{"gene":"NSA2","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2006,"finding":"Yeast Nsa2 is required for maturation of 27SB pre-rRNA and large (60S) ribosomal subunit biogenesis; its absence leads to accumulation of 27SB pre-rRNA and decreased 25S and 5.8S rRNA levels. Human TINP1 (NSA2 ortholog) complements yeast NSA2 repression, confirming functional conservation.","method":"Yeast genetic depletion, RNA analysis, complementation with human TINP1","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean loss-of-function with defined molecular phenotype (rRNA processing defect), complementation by human ortholog, replicated across multiple assays","pmids":["16861225"],"is_preprint":false},{"year":2006,"finding":"Nsa2 is a functional partner of the putative GTPase Nog1 on pre-60S particles: in the absence of Nog1, Nsa2 disappears from pre-60S complexes, but in the absence of Nsa2, Nog1 can still associate with (maturation-blocked) pre-60S complexes. This epistatic relationship places Nog1 upstream of Nsa2 association.","method":"Co-purification of pre-ribosomal complexes, genetic depletion epistasis experiments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal depletion epistasis with biochemical co-purification in same study, consistent genetic hierarchy established","pmids":["16861225"],"is_preprint":false},{"year":2006,"finding":"Nsa2 is an unstable (short half-life) protein whose cellular levels are tightly regulated: when ribosome biogenesis is blocked upstream of Nsa2, the protein is largely depleted, indicating its stability is coupled to active ribosome assembly.","method":"Genetic depletion of upstream ribosome biogenesis factors followed by Nsa2 protein level measurement","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — single lab, two genetic contexts tested, clean protein-level readout","pmids":["16861225"],"is_preprint":false},{"year":2009,"finding":"Human NSA2 localizes to the nucleolus; both putative nuclear localization signals (NLSs) also function as nucleolar localization signals (NoLSs) and are sufficient to direct nucleolar accumulation. Overexpression promotes G1/S cell cycle progression and cell growth; siRNA knockdown blocks G1/S transition and attenuates growth.","method":"GFP-fusion subcellular localization, deletion/mutation of NLS/NoLS, siRNA knockdown and overexpression with flow cytometry cell cycle analysis","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — direct localization with domain mapping, loss- and gain-of-function with cell cycle readout, single lab","pmids":["19932687"],"is_preprint":false},{"year":2013,"finding":"TINP1 (NSA2) overexpression promotes cell proliferation and S-phase entry while significantly reducing p53 and p21 protein/mRNA levels; TINP1 knockdown reduces cell viability. This places TINP1 upstream of p53/p21 in a proliferation-regulatory pathway.","method":"siRNA knockdown, overexpression, CCK-8 viability assay, luciferase reporter, flow cytometry, Western blot/RT-PCR for p53 and p21","journal":"Oncology reports","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single lab, multiple orthogonal readouts (viability, cell cycle, reporter, protein levels) but no direct interaction assay placing TINP1 on p53 pathway","pmids":["23912275"],"is_preprint":false},{"year":2012,"finding":"NSA2 protein is predominantly cytosolic in cultured renal mesangial cells (not nuclear as previously described in other cell types); exogenous TGFβ1 causes NSA2 to translocate from cytosol to nucleus. NSA2 knockdown by RNAi almost abolishes TGFβ1 mRNA/protein and activity and reduces fibronectin mRNA, placing NSA2 upstream of TGFβ1 transcriptional activity.","method":"Immunofluorescence subcellular localization, RNA interference, TGFβ1 ELISA/RT-PCR, fibronectin mRNA measurement","journal":"The international journal of biochemistry & cell biology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — direct localization with stimulus-dependent translocation, loss-of-function with defined pathway readouts, single lab","pmids":["23220173"],"is_preprint":false},{"year":2018,"finding":"Human NSA2 is required for 60S ribosomal subunit biogenesis and protein synthesis: NSA2 knockdown reduces rRNA synthesis, diminishes the 60S subunit, and suppresses overall protein synthesis. NSA2 depletion also inactivates the mTOR signaling pathway.","method":"siRNA knockdown, rRNA synthesis assay, ribosome profiling/subunit analysis, protein synthesis measurement, mTOR pathway western blot","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — single lab, multiple orthogonal assays (rRNA, subunit, protein synthesis, signaling pathway), clean loss-of-function","pmids":["30243719"],"is_preprint":false},{"year":2020,"finding":"Cryo-EM structures show Nsa2 docked via its C-terminal β-barrel domain to nuclear pre-60S particles, with an extended N-terminus (three α-helical segments) meandering between 25S rRNA helices and the extreme N-terminus near the Nog1 GTPase center. A conservative N-terminal mutation (Q3N) abolishes cell growth and impairs 60S biogenesis. The N-terminus is required to target Nsa2 to early pre-60S particles, and overexpression of the N-terminus (residues 1–58) arrests Nog1 on late cytoplasmic pre-60S particles, implicating a functional interaction between the Nsa2 N-terminus and Nog1 GTPase recycling.","method":"Cryo-EM structure, site-directed mutagenesis, yeast growth assay, biochemical fractionation of pre-60S particles","journal":"International journal of molecular sciences","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structural data combined with mutagenesis and biochemical epistasis in same study","pmids":["33266193"],"is_preprint":false},{"year":2025,"finding":"NSA2 forms an axis with EGFR that destabilizes wild-type p53: the camptothecin derivative 9c disrupts the NSA2-EGFR interaction, leading to p53 stabilization, cell cycle arrest, and apoptosis in NSCLC cells.","method":"Drug treatment with mechanistic follow-up (p53 western blot, cell cycle analysis, apoptosis assay, xenograft in vivo model)","journal":"International journal of molecular sciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, no direct binding assay between NSA2 and EGFR reported in abstract, mechanistic claim inferred from drug effects","pmids":["40076615"],"is_preprint":false}],"current_model":"NSA2 (TINP1) is a conserved nucleolar protein that functions as an assembly factor for the 60S ribosomal subunit — docking via its C-terminal β-barrel to pre-60S particles while its N-terminus contacts the Nog1 GTPase center — and is required for 27SB pre-rRNA maturation, rRNA synthesis, and protein synthesis; it also regulates G1/S cell cycle progression (partly through suppression of p53/p21), can translocate from cytosol to nucleus in response to TGFβ1 signaling, and acts upstream of TGFβ1 transcriptional activity and mTOR pathway activation."},"narrative":{"mechanistic_narrative":"NSA2 (TINP1) is a conserved nucleolar assembly factor for the large (60S) ribosomal subunit that is required for pre-rRNA maturation and ribosome production [PMID:16861225, PMID:30243719]. In yeast, Nsa2 is essential for processing 27SB pre-rRNA and for accumulation of mature 25S and 5.8S rRNA, and human TINP1 functionally complements yeast loss, establishing deep conservation of this role [PMID:16861225]. Cryo-EM places Nsa2 on nuclear pre-60S particles through its C-terminal β-barrel, while its extended N-terminus threads between 25S rRNA helices to reach the Nog1 GTPase center; the N-terminus targets Nsa2 to early pre-60S particles, and a conservative Q3N substitution abolishes growth and impairs 60S biogenesis [PMID:33266193]. Nsa2 works as a functional partner of the GTPase Nog1, which acts upstream to recruit Nsa2 to pre-60S complexes, and the Nsa2 N-terminus in turn influences Nog1 GTPase recycling on late cytoplasmic particles [PMID:16861225, PMID:33266193]. Nsa2 protein levels are coupled to active ribosome assembly, being depleted when upstream biogenesis is blocked [PMID:16861225]. In human cells NSA2 localizes to the nucleolus via dual NLS/NoLS signals and drives G1/S progression and cell growth [PMID:19932687], reduces ribosome production and protein synthesis along with mTOR pathway activity upon depletion [PMID:30243719], and promotes proliferation in part through suppression of p53 and p21 [PMID:23912275].","teleology":[{"year":2006,"claim":"Established NSA2's core molecular function by showing it is required for a specific step of 60S ribosomal subunit biogenesis and that this role is conserved from yeast to human.","evidence":"Yeast genetic depletion with rRNA processing analysis and complementation by human TINP1","pmids":["16861225"],"confidence":"High","gaps":["Did not define the structural basis of pre-60S docking","Mechanism by which Nsa2 promotes 27SB cleavage not resolved"]},{"year":2006,"claim":"Defined the genetic hierarchy of Nsa2 recruitment by placing the GTPase Nog1 upstream of Nsa2 association with pre-60S particles.","evidence":"Co-purification of pre-ribosomal complexes with reciprocal depletion epistasis in yeast","pmids":["16861225"],"confidence":"High","gaps":["Direct physical contact between Nsa2 and Nog1 not yet visualized at this stage","Functional consequence of the Nog1-Nsa2 partnership unresolved"]},{"year":2006,"claim":"Showed Nsa2 stability is coupled to ongoing ribosome assembly, implying its abundance reports on biogenesis flux.","evidence":"Depletion of upstream biogenesis factors followed by Nsa2 protein-level measurement","pmids":["16861225"],"confidence":"Medium","gaps":["Degradation pathway and turnover machinery not identified","Tested in only two genetic contexts in a single lab"]},{"year":2009,"claim":"Linked human NSA2 nucleolar targeting to cell cycle control, showing it promotes G1/S progression and growth.","evidence":"GFP-fusion localization with NLS/NoLS mapping, siRNA and overexpression with flow cytometry","pmids":["19932687"],"confidence":"Medium","gaps":["Whether cell cycle effect is a direct consequence of ribosome biogenesis defect not separated","Single lab"]},{"year":2012,"claim":"Identified a context-dependent cytosolic pool of NSA2 and a TGFβ1-driven nuclear translocation, placing NSA2 upstream of TGFβ1 transcriptional activity in mesangial cells.","evidence":"Immunofluorescence localization, RNAi, TGFβ1 ELISA/RT-PCR and fibronectin mRNA measurement","pmids":["23220173"],"confidence":"Medium","gaps":["Molecular mechanism connecting NSA2 to TGFβ1 transcription unknown","Apparent discrepancy with nucleolar localization in other cell types unresolved","Single lab"]},{"year":2013,"claim":"Connected NSA2-driven proliferation to suppression of the p53/p21 axis.","evidence":"siRNA/overexpression with viability, cell cycle, reporter and p53/p21 protein and mRNA readouts","pmids":["23912275"],"confidence":"Medium","gaps":["No direct interaction assay placing NSA2 on the p53 pathway","Whether p53 suppression is downstream of ribosome biogenesis stress not addressed"]},{"year":2018,"claim":"Confirmed in human cells that NSA2 supports rRNA synthesis, 60S formation and protein synthesis, and linked its loss to mTOR pathway inactivation.","evidence":"siRNA knockdown with rRNA synthesis, subunit analysis, protein synthesis and mTOR western blot","pmids":["30243719"],"confidence":"Medium","gaps":["Whether mTOR effect is direct or secondary to translation defect unresolved","Single lab"]},{"year":2020,"claim":"Provided the structural mechanism of NSA2 action, showing C-terminal β-barrel docking to pre-60S and an N-terminal extension reaching the Nog1 GTPase center that governs targeting and Nog1 recycling.","evidence":"Cryo-EM structure with site-directed mutagenesis (Q3N), growth assays and pre-60S fractionation","pmids":["33266193"],"confidence":"High","gaps":["Exact role in triggering 27SB cleavage not mechanistically defined","Structural data from yeast; human-specific features not resolved"]},{"year":2025,"claim":"Proposed an NSA2-EGFR axis that destabilizes wild-type p53 as a druggable vulnerability in NSCLC.","evidence":"Camptothecin derivative 9c treatment with p53 western blot, cell cycle, apoptosis and xenograft readouts","pmids":["40076615"],"confidence":"Low","gaps":["No direct NSA2-EGFR binding assay reported; interaction inferred from drug effects","Mechanism linking the axis to p53 destabilization not defined","Single lab"]},{"year":null,"claim":"How NSA2's ribosome biogenesis role mechanistically integrates with its reported effects on p53/p21, TGFβ1 and EGFR signaling remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No demonstrated physical link between NSA2 and p53 or EGFR","Whether signaling phenotypes are secondary to nucleolar/ribosomal stress untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[0,7]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[7]}],"localization":[{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[3]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[3,5]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[5]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,6]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[0,6,7]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[3,4]}],"complexes":["pre-60S ribosomal particle"],"partners":["NOG1","EGFR"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O95478","full_name":"Ribosome biogenesis protein NSA2 homolog","aliases":["Hairy cell leukemia protein 1","TGF-beta-inducible nuclear protein 1"],"length_aa":260,"mass_kda":30.1,"function":"Involved in the biogenesis of the 60S ribosomal subunit. May play a part in the quality control of pre-60S particles (By similarity)","subcellular_location":"Nucleus, nucleolus","url":"https://www.uniprot.org/uniprotkb/O95478/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/NSA2","classification":"Common Essential","n_dependent_lines":1206,"n_total_lines":1208,"dependency_fraction":0.9983443708609272},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000164346","cell_line_id":"CID001892","localizations":[{"compartment":"nucleolus_gc","grade":3}],"interactors":[{"gene":"TCERG1","stoichiometry":10.0},{"gene":"ATP6V1B2","stoichiometry":0.2},{"gene":"CD9","stoichiometry":0.2},{"gene":"IPO7","stoichiometry":0.2},{"gene":"LMNB1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001892","total_profiled":1310},"omim":[{"mim_id":"612497","title":"TRANSFORMING GROWTH FACTOR-BETA-INDUCIBLE NUCLEAR PROTEIN 1","url":"https://www.omim.org/entry/612497"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"},{"location":"Nucleoli","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/NSA2"},"hgnc":{"alias_symbol":["HUSSY-29","HCLG1","FLJ94393","TINP1"],"prev_symbol":[]},"alphafold":{"accession":"O95478","domains":[{"cath_id":"-","chopping":"1-49","consensus_level":"medium","plddt":92.6853,"start":1,"end":49},{"cath_id":"2.40.10.310","chopping":"128-257","consensus_level":"high","plddt":93.1629,"start":128,"end":257}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O95478","model_url":"https://alphafold.ebi.ac.uk/files/AF-O95478-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O95478-F1-predicted_aligned_error_v6.png","plddt_mean":87.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NSA2","jax_strain_url":"https://www.jax.org/strain/search?query=NSA2"},"sequence":{"accession":"O95478","fasta_url":"https://rest.uniprot.org/uniprotkb/O95478.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O95478/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O95478"}},"corpus_meta":[{"pmid":"16861225","id":"PMC_16861225","title":"Nsa2 is an unstable, conserved factor required for the maturation of 27 SB pre-rRNAs.","date":"2006","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16861225","citation_count":40,"is_preprint":false},{"pmid":"19932687","id":"PMC_19932687","title":"NSA2, a novel nucleolus protein regulates cell proliferation and cell cycle.","date":"2009","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/19932687","citation_count":28,"is_preprint":false},{"pmid":"23912275","id":"PMC_23912275","title":"A novel human TINP1 gene promotes cell proliferation through inhibition of p53 and p21 expression.","date":"2013","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/23912275","citation_count":8,"is_preprint":false},{"pmid":"23220173","id":"PMC_23220173","title":"Nop-7-associated 2 (NSA2), a candidate gene for diabetic nephropathy, is involved in the TGFβ1 pathway.","date":"2012","source":"The international journal of biochemistry & cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/23220173","citation_count":7,"is_preprint":false},{"pmid":"22095236","id":"PMC_22095236","title":"Elevated levels of renal and circulating Nop-7-associated 2 (NSA2) in rat and mouse models of diabetes, in mesangial cells in vitro and in patients with diabetic nephropathy.","date":"2011","source":"Diabetologia","url":"https://pubmed.ncbi.nlm.nih.gov/22095236","citation_count":6,"is_preprint":false},{"pmid":"33266193","id":"PMC_33266193","title":"Mutational Analysis of the Nsa2 N-Terminus Reveals Its Essential Role in Ribosomal 60S Subunit Assembly.","date":"2020","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/33266193","citation_count":6,"is_preprint":false},{"pmid":"30243719","id":"PMC_30243719","title":"Nop-7-associated 2 (NSA2) is required for ribosome biogenesis and protein synthesis.","date":"2018","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/30243719","citation_count":5,"is_preprint":false},{"pmid":"35139491","id":"PMC_35139491","title":"MiR-601 Promotes Cell Proliferation of Human Glioblastoma Cells by Suppressing TINP1 Expression.","date":"2022","source":"Alternative therapies in health and medicine","url":"https://pubmed.ncbi.nlm.nih.gov/35139491","citation_count":3,"is_preprint":false},{"pmid":"30913994","id":"PMC_30913994","title":"TINP1 homolog is required for planarian regeneration.","date":"2019","source":"Bioscience, biotechnology, and biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/30913994","citation_count":3,"is_preprint":false},{"pmid":"39396937","id":"PMC_39396937","title":"Co-regulation and synteny of GFM2 and NSA2 links ribosomal function in mitochondria and the cytosol with chronic kidney disease.","date":"2024","source":"Molecular medicine (Cambridge, Mass.)","url":"https://pubmed.ncbi.nlm.nih.gov/39396937","citation_count":1,"is_preprint":false},{"pmid":"40076615","id":"PMC_40076615","title":"Novel Camptothecin Derivative 9c with Enhanced Antitumor Activity via NSA2-EGFR-P53 Signaling Pathway.","date":"2025","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/40076615","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7326,"output_tokens":2333,"usd":0.028487,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9460,"output_tokens":3008,"usd":0.06125,"stage2_stop_reason":"end_turn"},"total_usd":0.089737,"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\": 2006,\n      \"finding\": \"Yeast Nsa2 is required for maturation of 27SB pre-rRNA and large (60S) ribosomal subunit biogenesis; its absence leads to accumulation of 27SB pre-rRNA and decreased 25S and 5.8S rRNA levels. Human TINP1 (NSA2 ortholog) complements yeast NSA2 repression, confirming functional conservation.\",\n      \"method\": \"Yeast genetic depletion, RNA analysis, complementation with human TINP1\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean loss-of-function with defined molecular phenotype (rRNA processing defect), complementation by human ortholog, replicated across multiple assays\",\n      \"pmids\": [\"16861225\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Nsa2 is a functional partner of the putative GTPase Nog1 on pre-60S particles: in the absence of Nog1, Nsa2 disappears from pre-60S complexes, but in the absence of Nsa2, Nog1 can still associate with (maturation-blocked) pre-60S complexes. This epistatic relationship places Nog1 upstream of Nsa2 association.\",\n      \"method\": \"Co-purification of pre-ribosomal complexes, genetic depletion epistasis experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal depletion epistasis with biochemical co-purification in same study, consistent genetic hierarchy established\",\n      \"pmids\": [\"16861225\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Nsa2 is an unstable (short half-life) protein whose cellular levels are tightly regulated: when ribosome biogenesis is blocked upstream of Nsa2, the protein is largely depleted, indicating its stability is coupled to active ribosome assembly.\",\n      \"method\": \"Genetic depletion of upstream ribosome biogenesis factors followed by Nsa2 protein level measurement\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — single lab, two genetic contexts tested, clean protein-level readout\",\n      \"pmids\": [\"16861225\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Human NSA2 localizes to the nucleolus; both putative nuclear localization signals (NLSs) also function as nucleolar localization signals (NoLSs) and are sufficient to direct nucleolar accumulation. Overexpression promotes G1/S cell cycle progression and cell growth; siRNA knockdown blocks G1/S transition and attenuates growth.\",\n      \"method\": \"GFP-fusion subcellular localization, deletion/mutation of NLS/NoLS, siRNA knockdown and overexpression with flow cytometry cell cycle analysis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — direct localization with domain mapping, loss- and gain-of-function with cell cycle readout, single lab\",\n      \"pmids\": [\"19932687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TINP1 (NSA2) overexpression promotes cell proliferation and S-phase entry while significantly reducing p53 and p21 protein/mRNA levels; TINP1 knockdown reduces cell viability. This places TINP1 upstream of p53/p21 in a proliferation-regulatory pathway.\",\n      \"method\": \"siRNA knockdown, overexpression, CCK-8 viability assay, luciferase reporter, flow cytometry, Western blot/RT-PCR for p53 and p21\",\n      \"journal\": \"Oncology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single lab, multiple orthogonal readouts (viability, cell cycle, reporter, protein levels) but no direct interaction assay placing TINP1 on p53 pathway\",\n      \"pmids\": [\"23912275\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"NSA2 protein is predominantly cytosolic in cultured renal mesangial cells (not nuclear as previously described in other cell types); exogenous TGFβ1 causes NSA2 to translocate from cytosol to nucleus. NSA2 knockdown by RNAi almost abolishes TGFβ1 mRNA/protein and activity and reduces fibronectin mRNA, placing NSA2 upstream of TGFβ1 transcriptional activity.\",\n      \"method\": \"Immunofluorescence subcellular localization, RNA interference, TGFβ1 ELISA/RT-PCR, fibronectin mRNA measurement\",\n      \"journal\": \"The international journal of biochemistry & cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — direct localization with stimulus-dependent translocation, loss-of-function with defined pathway readouts, single lab\",\n      \"pmids\": [\"23220173\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Human NSA2 is required for 60S ribosomal subunit biogenesis and protein synthesis: NSA2 knockdown reduces rRNA synthesis, diminishes the 60S subunit, and suppresses overall protein synthesis. NSA2 depletion also inactivates the mTOR signaling pathway.\",\n      \"method\": \"siRNA knockdown, rRNA synthesis assay, ribosome profiling/subunit analysis, protein synthesis measurement, mTOR pathway western blot\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — single lab, multiple orthogonal assays (rRNA, subunit, protein synthesis, signaling pathway), clean loss-of-function\",\n      \"pmids\": [\"30243719\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Cryo-EM structures show Nsa2 docked via its C-terminal β-barrel domain to nuclear pre-60S particles, with an extended N-terminus (three α-helical segments) meandering between 25S rRNA helices and the extreme N-terminus near the Nog1 GTPase center. A conservative N-terminal mutation (Q3N) abolishes cell growth and impairs 60S biogenesis. The N-terminus is required to target Nsa2 to early pre-60S particles, and overexpression of the N-terminus (residues 1–58) arrests Nog1 on late cytoplasmic pre-60S particles, implicating a functional interaction between the Nsa2 N-terminus and Nog1 GTPase recycling.\",\n      \"method\": \"Cryo-EM structure, site-directed mutagenesis, yeast growth assay, biochemical fractionation of pre-60S particles\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structural data combined with mutagenesis and biochemical epistasis in same study\",\n      \"pmids\": [\"33266193\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NSA2 forms an axis with EGFR that destabilizes wild-type p53: the camptothecin derivative 9c disrupts the NSA2-EGFR interaction, leading to p53 stabilization, cell cycle arrest, and apoptosis in NSCLC cells.\",\n      \"method\": \"Drug treatment with mechanistic follow-up (p53 western blot, cell cycle analysis, apoptosis assay, xenograft in vivo model)\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, no direct binding assay between NSA2 and EGFR reported in abstract, mechanistic claim inferred from drug effects\",\n      \"pmids\": [\"40076615\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NSA2 (TINP1) is a conserved nucleolar protein that functions as an assembly factor for the 60S ribosomal subunit — docking via its C-terminal β-barrel to pre-60S particles while its N-terminus contacts the Nog1 GTPase center — and is required for 27SB pre-rRNA maturation, rRNA synthesis, and protein synthesis; it also regulates G1/S cell cycle progression (partly through suppression of p53/p21), can translocate from cytosol to nucleus in response to TGFβ1 signaling, and acts upstream of TGFβ1 transcriptional activity and mTOR pathway activation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NSA2 (TINP1) is a conserved nucleolar assembly factor for the large (60S) ribosomal subunit that is required for pre-rRNA maturation and ribosome production [#0, #6]. In yeast, Nsa2 is essential for processing 27SB pre-rRNA and for accumulation of mature 25S and 5.8S rRNA, and human TINP1 functionally complements yeast loss, establishing deep conservation of this role [#0]. Cryo-EM places Nsa2 on nuclear pre-60S particles through its C-terminal β-barrel, while its extended N-terminus threads between 25S rRNA helices to reach the Nog1 GTPase center; the N-terminus targets Nsa2 to early pre-60S particles, and a conservative Q3N substitution abolishes growth and impairs 60S biogenesis [#7]. Nsa2 works as a functional partner of the GTPase Nog1, which acts upstream to recruit Nsa2 to pre-60S complexes, and the Nsa2 N-terminus in turn influences Nog1 GTPase recycling on late cytoplasmic particles [#1, #7]. Nsa2 protein levels are coupled to active ribosome assembly, being depleted when upstream biogenesis is blocked [#2]. In human cells NSA2 localizes to the nucleolus via dual NLS/NoLS signals and drives G1/S progression and cell growth [#3], reduces ribosome production and protein synthesis along with mTOR pathway activity upon depletion [#6], and promotes proliferation in part through suppression of p53 and p21 [#4].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Established NSA2's core molecular function by showing it is required for a specific step of 60S ribosomal subunit biogenesis and that this role is conserved from yeast to human.\",\n      \"evidence\": \"Yeast genetic depletion with rRNA processing analysis and complementation by human TINP1\",\n      \"pmids\": [\"16861225\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the structural basis of pre-60S docking\", \"Mechanism by which Nsa2 promotes 27SB cleavage not resolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defined the genetic hierarchy of Nsa2 recruitment by placing the GTPase Nog1 upstream of Nsa2 association with pre-60S particles.\",\n      \"evidence\": \"Co-purification of pre-ribosomal complexes with reciprocal depletion epistasis in yeast\",\n      \"pmids\": [\"16861225\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct physical contact between Nsa2 and Nog1 not yet visualized at this stage\", \"Functional consequence of the Nog1-Nsa2 partnership unresolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Showed Nsa2 stability is coupled to ongoing ribosome assembly, implying its abundance reports on biogenesis flux.\",\n      \"evidence\": \"Depletion of upstream biogenesis factors followed by Nsa2 protein-level measurement\",\n      \"pmids\": [\"16861225\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Degradation pathway and turnover machinery not identified\", \"Tested in only two genetic contexts in a single lab\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Linked human NSA2 nucleolar targeting to cell cycle control, showing it promotes G1/S progression and growth.\",\n      \"evidence\": \"GFP-fusion localization with NLS/NoLS mapping, siRNA and overexpression with flow cytometry\",\n      \"pmids\": [\"19932687\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether cell cycle effect is a direct consequence of ribosome biogenesis defect not separated\", \"Single lab\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identified a context-dependent cytosolic pool of NSA2 and a TGFβ1-driven nuclear translocation, placing NSA2 upstream of TGFβ1 transcriptional activity in mesangial cells.\",\n      \"evidence\": \"Immunofluorescence localization, RNAi, TGFβ1 ELISA/RT-PCR and fibronectin mRNA measurement\",\n      \"pmids\": [\"23220173\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism connecting NSA2 to TGFβ1 transcription unknown\", \"Apparent discrepancy with nucleolar localization in other cell types unresolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Connected NSA2-driven proliferation to suppression of the p53/p21 axis.\",\n      \"evidence\": \"siRNA/overexpression with viability, cell cycle, reporter and p53/p21 protein and mRNA readouts\",\n      \"pmids\": [\"23912275\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct interaction assay placing NSA2 on the p53 pathway\", \"Whether p53 suppression is downstream of ribosome biogenesis stress not addressed\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Confirmed in human cells that NSA2 supports rRNA synthesis, 60S formation and protein synthesis, and linked its loss to mTOR pathway inactivation.\",\n      \"evidence\": \"siRNA knockdown with rRNA synthesis, subunit analysis, protein synthesis and mTOR western blot\",\n      \"pmids\": [\"30243719\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether mTOR effect is direct or secondary to translation defect unresolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Provided the structural mechanism of NSA2 action, showing C-terminal β-barrel docking to pre-60S and an N-terminal extension reaching the Nog1 GTPase center that governs targeting and Nog1 recycling.\",\n      \"evidence\": \"Cryo-EM structure with site-directed mutagenesis (Q3N), growth assays and pre-60S fractionation\",\n      \"pmids\": [\"33266193\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Exact role in triggering 27SB cleavage not mechanistically defined\", \"Structural data from yeast; human-specific features not resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Proposed an NSA2-EGFR axis that destabilizes wild-type p53 as a druggable vulnerability in NSCLC.\",\n      \"evidence\": \"Camptothecin derivative 9c treatment with p53 western blot, cell cycle, apoptosis and xenograft readouts\",\n      \"pmids\": [\"40076615\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct NSA2-EGFR binding assay reported; interaction inferred from drug effects\", \"Mechanism linking the axis to p53 destabilization not defined\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How NSA2's ribosome biogenesis role mechanistically integrates with its reported effects on p53/p21, TGFβ1 and EGFR signaling remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No demonstrated physical link between NSA2 and p53 or EGFR\", \"Whether signaling phenotypes are secondary to nucleolar/ribosomal stress untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [0, 7]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [3, 5]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [0, 6, 7]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [3, 4]}\n    ],\n    \"complexes\": [\"pre-60S ribosomal particle\"],\n    \"partners\": [\"NOG1\", \"EGFR\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}