Catalytic subunit of the nuclear tRNA m1A58 methyltransferase. TRMT61A is a class I (Rossmann-fold) S-adenosyl-L-methionine-dependent methyltransferase that transfers a methyl group to N1 of adenosine 58 in the T-loop of cytosolic tRNAs, a modification conserved across bacteria, archaea and eukaryotes that stabilises initiator methionyl-tRNA and is required in human tRNA3(Lys) for correct termination of plus-strand strong-stop DNA synthesis during HIV-1 reverse transcription. It has no activity alone. The functional enzyme is a heterotetramer of two TRMT61A and two TRMT6 chains, in which TRMT6, a catalytically dead paralogue, binds the substrate tRNA and, acting in trans across the dimer interface, splays the T- and D-loops apart to deliver the otherwise buried A58 into the TRMT61A active site. Substrate recognition is therefore structural rather than sequence-based. The complex has also been reported to deposit m1A on a small number of cytosolic mRNAs that fold into tRNA T-loop-like elements, at low stoichiometry, and this mRNA substrate class is disputed. Biochemical validation of transcriptome-wide maps indicates that most mapped internal mRNA m1A sites are instead inosine produced by A-to-I editing, and that genuine mRNA m1A is restricted to a handful of sites.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005634 nucleus | IBA GO_REF:0000033 | ACCEPT | Summary: The tRNA m1A58 methyltransferase acts in the nucleus, where tRNA maturation occurs; the phylogenetic assertion is consistent with the yeast and human data. Reason: Correct compartment for the activity. Consistent with the direct HPA immunofluorescence localisation of the partner subunit TRMT6 to the nucleoplasm and with UniProt's subcellular location assignment for both chains. |
| GO:0160107 tRNA (adenine(58)-N1)-methyltransferase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic assertion of the family's defining catalytic activity, matching the direct human biochemistry. Reason: Sound node placement. The TRM61 family is deeply conserved and the activity is experimentally established in budding yeast, in fission yeast (PomBase SPAC9G1.12), in Mycobacterium (UniProtKB Q96FX7 aside, UniProtKB P9WFZ1) and in human. TRMT61A appearing in its own WITH/FROM list is expected and correct. It has an EXP annotation for this term, which is one of the descendant evidences the PAINT curator used to place the IBD, and it marks that experimental grounding exists on the target itself rather than indicating circularity. Supporting Evidence: PMID:16043508 Stable hTrm6p/hTrm61p complexes purified from yeast maintained tRNA m(1)A Mtase activity in vitro. |
| GO:0030488 tRNA methylation | IBA GO_REF:0000033 | ACCEPT | Summary: The biological process corresponding to the enzyme's catalytic activity. Reason: Core process, correct and appropriately specific. |
| GO:0031515 tRNA (m1A) methyltransferase complex | IBA GO_REF:0000033 | ACCEPT | Summary: TRMT61A is an obligate subunit of the TRMT6-TRMT61A heterotetramer. Reason: Complex membership is established structurally, not just phylogenetically. The crystal structure shows a dimer of heterodimers with two copies of each chain. Supporting Evidence: PMID:26470919 three structures of human tRNA m(1)A58 MTase in complex with human tRNA3(Lys) and the product S-adenosyl-L-homocysteine show a dimer of heterodimers in which each heterodimer comprises a catalytic chain, Trm61, and a homologous but noncatalytic chain, Trm6 |
| GO:0005634 nucleus | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic transfer of the UniProt subcellular location; agrees with the manual and phylogenetic assignments. Reason: Correct location, corroborated by three independent lines (IBA, ISS, NAS). |
| GO:0006396 RNA processing | IEA GO_REF:0000117 | MODIFY | Summary: An ARBA machine-learning assignment of a very general RNA-metabolism process. Reason: Correct in kind but far too general to be useful. GO:0030488 tRNA methylation is a descendant of GO:0006396 (verified against the QuickGO ancestors endpoint) and is already carried by this gene with better evidence, so the specific term should stand in its place. Proposed replacements: tRNA methylation |
| GO:0030488 tRNA methylation | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro signature IPR014816 (TRM61 family) mapped to tRNA methylation. Reason: The domain-to-process mapping is correct for this family and matches the experimental evidence. |
| GO:0031515 tRNA (m1A) methyltransferase complex | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro-based assignment of complex membership. Reason: Correct, and independently confirmed by the crystal structure and by ComplexPortal. |
| GO:0160107 tRNA (adenine(58)-N1)-methyltransferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined automated assignment from the InterPro signature plus the RHEA 43152 and EC 2.1.1.220 mappings. Reason: The EC and Rhea mappings are correct for TRMT61A and agree with the direct human biochemistry, so this is a well-grounded electronic annotation rather than an over-propagation. |
| GO:0005515 protein binding | IPI PMID:16043508 The bipartite structure of the tRNA m1A58 methyltransferase ... | REMOVE | Summary: IPI with TRMT6 (UniProtKB Q9UJA5), the obligate partner subunit. Reason: Bare protein binding carries no functional information. The interaction is real and important, but it is already stated far more informatively by GO:0031515 tRNA (m1A) methyltransferase complex, which this gene carries with IBA, IEA and IPI support. No more informative molecular function is supportable from this evidence, so the row is removed rather than rewritten. |
| GO:0005515 protein binding | IPI PMID:28514442 Architecture of the human interactome defines protein commun... | REMOVE | Summary: High-throughput interactome detection of the TRMT6 (UniProtKB Q9UJA5) interaction. Reason: Uninformative term; the TRMT6 interaction is captured properly by the complex annotation. No more informative molecular function is supportable from this evidence, so the row is removed rather than rewritten. Removal does not imply the reported interaction is false. |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | REMOVE | Summary: Binary interactome hit with TCL1A (UniProtKB P56279). Reason: Uninformative term, and unlike the TRMT6 rows this partner has no established functional relationship to tRNA methylation. A single large-scale binary screen hit with no follow-up. No more informative molecular function is supportable from this evidence, so the row is removed rather than rewritten. Removal does not imply the reported interaction is false. |
| GO:0005515 protein binding | IPI PMID:33961781 Dual proteome-scale networks reveal cell-specific remodeling... | REMOVE | Summary: Proteome-scale AP-MS detection of the TRMT6 (UniProtKB Q9UJA5) interaction. Reason: Uninformative term; the TRMT6 interaction is captured properly by the complex annotation. No more informative molecular function is supportable from this evidence, so the row is removed rather than rewritten. Removal does not imply the reported interaction is false. |
| GO:0005634 nucleus | ISS GO_REF:0000024 | ACCEPT | Summary: Sequence-similarity transfer of nuclear localisation from the S. cerevisiae orthologue Gcd14/Trm61 (UniProtKB P46959). Reason: The orthology is unambiguous and tRNA A58 methylation is a nuclear step of tRNA maturation in both organisms. |
| GO:0160107 tRNA (adenine(58)-N1)-methyltransferase activity | EXP PMID:16043508 The bipartite structure of the tRNA m1A58 methyltransferase ... | ACCEPT | Summary: Direct demonstration that the human TRMT6/TRMT61A complex methylates A58 of tRNA in vitro, with site specificity shown by loss of activity on an A58U substrate mutant. Reason: This is the defining, core molecular function of the gene product. The evidence is direct biochemistry on purified human protein, supported independently by the structure of the complex caught on its tRNA substrate, and it is untouched by the 2025-2026 dispute, which concerns only the mRNA substrate class. Supporting Evidence: PMID:16043508 Stable hTrm6p/hTrm61p complexes purified from yeast maintained tRNA m(1)A Mtase activity in vitro. PMID:16043508 The human m(1)A Mtase complex also exhibited substrate specificity--modifying wild-type yeast tRNA(i) (Met) but not an A58U mutant. |
| GO:0005634 nucleus | NAS PMID:16043508 The bipartite structure of the tRNA m1A58 methyltransferase ... | ACCEPT | Summary: ComplexPortal author statement placing the complex in the nucleus. Reason: Weak evidence code in itself, but the location is independently supported by IBA, IEA and ISS here and by direct immunofluorescence on the partner subunit. |
| GO:0031515 tRNA (m1A) methyltransferase complex | IPI PMID:26470919 Crystal Structure of the Human tRNA m(1)A58 Methyltransferas... | ACCEPT | Summary: ComplexPortal annotation based on the crystal structure of the human heterotetramer bound to tRNA3(Lys). Reason: The strongest evidence available for complex membership. The stoichiometry (two TRMT61A, two TRMT6) and the inter-subunit architecture are resolved directly. Supporting Evidence: PMID:26470919 tRNAs bind across the dimer interface such that Trm6 from the opposing heterodimer brings A58 into the active site of Trm61. |
| GO:0006397 mRNA processing | IDA PMID:29072297 The m1A landscape on cytosolic and mitochondrial mRNA at sin... | KEEP AS NON CORE | Summary: Base-resolution mapping assigned a small set of low-stoichiometry cytosolic mRNA m1A sites, lying in tRNA T-loop-like structures, to the TRMT6/TRMT61A complex. Reason: Retained but demoted, for the same reasons as the paired GO:0061953 annotation. Even on its own account this paper describes a rare, sub-stoichiometric modification that cells largely avoid, and the 2026 biochemical validation of transcriptome-wide m1A maps narrows the genuine mRNA m1A set further. Not removed, because this is an experimental annotation whose full text is not in the publication cache and the curator read it. Supporting Evidence: PMID:29072297 Within the cytosol, m1A is present in a low number of mRNAs, typically at low stoichiometries, and almost invariably in tRNA T-loop-like structures, where it is introduced by the TRMT6/TRMT61A complex. |
| GO:0006397 mRNA processing | IDA PMID:29107537 Base-Resolution Mapping Reveals Distinct m(1)A Methylome in ... | KEEP AS NON CORE | Summary: An independent base-resolution study likewise assigned a small, evenly distributed subset of m1A sites carrying a GUUCRA tRNA-like motif to TRMT6/61A. Reason: Same position as the other mRNA-derived annotations. The two 2017 studies agree with each other and are mechanistically coherent, since the complex recognises a T-loop fold and an mRNA that adopts that fold is an adventitious substrate, but the substrate class is contested by 2026 biochemistry and is in any case minor relative to tRNA. Supporting Evidence: PMID:29107537 A different, small subset of m1A exhibit a GUUCRA tRNA-like motif, are evenly distributed in the transcriptome, and are dependent on the methyltransferase TRMT6/61A. |
| GO:0061953 mRNA (adenine-N1-)-methyltransferase activity | IDA PMID:29072297 The m1A landscape on cytosolic and mitochondrial mRNA at sin... | KEEP AS NON CORE | Summary: The contested molecular function. Assigned from single-nucleotide-resolution m1A mapping showing TRMT6/TRMT61A-dependent methylation of a small number of cytosolic mRNAs at T-loop-like elements, and challenged in 2026 by biochemical validation showing that mapped internal mRNA m1A sites are largely inosine. Reason: GOA carries both this and GO:0160107, and the dispute is entirely about the substrate class, not about the enzyme. Two points argue for retaining it. First, the paper cited here is one of the sceptical, base-resolution studies that cut the earlier antibody-based maps down from thousands of sites to a handful, not one of the discredited antibody maps, and it reports the modification as rare and sub-stoichiometric. Second, the mechanism is coherent, because substrate recognition by this complex is structural (a splayed T-loop), so an mRNA folding into a T-loop-like element is a legitimate if adventitious substrate. Against retention as a core function, Nalavade et al. 2026 show that newly mapped internal mRNA m1A sites actually contain inosine from A-to-I editing and conclude that genuine mRNA m1A is restricted to PRUNE1, ND5 and MALAT1. That paper is a discriminating assay rather than a blanket debunk, since it confirmed the equally contested internal m7G at high stoichiometry. Note that it does not mention TRMT6 or TRMT61A and does not itself attribute the surviving PRUNE1/MALAT1 sites to this complex. Not REMOVE, because this is an experimental annotation whose full text is not in the publication cache, and the correct reading of the current evidence is that the substrate class is narrow, not absent. Supporting Evidence: PMID:29072297 Within the cytosol, m1A is present in a low number of mRNAs, typically at low stoichiometries, and almost invariably in tRNA T-loop-like structures, where it is introduced by the TRMT6/TRMT61A complex. PMID:42337368 Using SCARPET, we show that newly mapped internal m1A sites are not m1A, but instead contain inosine from A-to-I editing. PMID:42337368 These results strongly support the conclusion that m1A is not widespread in mammalian mRNAs but is instead largely restricted to the previously validated sites in PRUNE1, ND5 and MALAT1 RNA. |
| GO:0061953 mRNA (adenine-N1-)-methyltransferase activity | IDA PMID:29107537 Base-Resolution Mapping Reveals Distinct m(1)A Methylome in ... | KEEP AS NON CORE | Summary: Independent IDA for the same contested activity, from a different base-resolution method, identifying a GUUCRA tRNA-like motif class of TRMT6/61A-dependent mRNA sites. Reason: Same position as the companion GO:0061953 annotation, kept consistent by design. Two independent groups converged on the same narrow, structure-driven substrate class in 2017, which is the strongest argument for keeping the annotation; the 2026 biochemistry is the strongest argument for not treating it as core. A large cancer literature reports abundant and consequential TRMT6/TRMT61A-deposited mRNA m1A, but it rests on the same class of mapping methods now under challenge and reports mutually opposite effects on target mRNA stability, so it does not settle the question either way. Supporting Evidence: PMID:29107537 A different, small subset of m1A exhibit a GUUCRA tRNA-like motif, are evenly distributed in the transcriptome, and are dependent on the methyltransferase TRMT6/61A. PMID:42337368 Overall, our data suggest that m1A is not a widespread modification but instead is limited to just the PRUNE1 mRNA, ND5 mRNA and MALAT1 lncRNA in mammalian cells. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-6783492 | ACCEPT | Summary: Reactome places the TRMT6:TRMT61A methylation reaction in the nucleoplasm. Reason: Correct and more specific than the bare nucleus annotations; agrees with the direct immunofluorescence localisation of TRMT6 to the nucleoplasm. |
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Download this section (compressed HTML)Q: Are the two nuclear-encoded mRNA m1A sites that survive SCARPET validation, PRUNE1 A58 and MALAT1 A7945, actually deposited by the TRMT6-TRMT61A complex? PMID:42337368 confirms them biochemically but never mentions either subunit, so the attribution currently rests on inference from the 2017 mapping studies rather than on a stated result.
Suggested experts: Samie R Jaffrey, Schraga Schwartz, Chengqi Yi
Q: If internal mRNA m1A is as rare as the 2026 biochemistry indicates, what actually explains the reproducible tumour phenotypes seen on TRMT6 or TRMT61A knockdown? Is the relevant lesion tRNA A58 hypomodification and its downstream effects on translation, rather than loss of mRNA methylation at specific target transcripts?
Suggested experts: Huarong Chen, Peng Hou
Q: Does the complex have measurable in vitro activity on a synthetic mRNA folded into a T-loop-like element, and if so how does its kcat/Km compare with that on a genuine tRNA substrate? This would establish whether mRNA methylation is a real if minor activity or simply below the threshold at which the enzyme acts.
Suggested experts: Robert M Stroud, Janet Finer-Moore
Q: The GUUCRA tRNA-like motif class reported by PMID:29107537 and the T-loop-like class reported by PMID:29072297 were derived by different methods. Are they the same set of sites, and does either survive the A-to-I editing control that PMID:42337368 shows was missing from the later maps?
Suggested experts: Chengqi Yi, Modi Safra
Experiment: Apply SCARPET, or an equivalent site-specific biochemical assay with an inosine control, to the specific cytosolic mRNA sites assigned to TRMT6/TRMT61A by Safra et al. and Li et al., in parallel in wild-type cells and in TRMT61A and TRMT6 knockout cells. A site that gives an m1A spot in wild-type cells and loses it in either knockout is a validated substrate of the complex; a site that gives an inosine spot is an editing artefact.
Hypothesis: Genuine, TRMT6/TRMT61A-dependent m1A in cytosolic mRNA is restricted to a very small number of sites that fold into tRNA T-loop-like elements, and is absent from the bulk of sites reported by transcriptome-wide mapping.
Type: Site-specific biochemical validation with genetic dependency test
Experiment: Measure steady-state kinetics of the purified recombinant human TRMT6-TRMT61A heterotetramer on a panel of synthetic RNAs, comprising an authentic tRNA, an mRNA fragment predicted to fold into a T-loop-like element, the same fragment with the fold disrupted by compensatory mutations, and an unstructured adenosine-containing control, and compare kcat/Km across the panel.
Hypothesis: The complex methylates an mRNA only when that mRNA adopts a T-loop fold, so mRNA m1A is a structural spillover of tRNA recognition rather than a distinct activity.
Type: In vitro enzyme kinetics with structured-substrate panel
Experiment: In a TRMT61A-dependent cancer model, test whether the growth defect caused by TRMT61A knockdown is rescued by re-expression of a catalytically dead TRMT61A, of wild-type TRMT61A, and of an orthogonal tRNA m1A58 methyltransferase that cannot act on mRNA. Rescue by the orthogonal tRNA-only enzyme would localise the phenotype to tRNA hypomodification.
Hypothesis: Tumour phenotypes attributed to loss of mRNA m1A are in fact caused by tRNA A58 hypomodification.
Type: Genetic rescue with substrate-restricted enzyme variants
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: It is undetermined whether the TRMT6-TRMT61A complex has a genuine, physiologically meaningful mRNA substrate class, or whether cytosolic mRNA m1A is confined to a handful of T-loop-like sites at stoichiometries too low to regulate anything.
NARROWING BIOLOGY RESIDUAL_SUBGAP
What is known: The tRNA A58 activity is settled, by direct in vitro biochemistry on the purified human complex, by site specificity demonstrated with an A58U substrate mutant, and by a crystal structure of the enzyme engaged with its tRNA substrate. What is unsettled is only the second substrate class.
Significance: The mRNA-m1A assignment underpins a large and growing cancer literature that proposes TRMT61A and TRMT6 as drug targets. If internal mRNA m1A is essentially absent, the proposed mechanisms in those papers cannot be correct as stated, and any therapeutic effect of inhibiting the complex would have to be explained through tRNA hypomodification instead.
What would resolve it: Site-specific biochemical validation, by SCARPET or an equivalent orthogonal assay, of the specific mRNA sites that the 2017 base-resolution studies attributed to TRMT6/TRMT61A, performed in TRMT6 or TRMT61A knockout cells so that dependence on the complex is tested directly rather than inferred.
Provenance (the field's own admissions):
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