{"gene":"NOL9","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2010,"finding":"NOL9 (Nol9/Grc3 ortholog) is a polynucleotide 5'-kinase that sediments primarily with pre-60S ribosomal particles in HeLa nuclear extracts. Its kinase activity is required for efficient generation of 5.8S and 28S rRNAs from the 32S precursor, and its depletion causes a specific maturation defect at the 5' end of the predominant 5.8S short-form rRNA (5.8S-S), likely due to a requirement for 5'→3' exonucleolytic trimming.","method":"siRNA knockdown in HeLa cells, sucrose gradient sedimentation, in vitro polynucleotide kinase assay, Northern blot analysis of rRNA processing intermediates","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro kinase activity demonstrated, combined with loss-of-function cellular rRNA processing phenotype, two orthogonal methods in single rigorous study","pmids":["21063389"],"is_preprint":false},{"year":2019,"finding":"Human NOL9 associates with the Las1L endoribonuclease to form a higher-order ITS2 pre-rRNA endonuclease-kinase complex. NOL9 contains a nucleolar localization sequence (NoLS) that is responsible for nucleolar transport of the assembled Las1L-NOL9 complex, acting as a spatial regulator for ITS2 processing within the nucleolar sub-structure.","method":"Co-immunoprecipitation, deletion mapping of protein interaction domains, high-resolution electron microscopy structural imaging, fluorescence microscopy of GFP-tagged constructs with mutational analysis of the NoLS","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP, structural imaging, and mutagenesis of localization sequence in one study with multiple orthogonal methods","pmids":["31288032"],"is_preprint":false},{"year":2010,"finding":"Yeast Grc3 (NOL9 ortholog) is a polynucleotide kinase required for efficient transcription termination by RNA polymerase I in S. cerevisiae. It controls the phosphorylation status of the downstream Rnt1 cleavage product, regulating its accessibility to the torpedo exonuclease Rat1.","method":"Genetic analysis in S. cerevisiae, in vitro polynucleotide kinase assay, ChIP and transcription run-on assays","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro kinase activity combined with genetic epistasis placing Grc3 upstream of Rat1 in Pol I termination, two orthogonal methods","pmids":["20814424"],"is_preprint":false},{"year":2012,"finding":"Yeast Grc3 (NOL9 ortholog) is a major interacting partner of Las1, and the kinase activity of Grc3 is required for efficient pre-rRNA processing. Grc3 depletion leads to accumulation of 27S and 7S rRNA intermediates similar to Las1-depleted cells, placing Grc3 and Las1 in the same functional pathway for pre-60S ribosome biogenesis.","method":"Co-immunoprecipitation in S. cerevisiae, conditional depletion (anchor-away or GAL promoter shutoff), Northern blot analysis of rRNA intermediates, kinase-dead mutant analysis","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, kinase-dead mutant epistasis, and rRNA processing readout independently replicated across multiple organisms","pmids":["23175604"],"is_preprint":false},{"year":2017,"finding":"Grc3 (NOL9 ortholog) programs the Las1 endoribonuclease for specific C2 site cleavage of pre-rRNA, and Las1 reciprocally activates Grc3 kinase activity exclusively toward single-stranded RNA substrates. Together they assemble into a tetrameric complex required for competent rRNA processing, with mechanistic parallels to RNaseL/Ire1.","method":"In vitro reconstitution of kinase and endonuclease assays, site-directed mutagenesis, biochemical complex assembly, in vivo pre-rRNA cleavage assays in S. cerevisiae","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with mutagenesis plus in vivo genetic validation, multiple orthogonal methods establishing bidirectional activation mechanism","pmids":["28652339"],"is_preprint":false},{"year":2018,"finding":"Grc3 (NOL9 ortholog) has a distinct substrate preference for RNA over DNA substrates in vitro, unlike other polynucleotide kinase family members. Specific conserved residues at the Grc3 kinase active site are required to support Grc3-directed Las1-mediated pre-rRNA cleavage both in vitro and in vivo, establishing direct coupling of cleavage and phosphorylation during pre-rRNA processing.","method":"In vitro polynucleotide kinase assays with RNA and DNA substrates, active-site mutagenesis, in vivo pre-rRNA processing assays in S. cerevisiae","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assays with mutagenesis validated in vivo, two orthogonal methods in single rigorous study","pmids":["29440475"],"is_preprint":false},{"year":2011,"finding":"Fission yeast Grc3 (NOL9 ortholog) is required for both rRNA processing (25S rRNA maturation) and heterochromatic gene silencing. Grc3 physically associates with Las1 and components of the IPI complex (Rix1, Ipi1, Crb3). Its nuclear dot localization corresponds to heterochromatic regions, and this heterochromatic localization is dependent on the heterochromatic proteins Swi6 and Clr4.","method":"Co-immunoprecipitation in S. pombe, conditional mutant analysis, cytological analysis (fluorescence microscopy), Northern blotting for rRNA processing, gene silencing reporter assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP and multiple phenotypic readouts in single lab, fission yeast ortholog study","pmids":["21385875"],"is_preprint":false},{"year":2015,"finding":"Loss-of-function of nol9 in zebrafish causes a defect in 28S rRNA processing, impaired cell proliferation of hematopoietic stem/progenitor cells and pancreatic progenitor cells. Genetic loss of Tp53 rescued hematopoietic stem/progenitor cell defects but not pancreatic defects, placing Nol9 upstream of Tp53 in the hematopoietic lineage but indicating a Tp53-independent mechanism for pancreatic deficiency.","method":"Zebrafish loss-of-function mutant (nol9sa1022/sa1022), Northern blot for rRNA processing, genetic epistasis with tp53 mutant, L-Leucine/mTOR pathway activation experiment, flow cytometry for HSPC quantification","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis in vertebrate model with two pathway interventions and multiple cellular phenotype readouts, single lab","pmids":["26624285"],"is_preprint":false},{"year":2023,"finding":"Inhibition of the LAS1-NOL9 protein-protein interaction by covalent binding of compound HEN-463 to the C264 site of LAS1 causes cytoplasmic translocation of LAS1, thereby inhibiting 28S rRNA maturation and activating the NPM1-MDM2-p53 pathway leading to p53 stabilization.","method":"Covalent compound targeting with site-specific binding validation, cellular fractionation/localization assay, rRNA maturation assay, Western blot for p53 pathway components, AML cell line functional assays","journal":"Pharmacological research","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — compound-based disruption with localization readout and pathway activation, single lab, mechanistic follow-up is partial","pmids":["36796466"],"is_preprint":false},{"year":2025,"finding":"NOL9 expression in HCC cells is regulated by DNA methylation at specific CpG sites (involving DNMT1) and by the transcription factor ZNF384. NOL9-mediated cell proliferation is dependent on activation of the Wnt/β-catenin signaling pathway, as shown by NOL9 knockdown reducing Wnt/β-catenin activity and tumor growth.","method":"DNA methylation analysis, ChIP or reporter assays for ZNF384 regulation, siRNA knockdown and overexpression in HCC cell lines, in vivo xenograft tumor growth assay, Western blot for Wnt/β-catenin pathway components","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — loss-of-function with defined pathway readout and epigenetic regulation mechanism, single lab, limited mechanistic depth in abstract","pmids":["39955289"],"is_preprint":false}],"current_model":"NOL9 (also known as Grc3 in yeast) is a nucleolar polynucleotide 5'-kinase that forms a higher-order tetrameric complex with the Las1L endoribonuclease; NOL9 provides a nucleolar localization sequence that directs the assembled complex to the nucleolus, where it catalyzes RNA phosphorylation coupled to Las1L-mediated C2 endonucleolytic cleavage of ITS2 in pre-rRNA to drive production of the 5.8S and 28S mature rRNAs, with NOL9 kinase activity additionally required for 5'→3' exonucleolytic trimming of the 5.8S-S form and for RNA polymerase I transcription termination via phosphorylation of the Rat1 torpedo substrate."},"narrative":{"mechanistic_narrative":"NOL9 (the human ortholog of yeast Grc3) is a nucleolar polynucleotide 5'-kinase that drives the ITS2-processing arm of large ribosomal subunit biogenesis [PMID:21063389, PMID:23175604]. It assembles with the Las1L (yeast Las1) endoribonuclease into a higher-order tetrameric kinase–endonuclease complex, within which Las1 cleaves the pre-rRNA at the C2 site while NOL9 phosphorylates the resulting RNA ends; the two enzymes reciprocally activate one another, and NOL9 kinase activity is directed preferentially toward single-stranded RNA over DNA substrates [PMID:28652339, PMID:29440475]. NOL9 contributes a nucleolar localization sequence that targets the assembled complex to the nucleolus, providing spatial control over ITS2 cleavage [PMID:31288032]. Loss of NOL9 kinase function blocks generation of 5.8S and 28S rRNAs from the 32S precursor and produces a 5' maturation defect at the 5.8S short form consistent with a requirement for 5'→3' exonucleolytic trimming [PMID:21063389]. In yeast, the kinase additionally governs RNA polymerase I transcription termination by controlling the phosphorylation state of the Rnt1 cleavage product and thereby its accessibility to the Rat1 torpedo exonuclease [PMID:20814424]. Disruption of NOL9-dependent rRNA maturation engages the p53 surveillance axis: zebrafish nol9 loss impairs hematopoietic and pancreatic progenitor proliferation in part through Tp53 [PMID:26624285], and pharmacological severing of the LAS1–NOL9 interaction mislocalizes LAS1 to the cytoplasm and activates the NPM1–MDM2–p53 pathway [PMID:36796466].","teleology":[{"year":2010,"claim":"Establishing that NOL9 is an enzyme and not merely a structural factor answered what biochemical activity it contributes to ribosome assembly, identifying it as a polynucleotide 5'-kinase needed to make mature large-subunit rRNAs.","evidence":"in vitro kinase assay plus siRNA depletion with sucrose gradients and Northern blotting in HeLa cells","pmids":["21063389"],"confidence":"High","gaps":["Direct RNA substrate and exact phosphorylation site within the pre-rRNA not defined","Functional partner mediating coupling to cleavage not yet identified"]},{"year":2010,"claim":"Parallel yeast work answered how the kinase fits into transcription control, placing Grc3 upstream of the Rat1 torpedo exonuclease in RNA polymerase I termination via phosphorylation of the Rnt1 cleavage product.","evidence":"yeast genetics, in vitro kinase assay, ChIP and transcription run-on in S. cerevisiae","pmids":["20814424"],"confidence":"High","gaps":["Whether the human ortholog performs the same termination role not tested","Direct phosphorylation of the termination substrate not shown structurally"]},{"year":2012,"claim":"Identifying Grc3 as a major Las1 partner answered which pathway the kinase operates in, showing shared rRNA intermediate accumulation that places the two proteins in the same pre-60S processing route.","evidence":"co-IP, conditional depletion, kinase-dead mutant analysis and Northern blotting in S. cerevisiae","pmids":["23175604"],"confidence":"High","gaps":["Stoichiometry and architecture of the complex not resolved","Order of cleavage versus phosphorylation events not established"]},{"year":2017,"claim":"Reconstitution answered how cleavage and phosphorylation are mechanistically linked, revealing a tetrameric complex in which Grc3 programs Las1 for C2 cleavage and Las1 reciprocally activates Grc3 toward single-stranded RNA.","evidence":"in vitro reconstitution of kinase and endonuclease assays with mutagenesis and in vivo cleavage assays in S. cerevisiae","pmids":["28652339"],"confidence":"High","gaps":["High-resolution structure of the human complex not determined","How the complex selects the C2 site within ITS2 not fully defined"]},{"year":2018,"claim":"Defining substrate preference and active-site requirements answered whether the kinase activity itself is essential for cleavage, showing RNA-over-DNA selectivity and active-site residues required to support Las1-mediated cleavage in vivo.","evidence":"in vitro kinase assays with RNA/DNA substrates, active-site mutagenesis and in vivo pre-rRNA processing in S. cerevisiae","pmids":["29440475"],"confidence":"High","gaps":["Physiological 5'-phosphorylated product fate downstream not tracked","Coupling mechanism at atomic resolution not resolved"]},{"year":2019,"claim":"Mapping the human complex answered how it reaches its site of action, demonstrating that NOL9 supplies the nucleolar localization sequence that imports the assembled Las1L–NOL9 complex into the nucleolus.","evidence":"co-IP, deletion mapping, electron microscopy and NoLS mutational fluorescence microscopy in human cells","pmids":["31288032"],"confidence":"High","gaps":["Sub-nucleolar dynamics during the cell cycle not characterized","Regulation of complex assembly versus import not separated"]},{"year":2011,"claim":"Fission yeast work extended the role beyond rRNA, linking Grc3 to heterochromatic silencing and to the IPI complex via Swi6/Clr4-dependent localization.","evidence":"co-IP, conditional mutants, cytology, Northern blotting and silencing reporters in S. pombe","pmids":["21385875"],"confidence":"Medium","gaps":["Whether the human ortholog has any silencing function untested","Mechanistic link between rRNA processing and heterochromatin localization unclear"]},{"year":2015,"claim":"Vertebrate loss-of-function answered the physiological consequence of NOL9 deficiency, showing tissue-specific progenitor proliferation defects with a Tp53-dependent hematopoietic component and Tp53-independent pancreatic component.","evidence":"zebrafish nol9 mutant with Northern blot, tp53 epistasis and mTOR pathway intervention","pmids":["26624285"],"confidence":"Medium","gaps":["Molecular basis of the Tp53-independent pancreatic mechanism unknown","Direct relevance to human disease not established in this study"]},{"year":2023,"claim":"Pharmacological disruption answered whether the LAS1–NOL9 interaction is a tractable node, showing that severing it mislocalizes LAS1 to the cytoplasm, blocks 28S maturation and activates the NPM1–MDM2–p53 axis.","evidence":"covalent compound HEN-463 with site-specific binding, fractionation, rRNA maturation and p53 Western blot in AML cells","pmids":["36796466"],"confidence":"Medium","gaps":["Direct on-target effect of disrupting NOL9 specifically versus LAS1 not separated","Generality across cancer types not established"]},{"year":2025,"claim":"Cancer-context regulation answered how NOL9 expression is controlled and what downstream proliferative pathway it engages, linking DNMT1/ZNF384-mediated regulation to Wnt/β-catenin-dependent proliferation in HCC.","evidence":"DNA methylation analysis, ZNF384 regulation assays, knockdown/overexpression, xenograft growth and Wnt pathway Western blot in HCC cells","pmids":["39955289"],"confidence":"Medium","gaps":["Mechanistic link between NOL9 ribosome biogenesis function and Wnt signaling unresolved","Whether the effect requires NOL9 kinase activity not tested"]},{"year":null,"claim":"How NOL9's rRNA-processing activity is mechanistically connected to the diverse downstream outputs (p53 surveillance, Wnt signaling, heterochromatin) and whether these require its kinase activity remain open.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of the human Las1L–NOL9 complex","Causal chain from impaired ITS2 processing to Wnt activation unmapped","Human-specific termination and silencing roles untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,2,4,5]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[4,5]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[5]}],"localization":[{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[1]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[0,3,4]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[2]}],"complexes":["Las1L-NOL9 ITS2 endonuclease-kinase complex"],"partners":["LAS1L","LAS1","RAT1","RNT1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q5SY16","full_name":"Polynucleotide 5'-hydroxyl-kinase NOL9","aliases":["Nucleolar protein 9"],"length_aa":702,"mass_kda":79.3,"function":"Polynucleotide kinase that can phosphorylate the 5'-hydroxyl groups of single-stranded and double-stranded RNA and DNA substrates (PubMed:21063389). Involved in rRNA processing and its kinase activity is required for the processing of the 32S precursor into 5.8S and 28S rRNAs, more specifically for the generation of the major 5.8S(S) form (PubMed:21063389). Required for the efficient pre-rRNA processing of internal transcribed spacer 2 (ITS2) (PubMed:21063389). Associates with LAS1L to form an ITS2 pre-rRNA endonuclease-kinase complex and is responsible for the transport of this complex into the nucleolus (PubMed:31288032)","subcellular_location":"Nucleus; Nucleus, nucleolus","url":"https://www.uniprot.org/uniprotkb/Q5SY16/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/NOL9","classification":"Common Essential","n_dependent_lines":1168,"n_total_lines":1208,"dependency_fraction":0.9668874172185431},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"IPO5","stoichiometry":0.2},{"gene":"NPM1","stoichiometry":0.2},{"gene":"PSPC1","stoichiometry":0.2},{"gene":"SRP9","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/NOL9","total_profiled":1310},"omim":[{"mim_id":"620304","title":"NUCLEOLAR PROTEIN 9; NOL9","url":"https://www.omim.org/entry/620304"},{"mim_id":"609455","title":"PROLINE-, GLUTAMIC ACID-, AND LEUCINE-RICH PROTEIN 1; PELP1","url":"https://www.omim.org/entry/609455"},{"mim_id":"608757","title":"CLEAVAGE FACTOR POLYNUCLEOTIDE KINASE SUBUNIT 1; CLP1","url":"https://www.omim.org/entry/608757"},{"mim_id":"300964","title":"LAS1-LIKE RIBOSOME BIOGENESIS FACTOR; LAS1L","url":"https://www.omim.org/entry/300964"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoli","reliability":"Supported"},{"location":"Intermediate filaments","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/NOL9"},"hgnc":{"alias_symbol":["FLJ23323","NET6","Grc3"],"prev_symbol":[]},"alphafold":{"accession":"Q5SY16","domains":[{"cath_id":"2.60.120.1030","chopping":"117-274","consensus_level":"high","plddt":89.1404,"start":117,"end":274},{"cath_id":"3.40.50.300","chopping":"280-464_491-501","consensus_level":"high","plddt":92.8431,"start":280,"end":501},{"cath_id":"2.40.30.330","chopping":"544-653","consensus_level":"high","plddt":92.4416,"start":544,"end":653}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5SY16","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q5SY16-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q5SY16-F1-predicted_aligned_error_v6.png","plddt_mean":77.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NOL9","jax_strain_url":"https://www.jax.org/strain/search?query=NOL9"},"sequence":{"accession":"Q5SY16","fasta_url":"https://rest.uniprot.org/uniprotkb/Q5SY16.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q5SY16/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5SY16"}},"corpus_meta":[{"pmid":"23175604","id":"PMC_23175604","title":"Las1 interacts with Grc3 polynucleotide kinase and is required for ribosome synthesis in Saccharomyces cerevisiae.","date":"2012","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/23175604","citation_count":41,"is_preprint":false},{"pmid":"17965585","id":"PMC_17965585","title":"Expression and function of transmembrane-4 superfamily (tetraspanin) proteins in osteoclasts: reciprocal roles of Tspan-5 and NET-6 during osteoclastogenesis.","date":"2007","source":"Allergology international : official journal of the Japanese Society of Allergology","url":"https://pubmed.ncbi.nlm.nih.gov/17965585","citation_count":35,"is_preprint":false},{"pmid":"21063389","id":"PMC_21063389","title":"Nol9 is a novel polynucleotide 5'-kinase involved in ribosomal RNA processing.","date":"2010","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/21063389","citation_count":34,"is_preprint":false},{"pmid":"28652339","id":"PMC_28652339","title":"Grc3 programs the essential endoribonuclease Las1 for specific RNA cleavage.","date":"2017","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/28652339","citation_count":34,"is_preprint":false},{"pmid":"20814424","id":"PMC_20814424","title":"Role of the RNA/DNA kinase Grc3 in transcription termination by RNA polymerase I.","date":"2010","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/20814424","citation_count":33,"is_preprint":false},{"pmid":"17486367","id":"PMC_17486367","title":"The tetraspanin superfamily member NET-6 is a new tumor suppressor gene.","date":"2007","source":"Journal of cancer research and clinical oncology","url":"https://pubmed.ncbi.nlm.nih.gov/17486367","citation_count":25,"is_preprint":false},{"pmid":"26624285","id":"PMC_26624285","title":"The Ribosome Biogenesis Protein Nol9 Is Essential for Definitive Hematopoiesis and Pancreas Morphogenesis in Zebrafish.","date":"2015","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/26624285","citation_count":22,"is_preprint":false},{"pmid":"21385875","id":"PMC_21385875","title":"Roles of fission yeast Grc3 protein in ribosomal RNA processing and heterochromatic gene silencing.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21385875","citation_count":21,"is_preprint":false},{"pmid":"31004251","id":"PMC_31004251","title":"Expression of tetraspanins NET-6 and CD151 in breast cancer as a potential tumor biomarker.","date":"2019","source":"Clinical and experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/31004251","citation_count":15,"is_preprint":false},{"pmid":"31288032","id":"PMC_31288032","title":"Nol9 Is a Spatial Regulator for the Human ITS2 Pre-rRNA Endonuclease-Kinase Complex.","date":"2019","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/31288032","citation_count":13,"is_preprint":false},{"pmid":"29440475","id":"PMC_29440475","title":"Characterization of the molecular crosstalk within the essential Grc3/Las1 pre-rRNA processing complex.","date":"2018","source":"RNA (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/29440475","citation_count":13,"is_preprint":false},{"pmid":"39955289","id":"PMC_39955289","title":"Nucleolar NOL9 regulated by DNA methylation promotes hepatocellular carcinoma growth through activation of Wnt/β-catenin signaling pathway.","date":"2025","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/39955289","citation_count":5,"is_preprint":false},{"pmid":"36796466","id":"PMC_36796466","title":"Covalent targeting the LAS1-NOL9 axis for selective treatment in NPM1 mutant acute myeloid leukemia.","date":"2023","source":"Pharmacological research","url":"https://pubmed.ncbi.nlm.nih.gov/36796466","citation_count":5,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8347,"output_tokens":2899,"usd":0.034263,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10228,"output_tokens":3590,"usd":0.070445,"stage2_stop_reason":"end_turn"},"total_usd":0.104708,"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\": 2010,\n      \"finding\": \"NOL9 (Nol9/Grc3 ortholog) is a polynucleotide 5'-kinase that sediments primarily with pre-60S ribosomal particles in HeLa nuclear extracts. Its kinase activity is required for efficient generation of 5.8S and 28S rRNAs from the 32S precursor, and its depletion causes a specific maturation defect at the 5' end of the predominant 5.8S short-form rRNA (5.8S-S), likely due to a requirement for 5'→3' exonucleolytic trimming.\",\n      \"method\": \"siRNA knockdown in HeLa cells, sucrose gradient sedimentation, in vitro polynucleotide kinase assay, Northern blot analysis of rRNA processing intermediates\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro kinase activity demonstrated, combined with loss-of-function cellular rRNA processing phenotype, two orthogonal methods in single rigorous study\",\n      \"pmids\": [\"21063389\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Human NOL9 associates with the Las1L endoribonuclease to form a higher-order ITS2 pre-rRNA endonuclease-kinase complex. NOL9 contains a nucleolar localization sequence (NoLS) that is responsible for nucleolar transport of the assembled Las1L-NOL9 complex, acting as a spatial regulator for ITS2 processing within the nucleolar sub-structure.\",\n      \"method\": \"Co-immunoprecipitation, deletion mapping of protein interaction domains, high-resolution electron microscopy structural imaging, fluorescence microscopy of GFP-tagged constructs with mutational analysis of the NoLS\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP, structural imaging, and mutagenesis of localization sequence in one study with multiple orthogonal methods\",\n      \"pmids\": [\"31288032\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Yeast Grc3 (NOL9 ortholog) is a polynucleotide kinase required for efficient transcription termination by RNA polymerase I in S. cerevisiae. It controls the phosphorylation status of the downstream Rnt1 cleavage product, regulating its accessibility to the torpedo exonuclease Rat1.\",\n      \"method\": \"Genetic analysis in S. cerevisiae, in vitro polynucleotide kinase assay, ChIP and transcription run-on assays\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro kinase activity combined with genetic epistasis placing Grc3 upstream of Rat1 in Pol I termination, two orthogonal methods\",\n      \"pmids\": [\"20814424\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Yeast Grc3 (NOL9 ortholog) is a major interacting partner of Las1, and the kinase activity of Grc3 is required for efficient pre-rRNA processing. Grc3 depletion leads to accumulation of 27S and 7S rRNA intermediates similar to Las1-depleted cells, placing Grc3 and Las1 in the same functional pathway for pre-60S ribosome biogenesis.\",\n      \"method\": \"Co-immunoprecipitation in S. cerevisiae, conditional depletion (anchor-away or GAL promoter shutoff), Northern blot analysis of rRNA intermediates, kinase-dead mutant analysis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, kinase-dead mutant epistasis, and rRNA processing readout independently replicated across multiple organisms\",\n      \"pmids\": [\"23175604\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Grc3 (NOL9 ortholog) programs the Las1 endoribonuclease for specific C2 site cleavage of pre-rRNA, and Las1 reciprocally activates Grc3 kinase activity exclusively toward single-stranded RNA substrates. Together they assemble into a tetrameric complex required for competent rRNA processing, with mechanistic parallels to RNaseL/Ire1.\",\n      \"method\": \"In vitro reconstitution of kinase and endonuclease assays, site-directed mutagenesis, biochemical complex assembly, in vivo pre-rRNA cleavage assays in S. cerevisiae\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with mutagenesis plus in vivo genetic validation, multiple orthogonal methods establishing bidirectional activation mechanism\",\n      \"pmids\": [\"28652339\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Grc3 (NOL9 ortholog) has a distinct substrate preference for RNA over DNA substrates in vitro, unlike other polynucleotide kinase family members. Specific conserved residues at the Grc3 kinase active site are required to support Grc3-directed Las1-mediated pre-rRNA cleavage both in vitro and in vivo, establishing direct coupling of cleavage and phosphorylation during pre-rRNA processing.\",\n      \"method\": \"In vitro polynucleotide kinase assays with RNA and DNA substrates, active-site mutagenesis, in vivo pre-rRNA processing assays in S. cerevisiae\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assays with mutagenesis validated in vivo, two orthogonal methods in single rigorous study\",\n      \"pmids\": [\"29440475\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Fission yeast Grc3 (NOL9 ortholog) is required for both rRNA processing (25S rRNA maturation) and heterochromatic gene silencing. Grc3 physically associates with Las1 and components of the IPI complex (Rix1, Ipi1, Crb3). Its nuclear dot localization corresponds to heterochromatic regions, and this heterochromatic localization is dependent on the heterochromatic proteins Swi6 and Clr4.\",\n      \"method\": \"Co-immunoprecipitation in S. pombe, conditional mutant analysis, cytological analysis (fluorescence microscopy), Northern blotting for rRNA processing, gene silencing reporter assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP and multiple phenotypic readouts in single lab, fission yeast ortholog study\",\n      \"pmids\": [\"21385875\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Loss-of-function of nol9 in zebrafish causes a defect in 28S rRNA processing, impaired cell proliferation of hematopoietic stem/progenitor cells and pancreatic progenitor cells. Genetic loss of Tp53 rescued hematopoietic stem/progenitor cell defects but not pancreatic defects, placing Nol9 upstream of Tp53 in the hematopoietic lineage but indicating a Tp53-independent mechanism for pancreatic deficiency.\",\n      \"method\": \"Zebrafish loss-of-function mutant (nol9sa1022/sa1022), Northern blot for rRNA processing, genetic epistasis with tp53 mutant, L-Leucine/mTOR pathway activation experiment, flow cytometry for HSPC quantification\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis in vertebrate model with two pathway interventions and multiple cellular phenotype readouts, single lab\",\n      \"pmids\": [\"26624285\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Inhibition of the LAS1-NOL9 protein-protein interaction by covalent binding of compound HEN-463 to the C264 site of LAS1 causes cytoplasmic translocation of LAS1, thereby inhibiting 28S rRNA maturation and activating the NPM1-MDM2-p53 pathway leading to p53 stabilization.\",\n      \"method\": \"Covalent compound targeting with site-specific binding validation, cellular fractionation/localization assay, rRNA maturation assay, Western blot for p53 pathway components, AML cell line functional assays\",\n      \"journal\": \"Pharmacological research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — compound-based disruption with localization readout and pathway activation, single lab, mechanistic follow-up is partial\",\n      \"pmids\": [\"36796466\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NOL9 expression in HCC cells is regulated by DNA methylation at specific CpG sites (involving DNMT1) and by the transcription factor ZNF384. NOL9-mediated cell proliferation is dependent on activation of the Wnt/β-catenin signaling pathway, as shown by NOL9 knockdown reducing Wnt/β-catenin activity and tumor growth.\",\n      \"method\": \"DNA methylation analysis, ChIP or reporter assays for ZNF384 regulation, siRNA knockdown and overexpression in HCC cell lines, in vivo xenograft tumor growth assay, Western blot for Wnt/β-catenin pathway components\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — loss-of-function with defined pathway readout and epigenetic regulation mechanism, single lab, limited mechanistic depth in abstract\",\n      \"pmids\": [\"39955289\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NOL9 (also known as Grc3 in yeast) is a nucleolar polynucleotide 5'-kinase that forms a higher-order tetrameric complex with the Las1L endoribonuclease; NOL9 provides a nucleolar localization sequence that directs the assembled complex to the nucleolus, where it catalyzes RNA phosphorylation coupled to Las1L-mediated C2 endonucleolytic cleavage of ITS2 in pre-rRNA to drive production of the 5.8S and 28S mature rRNAs, with NOL9 kinase activity additionally required for 5'→3' exonucleolytic trimming of the 5.8S-S form and for RNA polymerase I transcription termination via phosphorylation of the Rat1 torpedo substrate.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NOL9 (the human ortholog of yeast Grc3) is a nucleolar polynucleotide 5'-kinase that drives the ITS2-processing arm of large ribosomal subunit biogenesis [#0, #3]. It assembles with the Las1L (yeast Las1) endoribonuclease into a higher-order tetrameric kinase–endonuclease complex, within which Las1 cleaves the pre-rRNA at the C2 site while NOL9 phosphorylates the resulting RNA ends; the two enzymes reciprocally activate one another, and NOL9 kinase activity is directed preferentially toward single-stranded RNA over DNA substrates [#4, #5]. NOL9 contributes a nucleolar localization sequence that targets the assembled complex to the nucleolus, providing spatial control over ITS2 cleavage [#1]. Loss of NOL9 kinase function blocks generation of 5.8S and 28S rRNAs from the 32S precursor and produces a 5' maturation defect at the 5.8S short form consistent with a requirement for 5'\\u21923' exonucleolytic trimming [#0]. In yeast, the kinase additionally governs RNA polymerase I transcription termination by controlling the phosphorylation state of the Rnt1 cleavage product and thereby its accessibility to the Rat1 torpedo exonuclease [#2]. Disruption of NOL9-dependent rRNA maturation engages the p53 surveillance axis: zebrafish nol9 loss impairs hematopoietic and pancreatic progenitor proliferation in part through Tp53 [#7], and pharmacological severing of the LAS1\\u2013NOL9 interaction mislocalizes LAS1 to the cytoplasm and activates the NPM1\\u2013MDM2\\u2013p53 pathway [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"Establishing that NOL9 is an enzyme and not merely a structural factor answered what biochemical activity it contributes to ribosome assembly, identifying it as a polynucleotide 5'-kinase needed to make mature large-subunit rRNAs.\",\n      \"evidence\": \"in vitro kinase assay plus siRNA depletion with sucrose gradients and Northern blotting in HeLa cells\",\n      \"pmids\": [\"21063389\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct RNA substrate and exact phosphorylation site within the pre-rRNA not defined\", \"Functional partner mediating coupling to cleavage not yet identified\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Parallel yeast work answered how the kinase fits into transcription control, placing Grc3 upstream of the Rat1 torpedo exonuclease in RNA polymerase I termination via phosphorylation of the Rnt1 cleavage product.\",\n      \"evidence\": \"yeast genetics, in vitro kinase assay, ChIP and transcription run-on in S. cerevisiae\",\n      \"pmids\": [\"20814424\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the human ortholog performs the same termination role not tested\", \"Direct phosphorylation of the termination substrate not shown structurally\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identifying Grc3 as a major Las1 partner answered which pathway the kinase operates in, showing shared rRNA intermediate accumulation that places the two proteins in the same pre-60S processing route.\",\n      \"evidence\": \"co-IP, conditional depletion, kinase-dead mutant analysis and Northern blotting in S. cerevisiae\",\n      \"pmids\": [\"23175604\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and architecture of the complex not resolved\", \"Order of cleavage versus phosphorylation events not established\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Reconstitution answered how cleavage and phosphorylation are mechanistically linked, revealing a tetrameric complex in which Grc3 programs Las1 for C2 cleavage and Las1 reciprocally activates Grc3 toward single-stranded RNA.\",\n      \"evidence\": \"in vitro reconstitution of kinase and endonuclease assays with mutagenesis and in vivo cleavage assays in S. cerevisiae\",\n      \"pmids\": [\"28652339\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"High-resolution structure of the human complex not determined\", \"How the complex selects the C2 site within ITS2 not fully defined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defining substrate preference and active-site requirements answered whether the kinase activity itself is essential for cleavage, showing RNA-over-DNA selectivity and active-site residues required to support Las1-mediated cleavage in vivo.\",\n      \"evidence\": \"in vitro kinase assays with RNA/DNA substrates, active-site mutagenesis and in vivo pre-rRNA processing in S. cerevisiae\",\n      \"pmids\": [\"29440475\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological 5'-phosphorylated product fate downstream not tracked\", \"Coupling mechanism at atomic resolution not resolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Mapping the human complex answered how it reaches its site of action, demonstrating that NOL9 supplies the nucleolar localization sequence that imports the assembled Las1L\\u2013NOL9 complex into the nucleolus.\",\n      \"evidence\": \"co-IP, deletion mapping, electron microscopy and NoLS mutational fluorescence microscopy in human cells\",\n      \"pmids\": [\"31288032\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Sub-nucleolar dynamics during the cell cycle not characterized\", \"Regulation of complex assembly versus import not separated\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Fission yeast work extended the role beyond rRNA, linking Grc3 to heterochromatic silencing and to the IPI complex via Swi6/Clr4-dependent localization.\",\n      \"evidence\": \"co-IP, conditional mutants, cytology, Northern blotting and silencing reporters in S. pombe\",\n      \"pmids\": [\"21385875\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the human ortholog has any silencing function untested\", \"Mechanistic link between rRNA processing and heterochromatin localization unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Vertebrate loss-of-function answered the physiological consequence of NOL9 deficiency, showing tissue-specific progenitor proliferation defects with a Tp53-dependent hematopoietic component and Tp53-independent pancreatic component.\",\n      \"evidence\": \"zebrafish nol9 mutant with Northern blot, tp53 epistasis and mTOR pathway intervention\",\n      \"pmids\": [\"26624285\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of the Tp53-independent pancreatic mechanism unknown\", \"Direct relevance to human disease not established in this study\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Pharmacological disruption answered whether the LAS1\\u2013NOL9 interaction is a tractable node, showing that severing it mislocalizes LAS1 to the cytoplasm, blocks 28S maturation and activates the NPM1\\u2013MDM2\\u2013p53 axis.\",\n      \"evidence\": \"covalent compound HEN-463 with site-specific binding, fractionation, rRNA maturation and p53 Western blot in AML cells\",\n      \"pmids\": [\"36796466\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct on-target effect of disrupting NOL9 specifically versus LAS1 not separated\", \"Generality across cancer types not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Cancer-context regulation answered how NOL9 expression is controlled and what downstream proliferative pathway it engages, linking DNMT1/ZNF384-mediated regulation to Wnt/\\u03b2-catenin-dependent proliferation in HCC.\",\n      \"evidence\": \"DNA methylation analysis, ZNF384 regulation assays, knockdown/overexpression, xenograft growth and Wnt pathway Western blot in HCC cells\",\n      \"pmids\": [\"39955289\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic link between NOL9 ribosome biogenesis function and Wnt signaling unresolved\", \"Whether the effect requires NOL9 kinase activity not tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How NOL9's rRNA-processing activity is mechanistically connected to the diverse downstream outputs (p53 surveillance, Wnt signaling, heterochromatin) and whether these require its kinase activity remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of the human Las1L\\u2013NOL9 complex\", \"Causal chain from impaired ITS2 processing to Wnt activation unmapped\", \"Human-specific termination and silencing roles untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 2, 4, 5]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [4, 5]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [0, 3, 4]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"complexes\": [\"Las1L-NOL9 ITS2 endonuclease-kinase complex\"],\n    \"partners\": [\"LAS1L\", \"Las1\", \"Rat1\", \"Rnt1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}