Affinage

CTU1

Cytoplasmic tRNA 2-thiolation protein 1 · UniProt Q7Z7A3

Length
348 aa
Mass
36.5 kDa
Annotated
2026-06-09
34 papers in source corpus 12 papers cited in narrative 12 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 5/5 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

CTU1 (NCS6/Ncs6) is the catalytic subunit of the eukaryotic CTU1/CTU2 complex that installs a 2-thiol group at the wobble uridine 34 (s2U34) of tRNALys(UUU), tRNAGlu(UUC), and tRNAGln(UUG), with CTU2 acting as a scaffold and CTU1 performing catalysis (PMID:19151091, PMID:39705244). Mechanistically, CTU1 activates the target uridine by adenylation and itself acts as a persulfide carrier, forming a persulfide adduct at an active-site cysteine (PMID:24530533), and its catalysis depends on an iron-sulfur cluster ([3Fe-4S]/[4Fe-4S]) coordinated by three conserved cysteines whose fourth, non-protein-bound iron positions a sulfur ligand for transfer to tRNA (PMID:27791189, PMID:37005440). Sulfur is supplied through a relay in which thiocarboxylated Urm1, generated by the Uba4/UBA4 E1-like enzyme, is received by CTU1 (PMID:19151091, PMID:39673271); CTU1 is additionally a target of Urm1 conjugation (urmylation) on lysine residues, a modification enhanced by oxidative stress (PMID:21209336). Functionally, this thiolation enables codon-biased translation of mRNAs enriched in AA-ending codons, driving production of the oncoprotein DEK and downstream LEF1 to support breast cancer invasion (PMID:27811057), sustaining HIF1A translation and glycolysis in melanoma where the activity cooperates with MAPK/PI3K signaling in BRAF therapy resistance (PMID:29925953), and becoming critically essential to ribosomal protein synthesis when mTORC1 is inactivated (PMID:40328729). CTU1-dependent thiolation is required for angiogenesis and embryonic development in vertebrates and for endothelial proliferation, migration, and tube formation (PMID:39705244).

Mechanistic history

Synthesis pass · year-by-year structured walk · 11 steps
  1. 2009 High

    Established that CTU1/Ncs6, with Ncs2, performs the terminal 2-thiolation step of wobble uridine, placing it at the end of a defined sulfur-relay pathway.

    Evidence In vitro sulfur-transfer reconstitution with recombinant proteins plus yeast deletion analysis

    PMID:19151091

    Open questions at the time
    • Catalytic mechanism of Ncs6/Ncs2 in sulfur incorporation not resolved
    • No structural basis for tRNA recognition
  2. 2011 Medium

    Showed human CTU1 is not only a thiolation enzyme but also a substrate of Urm1, linking the modifier to a post-translational lysine conjugation responsive to oxidative stress.

    Evidence Mass spectrometry of Urm1 conjugates and biochemical characterization of isopeptide bonds in mammalian cells

    PMID:21209336

    Open questions at the time
    • Functional consequence of CTU1 urmylation unknown
    • Single study/single lab
  3. 2014 Medium

    Defined the catalytic chemistry: CTU1/Ncs6 adenylates the uridine and carries sulfur as an active-site persulfide, resolving how sulfur reaches the tRNA.

    Evidence Biochemical persulfide adduct detection and in vitro adenylation assay on archaeal homolog Mmp1356

    PMID:24530533

    Open questions at the time
    • Demonstrated in archaeal homolog rather than human CTU1
    • Order of adenylation and sulfur transfer not fully resolved
  4. 2016 Medium

    Identified an iron-sulfur cluster as an essential catalytic cofactor of Ncs6/CTU1, revealing the chemical basis for sulfur activation.

    Evidence Spectroscopic and biochemical characterization of the Fe-S cluster with activity assays in archaeal/eukaryotic homologs

    PMID:27791189

    Open questions at the time
    • Cluster assembly machinery for cytosolic CTU1 not defined
    • Single study
  5. 2016 High

    Connected CTU1 thiolation to cancer phenotype by showing codon-biased translation of the oncoprotein DEK drives invasion and metastasis.

    Evidence Genetic ablation in PyMT model, codon-substitution rescue, IRES translation and invasion assays

    PMID:27811057

    Open questions at the time
    • Direct CTU1 catalytic loss vs Elp3 pathway contributions not separated
    • Full set of codon-dependent targets undefined
  6. 2018 High

    Extended the cancer role to metabolic reprogramming, showing CTU1/CTU2 sustain HIF1A translation and glycolysis and intersect with MAPK/PI3K signaling in BRAF therapy resistance.

    Evidence shRNA knockdown, pharmacological inhibition, polysome profiling, metabolic and codon-usage analysis in melanoma

    PMID:29925953

    Open questions at the time
    • Mechanism by which PI3K elevates CTU1/CTU2 expression not detailed
    • Generality across tumor types untested here
  7. 2019 Medium

    Confirmed CTU1/CTU2 as the human enzyme complex responsible for wobble uridine thiolation using patient cells with loss-of-function variants.

    Evidence Functional characterization of patient-derived cells with MS-based tRNA modification analysis and truncation variants

    PMID:31301155

    Open questions at the time
    • Variants studied were in CTU2 rather than CTU1
    • Phenotype-to-modification causality at organismal level not established
  8. 2023 High

    Provided the structural basis: a crystal structure of the NcsA homolog showed a dimer binding a [4Fe-4S] cluster via three cysteines with the fourth iron binding sulfide, and AlphaFold comparison supports conservation in human CTU1/CTU2.

    Evidence 2.8 Å crystal structure, spectroscopy, mutagenesis, and AlphaFold comparison with human CTU1/CTU2

    PMID:37005440

    Open questions at the time
    • No experimental structure of human CTU1/CTU2
    • tRNA-bound complex structure absent
  9. 2024 Medium

    Resolved how CTU1 receives sulfur, placing Ncs6/CTU1 downstream of the Uba4/Urm1 thiocarboxylation machinery via cryo-EM and pathway reconstitution.

    Evidence Cryo-EM of Uba4/Urm1 complex with mutagenesis and pathway reconstitution

    PMID:39673271

    Open questions at the time
    • Direct CTU1-Urm1 handoff interface not structurally resolved
    • Single study
  10. 2024 Medium

    Demonstrated a physiological requirement in vertebrates, showing CTU1 catalyzes s2U34 on three tRNAs and is needed for angiogenesis and embryonic development.

    Evidence Zebrafish morpholino knockdown, siRNA in human endothelial cells, scRNA-seq, and functional assays

    PMID:39705244

    Open questions at the time
    • Morpholino off-target effects not fully excluded
    • Codon-dependent target genes in endothelium not identified
  11. 2025 High

    Revealed a synthetic-lethal cooperation with mTORC1, showing U34 enzymes become essential for ribosomal protein synthesis when mTORC1 is inactivated.

    Evidence Genome-wide CRISPR screens, nascent and steady-state proteomics, ribosome profiling, and tumor models

    PMID:40328729

    Open questions at the time
    • Specific ribosomal protein mRNAs most codon-dependent not enumerated
    • Therapeutic window for combined mTORC1/U34 inhibition untested clinically

Open questions

Synthesis pass · forward-looking unresolved questions
  • The functional significance of CTU1 urmylation and the structure of the human CTU1/CTU2-tRNA complex during catalysis remain unresolved.
  • No experimental human CTU1/CTU2 structure
  • Consequence of CTU1 self-urmylation on activity unknown
  • Mechanism of Fe-S cluster assembly for cytosolic CTU1 undefined

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140098 catalytic activity, acting on RNA 4 GO:0003723 RNA binding 2 GO:0016740 transferase activity 1 GO:0140657 ATP-dependent activity 1
Localization
GO:0005829 cytosol 1
Pathway
R-HSA-8953854 Metabolism of RNA 3 R-HSA-1643685 Disease 2 R-HSA-392499 Metabolism of proteins 1
Partners
CTU2UBA4URM1
Complex memberships
CTU1/CTU2 complex

Evidence

Reading pass · 12 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2009 CTU1 (NCS6/Ncs6p in yeast) is required for 2-thiolation of the wobble uridine (mcm5s2U) at position 34 of tRNAs, acting together with NCS2/Ncs2p. In vitro reconstitution showed that the sulfur-relay pathway proceeds through Nfs1p cysteine desulfurase → Tum1p → Urm1p thiocarboxylation by Uba4p → Ncs2p/Ncs6p-mediated 2-thiouridine formation on tRNA. In vitro sulfur transfer reconstitution assay with recombinant proteins; genetic deletion analysis in S. cerevisiae Nucleic acids research High 19151091
2011 Human CTU1 (ATPBD3) is a substrate of Urm1 urmylation; Urm1 is covalently conjugated to CTU1 (and CTU2 and MOCS3) on lysine residues, forming an isopeptide bond, and this modification is enhanced by oxidative stress. Mass spectrometry identification of Urm1 conjugates in mammalian cells; biochemical characterization of the thioester intermediate and isopeptide bond formation Proceedings of the National Academy of Sciences of the United States of America Medium 21209336
2014 Ncs6/Ctu1 activates uridine by adenylation and directly participates in sulfur transfer as a persulfide carrier, forming a persulfide enzyme adduct at an active site cysteine residue, as shown in the archaeal Methanococcus maripaludis homolog Mmp1356. Biochemical characterization of persulfide adduct formation; transposon mutagenesis showing essentiality; in vitro adenylation assay FEBS letters Medium 24530533
2016 A [3Fe-4S] cluster in archaeal and eukaryotic Ncs6 homologs (the direct orthologs of human CTU1) is essential for tRNA thiolation activity. Eukaryotic cytosolic Ncs6 homologs possess this iron-sulfur cluster required for catalysis. Biochemical and spectroscopic characterization of iron-sulfur cluster; activity assays showing cluster requirement for thiolation Proceedings of the National Academy of Sciences of the United States of America Medium 27791189
2016 CTU1 and CTU2, partner enzymes in U34 mcm5s2-tRNA modification, support cellular invasion and metastasis in breast cancer by enabling efficient translation of the oncoprotein DEK, which in turn promotes IRES-dependent translation of LEF1. A DEK mutant with codon composition independent of U34 mcm5s2-tRNA modification escapes CTU1-dependent regulation. Genetic ablation (Elp3 knockout in PyMT model); codon-substitution rescue experiment; IRES-dependent translation assay; invasion assays The Journal of experimental medicine High 27811057
2018 CTU1 and CTU2, as U34 tRNA modification enzymes, promote glycolysis in melanoma cells through codon-dependent regulation of HIF1A mRNA translation, maintaining high HIF1α protein levels. Concurrent inhibition of MAPK signaling with CTU1/CTU2 synergistically kills melanoma cells. PI3K pathway activation, a mechanism of acquired BRAF therapy resistance, markedly increases CTU1/CTU2 expression. shRNA knockdown; pharmacological inhibition; polysome profiling; metabolic assays; codon-usage analysis Nature High 29925953
2019 The CTU1/CTU2 complex catalyzes thiolation of wobble uridine in tRNAs (tRNAGln, tRNAGlu, tRNALys); loss-of-function variants in CTU2 abolish this modification in patient-derived cells, establishing CTU1/CTU2 as the enzyme complex responsible for this conserved modification in humans. Patient-derived cell functional characterization; mass spectrometry-based tRNA modification analysis; protein truncation variants Human mutation Medium 31301155
2023 The archaeal NcsA (Ncs6/CTU1 homolog) is a dimer that binds a [4Fe-4S] cluster coordinated by three conserved cysteines, with the fourth non-protein-bonded iron serving as the binding site for a hydrogenosulfide ligand, supporting a mechanism in which the [4Fe-4S] cluster activates the sulfur atom for tRNA thiolation. Comparison of NcsA crystal structure with AlphaFold model of human CTU1/CTU2 complex shows close superposition of catalytic site residues including the cluster-coordinating cysteines. Crystal structure at 2.8 Å; spectroscopic characterization of [4Fe-4S] cluster; biochemical assays; mutagenesis; AlphaFold structural comparison with human CTU1/CTU2 Scientific reports High 37005440
2024 CTU2 serves as a scaffold protein while CTU1 catalyzes the 2-thiolation at the 34th wobble uridine (s2U34) of tRNAGlnUUG, tRNAGluUUC, and tRNALysUUU. CTU1 deficiency in zebrafish causes impaired angiogenesis and embryonic development, and in human endothelial cells impairs proliferation, migration, and tube formation. Antisense morpholino oligonucleotides in zebrafish; siRNA knockdown in human endothelial cells; single-cell RNA sequencing; functional assays (proliferation, migration, tube formation) PloS one Medium 39705244
2024 Cryo-EM structure of the Uba4/Urm1 complex reveals the interaction interface and shows that Ncs6 (CTU1 ortholog) is a downstream component that receives thiocarboxylated Urm1-SH in the tRNA thiolation pathway. Ncs6 interacts with Urm1 as part of the sulfur relay cascade leading to tRNA 2-thiolation. Cryo-EM structure of Uba4/Urm1 complex; in vitro and in vivo mutagenesis; biochemical pathway reconstitution Nucleic acids research Medium 39673271
2025 CTU1/CTU2 (U34-enzymes) become critically essential for cell growth when mTORC1 is inactivated. Loss of U34-enzymes particularly impairs synthesis of ribosomal proteins (identified by quantitative nascent proteomics, steady-state proteomics and ribosome profiling), and simultaneous suppression of mTORC1 and U34-enzymes depletes ribosomal proteins and globally inhibits translation. Genome-wide CRISPR screens; quantitative nascent proteomics; steady-state proteomics; ribosome profiling; in vitro and in vivo tumor models Nature communications High 40328729
2013 Fission yeast Ctu1-Ctu2 are essential for modification of wobble uridine in certain tRNAs and for normal tolerance to oxidative stress (H2O2). Loss of Ctu2 impairs translation of stress-induced mRNAs enriched in AAA lysine codons, including the transcription factors Atf1 and Pcr1, demonstrating codon-biased translation control downstream of tRNA thiolation. Genetic deletion; tRNA modification analysis; translation reporter assays; codon-substitution rescue (AAA-to-AAG atf1 mutant) PLoS genetics Medium 23874237

Source papers

Stage 0 corpus · 34 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2018 Codon-specific translation reprogramming promotes resistance to targeted therapy. Nature 215 29925953
2009 Mechanistic characterization of the sulfur-relay system for eukaryotic 2-thiouridine biogenesis at tRNA wobble positions. Nucleic acids research 192 19151091
2016 Elp3 links tRNA modification to IRES-dependent translation of LEF1 to sustain metastasis in breast cancer. The Journal of experimental medicine 146 27811057
2002 Differential expression of alternative oxidase genes in maize mitochondrial mutants. The Plant cell 141 12468742
2013 Modification of tRNA(Lys) UUU by elongator is essential for efficient translation of stress mRNAs. PLoS genetics 114 23874237
2003 Urmylation: a ubiquitin-like pathway that functions during invasive growth and budding in yeast. Molecular biology of the cell 95 14551258
2011 Role of the ubiquitin-like protein Urm1 as a noncanonical lysine-directed protein modifier. Proceedings of the National Academy of Sciences of the United States of America 93 21209336
2016 A [3Fe-4S] cluster is required for tRNA thiolation in archaea and eukaryotes. Proceedings of the National Academy of Sciences of the United States of America 58 27791189
2015 Loss of anticodon wobble uridine modifications affects tRNA(Lys) function and protein levels in Saccharomyces cerevisiae. PloS one 57 25747122
2015 Genome-wide association study for calving performance using high-density genotypes in dairy and beef cattle. Genetics, selection, evolution : GSE 50 26065883
2022 Dysfunctional tRNA reprogramming and codon-biased translation in cancer. Trends in molecular medicine 47 36241532
2009 Comparative genomics indicates the mammalian CD33rSiglec locus evolved by an ancient large-scale inverse duplication and suggests all Siglecs share a common ancestral region. Immunogenetics 37 19337729
1993 Analysis of Leaf Sectors in the NCS6 Mitochondrial Mutant of Maize. The Plant cell 37 12271093
2018 Recent Advances in Our Understanding of the Biosynthesis of Sulfur Modifications in tRNAs. Frontiers in microbiology 31 30450093
2014 Archaeal Tuc1/Ncs6 homolog required for wobble uridine tRNA thiolation is associated with ubiquitin-proteasome, translation, and RNA processing system homologs. PloS one 30 24906001
2020 Molten salt induced nitrogen-doped biochar nanosheets as highly efficient peroxymonosulfate catalyst for organic pollutant degradation. Environmental pollution (Barking, Essex : 1987) 28 31995772
1990 A partially deleted mitochondrial cytochrome oxidase gene in the NCS6 abnormal growth mutant of maize. The New biologist 28 1964592
2019 Genomic Landscape of Intramedullary Spinal Cord Gliomas. Scientific reports 26 31822682
2015 Elongator-dependent modification of cytoplasmic tRNALysUUU is required for mitochondrial function under stress conditions. Nucleic acids research 24 26240381
2019 Biallelic variants in CTU2 cause DREAM-PL syndrome and impair thiolation of tRNA wobble U34. Human mutation 23 31301155
2019 An ancient type of MnmA protein is an iron-sulfur cluster-dependent sulfurtransferase for tRNA anticodons. RNA (New York, N.Y.) 21 31801798
2005 Biochemical and molecular characterization of photosystem I deficiency in the NCS6 mitochondrial mutant of maize. Plant molecular biology 18 15821884
2014 The putative tRNA 2-thiouridine synthetase Ncs6 is an essential sulfur carrier in Methanococcus maripaludis. FEBS letters 16 24530533
1994 Rescue of a maize mitochondrial cytochrome oxidase mutant by tissue culture. The Plant journal : for cell and molecular biology 16 7849753
2025 De Novo Design of Efficient NIR-II-Activated Heavy-Atom-Free Type-I Photosensitizer for Anti-Tumor Photoimmunotherapy. Advanced materials (Deerfield Beach, Fla.) 15 40465313
2024 The tRNA thiolation-mediated translational control is essential for plant immunity. eLife 12 38284752
2023 The thiolation of uridine 34 in tRNA, which controls protein translation, depends on a [4Fe-4S] cluster in the archaeum Methanococcus maripaludis. Scientific reports 12 37005440
2025 tRNA thiolation optimizes appressorium-mediated infection by enhancing codon-specific translation in Magnaporthe oryzae. Nucleic acids research 7 39777460
2024 Molecular basis for thiocarboxylation and release of Urm1 by its E1-activating enzyme Uba4. Nucleic acids research 7 39673271
2022 Gene Promoter-Methylation Signature as Biomarker to Predict Cisplatin-Radiotherapy Sensitivity in Locally Advanced Cervical Cancer. Frontiers in oncology 5 35359376
2025 mTORC1 cooperates with tRNA wobble modification to sustain the protein synthesis machinery. Nature communications 1 40328729
2025 RBAD: The first database dedicated alterations of blood RNA in individuals with Alzheimer's disease and their clinical relevance. Neural regeneration research 0 40145964
2025 Proteomic Comparison between Hyphae and Spores Reveals Pathogenicity of Mucor Irregularis. Journal of proteome research 0 40692258
2024 The conserved wobble uridine tRNA thiolase Ctu1 is required for angiogenesis and embryonic development. PloS one 0 39705244

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