{"gene":"URM1","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2009,"finding":"Urm1p acts as a sulphur carrier in eukaryotic tRNA thiolation; Uba4p first adenylates and then directly transfers sulphur onto Urm1p to form a thiocarboxylate; Ncs6p binds tRNA as part of this conserved pathway; Ncs2p and Yor251cp are additional pathway components.","method":"In vitro sulphur-transfer assays, genetic screens (S. cerevisiae), tRNA thiolation assays, biochemical reconstitution","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with multiple pathway components, replicated by independent labs","pmids":["19145231"],"is_preprint":false},{"year":2008,"finding":"Urm1 is activated to a thiocarboxylate intermediate that serves as a sulfur donor in tRNA thiolation reactions, mechanistically reminiscent of prokaryotic sulfur carriers MoaD/ThiS; this activity is distinct from canonical ubiquitin-like protein conjugation.","method":"Functional proteomics, enzymatic activity assays, thiocarboxylate intermediate detection","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — enzymatic intermediate characterization with biochemical assays, independently corroborated","pmids":["19017811"],"is_preprint":false},{"year":2011,"finding":"Urm1 is conjugated to lysine residues of target proteins (urmylation) via a thioester intermediate forming an isopeptide bond; oxidative stress enhances urmylation in both S. cerevisiae and mammalian cells. Yeast peroxiredoxin Ahp1 is confirmed as a substrate. Mammalian targets include MOCS3, ATPBD3, CTU2, and cellular apoptosis susceptibility protein (CAS). Urmylation involves a C-terminal thiocarboxylate of Urm1 rather than canonical UBL mechanism.","method":"In vivo conjugation assays, site-directed mutagenesis, mass spectrometry identification of substrates, Co-IP","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (mutagenesis, MS, in vivo assays), confirmed in two organisms","pmids":["21209336"],"is_preprint":false},{"year":2006,"finding":"NMR solution structure of yeast Urm1 reveals a ubiquitin-fold most closely resembling prokaryotic MoaD sulfur-carrier proteins, placing Urm1 as a 'molecular fossil' linking sulfur-carrier proteins and ubiquitin-like modifiers. Structural and electrostatic surface similarities between Urm1-Uba4 and MoaD-MoeB suggest a conserved ATP-dependent activation mechanism.","method":"Solution NMR structure determination, phylogenetic and structural comparison","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure with functional/evolutionary validation in a single rigorous study","pmids":["16864801"],"is_preprint":false},{"year":2005,"finding":"NMR solution structure of mouse Urm1 (AAH26994.1) reveals close structural resemblance to bacterial MoaD sulfur-carrier proteins, supporting its role as an evolutionary link between sulfur-donor systems and ubiquitin-like modifiers.","method":"NMR spectroscopy, structural comparison","journal":"Protein science : a publication of the Protein Society","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — NMR structure determined, but functional validation limited to structural/evolutionary inference in a single study","pmids":["16046629"],"is_preprint":false},{"year":2018,"finding":"A critical thioester linkage between Urm1 and Uba4 residue Cys225 mediates intramolecular transfer of Urm1 between the E1-like (AD) and rhodanese (RHD) domains of Uba4; this thioester is indispensable for Urm1 thiocarboxylation and consequent tRNA thiolation in vivo.","method":"In vitro Urm1 thiocarboxylation assay, structure-function analysis, site-directed mutagenesis (Cys225), chemical profiling","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution combined with mutagenesis and in vivo validation in a single rigorous study","pmids":["29718331"],"is_preprint":false},{"year":2020,"finding":"Crystal structures of full-length Uba4 and the Uba4-Urm1 heterodimeric complex reveal how the AD and RHD domains of Uba4 orchestrate recognition, binding, and thiocarboxylation of the Urm1 C-terminus; a mechanism was identified by which Uba4 protects itself against self-conjugation with activated Urm1-COSH.","method":"X-ray crystallography, structural analysis of heterodimeric complex, mechanistic biochemical assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structures of full-length enzyme and substrate complex with mechanistic biochemical analysis in one rigorous study","pmids":["32901956"],"is_preprint":false},{"year":2022,"finding":"Urmylation of target proteins (including peroxiredoxin Ahp1) is reconstituted in vitro using thiocarboxylated Urm1 and is E2/E3-independent, requiring oxidative stress. Urmylation is accompanied by transfer of sulfur to cysteine residues in target proteins (cysteine persulfidation). Crystal structures of Ahp1 before and after Urm1 attachment were determined.","method":"In vitro reconstitution of urmylation, X-ray crystallography of Ahp1 ± Urm1, biochemical assays for persulfidation","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with crystal structures and mutagenesis in one rigorous study","pmids":["36102610"],"is_preprint":false},{"year":2024,"finding":"Cryo-EM structure of the Uba4/Urm1 complex reveals RHD domain positions after Urm1 binding; mutations at the Uba4-Urm1 interface impair thiocarboxylation in vitro and in vivo. Conserved cysteines of Uba4 are required for thioester formation and Urm1-SH generation. Urm1-SH release mechanism and interactions with upstream (Tum1) and downstream (Ncs6) pathway components are defined.","method":"Cryo-EM structural determination, site-directed mutagenesis, in vitro thiocarboxylation assays, in vivo complementation","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structure combined with mutagenesis and in vitro/in vivo functional assays in one rigorous study","pmids":["39673271"],"is_preprint":false},{"year":2015,"finding":"Human URM1 and UBA4/MOCS3 are functional orthologs of yeast Urm1 and Uba4; gene shuffle experiments show they support urmylation of peroxiredoxin Ahp1 and tRNA thiolation in yeast (albeit at reduced efficiency). Yeast Uba4 is itself modified by Urm1 and hURM1, revealing target overlap between eukaryal urmylation pathways.","method":"Yeast gene shuffle (complementation), biochemical urmylation assays, tRNA thiolation assays","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic complementation with biochemical validation, single lab, two orthogonal methods","pmids":["25747390"],"is_preprint":false},{"year":2015,"finding":"In Drosophila, Urm1 conjugation to target proteins including the conserved substrate Peroxiredoxin 5 (Prx5) requires the E1 activating enzyme Uba4. Loss of Urm1 is lethal; escapers show increased cytoprotective JNK signaling, and elevated JNK targets Jafrac1 and gstD1 confer oxidative stress resistance in Urm1 null mutants.","method":"Drosophila genetics (null mutants, genetic rescue), western blotting for urmylation, JNK pathway epistasis analysis","journal":"Cellular and molecular life sciences : CMLS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis combined with biochemical urmylation assays and molecular pathway analysis, single lab","pmids":["26715182"],"is_preprint":false},{"year":2016,"finding":"Urmylation of Ahp1 is suppressed at elevated temperatures or under sulfur starvation. The rhodanese domain (RHD) in Uba4, critical for Urm1-COSH formation, is required (but not absolutely essential) for both protein urmylation and tRNA thiolation, establishing that sulfur supply and thiocarboxylation chemically link the two branches of the URM1 pathway.","method":"Site-directed mutagenesis of Uba4 RHD, urmylation monitoring by western blot, tRNA thiolation assays, S. cerevisiae genetics","journal":"Microbial cell (Graz, Austria)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis with in vivo functional readouts across two pathway branches, single lab","pmids":["28357324"],"is_preprint":false},{"year":2024,"finding":"Covalent modification by Urm1 promotes phase separation of a wide range of proteins in yeast under stress. A drop in cellular pH triggered by stress drives Urm1 self-association and interaction with target proteins and Uba4. Urmylation of stress-sensitive proteins promotes their deposition into stress granules and nuclear condensates. Yeast cells lacking Urm1 exhibit condensate defects and reduced stress resilience.","method":"Live-cell imaging of condensates, genetic (Urm1 deletion), biochemical assays for Urm1 self-association, urmylation in stress conditions","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — live imaging, genetic KO with defined condensate phenotype, and biochemical assays for Urm1-driven phase separation in one rigorous study","pmids":["38942013"],"is_preprint":false},{"year":2018,"finding":"Urm1 conjugation (urmylation) of the HTLV-1 oncoprotein Tax redistributes Tax to the cytoplasm and increases transcription of NF-κB targets (Rantes and IL-6). In a tax-transgenic Drosophila model, Urm1 presence correlates with transcriptional output of the NF-κB target Diptericin, indicating evolutionary conservation of Urm1-dependent subcellular targeting of Tax.","method":"Co-immunoprecipitation, subcellular localization assays, NF-κB reporter assays, Drosophila transgenic model","journal":"Retrovirology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP plus functional reporter assays and Drosophila model, single lab","pmids":["29665857"],"is_preprint":false},{"year":2023,"finding":"URM-1 co-localizes and interacts with connexin 43 (Cx43) in breast cancer cell lines; URMylated Cx43 is increased upon cellular stress. Downregulation of URM-1 decreases Cx43 expression and affects SUMO-1-mediated SUMOylation of Cx43, while upregulation of URM-1 increases Cx43 expression and reverses EMT-associated processes.","method":"Co-immunoprecipitation, co-localization imaging, siRNA knockdown/overexpression, western blotting","journal":"International journal of molecular sciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP and co-localization with functional readouts, single lab, single study","pmids":["36769280"],"is_preprint":false},{"year":2024,"finding":"Using suspension bead loading (SBL) to deliver synthetic URM1 into live cells, oxidative stress was shown to alter both the subcellular localization and conjugation pattern of URM1, directly demonstrating stress-regulated urmylation and redistribution in living cells.","method":"Suspension bead loading of synthetic protein, live-cell imaging, subcellular fractionation, conjugation assays under redox stress","journal":"Angewandte Chemie (International ed. in English)","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct live-cell localization experiment with functional consequence, novel delivery method, single lab","pmids":["39246272"],"is_preprint":false},{"year":2026,"finding":"NAE1/UBA3 and UBE2M were identified as E1 and E2 enzymes, respectively, for the URM1 modification cascade in human cells under both normal and oxidative stress conditions. DCN1 (via the UBE2M-DCN1 module) may contribute to URM1 conjugation. Pharmacological inhibition of NAE1 by pevonedistat blocks protein urmylation in human cells and shows synergy with cisplatin in killing liver cancer cells.","method":"Activity-based URM1 probe for covalent capture of cysteine enzymes, proteomic characterization, cell-based validation, pharmacological inhibition (pevonedistat)","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Moderate — activity-based probe with proteomic identification and cell-based validation plus pharmacological perturbation, identifying previously unknown E1/E2 for mammalian urmylation","pmids":["42056084"],"is_preprint":false},{"year":2025,"finding":"Archaeal Urm1 (from Sulfolobus acidocaldarius) can conjugate to yeast peroxiredoxin Ahp1 (a bona fide urmylation target) when expressed in S. cerevisiae, but cannot support tRNA thiolation. Ahp1 conjugation by archaeal Urm1 requires sulfur transfer from yeast Uba4, establishing that thioactivation and urmylation-like conjugation are conserved between Sulfolobus and Saccharomyces.","method":"URM1 gene shuffle (Sulfolobus to yeast), urmylation assays, tRNA thiolation assays, genetic complementation","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic and biochemical cross-species complementation with two orthogonal functional readouts, single lab","pmids":["41276627"],"is_preprint":false}],"current_model":"URM1 is a dual-function ubiquitin-like protein that acts both as a sulfur carrier and a protein modifier: activated by its E1-like enzyme UBA4 (MOCS3 in humans) through sequential adenylation, thioesterification via Uba4-Cys225, and rhodanese-domain-mediated thiocarboxylation of Urm1's C-terminal glycine, the resulting thiocarboxylated Urm1 (Urm1-COSH) either donates sulfur for 2-thiolation of wobble uridine (U34) in cytoplasmic tRNAs (via downstream thiolases Ncs6/CTU1) or, under oxidative stress, conjugates covalently via an isopeptide (and/or persulfidation) bond to lysine residues of target proteins (including peroxiredoxin Ahp1, MOCS3, CAS, and Cx43) in an E2/E3-independent manner; in human cells, NAE1/UBA3 and UBE2M serve as canonical E1/E2 enzymes for the urmylation branch; stress-induced urmylation additionally promotes phase separation of target proteins into protective condensates, with Urm1 acting as a molecular 'adhesive' that drives stress granule and nuclear condensate formation."},"narrative":{"mechanistic_narrative":"URM1 is a dual-function ubiquitin-like protein that serves both as a sulfur carrier in tRNA modification and as a covalent protein modifier under oxidative stress [PMID:19145231, PMID:21209336]. Its NMR solution structure adopts a ubiquitin fold most closely resembling the prokaryotic MoaD/ThiS sulfur-carrier proteins, positioning Urm1 as an evolutionary link between bacterial sulfur-donor systems and eukaryotic ubiquitin-like modifiers [PMID:16864801, PMID:16046629]. Activation proceeds through the E1-like enzyme Uba4 (MOCS3 in humans): Uba4 adenylates the Urm1 C-terminus and, via a thioester linkage at Cys225, shuttles Urm1 between its adenylation and rhodanese domains to generate a C-terminal thiocarboxylate (Urm1-COSH), the chemistry that drives both pathway branches [PMID:19145231, PMID:29718331, PMID:32901956, PMID:39673271]. The thiocarboxylated species donates sulfur for 2-thiolation of wobble uridines in cytoplasmic tRNAs through downstream thiolases including Ncs6 [PMID:19145231, PMID:39673271]. The same thiocarboxylate intermediate enables urmylation: under oxidative stress Urm1 conjugates to lysine residues of target proteins—including the peroxiredoxin Ahp1—via an E2/E3-independent mechanism accompanied by cysteine persulfidation, with substrates spanning yeast Ahp1 and mammalian MOCS3, CTU2, and CAS [PMID:21209336, PMID:36102610]. Sulfur supply mechanistically couples the tRNA-thiolation and protein-conjugation branches, both depending on the Uba4 rhodanese domain [PMID:28357324]. Beyond modification chemistry, urmylation promotes stress-induced phase separation, driving deposition of stress-sensitive proteins into stress granules and nuclear condensates and conferring stress resilience [PMID:38942013]. In human cells the canonical neddylation enzymes NAE1/UBA3 and UBE2M act as E1 and E2 for the urmylation cascade, and pharmacological NAE1 inhibition by pevonedistat blocks urmylation and synergizes with cisplatin in liver cancer cells [PMID:42056084].","teleology":[{"year":2005,"claim":"Establishing the Urm1 fold answered whether this ubiquitin-like protein is built like a conjugation modifier or a sulfur carrier, revealing its closest structural kinship to bacterial MoaD.","evidence":"NMR solution structures of mouse and yeast Urm1 with structural/phylogenetic comparison","pmids":["16046629","16864801"],"confidence":"Medium","gaps":["Structure alone did not demonstrate which biochemical activity (sulfur transfer vs conjugation) dominates in vivo","No partner enzyme captured in the structural studies"]},{"year":2008,"claim":"Detection of a thiocarboxylate intermediate defined Urm1's first biochemical activity as a sulfur donor distinct from canonical UBL conjugation, mechanistically aligning it with prokaryotic sulfur carriers.","evidence":"Functional proteomics and enzymatic intermediate detection","pmids":["19017811"],"confidence":"High","gaps":["Did not resolve the full enzymatic route of thiocarboxylate formation","Downstream tRNA targets not yet mapped at this stage"]},{"year":2009,"claim":"Reconstitution placed Urm1 in a defined tRNA-thiolation pathway, showing Uba4 adenylates then transfers sulfur to Urm1 and that Ncs6 binds tRNA, establishing the sulfur-relay architecture.","evidence":"In vitro sulfur-transfer and tRNA thiolation assays with genetic screens in S. cerevisiae","pmids":["19145231"],"confidence":"High","gaps":["Atomic mechanism of intramolecular sulfur transfer within Uba4 not yet defined","Did not address a protein-conjugation role"]},{"year":2011,"claim":"Discovery of urmylation answered whether Urm1 also acts as a protein modifier, showing oxidative-stress-driven isopeptide conjugation to lysines using the C-terminal thiocarboxylate rather than canonical UBL chemistry.","evidence":"In vivo conjugation assays, mutagenesis, MS substrate identification and Co-IP in yeast and mammalian cells","pmids":["21209336"],"confidence":"High","gaps":["E2/E3 requirements and enzymatic route of conjugation unresolved","Functional consequence of modifying each substrate not defined"]},{"year":2015,"claim":"Cross-species complementation established that human URM1/MOCS3 and a Drosophila pathway are functional orthologs, demonstrating conservation of both branches and substrate overlap.","evidence":"Yeast gene-shuffle complementation, urmylation and tRNA thiolation assays; Drosophila null mutants and JNK epistasis","pmids":["25747390","26715182"],"confidence":"Medium","gaps":["Reduced efficiency of human enzymes in yeast leaves native human kinetics unaddressed","Connection between urmylation loss and JNK induction not mechanistically dissected"]},{"year":2018,"claim":"Identifying the Uba4 Cys225 thioester resolved how Urm1 is handed between Uba4's domains, defining the indispensable step linking activation to thiocarboxylation and tRNA thiolation.","evidence":"In vitro thiocarboxylation assays, Cys225 mutagenesis and in vivo validation","pmids":["29718331"],"confidence":"High","gaps":["Did not provide a full structural view of the heterodimer","Release of Urm1-SH to downstream factors not defined"]},{"year":2018,"claim":"A virally relevant urmylation event was characterized: modification of HTLV-1 Tax redirects it to the cytoplasm and tunes NF-kB target transcription, extending urmylation to host-pathogen signaling.","evidence":"Co-IP, subcellular localization, NF-kB reporter assays and a tax-transgenic Drosophila model","pmids":["29665857"],"confidence":"Medium","gaps":["Single-lab study without reciprocal validation of the conjugation site","Direct biochemical demonstration of Tax urmylation chemistry limited"]},{"year":2020,"claim":"Crystal structures of full-length Uba4 and the Uba4-Urm1 complex revealed how the adenylation and rhodanese domains recognize and thiocarboxylate Urm1 and how Uba4 avoids self-conjugation.","evidence":"X-ray crystallography of full-length enzyme and heterodimer with mechanistic biochemistry","pmids":["32901956"],"confidence":"High","gaps":["Dynamic positioning of the rhodanese domain during catalysis not captured","Did not address mammalian enzyme architecture"]},{"year":2022,"claim":"In vitro reconstitution proved urmylation is E2/E3-independent and oxidative-stress-dependent, and showed it transfers sulfur to target cysteines (persulfidation), unifying the sulfur-carrier and modifier roles at the chemical level.","evidence":"In vitro urmylation reconstitution, crystal structures of Ahp1 +/- Urm1, persulfidation assays","pmids":["36102610"],"confidence":"High","gaps":["Whether mammalian urmylation requires dedicated enzymes left open","Substrate-selection rules for which lysines are modified not defined"]},{"year":2024,"claim":"Demonstrating Urm1-driven phase separation answered what cellular outcome urmylation produces under stress, showing pH-triggered Urm1 self-association deposits target proteins into protective condensates.","evidence":"Live-cell condensate imaging, Urm1 deletion phenotyping, biochemical self-association and stress urmylation assays","pmids":["38942013"],"confidence":"High","gaps":["Structural basis of Urm1 self-association not resolved","Generality of condensate role beyond yeast not established"]},{"year":2024,"claim":"A cryo-EM structure plus live-cell delivery of synthetic URM1 refined the activation mechanism and directly visualized stress-regulated URM1 redistribution and conjugation in cells.","evidence":"Cryo-EM of Uba4/Urm1, interface mutagenesis with in vitro/in vivo assays; suspension bead loading with live-cell imaging under redox stress","pmids":["39673271","39246272"],"confidence":"High","gaps":["Bead-loading study is single-lab with a novel delivery method","Precise compartments of stress-induced redistribution not fully mapped"]},{"year":2026,"claim":"Identification of NAE1/UBA3 and UBE2M as human E1/E2 for urmylation answered the long-standing question of mammalian conjugation enzymes and opened a therapeutic angle via pevonedistat.","evidence":"Activity-based URM1 probe, proteomics, cell-based validation and pharmacological NAE1 inhibition in liver cancer cells","pmids":["42056084"],"confidence":"High","gaps":["Reconciliation with the E2/E3-independent yeast mechanism not addressed","Role of DCN1 module remains tentative"]},{"year":null,"claim":"How urmylation substrate specificity is encoded, and how the apparently E2/E3-independent yeast chemistry reconciles with a dedicated human E1/E2 cascade, remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No unified model of lysine-site selection across substrates","Mechanistic reconciliation of E2/E3-independent vs NAE1/UBE2M-dependent conjugation lacking","Disease relevance of mammalian urmylation beyond cancer cell models unexplored"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0031386","term_label":"protein tag activity","supporting_discovery_ids":[2,7,12]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[2,7]},{"term_id":"GO:0140104","term_label":"molecular carrier activity","supporting_discovery_ids":[0,1,8]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[12,13,15]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[12]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,8]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[2,7,16]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[2,12,15]}],"complexes":["Uba4-Urm1 (MOCS3-URM1) activation complex"],"partners":["UBA4","MOCS3","NCS6","TUM1","AHP1","NAE1","UBE2M","CAS"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9BTM9","full_name":"Ubiquitin-related modifier 1","aliases":[],"length_aa":101,"mass_kda":11.4,"function":"Acts as a sulfur carrier required for 2-thiolation of mcm(5)S(2)U at tRNA wobble positions of cytosolic tRNA(Lys), tRNA(Glu) and tRNA(Gln) (PubMed:19017811). Serves as sulfur donor in tRNA 2-thiolation reaction by being thiocarboxylated (-COSH) at its C-terminus by MOCS3 (PubMed:19017811, PubMed:22453920). The sulfur is then transferred to tRNA to form 2-thiolation of mcm(5)S(2)U (PubMed:19017811, PubMed:22453920). Also acts as a ubiquitin-like protein (UBL) that is covalently conjugated via an isopeptide bond to lysine residues of target proteins such as MOCS3, ATPBD3, CTU2, USP15 and CAS (PubMed:21209336). The thiocarboxylated form serves as substrate for conjugation and oxidative stress specifically induces the formation of UBL-protein conjugates (PubMed:21209336)","subcellular_location":"Cytoplasm, cytosol; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9BTM9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/URM1","classification":"Common Essential","n_dependent_lines":993,"n_total_lines":1208,"dependency_fraction":0.8220198675496688},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/URM1","total_profiled":1310},"omim":[{"mim_id":"617057","title":"CYTOSOLIC THIOURIDYLASE, SUBUNIT 2; CTU2","url":"https://www.omim.org/entry/617057"},{"mim_id":"612694","title":"CYTOSOLIC THIOURIDYLASE, SUBUNIT 1; CTU1","url":"https://www.omim.org/entry/612694"},{"mim_id":"612693","title":"UBIQUITIN-RELATED MODIFIER 1; URM1","url":"https://www.omim.org/entry/612693"},{"mim_id":"609277","title":"MOLYBDENUM COFACTOR SYNTHESIS 3; MOCS3","url":"https://www.omim.org/entry/609277"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Vesicles","reliability":"Approved"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/URM1"},"hgnc":{"alias_symbol":["MGC2668"],"prev_symbol":["C9orf74"]},"alphafold":{"accession":"Q9BTM9","domains":[{"cath_id":"3.10.20.30","chopping":"4-95","consensus_level":"high","plddt":96.5502,"start":4,"end":95}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BTM9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BTM9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BTM9-F1-predicted_aligned_error_v6.png","plddt_mean":94.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=URM1","jax_strain_url":"https://www.jax.org/strain/search?query=URM1"},"sequence":{"accession":"Q9BTM9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BTM9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BTM9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BTM9"}},"corpus_meta":[{"pmid":"19145231","id":"PMC_19145231","title":"Ubiquitin-related modifier Urm1 acts as a sulphur carrier in thiolation of eukaryotic transfer RNA.","date":"2009","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/19145231","citation_count":242,"is_preprint":false},{"pmid":"19017811","id":"PMC_19017811","title":"A functional proteomics approach links the ubiquitin-related modifier Urm1 to a tRNA modification pathway.","date":"2008","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/19017811","citation_count":98,"is_preprint":false},{"pmid":"21209336","id":"PMC_21209336","title":"Role of the ubiquitin-like protein Urm1 as a noncanonical lysine-directed protein modifier.","date":"2011","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/21209336","citation_count":93,"is_preprint":false},{"pmid":"16864801","id":"PMC_16864801","title":"Solution structure of Urm1 and its implications for the origin of protein modifiers.","date":"2006","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/16864801","citation_count":61,"is_preprint":false},{"pmid":"19047990","id":"PMC_19047990","title":"Urm1 at the crossroad of modifications. 'Protein Modifications: Beyond the Usual Suspects' Review Series.","date":"2008","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/19047990","citation_count":52,"is_preprint":false},{"pmid":"28357324","id":"PMC_28357324","title":"Sulfur transfer and activation by ubiquitin-like modifier system Uba4•Urm1 link protein urmylation and tRNA thiolation in yeast.","date":"2016","source":"Microbial cell (Graz, Austria)","url":"https://pubmed.ncbi.nlm.nih.gov/28357324","citation_count":38,"is_preprint":false},{"pmid":"21904977","id":"PMC_21904977","title":"The dual role of ubiquitin-like protein Urm1 as a protein modifier and sulfur carrier.","date":"2011","source":"Protein & cell","url":"https://pubmed.ncbi.nlm.nih.gov/21904977","citation_count":29,"is_preprint":false},{"pmid":"29718331","id":"PMC_29718331","title":"The Uba4 domain interplay is mediated via a thioester that is critical for tRNA thiolation through Urm1 thiocarboxylation.","date":"2018","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/29718331","citation_count":28,"is_preprint":false},{"pmid":"26715182","id":"PMC_26715182","title":"Urm1: an essential regulator of JNK signaling and oxidative stress in Drosophila melanogaster.","date":"2015","source":"Cellular and molecular life sciences : CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/26715182","citation_count":27,"is_preprint":false},{"pmid":"32901956","id":"PMC_32901956","title":"Molecular basis for the bifunctional Uba4-Urm1 sulfur-relay system in tRNA thiolation and ubiquitin-like conjugation.","date":"2020","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/32901956","citation_count":26,"is_preprint":false},{"pmid":"25747390","id":"PMC_25747390","title":"Urmylation and tRNA thiolation functions of ubiquitin-like Uba4·Urm1 systems are conserved from yeast to man.","date":"2015","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/25747390","citation_count":26,"is_preprint":false},{"pmid":"16046629","id":"PMC_16046629","title":"Three-dimensional structure of the AAH26994.1 protein from Mus musculus, a putative eukaryotic Urm1.","date":"2005","source":"Protein science : a publication of the Protein Society","url":"https://pubmed.ncbi.nlm.nih.gov/16046629","citation_count":21,"is_preprint":false},{"pmid":"38942013","id":"PMC_38942013","title":"Stress-dependent condensate formation regulated by the ubiquitin-related modifier Urm1.","date":"2024","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/38942013","citation_count":19,"is_preprint":false},{"pmid":"36102610","id":"PMC_36102610","title":"E2/E3-independent ubiquitin-like protein conjugation by Urm1 is directly coupled to cysteine persulfidation.","date":"2022","source":"The EMBO 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Non-Canonical UBL.","date":"2021","source":"Biomolecules","url":"https://pubmed.ncbi.nlm.nih.gov/33499055","citation_count":12,"is_preprint":false},{"pmid":"31551975","id":"PMC_31551975","title":"URM1-Mediated Ubiquitin-Like Modification Is Required for Oxidative Stress Adaptation During Infection of the Rice Blast Fungus.","date":"2019","source":"Frontiers in microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/31551975","citation_count":11,"is_preprint":false},{"pmid":"33548388","id":"PMC_33548388","title":"The emerging roles of ubiquitin-like protein Urm1 in eukaryotes.","date":"2021","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/33548388","citation_count":10,"is_preprint":false},{"pmid":"39673271","id":"PMC_39673271","title":"Molecular basis for thiocarboxylation and release of Urm1 by its E1-activating enzyme Uba4.","date":"2024","source":"Nucleic acids 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English)","url":"https://pubmed.ncbi.nlm.nih.gov/39246272","citation_count":5,"is_preprint":false},{"pmid":"36142209","id":"PMC_36142209","title":"Uba1: A Potential Ubiquitin-like Activator Protein of Urm1 in Toxoplasma gondii.","date":"2022","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/36142209","citation_count":4,"is_preprint":false},{"pmid":"39705796","id":"PMC_39705796","title":"Evaluation of the toxoplasma Urm1 gene deletion mutant (PruΔUrm1) as a promising vaccine candidate against toxoplasmosis in mice.","date":"2024","source":"Vaccine","url":"https://pubmed.ncbi.nlm.nih.gov/39705796","citation_count":2,"is_preprint":false},{"pmid":"36769280","id":"PMC_36769280","title":"Ubiquitin-Related Modifier 1 (URM-1) Modulates Cx43 in Breast Cancer Cell Lines.","date":"2023","source":"International journal of molecular 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communications","url":"https://pubmed.ncbi.nlm.nih.gov/42056084","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":15645,"output_tokens":4653,"usd":0.058365,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12650,"output_tokens":4199,"usd":0.084113,"stage2_stop_reason":"end_turn"},"total_usd":0.142478,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2009,\n      \"finding\": \"Urm1p acts as a sulphur carrier in eukaryotic tRNA thiolation; Uba4p first adenylates and then directly transfers sulphur onto Urm1p to form a thiocarboxylate; Ncs6p binds tRNA as part of this conserved pathway; Ncs2p and Yor251cp are additional pathway components.\",\n      \"method\": \"In vitro sulphur-transfer assays, genetic screens (S. cerevisiae), tRNA thiolation assays, biochemical reconstitution\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with multiple pathway components, replicated by independent labs\",\n      \"pmids\": [\"19145231\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Urm1 is activated to a thiocarboxylate intermediate that serves as a sulfur donor in tRNA thiolation reactions, mechanistically reminiscent of prokaryotic sulfur carriers MoaD/ThiS; this activity is distinct from canonical ubiquitin-like protein conjugation.\",\n      \"method\": \"Functional proteomics, enzymatic activity assays, thiocarboxylate intermediate detection\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — enzymatic intermediate characterization with biochemical assays, independently corroborated\",\n      \"pmids\": [\"19017811\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Urm1 is conjugated to lysine residues of target proteins (urmylation) via a thioester intermediate forming an isopeptide bond; oxidative stress enhances urmylation in both S. cerevisiae and mammalian cells. Yeast peroxiredoxin Ahp1 is confirmed as a substrate. Mammalian targets include MOCS3, ATPBD3, CTU2, and cellular apoptosis susceptibility protein (CAS). Urmylation involves a C-terminal thiocarboxylate of Urm1 rather than canonical UBL mechanism.\",\n      \"method\": \"In vivo conjugation assays, site-directed mutagenesis, mass spectrometry identification of substrates, Co-IP\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (mutagenesis, MS, in vivo assays), confirmed in two organisms\",\n      \"pmids\": [\"21209336\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"NMR solution structure of yeast Urm1 reveals a ubiquitin-fold most closely resembling prokaryotic MoaD sulfur-carrier proteins, placing Urm1 as a 'molecular fossil' linking sulfur-carrier proteins and ubiquitin-like modifiers. Structural and electrostatic surface similarities between Urm1-Uba4 and MoaD-MoeB suggest a conserved ATP-dependent activation mechanism.\",\n      \"method\": \"Solution NMR structure determination, phylogenetic and structural comparison\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure with functional/evolutionary validation in a single rigorous study\",\n      \"pmids\": [\"16864801\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"NMR solution structure of mouse Urm1 (AAH26994.1) reveals close structural resemblance to bacterial MoaD sulfur-carrier proteins, supporting its role as an evolutionary link between sulfur-donor systems and ubiquitin-like modifiers.\",\n      \"method\": \"NMR spectroscopy, structural comparison\",\n      \"journal\": \"Protein science : a publication of the Protein Society\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — NMR structure determined, but functional validation limited to structural/evolutionary inference in a single study\",\n      \"pmids\": [\"16046629\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"A critical thioester linkage between Urm1 and Uba4 residue Cys225 mediates intramolecular transfer of Urm1 between the E1-like (AD) and rhodanese (RHD) domains of Uba4; this thioester is indispensable for Urm1 thiocarboxylation and consequent tRNA thiolation in vivo.\",\n      \"method\": \"In vitro Urm1 thiocarboxylation assay, structure-function analysis, site-directed mutagenesis (Cys225), chemical profiling\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution combined with mutagenesis and in vivo validation in a single rigorous study\",\n      \"pmids\": [\"29718331\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Crystal structures of full-length Uba4 and the Uba4-Urm1 heterodimeric complex reveal how the AD and RHD domains of Uba4 orchestrate recognition, binding, and thiocarboxylation of the Urm1 C-terminus; a mechanism was identified by which Uba4 protects itself against self-conjugation with activated Urm1-COSH.\",\n      \"method\": \"X-ray crystallography, structural analysis of heterodimeric complex, mechanistic biochemical assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structures of full-length enzyme and substrate complex with mechanistic biochemical analysis in one rigorous study\",\n      \"pmids\": [\"32901956\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Urmylation of target proteins (including peroxiredoxin Ahp1) is reconstituted in vitro using thiocarboxylated Urm1 and is E2/E3-independent, requiring oxidative stress. Urmylation is accompanied by transfer of sulfur to cysteine residues in target proteins (cysteine persulfidation). Crystal structures of Ahp1 before and after Urm1 attachment were determined.\",\n      \"method\": \"In vitro reconstitution of urmylation, X-ray crystallography of Ahp1 ± Urm1, biochemical assays for persulfidation\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with crystal structures and mutagenesis in one rigorous study\",\n      \"pmids\": [\"36102610\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Cryo-EM structure of the Uba4/Urm1 complex reveals RHD domain positions after Urm1 binding; mutations at the Uba4-Urm1 interface impair thiocarboxylation in vitro and in vivo. Conserved cysteines of Uba4 are required for thioester formation and Urm1-SH generation. Urm1-SH release mechanism and interactions with upstream (Tum1) and downstream (Ncs6) pathway components are defined.\",\n      \"method\": \"Cryo-EM structural determination, site-directed mutagenesis, in vitro thiocarboxylation assays, in vivo complementation\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structure combined with mutagenesis and in vitro/in vivo functional assays in one rigorous study\",\n      \"pmids\": [\"39673271\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Human URM1 and UBA4/MOCS3 are functional orthologs of yeast Urm1 and Uba4; gene shuffle experiments show they support urmylation of peroxiredoxin Ahp1 and tRNA thiolation in yeast (albeit at reduced efficiency). Yeast Uba4 is itself modified by Urm1 and hURM1, revealing target overlap between eukaryal urmylation pathways.\",\n      \"method\": \"Yeast gene shuffle (complementation), biochemical urmylation assays, tRNA thiolation assays\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic complementation with biochemical validation, single lab, two orthogonal methods\",\n      \"pmids\": [\"25747390\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In Drosophila, Urm1 conjugation to target proteins including the conserved substrate Peroxiredoxin 5 (Prx5) requires the E1 activating enzyme Uba4. Loss of Urm1 is lethal; escapers show increased cytoprotective JNK signaling, and elevated JNK targets Jafrac1 and gstD1 confer oxidative stress resistance in Urm1 null mutants.\",\n      \"method\": \"Drosophila genetics (null mutants, genetic rescue), western blotting for urmylation, JNK pathway epistasis analysis\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis combined with biochemical urmylation assays and molecular pathway analysis, single lab\",\n      \"pmids\": [\"26715182\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Urmylation of Ahp1 is suppressed at elevated temperatures or under sulfur starvation. The rhodanese domain (RHD) in Uba4, critical for Urm1-COSH formation, is required (but not absolutely essential) for both protein urmylation and tRNA thiolation, establishing that sulfur supply and thiocarboxylation chemically link the two branches of the URM1 pathway.\",\n      \"method\": \"Site-directed mutagenesis of Uba4 RHD, urmylation monitoring by western blot, tRNA thiolation assays, S. cerevisiae genetics\",\n      \"journal\": \"Microbial cell (Graz, Austria)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis with in vivo functional readouts across two pathway branches, single lab\",\n      \"pmids\": [\"28357324\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Covalent modification by Urm1 promotes phase separation of a wide range of proteins in yeast under stress. A drop in cellular pH triggered by stress drives Urm1 self-association and interaction with target proteins and Uba4. Urmylation of stress-sensitive proteins promotes their deposition into stress granules and nuclear condensates. Yeast cells lacking Urm1 exhibit condensate defects and reduced stress resilience.\",\n      \"method\": \"Live-cell imaging of condensates, genetic (Urm1 deletion), biochemical assays for Urm1 self-association, urmylation in stress conditions\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live imaging, genetic KO with defined condensate phenotype, and biochemical assays for Urm1-driven phase separation in one rigorous study\",\n      \"pmids\": [\"38942013\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Urm1 conjugation (urmylation) of the HTLV-1 oncoprotein Tax redistributes Tax to the cytoplasm and increases transcription of NF-κB targets (Rantes and IL-6). In a tax-transgenic Drosophila model, Urm1 presence correlates with transcriptional output of the NF-κB target Diptericin, indicating evolutionary conservation of Urm1-dependent subcellular targeting of Tax.\",\n      \"method\": \"Co-immunoprecipitation, subcellular localization assays, NF-κB reporter assays, Drosophila transgenic model\",\n      \"journal\": \"Retrovirology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP plus functional reporter assays and Drosophila model, single lab\",\n      \"pmids\": [\"29665857\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"URM-1 co-localizes and interacts with connexin 43 (Cx43) in breast cancer cell lines; URMylated Cx43 is increased upon cellular stress. Downregulation of URM-1 decreases Cx43 expression and affects SUMO-1-mediated SUMOylation of Cx43, while upregulation of URM-1 increases Cx43 expression and reverses EMT-associated processes.\",\n      \"method\": \"Co-immunoprecipitation, co-localization imaging, siRNA knockdown/overexpression, western blotting\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP and co-localization with functional readouts, single lab, single study\",\n      \"pmids\": [\"36769280\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Using suspension bead loading (SBL) to deliver synthetic URM1 into live cells, oxidative stress was shown to alter both the subcellular localization and conjugation pattern of URM1, directly demonstrating stress-regulated urmylation and redistribution in living cells.\",\n      \"method\": \"Suspension bead loading of synthetic protein, live-cell imaging, subcellular fractionation, conjugation assays under redox stress\",\n      \"journal\": \"Angewandte Chemie (International ed. in English)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct live-cell localization experiment with functional consequence, novel delivery method, single lab\",\n      \"pmids\": [\"39246272\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"NAE1/UBA3 and UBE2M were identified as E1 and E2 enzymes, respectively, for the URM1 modification cascade in human cells under both normal and oxidative stress conditions. DCN1 (via the UBE2M-DCN1 module) may contribute to URM1 conjugation. Pharmacological inhibition of NAE1 by pevonedistat blocks protein urmylation in human cells and shows synergy with cisplatin in killing liver cancer cells.\",\n      \"method\": \"Activity-based URM1 probe for covalent capture of cysteine enzymes, proteomic characterization, cell-based validation, pharmacological inhibition (pevonedistat)\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — activity-based probe with proteomic identification and cell-based validation plus pharmacological perturbation, identifying previously unknown E1/E2 for mammalian urmylation\",\n      \"pmids\": [\"42056084\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Archaeal Urm1 (from Sulfolobus acidocaldarius) can conjugate to yeast peroxiredoxin Ahp1 (a bona fide urmylation target) when expressed in S. cerevisiae, but cannot support tRNA thiolation. Ahp1 conjugation by archaeal Urm1 requires sulfur transfer from yeast Uba4, establishing that thioactivation and urmylation-like conjugation are conserved between Sulfolobus and Saccharomyces.\",\n      \"method\": \"URM1 gene shuffle (Sulfolobus to yeast), urmylation assays, tRNA thiolation assays, genetic complementation\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic and biochemical cross-species complementation with two orthogonal functional readouts, single lab\",\n      \"pmids\": [\"41276627\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"URM1 is a dual-function ubiquitin-like protein that acts both as a sulfur carrier and a protein modifier: activated by its E1-like enzyme UBA4 (MOCS3 in humans) through sequential adenylation, thioesterification via Uba4-Cys225, and rhodanese-domain-mediated thiocarboxylation of Urm1's C-terminal glycine, the resulting thiocarboxylated Urm1 (Urm1-COSH) either donates sulfur for 2-thiolation of wobble uridine (U34) in cytoplasmic tRNAs (via downstream thiolases Ncs6/CTU1) or, under oxidative stress, conjugates covalently via an isopeptide (and/or persulfidation) bond to lysine residues of target proteins (including peroxiredoxin Ahp1, MOCS3, CAS, and Cx43) in an E2/E3-independent manner; in human cells, NAE1/UBA3 and UBE2M serve as canonical E1/E2 enzymes for the urmylation branch; stress-induced urmylation additionally promotes phase separation of target proteins into protective condensates, with Urm1 acting as a molecular 'adhesive' that drives stress granule and nuclear condensate formation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"URM1 is a dual-function ubiquitin-like protein that serves both as a sulfur carrier in tRNA modification and as a covalent protein modifier under oxidative stress [#0, #2]. Its NMR solution structure adopts a ubiquitin fold most closely resembling the prokaryotic MoaD/ThiS sulfur-carrier proteins, positioning Urm1 as an evolutionary link between bacterial sulfur-donor systems and eukaryotic ubiquitin-like modifiers [#3, #4]. Activation proceeds through the E1-like enzyme Uba4 (MOCS3 in humans): Uba4 adenylates the Urm1 C-terminus and, via a thioester linkage at Cys225, shuttles Urm1 between its adenylation and rhodanese domains to generate a C-terminal thiocarboxylate (Urm1-COSH), the chemistry that drives both pathway branches [#0, #5, #6, #8]. The thiocarboxylated species donates sulfur for 2-thiolation of wobble uridines in cytoplasmic tRNAs through downstream thiolases including Ncs6 [#0, #8]. The same thiocarboxylate intermediate enables urmylation: under oxidative stress Urm1 conjugates to lysine residues of target proteins—including the peroxiredoxin Ahp1—via an E2/E3-independent mechanism accompanied by cysteine persulfidation, with substrates spanning yeast Ahp1 and mammalian MOCS3, CTU2, and CAS [#2, #7]. Sulfur supply mechanistically couples the tRNA-thiolation and protein-conjugation branches, both depending on the Uba4 rhodanese domain [#11]. Beyond modification chemistry, urmylation promotes stress-induced phase separation, driving deposition of stress-sensitive proteins into stress granules and nuclear condensates and conferring stress resilience [#12]. In human cells the canonical neddylation enzymes NAE1/UBA3 and UBE2M act as E1 and E2 for the urmylation cascade, and pharmacological NAE1 inhibition by pevonedistat blocks urmylation and synergizes with cisplatin in liver cancer cells [#16].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Establishing the Urm1 fold answered whether this ubiquitin-like protein is built like a conjugation modifier or a sulfur carrier, revealing its closest structural kinship to bacterial MoaD.\",\n      \"evidence\": \"NMR solution structures of mouse and yeast Urm1 with structural/phylogenetic comparison\",\n      \"pmids\": [\"16046629\", \"16864801\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structure alone did not demonstrate which biochemical activity (sulfur transfer vs conjugation) dominates in vivo\", \"No partner enzyme captured in the structural studies\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Detection of a thiocarboxylate intermediate defined Urm1's first biochemical activity as a sulfur donor distinct from canonical UBL conjugation, mechanistically aligning it with prokaryotic sulfur carriers.\",\n      \"evidence\": \"Functional proteomics and enzymatic intermediate detection\",\n      \"pmids\": [\"19017811\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the full enzymatic route of thiocarboxylate formation\", \"Downstream tRNA targets not yet mapped at this stage\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Reconstitution placed Urm1 in a defined tRNA-thiolation pathway, showing Uba4 adenylates then transfers sulfur to Urm1 and that Ncs6 binds tRNA, establishing the sulfur-relay architecture.\",\n      \"evidence\": \"In vitro sulfur-transfer and tRNA thiolation assays with genetic screens in S. cerevisiae\",\n      \"pmids\": [\"19145231\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic mechanism of intramolecular sulfur transfer within Uba4 not yet defined\", \"Did not address a protein-conjugation role\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Discovery of urmylation answered whether Urm1 also acts as a protein modifier, showing oxidative-stress-driven isopeptide conjugation to lysines using the C-terminal thiocarboxylate rather than canonical UBL chemistry.\",\n      \"evidence\": \"In vivo conjugation assays, mutagenesis, MS substrate identification and Co-IP in yeast and mammalian cells\",\n      \"pmids\": [\"21209336\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"E2/E3 requirements and enzymatic route of conjugation unresolved\", \"Functional consequence of modifying each substrate not defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Cross-species complementation established that human URM1/MOCS3 and a Drosophila pathway are functional orthologs, demonstrating conservation of both branches and substrate overlap.\",\n      \"evidence\": \"Yeast gene-shuffle complementation, urmylation and tRNA thiolation assays; Drosophila null mutants and JNK epistasis\",\n      \"pmids\": [\"25747390\", \"26715182\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reduced efficiency of human enzymes in yeast leaves native human kinetics unaddressed\", \"Connection between urmylation loss and JNK induction not mechanistically dissected\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identifying the Uba4 Cys225 thioester resolved how Urm1 is handed between Uba4's domains, defining the indispensable step linking activation to thiocarboxylation and tRNA thiolation.\",\n      \"evidence\": \"In vitro thiocarboxylation assays, Cys225 mutagenesis and in vivo validation\",\n      \"pmids\": [\"29718331\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not provide a full structural view of the heterodimer\", \"Release of Urm1-SH to downstream factors not defined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"A virally relevant urmylation event was characterized: modification of HTLV-1 Tax redirects it to the cytoplasm and tunes NF-kB target transcription, extending urmylation to host-pathogen signaling.\",\n      \"evidence\": \"Co-IP, subcellular localization, NF-kB reporter assays and a tax-transgenic Drosophila model\",\n      \"pmids\": [\"29665857\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study without reciprocal validation of the conjugation site\", \"Direct biochemical demonstration of Tax urmylation chemistry limited\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Crystal structures of full-length Uba4 and the Uba4-Urm1 complex revealed how the adenylation and rhodanese domains recognize and thiocarboxylate Urm1 and how Uba4 avoids self-conjugation.\",\n      \"evidence\": \"X-ray crystallography of full-length enzyme and heterodimer with mechanistic biochemistry\",\n      \"pmids\": [\"32901956\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dynamic positioning of the rhodanese domain during catalysis not captured\", \"Did not address mammalian enzyme architecture\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"In vitro reconstitution proved urmylation is E2/E3-independent and oxidative-stress-dependent, and showed it transfers sulfur to target cysteines (persulfidation), unifying the sulfur-carrier and modifier roles at the chemical level.\",\n      \"evidence\": \"In vitro urmylation reconstitution, crystal structures of Ahp1 +/- Urm1, persulfidation assays\",\n      \"pmids\": [\"36102610\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether mammalian urmylation requires dedicated enzymes left open\", \"Substrate-selection rules for which lysines are modified not defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrating Urm1-driven phase separation answered what cellular outcome urmylation produces under stress, showing pH-triggered Urm1 self-association deposits target proteins into protective condensates.\",\n      \"evidence\": \"Live-cell condensate imaging, Urm1 deletion phenotyping, biochemical self-association and stress urmylation assays\",\n      \"pmids\": [\"38942013\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of Urm1 self-association not resolved\", \"Generality of condensate role beyond yeast not established\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"A cryo-EM structure plus live-cell delivery of synthetic URM1 refined the activation mechanism and directly visualized stress-regulated URM1 redistribution and conjugation in cells.\",\n      \"evidence\": \"Cryo-EM of Uba4/Urm1, interface mutagenesis with in vitro/in vivo assays; suspension bead loading with live-cell imaging under redox stress\",\n      \"pmids\": [\"39673271\", \"39246272\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Bead-loading study is single-lab with a novel delivery method\", \"Precise compartments of stress-induced redistribution not fully mapped\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identification of NAE1/UBA3 and UBE2M as human E1/E2 for urmylation answered the long-standing question of mammalian conjugation enzymes and opened a therapeutic angle via pevonedistat.\",\n      \"evidence\": \"Activity-based URM1 probe, proteomics, cell-based validation and pharmacological NAE1 inhibition in liver cancer cells\",\n      \"pmids\": [\"42056084\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reconciliation with the E2/E3-independent yeast mechanism not addressed\", \"Role of DCN1 module remains tentative\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How urmylation substrate specificity is encoded, and how the apparently E2/E3-independent yeast chemistry reconciles with a dedicated human E1/E2 cascade, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No unified model of lysine-site selection across substrates\", \"Mechanistic reconciliation of E2/E3-independent vs NAE1/UBE2M-dependent conjugation lacking\", \"Disease relevance of mammalian urmylation beyond cancer cell models unexplored\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0031386\", \"supporting_discovery_ids\": [2, 7, 12]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [2, 7]},\n      {\"term_id\": \"GO:0140104\", \"supporting_discovery_ids\": [0, 1, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [12, 13, 15]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [12]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 8]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [2, 7, 16]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [2, 12, 15]}\n    ],\n    \"complexes\": [\"Uba4-Urm1 (MOCS3-URM1) activation complex\"],\n    \"partners\": [\"UBA4\", \"MOCS3\", \"NCS6\", \"TUM1\", \"AHP1\", \"NAE1\", \"UBE2M\", \"CAS\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}