{"gene":"NMD3","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":1999,"finding":"NMD3 (yeast Nmd3p) is an essential cytoplasmic protein required for stable 60S ribosomal subunits; loss-of-function causes rapid degradation of mature 25S rRNA and formation of half-mer polysomes, indicating a role in a late cytoplasmic assembly step of the 60S subunit. Nmd3p fractionates as a cytoplasmic protein and co-sediments with free 60S subunits in sucrose gradients.","method":"Temperature-sensitive allele (loss-of-function), sucrose gradient sedimentation, pulse-chase rRNA analysis, cell fractionation","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (genetic, biochemical fractionation, pulse-chase) in a foundational study","pmids":["10022925"],"is_preprint":false},{"year":2003,"finding":"Human NMD3 (hNMD3) acts as an adaptor for CRM1/Ran.GTP-mediated nuclear export of 60S ribosomal subunits. hNMD3 contains a CRM1-dependent leucine-rich nuclear export signal (NES) and a complex nuclear localization signal (NLS); the basic region of the NLS is also required for nucleolar accumulation. Export-defective NES-mutant hNMD3 binds nascent nuclear 60S pre-export particles and acts as a dominant negative, blocking 60S subunit export from Xenopus oocyte nuclei.","method":"Xenopus oocyte microinjection, dominant-negative NES mutant analysis, nuclear export assays, co-sedimentation with 60S particles","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — dominant-negative mutagenesis in Xenopus oocytes plus biochemical co-sedimentation; replicated in vertebrate context, consistent with yeast data","pmids":["12773398"],"is_preprint":false},{"year":2006,"finding":"Mutational analysis of yeast Nmd3p identified two distinct regions required for binding to 60S subunits, indicating multivalent interaction with the pre-60S particle. Separate domains govern nucleocytoplasmic shuttling (NES for CRM1 recognition) and ribosome binding.","method":"Site-directed mutagenesis, in vivo ribosome binding assays, nuclear export assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic mutagenesis with functional binding readouts, single lab","pmids":["17015443"],"is_preprint":false},{"year":2007,"finding":"Nmd3 mutants impaired for 60S subunit binding accumulate at the nuclear envelope and show enhanced binding to Crm1 independently of RanGTP. Interaction with the NPC is NES/Crm1-dependent. Components of the cytoplasmic-face Nup82 complex co-purify with mutant Nmd3, and mutations in Nup82 complex components cause wild-type Nmd3 to accumulate in the nucleoplasm, indicating that the Nup82 complex is a terminal binding site required for Nmd3 release from Crm1 at the NPC.","method":"In vivo/in vitro Crm1 binding assays, co-purification with GFP-tagged nucleoporins, NPC mutant analysis, fluorescence microscopy","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple methods (co-purification, in vitro binding, mutant phenotypes) in a single lab study","pmids":["17347149"],"is_preprint":false},{"year":2010,"finding":"Cryo-EM reconstruction of the yeast 60S–Nmd3 complex reveals that Nmd3 binds to the intersubunit face of the large subunit, contacting regions around helices 38, 69, and 95 of 25S rRNA, adjacent to ribosomal protein Rpl10. This binding site is blocked in 80S ribosomes. rRNA protection experiments corroborated the structural binding site.","method":"Cryo-electron microscopy, rRNA protection assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structure with orthogonal rRNA footprinting validation; single lab but two independent methods","pmids":["20584915"],"is_preprint":false},{"year":2013,"finding":"In human cells, CRM1 and NMD3 co-localize with nucleolar marker proteins; CRM1 nucleolar localization depends on CRM1 activity and NMD3 expression, whereas NMD3 nucleolar localization is independent of CRM1, indicating NMD3 provides nucleolar tethering of CRM1. NMD3 depletion reduced the rate of pre-47S rRNA synthesis but did not affect 28S rRNA processing (unlike CRM1 inhibition), and did not cause nucleolar disintegration.","method":"siRNA depletion, immunofluorescence co-localization, leptomycin B inhibition, rRNA synthesis rate measurement","journal":"Nucleus (Austin, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical and imaging analyses with genetic/pharmacological perturbations, single lab","pmids":["23782956"],"is_preprint":false},{"year":2017,"finding":"Cryo-EM of cytoplasmic pre-60S particles purified via tagged Nmd3 reveals that Nmd3 and Lsg1 occupy positions near the peptidyl-transferase center (PTC), with Nmd3 recognizing the PTC in its near-mature conformation. Reh1 anchors to the polypeptide tunnel exit with its C-terminus inserted into the tunnel. The structural data support a checkpoint role for Nmd3 in monitoring PTC assembly before final maturation.","method":"Cryo-electron microscopy of affinity-purified pre-60S particles","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM structure of the native complex, multiple assembly factors localized simultaneously","pmids":["28112732"],"is_preprint":false},{"year":2017,"finding":"The T-ALL-associated Rpl10-R98S mutation traps Nmd3 in the ribosomal P site, blocking its release. Suppressor mutations in Nmd3 that disrupt its interactions with the ribosome or with Tif6 bypass this block. Using purified components in vitro, Nmd3 inhibited Sdo1-stimulated Efl1 GTPase activity on rpl10-R98S mutant 60S subunits but not wild-type subunits; Nmd3 suppressor mutations reversed this inhibition, establishing that Nmd3 must vacate the P site to allow Sdo1-mediated Efl1 activation.","method":"Genetic suppressor screen, in vitro reconstitution with purified components, GTPase activity assay, cryo-EM (referenced from prior work)","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified components plus corroborating in vivo suppressor genetics and mutagenesis","pmids":["28715419"],"is_preprint":false},{"year":2025,"finding":"Simultaneous bypass mutations in Nmd3 and Tif6 release defective uL16-mutant 60S subunits into the translating pool, demonstrating that Nmd3 (together with Tif6) acts as a quality control checkpoint during late cytoplasmic pre-60S maturation. Cryo-EM and selective ribosome profiling of these escaped ribosomes show they can form peptide bonds but stall at early codons and are degraded if they enter translation. Reh1 is identified as required for the surveillance pathway that detects and degrades defective ribosomes during biogenesis.","method":"Genetic bypass mutations, cryo-EM, selective ribosome profiling, in vivo degradation assays","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — cryo-EM and ribosome profiling with genetic evidence; preprint, not yet peer-reviewed","pmids":["bio_10.1101_2025.10.29.685433"],"is_preprint":true}],"current_model":"NMD3 encodes a conserved nucleocytoplasmic shuttling adaptor protein that binds to the intersubunit face of nascent 60S ribosomal subunits (near helices 38, 69, and 95 of 25S rRNA, adjacent to uL16/Rpl10) in the nucleus, recruits the export receptor CRM1 via its leucine-rich NES to drive RanGTP-dependent 60S subunit nuclear export through the NPC (engaging the cytoplasmic Nup82 complex for release), and then in the cytoplasm must be displaced from the P site by the concerted action of Rpl10/uL16 and the GTPase Lsg1 (assisted by Efl1/Sdo1) to complete 60S maturation; structurally, Nmd3 recognizes the near-mature peptidyl-transferase center and thereby acts as a quality control checkpoint ensuring PTC assembly before permitting ribosome entry into translation."},"narrative":{"mechanistic_narrative":"NMD3 is a conserved nucleocytoplasmic shuttling adaptor that drives the late nuclear export and final cytoplasmic maturation of the 60S ribosomal subunit and acts as a quality-control checkpoint over peptidyl-transferase center assembly [PMID:10022925, PMID:28112732]. It was first defined in yeast as an essential factor required for stable 60S subunits, where its loss triggers rapid degradation of mature 25S rRNA and accumulation of half-mer polysomes, placing it at a late assembly step on free 60S subunits [PMID:10022925]. NMD3 binds the intersubunit face of the large subunit, contacting helices 38, 69, and 95 of 25S rRNA adjacent to Rpl10/uL16 near the PTC — a site occluded in assembled 80S ribosomes — and engages the particle through multiple separable ribosome-binding regions [PMID:17015443, PMID:20584915]. For export, NMD3 supplies a CRM1-dependent leucine-rich NES and a complex NLS, serving as the adaptor for CRM1/Ran.GTP-mediated transport of pre-60S particles; export-defective NES mutants bind nascent nuclear 60S and act as dominant negatives, and in human cells NMD3 tethers CRM1 to the nucleolus [PMID:12773398, PMID:23782956]. Release from CRM1 at the cytoplasmic face of the NPC requires the Nup82 complex as a terminal binding site [PMID:17347149]. In the cytoplasm NMD3 must vacate the P site to permit maturation: the T-ALL-associated Rpl10-R98S mutation traps NMD3 in the P site and blocks Sdo1-stimulated Efl1 GTPase activation, while NMD3 suppressor mutations restore it, establishing that NMD3 displacement gates the final GTPase-driven step [PMID:28715419]. Together with Tif6, NMD3 enforces a surveillance checkpoint that prevents subunits with defective PTC assembly from entering translation; bypass mutations release defective uL16-mutant subunits that stall at early codons and are degraded [PMID:bio_10.1101_2025.10.29.685433].","teleology":[{"year":1999,"claim":"Establishing where NMD3 acts: it was unknown which step of ribosome assembly required NMD3, and genetic depletion placed it at a late cytoplasmic 60S maturation step.","evidence":"Temperature-sensitive allele with sucrose gradient sedimentation, pulse-chase rRNA analysis, and cell fractionation in yeast","pmids":["10022925"],"confidence":"High","gaps":["Molecular binding site on the subunit not resolved","Mechanism linking NMD3 loss to 25S rRNA degradation unclear"]},{"year":2003,"claim":"Defining NMD3's transport function: whether NMD3 mediates 60S nuclear export was unknown, and it was shown to be a CRM1/Ran.GTP export adaptor with a leucine-rich NES.","evidence":"Xenopus oocyte microinjection, dominant-negative NES-mutant analysis, and co-sedimentation with 60S particles","pmids":["12773398"],"confidence":"High","gaps":["How NMD3 is loaded onto nascent 60S in the nucleus not defined","NPC release mechanism not addressed"]},{"year":2006,"claim":"Dissecting NMD3 architecture: the relationship between ribosome binding and shuttling was unknown, and mutagenesis showed two distinct 60S-binding regions separable from the export determinants.","evidence":"Site-directed mutagenesis with in vivo ribosome binding and nuclear export assays in yeast","pmids":["17015443"],"confidence":"Medium","gaps":["Structural basis of multivalent binding not resolved","Single-lab functional readouts"]},{"year":2007,"claim":"Resolving NPC release: how NMD3 is discharged from CRM1 after translocation was unknown, and the Nup82 complex was identified as the terminal cytoplasmic binding site required for release.","evidence":"In vivo/in vitro Crm1 binding assays, co-purification with tagged nucleoporins, and NPC mutant microscopy in yeast","pmids":["17347149"],"confidence":"Medium","gaps":["Direct biochemical mechanism of CRM1 disassembly not reconstituted","Order of release relative to ribosome remodeling unclear"]},{"year":2010,"claim":"Mapping the binding site: where NMD3 contacts the subunit was unknown, and cryo-EM placed it on the intersubunit face near 25S rRNA helices 38/69/95 adjacent to Rpl10.","evidence":"Cryo-EM reconstruction of the yeast 60S–Nmd3 complex with rRNA protection assays","pmids":["20584915"],"confidence":"High","gaps":["Near-atomic detail of PTC recognition not resolved at this stage","Functional consequence of the contacts not tested"]},{"year":2013,"claim":"Extending to human cells: the relationship between NMD3 and CRM1 nucleolar behavior was unknown, and NMD3 was shown to tether CRM1 to the nucleolus and influence pre-47S rRNA synthesis.","evidence":"siRNA depletion, immunofluorescence co-localization, leptomycin B inhibition, and rRNA synthesis measurement in human cells","pmids":["23782956"],"confidence":"Medium","gaps":["Mechanism linking NMD3 to rRNA synthesis rate unclear","Whether tethering is direct not established"]},{"year":2017,"claim":"Defining the cytoplasmic checkpoint structurally and biochemically: it was unknown how NMD3 monitors PTC maturity and how it is released, and structures plus reconstitution showed it recognizes the near-mature PTC and must vacate the P site to allow Sdo1-stimulated Efl1 GTPase activation.","evidence":"Cryo-EM of affinity-purified pre-60S particles, genetic suppressor screen, and in vitro GTPase reconstitution with purified components","pmids":["28112732","28715419"],"confidence":"High","gaps":["Precise trigger that signals PTC maturity to NMD3 not defined","How Rpl10/uL16 mechanically displaces NMD3 not fully resolved"]},{"year":2025,"claim":"Demonstrating surveillance function: whether NMD3 actively rejects defective subunits was unknown, and bypass mutations showed that NMD3 with Tif6 prevents PTC-defective subunits from entering and persisting in translation.","evidence":"Genetic bypass mutations, cryo-EM, selective ribosome profiling, and in vivo degradation assays in yeast (preprint)","pmids":["bio_10.1101_2025.10.29.685433"],"confidence":"Medium","gaps":["Preprint, not yet peer-reviewed","How defective subunits are routed to degradation downstream of escape unclear"]},{"year":null,"claim":"The molecular signal that licenses NMD3 release from the mature P site, and how this is coordinated across species, remains incompletely defined.","evidence":"","pmids":[],"confidence":"High","gaps":["Conformational trigger distinguishing mature from immature PTC not resolved","Human reconstitution of the Lsg1/Efl1-equivalent release step absent from the corpus"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,3]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[4]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0]},{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[1,5]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1]},{"term_id":"GO:0005635","term_label":"nuclear envelope","supporting_discovery_ids":[3]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,6]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[1,3]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[0,6]}],"complexes":["pre-60S ribosomal subunit"],"partners":["CRM1","RPL10","LSG1","EFL1","SDO1","TIF6","NUP82"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96D46","full_name":"60S ribosomal export protein NMD3","aliases":[],"length_aa":503,"mass_kda":57.6,"function":"Acts as an adapter for the XPO1/CRM1-mediated export of the 60S ribosomal subunit","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q96D46/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/NMD3","classification":"Common Essential","n_dependent_lines":1194,"n_total_lines":1208,"dependency_fraction":0.9884105960264901},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000169251","cell_line_id":"CID001103","localizations":[{"compartment":"nucleolus_gc","grade":3},{"compartment":"cytoplasmic","grade":2},{"compartment":"nucleoplasm","grade":1}],"interactors":[{"gene":"HAT1","stoichiometry":0.2},{"gene":"PSPC1","stoichiometry":0.2},{"gene":"RPS16","stoichiometry":0.2},{"gene":"SRP9","stoichiometry":0.2},{"gene":"VAPA","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001103","total_profiled":1310},"omim":[{"mim_id":"611021","title":"NMD3 RIBOSOME EXPORT ADAPTOR; NMD3","url":"https://www.omim.org/entry/611021"},{"mim_id":"609365","title":"GUANINE NUCLEOTIDE-BINDING PROTEIN-LIKE 2; GNL2","url":"https://www.omim.org/entry/609365"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Nucleoli","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/NMD3"},"hgnc":{"alias_symbol":["CGI-07"],"prev_symbol":[]},"alphafold":{"accession":"Q96D46","domains":[{"cath_id":"3.10.450","chopping":"25-139","consensus_level":"high","plddt":89.2901,"start":25,"end":139},{"cath_id":"-","chopping":"254-408","consensus_level":"high","plddt":90.6301,"start":254,"end":408},{"cath_id":"-","chopping":"422-467_476-483","consensus_level":"medium","plddt":81.1996,"start":422,"end":483},{"cath_id":"3.30.70","chopping":"144-247","consensus_level":"medium","plddt":90.9994,"start":144,"end":247}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96D46","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96D46-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96D46-F1-predicted_aligned_error_v6.png","plddt_mean":86.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NMD3","jax_strain_url":"https://www.jax.org/strain/search?query=NMD3"},"sequence":{"accession":"Q96D46","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96D46.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96D46/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96D46"}},"corpus_meta":[{"pmid":"12773398","id":"PMC_12773398","title":"Coordinated nuclear export of 60S ribosomal subunits and NMD3 in vertebrates.","date":"2003","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/12773398","citation_count":112,"is_preprint":false},{"pmid":"10022925","id":"PMC_10022925","title":"NMD3 encodes an essential cytoplasmic protein required for stable 60S ribosomal subunits in Saccharomyces cerevisiae.","date":"1999","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/10022925","citation_count":86,"is_preprint":false},{"pmid":"28112732","id":"PMC_28112732","title":"Structural snapshot of cytoplasmic pre-60S ribosomal particles bound by Nmd3, Lsg1, Tif6 and Reh1.","date":"2017","source":"Nature structural & molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/28112732","citation_count":83,"is_preprint":false},{"pmid":"20584915","id":"PMC_20584915","title":"Characterization of the nuclear export adaptor protein Nmd3 in association with the 60S ribosomal subunit.","date":"2010","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/20584915","citation_count":59,"is_preprint":false},{"pmid":"23782956","id":"PMC_23782956","title":"CRM1 and its ribosome export adaptor NMD3 localize to the nucleolus and affect rRNA synthesis.","date":"2013","source":"Nucleus (Austin, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/23782956","citation_count":36,"is_preprint":false},{"pmid":"25873624","id":"PMC_25873624","title":"NMD3 regulates both mRNA and rRNA nuclear export in African trypanosomes via an XPOI-linked pathway.","date":"2015","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/25873624","citation_count":29,"is_preprint":false},{"pmid":"17015443","id":"PMC_17015443","title":"Mapping the functional domains of yeast NMD3, the nuclear export adapter for the 60 S ribosomal subunit.","date":"2006","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17015443","citation_count":20,"is_preprint":false},{"pmid":"28715419","id":"PMC_28715419","title":"The T-cell leukemia related rpl10-R98S mutant traps the 60S export adapter Nmd3 in the ribosomal P site in yeast.","date":"2017","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/28715419","citation_count":19,"is_preprint":false},{"pmid":"29564728","id":"PMC_29564728","title":"Determining the Effect of the HNMT, STK39, and NMD3 Polymorphisms on the Incidence of Parkinson's Disease, Amyotrophic Lateral Sclerosis, and Multiple System Atrophy in Chinese Populations.","date":"2018","source":"Journal of molecular neuroscience : MN","url":"https://pubmed.ncbi.nlm.nih.gov/29564728","citation_count":11,"is_preprint":false},{"pmid":"17347149","id":"PMC_17347149","title":"Novel interaction of the 60S ribosomal subunit export adapter Nmd3 at the nuclear pore complex.","date":"2007","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17347149","citation_count":10,"is_preprint":false},{"pmid":"20584913","id":"PMC_20584913","title":"Joining the interface: a site for Nmd3 association on 60S ribosome subunits.","date":"2010","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/20584913","citation_count":5,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.10.29.685433","title":"A late cytoplasmic surveillance pathway ensures ribosome integrity","date":"2025-10-29","source":"bioRxiv","url":"https://doi.org/10.1101/2025.10.29.685433","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7262,"output_tokens":2645,"usd":0.030731,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9819,"output_tokens":3196,"usd":0.064497,"stage2_stop_reason":"end_turn"},"total_usd":0.095228,"stage1_batch_id":"msgbatch_013a8chtv3AWyTqv4z4sEF4C","stage2_batch_id":"msgbatch_01AHsuW63vinsvuTgzmQ3n62","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1999,\n      \"finding\": \"NMD3 (yeast Nmd3p) is an essential cytoplasmic protein required for stable 60S ribosomal subunits; loss-of-function causes rapid degradation of mature 25S rRNA and formation of half-mer polysomes, indicating a role in a late cytoplasmic assembly step of the 60S subunit. Nmd3p fractionates as a cytoplasmic protein and co-sediments with free 60S subunits in sucrose gradients.\",\n      \"method\": \"Temperature-sensitive allele (loss-of-function), sucrose gradient sedimentation, pulse-chase rRNA analysis, cell fractionation\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (genetic, biochemical fractionation, pulse-chase) in a foundational study\",\n      \"pmids\": [\"10022925\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Human NMD3 (hNMD3) acts as an adaptor for CRM1/Ran.GTP-mediated nuclear export of 60S ribosomal subunits. hNMD3 contains a CRM1-dependent leucine-rich nuclear export signal (NES) and a complex nuclear localization signal (NLS); the basic region of the NLS is also required for nucleolar accumulation. Export-defective NES-mutant hNMD3 binds nascent nuclear 60S pre-export particles and acts as a dominant negative, blocking 60S subunit export from Xenopus oocyte nuclei.\",\n      \"method\": \"Xenopus oocyte microinjection, dominant-negative NES mutant analysis, nuclear export assays, co-sedimentation with 60S particles\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — dominant-negative mutagenesis in Xenopus oocytes plus biochemical co-sedimentation; replicated in vertebrate context, consistent with yeast data\",\n      \"pmids\": [\"12773398\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Mutational analysis of yeast Nmd3p identified two distinct regions required for binding to 60S subunits, indicating multivalent interaction with the pre-60S particle. Separate domains govern nucleocytoplasmic shuttling (NES for CRM1 recognition) and ribosome binding.\",\n      \"method\": \"Site-directed mutagenesis, in vivo ribosome binding assays, nuclear export assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic mutagenesis with functional binding readouts, single lab\",\n      \"pmids\": [\"17015443\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Nmd3 mutants impaired for 60S subunit binding accumulate at the nuclear envelope and show enhanced binding to Crm1 independently of RanGTP. Interaction with the NPC is NES/Crm1-dependent. Components of the cytoplasmic-face Nup82 complex co-purify with mutant Nmd3, and mutations in Nup82 complex components cause wild-type Nmd3 to accumulate in the nucleoplasm, indicating that the Nup82 complex is a terminal binding site required for Nmd3 release from Crm1 at the NPC.\",\n      \"method\": \"In vivo/in vitro Crm1 binding assays, co-purification with GFP-tagged nucleoporins, NPC mutant analysis, fluorescence microscopy\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple methods (co-purification, in vitro binding, mutant phenotypes) in a single lab study\",\n      \"pmids\": [\"17347149\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Cryo-EM reconstruction of the yeast 60S–Nmd3 complex reveals that Nmd3 binds to the intersubunit face of the large subunit, contacting regions around helices 38, 69, and 95 of 25S rRNA, adjacent to ribosomal protein Rpl10. This binding site is blocked in 80S ribosomes. rRNA protection experiments corroborated the structural binding site.\",\n      \"method\": \"Cryo-electron microscopy, rRNA protection assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structure with orthogonal rRNA footprinting validation; single lab but two independent methods\",\n      \"pmids\": [\"20584915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"In human cells, CRM1 and NMD3 co-localize with nucleolar marker proteins; CRM1 nucleolar localization depends on CRM1 activity and NMD3 expression, whereas NMD3 nucleolar localization is independent of CRM1, indicating NMD3 provides nucleolar tethering of CRM1. NMD3 depletion reduced the rate of pre-47S rRNA synthesis but did not affect 28S rRNA processing (unlike CRM1 inhibition), and did not cause nucleolar disintegration.\",\n      \"method\": \"siRNA depletion, immunofluorescence co-localization, leptomycin B inhibition, rRNA synthesis rate measurement\",\n      \"journal\": \"Nucleus (Austin, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical and imaging analyses with genetic/pharmacological perturbations, single lab\",\n      \"pmids\": [\"23782956\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Cryo-EM of cytoplasmic pre-60S particles purified via tagged Nmd3 reveals that Nmd3 and Lsg1 occupy positions near the peptidyl-transferase center (PTC), with Nmd3 recognizing the PTC in its near-mature conformation. Reh1 anchors to the polypeptide tunnel exit with its C-terminus inserted into the tunnel. The structural data support a checkpoint role for Nmd3 in monitoring PTC assembly before final maturation.\",\n      \"method\": \"Cryo-electron microscopy of affinity-purified pre-60S particles\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM structure of the native complex, multiple assembly factors localized simultaneously\",\n      \"pmids\": [\"28112732\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The T-ALL-associated Rpl10-R98S mutation traps Nmd3 in the ribosomal P site, blocking its release. Suppressor mutations in Nmd3 that disrupt its interactions with the ribosome or with Tif6 bypass this block. Using purified components in vitro, Nmd3 inhibited Sdo1-stimulated Efl1 GTPase activity on rpl10-R98S mutant 60S subunits but not wild-type subunits; Nmd3 suppressor mutations reversed this inhibition, establishing that Nmd3 must vacate the P site to allow Sdo1-mediated Efl1 activation.\",\n      \"method\": \"Genetic suppressor screen, in vitro reconstitution with purified components, GTPase activity assay, cryo-EM (referenced from prior work)\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified components plus corroborating in vivo suppressor genetics and mutagenesis\",\n      \"pmids\": [\"28715419\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Simultaneous bypass mutations in Nmd3 and Tif6 release defective uL16-mutant 60S subunits into the translating pool, demonstrating that Nmd3 (together with Tif6) acts as a quality control checkpoint during late cytoplasmic pre-60S maturation. Cryo-EM and selective ribosome profiling of these escaped ribosomes show they can form peptide bonds but stall at early codons and are degraded if they enter translation. Reh1 is identified as required for the surveillance pathway that detects and degrades defective ribosomes during biogenesis.\",\n      \"method\": \"Genetic bypass mutations, cryo-EM, selective ribosome profiling, in vivo degradation assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — cryo-EM and ribosome profiling with genetic evidence; preprint, not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.10.29.685433\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"NMD3 encodes a conserved nucleocytoplasmic shuttling adaptor protein that binds to the intersubunit face of nascent 60S ribosomal subunits (near helices 38, 69, and 95 of 25S rRNA, adjacent to uL16/Rpl10) in the nucleus, recruits the export receptor CRM1 via its leucine-rich NES to drive RanGTP-dependent 60S subunit nuclear export through the NPC (engaging the cytoplasmic Nup82 complex for release), and then in the cytoplasm must be displaced from the P site by the concerted action of Rpl10/uL16 and the GTPase Lsg1 (assisted by Efl1/Sdo1) to complete 60S maturation; structurally, Nmd3 recognizes the near-mature peptidyl-transferase center and thereby acts as a quality control checkpoint ensuring PTC assembly before permitting ribosome entry into translation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NMD3 is a conserved nucleocytoplasmic shuttling adaptor that drives the late nuclear export and final cytoplasmic maturation of the 60S ribosomal subunit and acts as a quality-control checkpoint over peptidyl-transferase center assembly [#0, #6]. It was first defined in yeast as an essential factor required for stable 60S subunits, where its loss triggers rapid degradation of mature 25S rRNA and accumulation of half-mer polysomes, placing it at a late assembly step on free 60S subunits [#0]. NMD3 binds the intersubunit face of the large subunit, contacting helices 38, 69, and 95 of 25S rRNA adjacent to Rpl10/uL16 near the PTC — a site occluded in assembled 80S ribosomes — and engages the particle through multiple separable ribosome-binding regions [#2, #4]. For export, NMD3 supplies a CRM1-dependent leucine-rich NES and a complex NLS, serving as the adaptor for CRM1/Ran.GTP-mediated transport of pre-60S particles; export-defective NES mutants bind nascent nuclear 60S and act as dominant negatives, and in human cells NMD3 tethers CRM1 to the nucleolus [#1, #5]. Release from CRM1 at the cytoplasmic face of the NPC requires the Nup82 complex as a terminal binding site [#3]. In the cytoplasm NMD3 must vacate the P site to permit maturation: the T-ALL-associated Rpl10-R98S mutation traps NMD3 in the P site and blocks Sdo1-stimulated Efl1 GTPase activation, while NMD3 suppressor mutations restore it, establishing that NMD3 displacement gates the final GTPase-driven step [#7]. Together with Tif6, NMD3 enforces a surveillance checkpoint that prevents subunits with defective PTC assembly from entering translation; bypass mutations release defective uL16-mutant subunits that stall at early codons and are degraded [#8].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Establishing where NMD3 acts: it was unknown which step of ribosome assembly required NMD3, and genetic depletion placed it at a late cytoplasmic 60S maturation step.\",\n      \"evidence\": \"Temperature-sensitive allele with sucrose gradient sedimentation, pulse-chase rRNA analysis, and cell fractionation in yeast\",\n      \"pmids\": [\"10022925\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular binding site on the subunit not resolved\", \"Mechanism linking NMD3 loss to 25S rRNA degradation unclear\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defining NMD3's transport function: whether NMD3 mediates 60S nuclear export was unknown, and it was shown to be a CRM1/Ran.GTP export adaptor with a leucine-rich NES.\",\n      \"evidence\": \"Xenopus oocyte microinjection, dominant-negative NES-mutant analysis, and co-sedimentation with 60S particles\",\n      \"pmids\": [\"12773398\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How NMD3 is loaded onto nascent 60S in the nucleus not defined\", \"NPC release mechanism not addressed\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Dissecting NMD3 architecture: the relationship between ribosome binding and shuttling was unknown, and mutagenesis showed two distinct 60S-binding regions separable from the export determinants.\",\n      \"evidence\": \"Site-directed mutagenesis with in vivo ribosome binding and nuclear export assays in yeast\",\n      \"pmids\": [\"17015443\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of multivalent binding not resolved\", \"Single-lab functional readouts\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Resolving NPC release: how NMD3 is discharged from CRM1 after translocation was unknown, and the Nup82 complex was identified as the terminal cytoplasmic binding site required for release.\",\n      \"evidence\": \"In vivo/in vitro Crm1 binding assays, co-purification with tagged nucleoporins, and NPC mutant microscopy in yeast\",\n      \"pmids\": [\"17347149\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct biochemical mechanism of CRM1 disassembly not reconstituted\", \"Order of release relative to ribosome remodeling unclear\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Mapping the binding site: where NMD3 contacts the subunit was unknown, and cryo-EM placed it on the intersubunit face near 25S rRNA helices 38/69/95 adjacent to Rpl10.\",\n      \"evidence\": \"Cryo-EM reconstruction of the yeast 60S–Nmd3 complex with rRNA protection assays\",\n      \"pmids\": [\"20584915\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Near-atomic detail of PTC recognition not resolved at this stage\", \"Functional consequence of the contacts not tested\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Extending to human cells: the relationship between NMD3 and CRM1 nucleolar behavior was unknown, and NMD3 was shown to tether CRM1 to the nucleolus and influence pre-47S rRNA synthesis.\",\n      \"evidence\": \"siRNA depletion, immunofluorescence co-localization, leptomycin B inhibition, and rRNA synthesis measurement in human cells\",\n      \"pmids\": [\"23782956\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking NMD3 to rRNA synthesis rate unclear\", \"Whether tethering is direct not established\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Defining the cytoplasmic checkpoint structurally and biochemically: it was unknown how NMD3 monitors PTC maturity and how it is released, and structures plus reconstitution showed it recognizes the near-mature PTC and must vacate the P site to allow Sdo1-stimulated Efl1 GTPase activation.\",\n      \"evidence\": \"Cryo-EM of affinity-purified pre-60S particles, genetic suppressor screen, and in vitro GTPase reconstitution with purified components\",\n      \"pmids\": [\"28112732\", \"28715419\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise trigger that signals PTC maturity to NMD3 not defined\", \"How Rpl10/uL16 mechanically displaces NMD3 not fully resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrating surveillance function: whether NMD3 actively rejects defective subunits was unknown, and bypass mutations showed that NMD3 with Tif6 prevents PTC-defective subunits from entering and persisting in translation.\",\n      \"evidence\": \"Genetic bypass mutations, cryo-EM, selective ribosome profiling, and in vivo degradation assays in yeast (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.10.29.685433\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not yet peer-reviewed\", \"How defective subunits are routed to degradation downstream of escape unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The molecular signal that licenses NMD3 release from the mature P site, and how this is coordinated across species, remains incompletely defined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conformational trigger distinguishing mature from immature PTC not resolved\", \"Human reconstitution of the Lsg1/Efl1-equivalent release step absent from the corpus\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 3]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [1, 5]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005635\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [1, 3]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [0, 6]}\n    ],\n    \"complexes\": [\"pre-60S ribosomal subunit\"],\n    \"partners\": [\"CRM1\", \"RPL10\", \"LSG1\", \"EFL1\", \"SDO1\", \"TIF6\", \"Nup82\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}