs. Re-expression of WT METTL8 rescues m3C loss, but a variant lacking the N-terminal mitochondrial localization signal does not. Loss of METTL8 alters native migration pattern of mt-tRNA-Ser-UGA, suggesting m3C influences tRNA folding. METTL8-deficient human cells, m3C detection by sequencing, Co-IP with mitochondrial seryl-tRNA synthetase, rescue with MLS-deletion variant, native gel analysis of tRNA migration The Journal of biological chemistry High 35247384
2022 METTL8 crosslinks to the anticodon stem loop (ASL) of many mt-tRNAs, but methylation target specificity is determined by U34G35 combined with t6A37/(ms2)i6A37, present only in mt-tRNAThr and mt-tRNASer(UCN). m3C32 modification influences the structure of these mt-tRNAs, though mt-tRNAs lacking m3C32 are still efficiently aminoacylated and associate with mitochondrial ribosomes. Mitochondrial translation is mildly impaired without METTL8. METTL8 crosslinking to mt-tRNAs, dissection of tRNA recognition elements by mutagenesis, structural probing of mt-tRNAs, aminoacylation assays, mitoribosome association assays, translation assays in METTL8 KO cells Nature communications High 35017528
2023 Mettl8-Iso4 (nucleolar isoform) is catalytically inactive for m3C32 generation due to lack of the N-terminal extension (N-extension), which contains absolutely conserved modification-critical residues. These residues are also essential for cytoplasmic m3C32 by METTL2A and yeast Trm140. METTL8-Iso1 can modify several cytoplasmic or bacterial tRNAs in vitro. METTL8-Iso1 also interacts with mitochondrial threonyl-tRNA synthetase (TARS2) in addition to SARS2, and substantially stimulates aminoacylation activities of both SARS2 and TARS2 in vitro. In vitro m3C32 methylation assays with Iso1 and Iso4, mutagenesis of N-extension residues, cross-species tRNA modification assays, Co-IP with TARS2, aminoacylation activity assays Science bulletin High 37573249
2020 METTL8 forms a large SUMOylated nuclear RNA-binding protein complex (~0.8 megadaltons) containing R-loop-related factors in the nucleus. Genetic ablation of METTL8 results in overall reduction of R-loops in cells. METTL8 binds to RNAs and stabilizes R-loops on selected gene regions through its methyltransferase activity on m3C. Biochemical fractionation, pulldown/interaction assays, R-loop quantification after METTL8 knockout, RNA-binding assays iScience Medium 32199293
2018 METTL8 is a STAT3 transcriptional target in mouse ESCs. METTL8 interacts with Mapkbp1 mRNA (an intermediate in JNK signaling) and inhibits translation of that mRNA, thereby suppressing JNK pathway activation and enhancing ESC differentiation. STAT3 ChIP/reporter assays, RNA-IP of Mapkbp1 mRNA, translation assays, JNK pathway activity measurement in METTL8 KO/OE cells Stem cell reports Medium 29706498
2023 Mettl8 is localized in mitochondria of mouse embryonic cortical neural stem cells and installs m3C specifically on mitochondrial tRNAThr/Ser(UCN). Conditional Mettl8 deletion reduces mitochondrial protein translation and respiration activity, leading to impaired embryonic cortical neural stem cell maintenance in vivo. Pharmacological enhancement of mitochondrial function rescues the neural stem cell maintenance defect caused by Mettl8 loss. Conditional knockout in mice, mitochondrial protein translation assays, respiration measurement, neural stem cell quantification in vivo, pharmacological rescue, human forebrain cortical organoids Cell stem cell High 36764294
2021 METTL8 expression is regulated by transcription factor YY1 in breast cancer cells. METTL8 protein directly binds ARID1A mRNA, and METTL8 knockdown increases ARID1A protein levels without changing ARID1A mRNA levels (suggesting translational repression). METTL8 knockdown strongly blocks tumor cell migration. YY1 knockdown with METTL8 expression measurement, RNA-IP of ARID1A mRNA, METTL8 knockdown with ARID1A protein/mRNA quantification, cell migration assays International journal of molecular sciences Medium 34063990
2024 In glioblastoma stem cells (GSCs), METTL8 is exclusively localized to the mitochondrial matrix where it installs m3C on mt-tRNAThr/Ser(UCN) for mitochondrial translation and respiration. METTL8 depletion decreases HIF1α protein levels, which reduces transcription of RTK genes and inactivates the RTK/Akt signaling axis. High METTL8 expression in GBM is attributed to histone variant H2AZ-mediated chromatin accessibility of HIF1α. Subcellular fractionation, m3C detection, METTL8 KD with HIF1α/RTK/Akt pathway analysis, intracranial xenograft model, chromatin accessibility assay (H2AZ) Cell death & disease Medium 38744809
2025 Mettl8 stabilizes Tcf7 mRNA via m3C modification, enhancing Tcf1 protein expression in CD8+ TPEX cells. Additionally, Mettl8 interacts with Tcf1 protein to facilitate chromatin looping at the Tox locus, maintaining TPEX stemness. Mettl8 deletion drives TPEX differentiation into effective Int-TEX cells and restrains tumor progression. Mettl8 deletion in murine models, m3C modification of Tcf7 mRNA, Co-IP of Mettl8-Tcf1, chromatin conformation assay at Tox locus, T cell subset analysis, pharmacological inhibition The Journal of experimental medicine Medium 41891923
2026 Mettl8 negatively regulates the Akt/mTOR/4E-BP1 pathway in hippocampal neural stem cells. Mettl8 knockdown activates this pathway (increased mTOR/4E-BP1 phosphorylation), induces G0/G1 arrest, and promotes neuronal and astrocytic differentiation markers. Rapamycin (mTOR inhibitor) reverses the enhanced mTOR/4E-BP1 phosphorylation and neuronal differentiation caused by Mettl8 loss. Lentiviral KD/OE in NSCs, EdU proliferation assay, flow cytometry cell cycle analysis, RNA-seq, Western blot of mTOR/4E-BP1 phosphorylation, rapamycin rescue CNS neuroscience & therapeutics Medium 42178900
2025 METTL8 catalyzes m3C modification on mitochondrial mRNAs (mt-mRNAs), particularly those encoding complex I subunits, in addition to its known mt-tRNA activity. METTL8 depletion impairs cell migration in vitro and reduces tumor growth in mouse xenografts. Transcriptome-wide m3C mapping, METTL8 depletion with mt-mRNA m3C quantification, cell migration assays, mouse xenograft tumor growth assays bioRxivpreprint Low bio_10.1101_2025.01.15.633161
2022 miR-208b directly targets and inhibits Mettl8 expression (confirmed by dual luciferase assay). Mettl8 knockdown in C2C12 cells increases Myh7 (slow-twitch) and decreases Myh4 (fast-twitch) expression, indicating Mettl8 promotes fast muscle fiber formation. Mettl8 knockout in mice inhibits formation of fast muscle fibers. Dual luciferase assay confirming miR-208b targeting of Mettl8 3'UTR, siRNA knockdown of Mettl8 in C2C12 cells with myosin heavy chain isoform analysis, Mettl8 knockout mice Frontiers in genetics Low 35281804

Source papers

Stage 0 corpus · 16 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2021 Balancing of mitochondrial translation through METTL8-mediated m3C modification of mitochondrial tRNAs. Molecular cell 73 34774131
2020 The SUMOylated METTL8 Induces R-loop and Tumorigenesis via m3C. iScience 52 32199293
2017 Frameshift Mutations in Repeat Sequences of ANK3, HACD4, TCP10L, TP53BP1, MFN1, LCMT2, RNMT, TRMT6, METTL8 and METTL16 Genes in Colon Cancers. Pathology oncology research : POR 47 28803425
2022 The RNA methyltransferase METTL8 installs m3C32 in mitochondrial tRNAsThr/Ser(UCN) to optimise tRNA structure and mitochondrial translation. Nature communications 44 35017528
2023 Epitranscriptomic regulation of cortical neurogenesis via Mettl8-dependent mitochondrial tRNA m3C modification. Cell stem cell 37 36764294
2022 Molecular basis for human mitochondrial tRNA m3C modification by alternatively spliced METTL8. Nucleic acids research 35 35357504
2022 Methyltransferase METTL8 is required for 3-methylcytosine modification in human mitochondrial tRNAs. The Journal of biological chemistry 27 35247384
2022 MiR-208b Regulates the Conversion of Skeletal Muscle Fiber Types by Inhibiting Mettl8 Expression. Frontiers in genetics 15 35281804
2018 The STAT3 Target Mettl8 Regulates Mouse ESC Differentiation via Inhibiting the JNK Pathway. Stem cell reports 15 29706498
2023 Mitochondrial RNA m3C methyltransferase METTL8 relies on an isoform-specific N-terminal extension and modifies multiple heterogenous tRNAs. Science bulletin 14 37573249
2021 METTL8 mRNA Methyltransferase Enhances Cancer Cell Migration via Direct Binding to ARID1A. International journal of molecular sciences 14 34063990
2024 METTL8 links mt-tRNA m3C modification to the HIF1α/RTK/Akt axis to sustain GBM stemness and tumorigenicity. Cell death & disease 12 38744809
2021 METTLing in the right place: METTL8 is a mitochondrial tRNA-specific methyltransferase. Molecular cell 8 34861186
2024 Targeting METTL8 with Rabdosiin overcomes lenvatinib resistance in hepatocellular carcinoma. Experimental cell research 4 39701355
2026 Targeting Mettl8-Tcf1 axis promotes CD8+ TPEX differentiation and antitumor immunity. The Journal of experimental medicine 1 41891923
2026 Mettl8 Regulates Hippocampal Neural Stem Cell Proliferation and Neurogenesis via mTOR/4E-BP1 Signaling. CNS neuroscience & therapeutics 0 42178900

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