{"gene":"NOL10","run_date":"2026-04-29T11:37:57","timeline":{"discoveries":[{"year":2016,"finding":"NOL10 forms a salt-stable trimeric protein complex with AATF and NGDN (the ANN complex) in human cells. All three subunits localize to nucleoli and show mutual dependence for protein stability. The WD40 repeats of NOL10 and the UTP3/SAS10 domain of NGDN are required for complex formation. The ANN complex is required for nucleolar steps of 40S ribosomal subunit biosynthesis, specifically for 18S rRNA maturation; depletion of any single member affects the same cleavage steps in the 5'ETS and ITS1 regions of the ribosomal RNA precursor.","method":"Immunoprecipitation, domain-mapping mutagenesis, siRNA knockdown, rRNA processing assays (Northern blot), nucleolar localization by microscopy","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP with domain mapping, clean KD with defined rRNA processing phenotype, multiple orthogonal methods in single study","pmids":["27599843"],"is_preprint":false},{"year":2023,"finding":"PQBP5/NOL10 is an intrinsically disordered protein that constitutes the skeletal granule meshwork of the granular component of the nucleolus (distinct from the fibrillar center and dense fibrillar component). Unlike other nucleolar proteins that disperse to the nucleoplasm under osmotic stress, NOL10 remains in the nucleolus and functions as an anchor for reassembly of other nucleolar proteins after stress. Its biophysical properties remain stable under stress conditions. In polyglutamine disease models, NOL10 is sequestered by polyglutamine proteins, leading to pathological nucleolar deformity.","method":"High-speed atomic force microscopy (HS-AFM), super-resolution microscopy (STED), correlative light-electron microscopy (CLEM), droplet assay, thermal shift assay, osmotic stress experiments with live imaging","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal structural and biophysical methods with direct functional consequence (nucleolar anchoring) validated in vitro and in vivo","pmids":["36599853"],"is_preprint":false},{"year":2014,"finding":"GFP-NOL10 localizes to the dense fibrillar component and granular component of nucleoli in living HeLa cells. Its mobility is very low under normal conditions, indicating tight binding to macroprotein complexes, consistent with a scaffold or core role. When rRNA transcription is suppressed, mobility increases but remains slow.","method":"GFP-fusion live-cell imaging, FRAP in living cells, suppression of rRNA transcription by ActD treatment","journal":"Biochemistry and cell biology","confidence":"Medium","confidence_rationale":"Tier 2 — direct localization experiment with functional context (tight complex binding, rRNA-transcription dependence); single lab","pmids":["24754225"],"is_preprint":false},{"year":2025,"finding":"A homozygous missense variant in the WD-repeat domain of NOL10 (p.Asn228His) causes nucleoplasmic mislocalization of the NOL10 protein, loss of interaction with AATF and NGDN, specific impairment of 40S ribosomal subunit maturation (reduced 40S, 80S, and polysome content), G0/G1 cell cycle arrest, and increased cell death in patient-derived fibroblasts. Structural modeling predicts N228H is strongly destabilizing.","method":"Exome sequencing, immunofluorescence (localization), co-immunoprecipitation (interaction loss), polysome profiling, cell cycle analysis, structural modeling and ΔΔG calculations","journal":"Journal of human genetics","confidence":"High","confidence_rationale":"Tier 1–2 — patient variant with multiple orthogonal functional readouts (mislocalization, lost interactions, polysome profiling, cell cycle) in primary cells","pmids":["41093997"],"is_preprint":false},{"year":2025,"finding":"NOL10 physically interacts with a 24-amino-acid region within the DDX10 moiety of the NUP98::DDX10 leukemia fusion protein. NOL10 is a critical dependency of NUP98::DDX10 leukemia; loss of Nol10 in a mouse model impairs disease progression and improves survival. NOL10 acts cooperatively with NUP98::DDX10 to regulate the serine biosynthesis pathway and stabilize ATF4 mRNA.","method":"Co-immunoprecipitation (interaction mapping), mouse leukemia model (Nol10 knockout), gene expression analysis (serine biosynthesis pathway), mRNA stability assays (ATF4)","journal":"Leukemia","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP interaction mapping plus in vivo mouse model with survival readout and pathway analysis; single lab","pmids":["40263434"],"is_preprint":false},{"year":2025,"finding":"The risk allele A of rs4519489 (in the NOL10 locus) exhibits enhanced binding to the transcription factor USF1, resulting in elevated NOL10 expression. NOL10 overexpression promotes cell cycle progression in prostate cancer cells. USF1 thus regulates NOL10 transcription through allele-specific binding at the 2p25 locus.","method":"SNPs-seq (allele-specific protein binding), unbiased proteomics (allele-specific capture), functional cell cycle assays, clinical cohort correlation","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 — allele-specific proteomics with functional follow-up (cell cycle assays); single lab study","pmids":["41062477"],"is_preprint":false},{"year":2025,"finding":"In fission yeast, Enp2/NOL10 forms a complex with a novel non-coding RNA (RiboCop) and RNase H1 in response to improper pre-rRNA processing by Dicer during quiescence. This complex triggers rDNA repeat silencing via Sir2, RENT, and H3K9 methylation specifically during cellular dormancy.","method":"RNA co-immunoprecipitation, genetic mutant analysis (Dicer mutants), chromatin immunoprecipitation, epistasis analysis (Sir2/RENT/H3K9me pathway)","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 2 method but preprint, yeast ortholog, single study","pmids":["41000809"],"is_preprint":true}],"current_model":"NOL10/PQBP5 is an intrinsically disordered nucleolar scaffold protein that forms the granule meshwork of the granular component; it assembles with AATF and NGDN (via its WD40 repeats) into the ANN complex required for 18S rRNA maturation and 40S ribosomal subunit biogenesis, anchors the nucleolus under osmotic stress, and its mislocalization or loss—whether by disease-causing WD40 mutations, polyglutamine sequestration, or cooperative action with oncofusion proteins—disrupts ribosome biogenesis and cell viability."},"narrative":{"teleology":[{"year":2014,"claim":"Establishing NOL10 as a stably incorporated nucleolar component resolved its subnuclear address and raised the question of what macromolecular complex it participates in.","evidence":"GFP-NOL10 live-cell imaging and FRAP in HeLa cells showed nucleolar localization (DFC/GC) and very low mobility indicating tight complex association","pmids":["24754225"],"confidence":"Medium","gaps":["Identity of the complex(es) retaining NOL10 in the nucleolus was unknown","Functional role in rRNA processing not yet tested"]},{"year":2016,"claim":"Identification of the ANN complex (NOL10–AATF–NGDN) and its requirement for 18S rRNA maturation defined NOL10's molecular function in ribosome biogenesis and showed that its WD40 repeats mediate complex assembly.","evidence":"Reciprocal co-IP, domain-mapping mutagenesis, siRNA knockdown with Northern blot rRNA processing assays in human cells","pmids":["27599843"],"confidence":"High","gaps":["Structural basis of ANN complex assembly unresolved","Whether NOL10 has functions beyond rRNA processing not addressed","No in vivo disease model yet linked to ANN disruption"]},{"year":2023,"claim":"Biophysical characterization revealed that NOL10 is an intrinsically disordered protein forming the granular-component meshwork and acting as a nucleolar stress anchor, explaining its scaffold role and linking its sequestration to polyglutamine disease pathology.","evidence":"HS-AFM, STED super-resolution, CLEM, droplet assays, osmotic stress live imaging, and polyglutamine disease cell models","pmids":["36599853"],"confidence":"High","gaps":["Molecular determinants of stress-resistant anchoring are not mapped","Causal role in polyglutamine disease progression not demonstrated in animal models"]},{"year":2025,"claim":"A patient-derived WD40 missense variant (N228H) proved that ANN complex integrity is essential for human 40S subunit production and cell viability, establishing NOL10 deficiency as a ribosomopathy.","evidence":"Exome sequencing, immunofluorescence, co-IP, polysome profiling, and cell cycle analysis in patient fibroblasts","pmids":["41093997"],"confidence":"High","gaps":["Full clinical spectrum and prevalence of NOL10-associated disease remain undefined","Whether residual NOL10 function persists or variant is a complete loss-of-function is unclear"]},{"year":2025,"claim":"Discovery that NOL10 physically interacts with the NUP98::DDX10 oncofusion and is a critical dependency for leukemia progression expanded NOL10's relevance beyond normal ribosome biogenesis into oncogenic mechanisms.","evidence":"Co-IP domain mapping, Nol10-knockout mouse leukemia model with survival analysis, ATF4 mRNA stability and serine biosynthesis pathway analysis","pmids":["40263434"],"confidence":"Medium","gaps":["Whether the leukemia dependency reflects canonical ribosome biogenesis or a neomorphic function is unresolved","Therapeutic vulnerability of the NOL10–DDX10 interaction not tested"]},{"year":2025,"claim":"A prostate cancer risk SNP at the NOL10 locus was shown to increase NOL10 transcription via allele-specific USF1 binding, and NOL10 overexpression promoted cell cycle progression, linking NOL10 dosage to cancer proliferation.","evidence":"SNPs-seq, allele-specific proteomics, functional cell cycle assays in prostate cancer cells","pmids":["41062477"],"confidence":"Medium","gaps":["Whether cell cycle effects are mediated through ribosome biogenesis or an independent pathway is unclear","In vivo validation of NOL10 overexpression driving tumor growth is lacking"]},{"year":null,"claim":"Key unresolved questions include the atomic structure of the ANN complex, the mechanism by which NOL10's intrinsic disorder confers stress-resistant nucleolar anchoring, and whether NOL10's roles in leukemia and prostate cancer are ribosome biogenesis-dependent or involve distinct moonlighting functions.","evidence":"","pmids":[],"confidence":"Low","gaps":["No high-resolution structure of the ANN complex or NOL10 alone","Mechanistic basis of stress-resistant anchoring not mapped to specific disordered regions","Ribosome biogenesis-dependent vs. independent contributions to cancer biology not delineated"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[1,2]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[2,3]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,3]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,3]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[4,5]}],"complexes":["ANN complex (NOL10–AATF–NGDN)"],"partners":["AATF","NGDN","NUP98::DDX10","USF1"],"other_free_text":[]},"mechanistic_narrative":"NOL10 is a nucleolar scaffold protein that assembles with AATF and NGDN into the trimeric ANN complex via its WD40 repeats, and this complex is essential for 18S rRNA maturation and 40S ribosomal subunit biogenesis [PMID:27599843, PMID:41093997]. As an intrinsically disordered protein, NOL10 constitutes the skeletal granule meshwork of the nucleolar granular component, remains anchored in the nucleolus under osmotic stress, and serves as a platform for reassembly of other nucleolar proteins after stress; sequestration by polyglutamine-expanded proteins causes pathological nucleolar deformity [PMID:36599853]. A homozygous WD40-domain missense mutation (p.Asn228His) abolishes AATF/NGDN interaction, mislocalizes NOL10 to the nucleoplasm, impairs 40S subunit production, and causes G0/G1 arrest and cell death in patient fibroblasts, establishing NOL10 deficiency as a cause of a ribosomopathy [PMID:41093997]. NOL10 also physically interacts with the DDX10 moiety of the NUP98::DDX10 leukemia fusion protein, and its loss impairs leukemia progression in a mouse model [PMID:40263434]."},"prefetch_data":{"uniprot":{"accession":"Q9BSC4","full_name":"Nucleolar protein 10","aliases":[],"length_aa":688,"mass_kda":80.3,"function":"","subcellular_location":"Nucleus, nucleolus","url":"https://www.uniprot.org/uniprotkb/Q9BSC4/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/NOL10","classification":"Common Essential","n_dependent_lines":1202,"n_total_lines":1208,"dependency_fraction":0.9950331125827815},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000115761","cell_line_id":"CID001112","localizations":[{"compartment":"nucleolus_gc","grade":3}],"interactors":[{"gene":"ZNF644","stoichiometry":10.0},{"gene":"SRI","stoichiometry":10.0}],"url":"https://opencell.sf.czbiohub.org/target/CID001112","total_profiled":1310},"omim":[{"mim_id":"616197","title":"NUCLEOLAR PROTEIN 10; NOL10","url":"https://www.omim.org/entry/616197"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoli","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/NOL10"},"hgnc":{"alias_symbol":["FLJ14075"],"prev_symbol":["PQBP5"]},"alphafold":{"accession":"Q9BSC4","domains":[{"cath_id":"2.130.10.10","chopping":"1-362","consensus_level":"medium","plddt":88.7197,"start":1,"end":362},{"cath_id":"-","chopping":"372-415","consensus_level":"high","plddt":84.5545,"start":372,"end":415}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BSC4","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BSC4-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BSC4-F1-predicted_aligned_error_v6.png","plddt_mean":77.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NOL10","jax_strain_url":"https://www.jax.org/strain/search?query=NOL10"},"sequence":{"accession":"Q9BSC4","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BSC4.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BSC4/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BSC4"}},"corpus_meta":[{"pmid":"30693177","id":"PMC_30693177","title":"Circular RNA circNOL10 Inhibits Lung Cancer Development by Promoting SCLM1-Mediated Transcriptional Regulation of the Humanin Polypeptide Family.","date":"2018","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/30693177","citation_count":87,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"35035657","id":"PMC_35035657","title":"Methylation Pattern Mediated by m6A Regulator and Tumor Microenvironment Invasion in Lung Adenocarcinoma.","date":"2022","source":"Oxidative medicine and cellular longevity","url":"https://pubmed.ncbi.nlm.nih.gov/35035657","citation_count":30,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"27599843","id":"PMC_27599843","title":"Human AATF/Che-1 forms a nucleolar protein complex with NGDN and NOL10 required for 40S ribosomal subunit synthesis.","date":"2016","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/27599843","citation_count":23,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"34729247","id":"PMC_34729247","title":"circ-NOL10 regulated by MTDH/CASC3 inhibits breast cancer progression and metastasis via multiple miRNAs and PDCD4.","date":"2021","source":"Molecular therapy. 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reports","url":"https://pubmed.ncbi.nlm.nih.gov/27926873","citation_count":137,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9169,"output_tokens":1886,"usd":0.027898},"stage2":{"model":"claude-opus-4-6","input_tokens":5145,"output_tokens":2277,"usd":0.123975},"total_usd":0.314618,"stage1_batch_id":"msgbatch_012ZiiCUBfB62NCTQd4Bk1Jo","stage2_batch_id":"msgbatch_01Xuqq9aUHoQouS7UVGZNStZ","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":21407,"output_tokens":1998,"usd":0.047095},"round2_rules_fired":"R3","round2_stage2":{"model":"claude-opus-4-6","input_tokens":5495,"output_tokens":1985,"usd":0.11565}},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2016,\n      \"finding\": \"NOL10 forms a stable trimeric protein complex (ANN complex) with AATF and NGDN in human cells; the WD40 repeats of NOL10 are required for complex formation, and all three members show mutual dependence for protein stability. The ANN complex localizes to nucleoli and is required for 18S rRNA maturation and nucleolar cleavage steps in the 5'ETS and ITS1 regions of the rRNA precursor, supporting 40S ribosomal subunit biosynthesis.\",\n      \"method\": \"Immunoprecipitation, protein-protein interaction domain mapping, rRNA processing analysis, siRNA depletion with Northern blot/pulse-chase assays, nucleolar localization by microscopy\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP, domain mapping, functional depletion with defined rRNA processing phenotype, replicated across all three complex members\",\n      \"pmids\": [\"27599843\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PQBP5/NOL10 is an intrinsically disordered protein that constitutes the granule meshwork of the granular component area of the nucleolus, forming the skeletal structure. Unlike other nucleolar proteins, NOL10 remains in the nucleolus under osmotic stress and functions as an anchor for reassembly of other nucleolar proteins. Its biophysical properties remain stable under stress. Sequestration by polyglutamine disease proteins depletes functional NOL10 and causes nucleolar deformity or disappearance.\",\n      \"method\": \"High-speed atomic force microscopy, super-resolution microscopy, correlative light and electron microscopy, droplet assays, thermal shift assays, in vitro and in vivo polyglutamine sequestration experiments\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal structural and biophysical methods with functional validation in a single rigorous study\",\n      \"pmids\": [\"36599853\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"GFP-NOL10 localizes to the granular component and dense fibrillar component regions of nucleoli in living HeLa cells, exhibits very low mobility (tight association with macro-protein complexes), and when rRNA transcription is suppressed its mobility increases but remains slow, suggesting it acts as a scaffold or core component of nucleolar complexes.\",\n      \"method\": \"GFP fusion live-cell imaging, FRAP in HeLa cells, rRNA transcription suppression experiments\",\n      \"journal\": \"Biochemistry and cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct live-cell localization and dynamics with functional inference, single lab\",\n      \"pmids\": [\"24754225\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"A homozygous NOL10 variant (p.Asn228His) within the WD-repeat domain causes nucleoplasmic mislocalization of NOL10 protein, loss of interaction with AATF and NGDN, specific impairment of 40S ribosomal subunit maturation (reduced 40S, 80S, and polysome content), G0/G1 cell cycle arrest, and increased cell death in patient fibroblasts, establishing NOL10 as required for small ribosomal subunit biogenesis via the ANN complex.\",\n      \"method\": \"Exome sequencing, co-immunoprecipitation, immunofluorescence localization, polysome profiling, cell cycle analysis, structural modeling and ΔΔG calculation in patient-derived fibroblasts\",\n      \"journal\": \"Journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — patient variant with multiple orthogonal functional readouts confirming ANN complex interaction and 40S biogenesis role\",\n      \"pmids\": [\"41093997\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NOL10 physically interacts with a 24-amino-acid region within the DDX10 moiety of the NUP98::DDX10 fusion protein. NOL10 acts cooperatively with NUP98::DDX10 to regulate serine biosynthesis pathways and stabilize ATF4 mRNA. Loss of Nol10 in a mouse model of NUP98::DDX10 leukemia impaired disease progression and improved survival.\",\n      \"method\": \"Co-immunoprecipitation with deletion constructs, mouse leukemia model with Nol10 knockout, gene expression and metabolic pathway analysis\",\n      \"journal\": \"Leukemia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP with defined interaction domain, in vivo genetic loss-of-function, but novel function not independently replicated\",\n      \"pmids\": [\"40263434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The risk allele A of rs4519489 at the 2p25 locus exhibits enhanced binding to USF1 transcription factor, resulting in elevated NOL10 expression. NOL10 regulates cell cycle pathways to promote prostate cancer cell progression.\",\n      \"method\": \"SNPs-seq allele-specific protein binding assay, unbiased proteomics, cell cycle analysis upon NOL10 modulation\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — allele-specific proteomics identifies USF1 as transcriptional regulator of NOL10; cell cycle phenotype upon NOL10 perturbation, single lab\",\n      \"pmids\": [\"41062477\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The fission yeast ortholog Enp2/NOL10 forms a complex with a novel non-coding RNA (RiboCop) and RNase H1; this complex is involved in pre-rRNA surveillance in quiescence, and improper rRNA processing triggers a nucleolar stress response leading to rDNA silencing via Sir2, RENT, and H3K9 methylation.\",\n      \"method\": \"Genetic and biochemical analysis in fission yeast, complex identification, rRNA processing assays, chromatin silencing assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ortholog in fission yeast with defined complex and pathway, but preprint and not yet peer-reviewed\",\n      \"pmids\": [\"41000809\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"NOL10 (PQBP5) is a nucleolar WD-repeat-containing intrinsically disordered protein that forms the ANN complex with AATF and NGDN via its WD40 domain, functioning as an essential scaffold for nucleolar structure and for the maturation of 40S ribosomal subunits through nucleolar rRNA cleavage steps in the 5'ETS and ITS1; it also anchors the nucleolus under stress conditions and, in a disease context, cooperates with the NUP98::DDX10 oncoprotein to regulate serine biosynthesis and ATF4 mRNA stability.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2016,\n      \"finding\": \"NOL10 forms a salt-stable trimeric protein complex with AATF and NGDN (the ANN complex) in human cells. All three subunits localize to nucleoli and show mutual dependence for protein stability. The WD40 repeats of NOL10 and the UTP3/SAS10 domain of NGDN are required for complex formation. The ANN complex is required for nucleolar steps of 40S ribosomal subunit biosynthesis, specifically for 18S rRNA maturation; depletion of any single member affects the same cleavage steps in the 5'ETS and ITS1 regions of the ribosomal RNA precursor.\",\n      \"method\": \"Immunoprecipitation, domain-mapping mutagenesis, siRNA knockdown, rRNA processing assays (Northern blot), nucleolar localization by microscopy\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP with domain mapping, clean KD with defined rRNA processing phenotype, multiple orthogonal methods in single study\",\n      \"pmids\": [\"27599843\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PQBP5/NOL10 is an intrinsically disordered protein that constitutes the skeletal granule meshwork of the granular component of the nucleolus (distinct from the fibrillar center and dense fibrillar component). Unlike other nucleolar proteins that disperse to the nucleoplasm under osmotic stress, NOL10 remains in the nucleolus and functions as an anchor for reassembly of other nucleolar proteins after stress. Its biophysical properties remain stable under stress conditions. In polyglutamine disease models, NOL10 is sequestered by polyglutamine proteins, leading to pathological nucleolar deformity.\",\n      \"method\": \"High-speed atomic force microscopy (HS-AFM), super-resolution microscopy (STED), correlative light-electron microscopy (CLEM), droplet assay, thermal shift assay, osmotic stress experiments with live imaging\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal structural and biophysical methods with direct functional consequence (nucleolar anchoring) validated in vitro and in vivo\",\n      \"pmids\": [\"36599853\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"GFP-NOL10 localizes to the dense fibrillar component and granular component of nucleoli in living HeLa cells. Its mobility is very low under normal conditions, indicating tight binding to macroprotein complexes, consistent with a scaffold or core role. When rRNA transcription is suppressed, mobility increases but remains slow.\",\n      \"method\": \"GFP-fusion live-cell imaging, FRAP in living cells, suppression of rRNA transcription by ActD treatment\",\n      \"journal\": \"Biochemistry and cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct localization experiment with functional context (tight complex binding, rRNA-transcription dependence); single lab\",\n      \"pmids\": [\"24754225\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"A homozygous missense variant in the WD-repeat domain of NOL10 (p.Asn228His) causes nucleoplasmic mislocalization of the NOL10 protein, loss of interaction with AATF and NGDN, specific impairment of 40S ribosomal subunit maturation (reduced 40S, 80S, and polysome content), G0/G1 cell cycle arrest, and increased cell death in patient-derived fibroblasts. Structural modeling predicts N228H is strongly destabilizing.\",\n      \"method\": \"Exome sequencing, immunofluorescence (localization), co-immunoprecipitation (interaction loss), polysome profiling, cell cycle analysis, structural modeling and ΔΔG calculations\",\n      \"journal\": \"Journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — patient variant with multiple orthogonal functional readouts (mislocalization, lost interactions, polysome profiling, cell cycle) in primary cells\",\n      \"pmids\": [\"41093997\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NOL10 physically interacts with a 24-amino-acid region within the DDX10 moiety of the NUP98::DDX10 leukemia fusion protein. NOL10 is a critical dependency of NUP98::DDX10 leukemia; loss of Nol10 in a mouse model impairs disease progression and improves survival. NOL10 acts cooperatively with NUP98::DDX10 to regulate the serine biosynthesis pathway and stabilize ATF4 mRNA.\",\n      \"method\": \"Co-immunoprecipitation (interaction mapping), mouse leukemia model (Nol10 knockout), gene expression analysis (serine biosynthesis pathway), mRNA stability assays (ATF4)\",\n      \"journal\": \"Leukemia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP interaction mapping plus in vivo mouse model with survival readout and pathway analysis; single lab\",\n      \"pmids\": [\"40263434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The risk allele A of rs4519489 (in the NOL10 locus) exhibits enhanced binding to the transcription factor USF1, resulting in elevated NOL10 expression. NOL10 overexpression promotes cell cycle progression in prostate cancer cells. USF1 thus regulates NOL10 transcription through allele-specific binding at the 2p25 locus.\",\n      \"method\": \"SNPs-seq (allele-specific protein binding), unbiased proteomics (allele-specific capture), functional cell cycle assays, clinical cohort correlation\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — allele-specific proteomics with functional follow-up (cell cycle assays); single lab study\",\n      \"pmids\": [\"41062477\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In fission yeast, Enp2/NOL10 forms a complex with a novel non-coding RNA (RiboCop) and RNase H1 in response to improper pre-rRNA processing by Dicer during quiescence. This complex triggers rDNA repeat silencing via Sir2, RENT, and H3K9 methylation specifically during cellular dormancy.\",\n      \"method\": \"RNA co-immunoprecipitation, genetic mutant analysis (Dicer mutants), chromatin immunoprecipitation, epistasis analysis (Sir2/RENT/H3K9me pathway)\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 2 method but preprint, yeast ortholog, single study\",\n      \"pmids\": [\"41000809\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"NOL10/PQBP5 is an intrinsically disordered nucleolar scaffold protein that forms the granule meshwork of the granular component; it assembles with AATF and NGDN (via its WD40 repeats) into the ANN complex required for 18S rRNA maturation and 40S ribosomal subunit biogenesis, anchors the nucleolus under osmotic stress, and its mislocalization or loss—whether by disease-causing WD40 mutations, polyglutamine sequestration, or cooperative action with oncofusion proteins—disrupts ribosome biogenesis and cell viability.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"NOL10 is a nucleolar WD-repeat-containing intrinsically disordered protein that functions as a core structural scaffold of the nucleolus and an essential factor in 40S ribosomal subunit biogenesis. It forms the trimeric ANN complex with AATF and NGDN via its WD40 domain, and this complex is required for pre-rRNA cleavage steps in the 5'ETS and ITS1 regions that generate mature 18S rRNA; a disease-causing missense variant (p.Asn228His) in the WD-repeat domain abolishes ANN complex formation, causes nucleoplasmic mislocalization, and specifically impairs 40S subunit production with consequent cell cycle arrest [PMID:27599843, PMID:41093997]. NOL10 constitutes the granule meshwork of the nucleolar granular component, exhibits extremely low mobility consistent with a scaffold role, and uniquely persists in the nucleolus under stress to anchor reassembly of other nucleolar proteins; sequestration by polyglutamine disease proteins depletes functional NOL10 and causes nucleolar collapse [PMID:36599853, PMID:24754225]. NOL10 also physically interacts with the DDX10 moiety of the NUP98::DDX10 oncoprotein, cooperatively regulating serine biosynthesis and ATF4 mRNA stability, and loss of Nol10 impairs NUP98::DDX10-driven leukemia progression in mice [PMID:40263434].\",\n  \"teleology\": [\n    {\n      \"year\": 2014,\n      \"claim\": \"Establishing NOL10 as a stable nucleolar scaffold: live-cell imaging revealed that GFP-NOL10 localizes to the granular and dense fibrillar components of nucleoli with exceptionally low mobility, indicating it is a tightly integrated core component rather than a transient visitor.\",\n      \"evidence\": \"GFP-NOL10 fusion FRAP and live-cell imaging in HeLa cells with rRNA transcription inhibition\",\n      \"pmids\": [\"24754225\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single-lab observation without independent replication\",\n        \"No identification of the molecular partners responsible for the low mobility\",\n        \"Functional consequence of nucleolar scaffolding not yet demonstrated\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defining the molecular function of NOL10: discovery of the ANN complex (AATF–NOL10–NGDN) demonstrated that NOL10's WD40 domain mediates trimeric complex formation, that the three subunits are mutually dependent for stability, and that the complex is required for 18S rRNA maturation through 5'ETS and ITS1 cleavage steps, placing NOL10 directly in the 40S ribosomal subunit biogenesis pathway.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation, WD40 domain deletion mapping, siRNA depletion with Northern blot and pulse-chase rRNA analysis in human cells\",\n      \"pmids\": [\"27599843\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether NOL10 directly contacts pre-rRNA or acts solely as a protein scaffold\",\n        \"No structural model of the ANN complex at atomic resolution\",\n        \"Mechanism by which the ANN complex promotes specific cleavage events unknown\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Revealing NOL10 as the structural skeleton of the nucleolus: biophysical analyses showed that NOL10, as an intrinsically disordered protein, forms the granule meshwork of the granular component, remains anchored in the nucleolus under osmotic stress while other proteins disperse, and serves as the platform for nucleolar reassembly — establishing its unique structural role distinct from phase-separating nucleolar proteins.\",\n      \"evidence\": \"High-speed atomic force microscopy, super-resolution and correlative light-electron microscopy, droplet assays, thermal shift assays, polyglutamine sequestration experiments\",\n      \"pmids\": [\"36599853\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The biophysical basis for stress-resistant nucleolar retention is not fully resolved\",\n        \"Whether NOL10 scaffolding is required for nucleolar function beyond rRNA processing\",\n        \"Relevance of polyglutamine sequestration to human polyglutamine disease pathology in vivo not demonstrated\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Genetic validation of NOL10 in human disease: a homozygous WD-domain missense variant (p.Asn228His) in a patient abolished AATF/NGDN interaction, caused nucleoplasmic mislocalization, and specifically reduced 40S subunit levels with G0/G1 arrest and cell death, confirming NOL10 as essential for small subunit biogenesis in humans and linking it to a ribosomopathy.\",\n      \"evidence\": \"Exome sequencing, co-IP, immunofluorescence, polysome profiling, and cell cycle analysis in patient-derived fibroblasts\",\n      \"pmids\": [\"41093997\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Single family reported; additional cases needed to define the full clinical phenotype\",\n        \"Whether partial loss of NOL10 function produces intermediate phenotypes\",\n        \"Rescue experiment with wild-type NOL10 not reported\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Expanding NOL10 function beyond ribosome biogenesis: NOL10 physically interacts with the DDX10 moiety of NUP98::DDX10 and cooperatively regulates serine biosynthesis and ATF4 mRNA stability, with Nol10 knockout impairing leukemia progression in a mouse model — revealing a non-canonical role in oncogenic transcriptional and metabolic programs.\",\n      \"evidence\": \"Co-immunoprecipitation with deletion mapping, Nol10 knockout mouse leukemia model, gene expression and metabolic pathway analysis\",\n      \"pmids\": [\"40263434\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Novel non-ribosomal function not independently replicated\",\n        \"Whether ATF4 mRNA stabilization is direct or mediated through ribosome biogenesis defects\",\n        \"Mechanism by which NOL10 influences serine biosynthesis gene expression not defined\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Linking NOL10 expression regulation to cancer: a prostate cancer risk allele at rs4519489 enhances USF1 binding to increase NOL10 transcription, and elevated NOL10 promotes cell cycle progression in prostate cancer cells, connecting NOL10 levels to proliferative control.\",\n      \"evidence\": \"SNPs-seq allele-specific protein binding, proteomics for USF1 identification, cell cycle analysis upon NOL10 modulation\",\n      \"pmids\": [\"41062477\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single-lab finding not independently replicated\",\n        \"Whether cell cycle effects are mediated through 40S biogenesis or a distinct pathway\",\n        \"In vivo tumor model validation absent\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: the atomic structure of the ANN complex and how it engages pre-rRNA; whether NOL10's structural scaffolding and rRNA processing functions are separable; the full clinical spectrum of NOL10 deficiency in humans; and the mechanistic basis for non-canonical roles in mRNA stabilization and metabolic regulation.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No high-resolution structure of NOL10 or the ANN complex\",\n        \"Separation-of-function analysis for scaffold versus rRNA processing roles not performed\",\n        \"In vivo validation of NOL10 stress-anchoring function in mammalian models lacking\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 3]}\n    ],\n    \"complexes\": [\n      \"ANN complex (AATF-NOL10-NGDN)\"\n    ],\n    \"partners\": [\n      \"AATF\",\n      \"NGDN\",\n      \"DDX10\",\n      \"USF1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"NOL10 is a nucleolar scaffold protein that assembles with AATF and NGDN into the trimeric ANN complex via its WD40 repeats, and this complex is essential for 18S rRNA maturation and 40S ribosomal subunit biogenesis [PMID:27599843, PMID:41093997]. As an intrinsically disordered protein, NOL10 constitutes the skeletal granule meshwork of the nucleolar granular component, remains anchored in the nucleolus under osmotic stress, and serves as a platform for reassembly of other nucleolar proteins after stress; sequestration by polyglutamine-expanded proteins causes pathological nucleolar deformity [PMID:36599853]. A homozygous WD40-domain missense mutation (p.Asn228His) abolishes AATF/NGDN interaction, mislocalizes NOL10 to the nucleoplasm, impairs 40S subunit production, and causes G0/G1 arrest and cell death in patient fibroblasts, establishing NOL10 deficiency as a cause of a ribosomopathy [PMID:41093997]. NOL10 also physically interacts with the DDX10 moiety of the NUP98::DDX10 leukemia fusion protein, and its loss impairs leukemia progression in a mouse model [PMID:40263434].\",\n  \"teleology\": [\n    {\n      \"year\": 2014,\n      \"claim\": \"Establishing NOL10 as a stably incorporated nucleolar component resolved its subnuclear address and raised the question of what macromolecular complex it participates in.\",\n      \"evidence\": \"GFP-NOL10 live-cell imaging and FRAP in HeLa cells showed nucleolar localization (DFC/GC) and very low mobility indicating tight complex association\",\n      \"pmids\": [\"24754225\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Identity of the complex(es) retaining NOL10 in the nucleolus was unknown\",\n        \"Functional role in rRNA processing not yet tested\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identification of the ANN complex (NOL10–AATF–NGDN) and its requirement for 18S rRNA maturation defined NOL10's molecular function in ribosome biogenesis and showed that its WD40 repeats mediate complex assembly.\",\n      \"evidence\": \"Reciprocal co-IP, domain-mapping mutagenesis, siRNA knockdown with Northern blot rRNA processing assays in human cells\",\n      \"pmids\": [\"27599843\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of ANN complex assembly unresolved\",\n        \"Whether NOL10 has functions beyond rRNA processing not addressed\",\n        \"No in vivo disease model yet linked to ANN disruption\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Biophysical characterization revealed that NOL10 is an intrinsically disordered protein forming the granular-component meshwork and acting as a nucleolar stress anchor, explaining its scaffold role and linking its sequestration to polyglutamine disease pathology.\",\n      \"evidence\": \"HS-AFM, STED super-resolution, CLEM, droplet assays, osmotic stress live imaging, and polyglutamine disease cell models\",\n      \"pmids\": [\"36599853\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Molecular determinants of stress-resistant anchoring are not mapped\",\n        \"Causal role in polyglutamine disease progression not demonstrated in animal models\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"A patient-derived WD40 missense variant (N228H) proved that ANN complex integrity is essential for human 40S subunit production and cell viability, establishing NOL10 deficiency as a ribosomopathy.\",\n      \"evidence\": \"Exome sequencing, immunofluorescence, co-IP, polysome profiling, and cell cycle analysis in patient fibroblasts\",\n      \"pmids\": [\"41093997\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Full clinical spectrum and prevalence of NOL10-associated disease remain undefined\",\n        \"Whether residual NOL10 function persists or variant is a complete loss-of-function is unclear\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Discovery that NOL10 physically interacts with the NUP98::DDX10 oncofusion and is a critical dependency for leukemia progression expanded NOL10's relevance beyond normal ribosome biogenesis into oncogenic mechanisms.\",\n      \"evidence\": \"Co-IP domain mapping, Nol10-knockout mouse leukemia model with survival analysis, ATF4 mRNA stability and serine biosynthesis pathway analysis\",\n      \"pmids\": [\"40263434\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether the leukemia dependency reflects canonical ribosome biogenesis or a neomorphic function is unresolved\",\n        \"Therapeutic vulnerability of the NOL10–DDX10 interaction not tested\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"A prostate cancer risk SNP at the NOL10 locus was shown to increase NOL10 transcription via allele-specific USF1 binding, and NOL10 overexpression promoted cell cycle progression, linking NOL10 dosage to cancer proliferation.\",\n      \"evidence\": \"SNPs-seq, allele-specific proteomics, functional cell cycle assays in prostate cancer cells\",\n      \"pmids\": [\"41062477\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether cell cycle effects are mediated through ribosome biogenesis or an independent pathway is unclear\",\n        \"In vivo validation of NOL10 overexpression driving tumor growth is lacking\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the atomic structure of the ANN complex, the mechanism by which NOL10's intrinsic disorder confers stress-resistant nucleolar anchoring, and whether NOL10's roles in leukemia and prostate cancer are ribosome biogenesis-dependent or involve distinct moonlighting functions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No high-resolution structure of the ANN complex or NOL10 alone\",\n        \"Mechanistic basis of stress-resistant anchoring not mapped to specific disordered regions\",\n        \"Ribosome biogenesis-dependent vs. independent contributions to cancer biology not delineated\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [4, 5]}\n    ],\n    \"complexes\": [\n      \"ANN complex (NOL10–AATF–NGDN)\"\n    ],\n    \"partners\": [\n      \"AATF\",\n      \"NGDN\",\n      \"NUP98::DDX10\",\n      \"USF1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}