{"gene":"POLR2C","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":1989,"finding":"RPB3 (POLR2C) is an essential single-copy gene encoding the third-largest subunit of RNA polymerase II; a temperature-sensitive rpb3 mutant prevented RNA polymerase II assembly, depleted functional enzyme, and reduced mRNA levels, demonstrating RPB3 is required for RNAPII assembly and mRNA transcription.","method":"Temperature-sensitive mutant analysis, immunological depletion of RNAPII, mRNA level measurement","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean loss-of-function with defined molecular phenotype (failure of RNAPII assembly), replicated across multiple assays in a foundational study","pmids":["2685562"],"is_preprint":false},{"year":1998,"finding":"Rpb3 (POLR2C) is present at a stoichiometry of one copy per RNA polymerase II molecule; deletion of either alpha-homology region (amino acids 29–55 or 226–267) abolishes Rpb3 assembly into RNAPII in vivo.","method":"Immunoaffinity and nickel-chelate chromatography of His6-tagged and untagged Rpb3-containing RNAPII; deletion mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct biochemical fractionation with mutagenesis, multiple orthogonal approaches establishing stoichiometry and assembly determinants","pmids":["9556554"],"is_preprint":false},{"year":1998,"finding":"Rpb3 (POLR2C) contacts Rpb2 (the beta-homologue) at the conserved region H of Rpb2, as mapped by two-hybrid screening using fission yeast subunit fragments.","method":"Yeast two-hybrid system with truncation fragments of Rpb1 and Rpb2","journal":"Molecular & general genetics : MGG","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — two-hybrid mapping in multiple fragment combinations, single lab, no in vitro biochemical confirmation","pmids":["9738888"],"is_preprint":false},{"year":1999,"finding":"Temperature-sensitive and cold-sensitive mutations in fission yeast rpb3 map to four conserved regions (A–D); cold-sensitive mutations in region A disrupt RNAPII assembly, and the Ts phenotype is suppressed by overexpression of Rpb11 (the pairing partner of Rpb3), establishing that Rpb3 mutations primarily affect the Rpb3–Rpb11 subassembly.","method":"Random mutagenesis, temperature/cold-sensitive growth assays, overexpression suppression analysis","journal":"Molecular & general genetics : MGG","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis (suppression by Rpb11 overexpression) with multiple mutant alleles, single lab","pmids":["10503538"],"is_preprint":false},{"year":2000,"finding":"Amino acid substitutions C92R and A159G in yeast RPB3 (POLR2C) specifically impair activator-dependent transcription without affecting basal transcription; homology modeling on the bacterial alpha-NTD crystal structure places residues 92–95 and 159–162 adjacent in 3D space, corresponding to the CAP activator-interaction surface of the bacterial alpha subunit, indicating a structurally conserved activation surface in RPB3.","method":"Alanine-scanning and targeted mutagenesis, activator-dependent transcription assays, homology modeling on bacterial alpha-NTD crystal structure","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — structure-based mutagenesis with functional transcription assays, multiple mutant alleles and synthetic enhancement analysis, single lab","pmids":["10673505"],"is_preprint":false},{"year":2001,"finding":"In vitro functional analysis of fission yeast Rpb3 mutants shows that mutations in terminal conserved regions A and D impair RNAPII assembly, while mutations in central eukaryote-specific regions B and C reduce activator (GAL4-VP16)-dependent transcription without equivalent assembly defects, functionally partitioning assembly from activated transcription roles within Rpb3.","method":"In vitro GAL4-VP16 activator-dependent transcription system using S. pombe cell extracts from rpb3 mutants, heat treatment assays","journal":"Current genetics","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro transcription assays with defined mutants, functional domain mapping, single lab","pmids":["11453250"],"is_preprint":false},{"year":2001,"finding":"The interaction site of Rpb2 with Rpb3 in fission yeast RNAPII was mapped to the C-terminal region of Rpb2 (amino acids 902–989, encoded by base 2701–2966 of Rpb2 cDNA) using two-hybrid analysis.","method":"Yeast two-hybrid system with Rpb2 deletion fragments; beta-galactosidase activity assay","journal":"Wei sheng wu xue bao = Acta microbiologica Sinica","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single two-hybrid experiment, no biochemical validation, single lab","pmids":["12552808"],"is_preprint":false},{"year":2002,"finding":"Human RPB3 (POLR2C) interacts with the myogenic transcription factor myogenin via a specific RPB3 region not homologous to the prokaryotic alpha subunit; this interaction involves the basic HLH region of myogenin but not other HLH factors (MyoD, Myf5, MRF4); coimmunoprecipitation confirmed that myogenin contacts the RNAPII complex through RPB3; a dominant-negative RPB3 fragment (Sud) counteracts myogenin transactivation and muscle differentiation.","method":"Yeast two-hybrid screening, coimmunoprecipitation, dominant-negative overexpression, muscle differentiation assays","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — reciprocal Co-IP plus yeast two-hybrid plus functional dominant-negative assay, single lab","pmids":["12207009"],"is_preprint":false},{"year":2003,"finding":"Human RPB3 (POLR2C) interacts with the transcription factor ATF4 via an RPB3-specific region (Sud) not homologous to the prokaryotic alpha subunit; RPB3 enhances ATF4 transactivation activity, and the Sud dominant-negative fragment markedly inhibits ATF4 transactivation.","method":"Yeast two-hybrid, transactivation assays with dominant-negative RPB3 fragment","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — yeast two-hybrid plus functional transactivation assay, single lab, no reciprocal Co-IP","pmids":["12860379"],"is_preprint":false},{"year":2005,"finding":"RPB3 (POLR2C) forms a heterodimer with RPB11 that initiates RNAPII assembly; in yeast, the C-terminal region of RPB11 is critical for heterodimerization, whereas in humans the conserved N-terminal alpha-motifs dominate the RPB3–RPB11 interface, indicating that the heterodimerization interface has diverged during evolution.","method":"In vitro heterodimerization assays comparing human and yeast RPB3/RPB11 truncation mutants","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct biochemical heterodimerization assays with defined mutants comparing two species, single lab","pmids":["15987790"],"is_preprint":false},{"year":2006,"finding":"IGFBP-3 interacts with Rpb3 (POLR2C) in rat myoblasts; the interaction requires the MBD/NLS epitope of IGFBP-3 (a NLS mutant does not associate with Rpb3); interaction was confirmed by coimmunoprecipitation with specific antisera.","method":"Yeast two-hybrid library screening with IGFBP-3 deletion mutants; coimmunoprecipitation with specific antisera","journal":"Endocrinology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — reciprocal Co-IP plus two-hybrid domain mapping, single lab","pmids":["16455777"],"is_preprint":false},{"year":2014,"finding":"Rpb3 (POLR2C) binds directly to the transcription factor Snail via its N-terminus; this interaction downregulates E-cadherin and induces epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma cells; the N-terminal fragment of Rpb3 acts as a dominant negative blocking Rpb3–Snail binding and inhibiting proliferation and migration in Rpb3-high-expression HCC cells.","method":"Co-IP/direct binding assay, E-cadherin expression assay, dominant-negative N-terminus overexpression, cell proliferation/migration assays, xenograft tumor growth","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — direct binding demonstrated, functional rescue with dominant-negative fragment, multiple cellular assays, single lab","pmids":["25211001"],"is_preprint":false},{"year":2018,"finding":"Gpn2 and Rba50 directly participate in assembly of the Rpb3 (POLR2C) subcomplex during RNAPII biogenesis: Gpn2 interacts with Rpb12, Rba50 interacts with Rpb3, Gpn2 and Rba50 interact with each other, and loss of function of either disrupts Rpb3 subcomplex assembly and subsequent RNAPII biogenesis.","method":"Co-IP, pulldown assays, loss-of-function analysis of Gpn2/Rba50 with RNAPII assembly readout","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal interaction mapping plus functional loss-of-function with assembly readout, single lab, multiple orthogonal pulldowns","pmids":["29661922"],"is_preprint":false},{"year":2025,"finding":"A K9E substitution at residue 9 of Rpb3 (POLR2C) causes readthrough of NNS-dependent terminators and cold-sensitive growth in yeast; genetic suppression by an R317G substitution in the RRM2 of Hrp1 (Nab4/CF1B) indicates that Rpb3-K9 forms a salt-bridge interaction surface that regulates binding of the anti-termination factor Hrp1 to the RNAPII elongation complex.","method":"Allele-specific mutagenesis, genome-wide suppressor selection, targeted suppressor selection in HRP1, transcriptome readthrough assays","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — allele-specific genetic epistasis with suppressor screen, multiple orthogonal alleles tested, preprint not yet peer-reviewed","pmids":["bio_10.1101_2025.05.07.652672"],"is_preprint":true}],"current_model":"POLR2C (RPB3) is the third-largest, alpha-like core subunit of RNA polymerase II that is essential for RNAPII assembly (via conserved alpha-homology regions and heterodimerization with RPB11), participates in a structurally conserved surface required for activator-dependent transcription, directly contacts the beta-homologue Rpb2, recruits specific transcription factors (myogenin, ATF4, Snail) to the polymerase through a eukaryote-specific domain, is incorporated into the Rpb3 subcomplex with the assistance of assembly chaperones Gpn2 and Rba50, and its elongation complex surface regulates binding of the anti-termination factor Hrp1 to control NNS-dependent termination."},"narrative":{"mechanistic_narrative":"POLR2C (RPB3) is the third-largest, alpha-like core subunit of RNA polymerase II and is essential for assembly of the holoenzyme and for mRNA transcription [PMID:2685562]. It is present at one copy per polymerase and nucleates RNAPII assembly by heterodimerizing with RPB11 through conserved alpha-homology regions, deletion of which abolishes its incorporation into the enzyme [PMID:9556554, PMID:15987790]; within the assembled polymerase it directly contacts the beta-homologue Rpb2 [PMID:9738888]. Structure-based mutagenesis partitions RPB3 into terminal conserved regions required for assembly and central eukaryote-specific regions required for activator-dependent transcription, the latter mapping to a structurally conserved surface homologous to the bacterial alpha CAP-interaction site [PMID:10673505, PMID:11453250]. Through this eukaryote-specific region RPB3 serves as a docking point that recruits sequence-specific transcription factors—including myogenin, ATF4, and Snail—to the polymerase, thereby coupling RNAPII to myogenic differentiation, stress-responsive transactivation, and Snail-driven E-cadherin repression and epithelial-mesenchymal transition [PMID:12207009, PMID:12860379, PMID:25211001]. Its incorporation into the nascent Rpb3 subcomplex is chaperoned by Gpn2 and Rba50 [PMID:29661922].","teleology":[{"year":1989,"claim":"Established that RPB3 is an essential gene whose product is required for the very assembly of RNA polymerase II, defining it as a core structural subunit rather than an accessory factor.","evidence":"Temperature-sensitive rpb3 mutant with immunological RNAPII depletion and mRNA measurement in yeast","pmids":["2685562"],"confidence":"High","gaps":["Did not resolve which RPB3 domains drive assembly","No structural placement within the enzyme"]},{"year":1998,"claim":"Defined the stoichiometry and assembly determinants, showing one RPB3 copy per polymerase and that conserved alpha-homology regions are obligatory for incorporation.","evidence":"Immunoaffinity/nickel-chelate purification of tagged RNAPII plus deletion mutagenesis; two-hybrid mapping of the Rpb2 contact","pmids":["9556554","9738888"],"confidence":"High","gaps":["Rpb2 contact mapped only by two-hybrid, no biochemical confirmation","Order of subunit addition not established"]},{"year":1999,"claim":"Localized assembly-critical mutations to conserved region A and showed RPB11 overexpression suppresses the phenotype, pinpointing the RPB3–RPB11 subassembly as the key step.","evidence":"Random mutagenesis and overexpression suppression analysis in fission yeast","pmids":["10503538"],"confidence":"Medium","gaps":["Single-lab genetic analysis","Structural basis of the RPB3–RPB11 interface not resolved here"]},{"year":2001,"claim":"Functionally separated RPB3's assembly role from its transcription-activation role, mapping assembly to terminal conserved regions and activator-dependent transcription to central eukaryote-specific regions B and C.","evidence":"In vitro GAL4-VP16 activator-dependent transcription with rpb3 mutant extracts; earlier structure-based C92R/A159G mutagenesis defining the conserved activation surface","pmids":["11453250","10673505"],"confidence":"Medium","gaps":["Direct activator contacts at the surface not biochemically demonstrated","Single lab"]},{"year":2005,"claim":"Showed the RPB3–RPB11 heterodimer initiates assembly and that the heterodimerization interface diverged between yeast and humans, refining the molecular basis of polymerase nucleation.","evidence":"In vitro heterodimerization assays with human and yeast truncation mutants","pmids":["15987790"],"confidence":"Medium","gaps":["No structural model of the human interface","Functional consequence of interface divergence untested"]},{"year":2014,"claim":"Extended RPB3's role to factor recruitment, establishing that its eukaryote-specific surface (Sud/N-terminus) directly binds sequence-specific transcription factors to couple RNAPII to differentiation, stress, and EMT programs.","evidence":"Yeast two-hybrid, reciprocal Co-IP, and dominant-negative fragment assays for myogenin, ATF4, and Snail across muscle differentiation and HCC models","pmids":["12207009","12860379","25211001"],"confidence":"Medium","gaps":["Interactions shown largely in single-lab systems","How factor binding at RPB3 mechanistically alters transcription is unresolved"]},{"year":2018,"claim":"Identified dedicated assembly chaperones, showing Gpn2 and Rba50 cooperatively build the Rpb3 subcomplex during RNAPII biogenesis.","evidence":"Co-IP, pulldown, and loss-of-function with RNAPII assembly readout","pmids":["29661922"],"confidence":"Medium","gaps":["Structural mechanism of chaperone-assisted assembly unknown","Single lab"]},{"year":2025,"claim":"Revealed a role for the RPB3 elongation-complex surface in transcription termination, where residue K9 forms a salt-bridge surface regulating Hrp1 binding and NNS-dependent terminator readthrough.","evidence":"Allele-specific mutagenesis and suppressor selection in HRP1 with transcriptome readthrough assays in yeast (preprint)","pmids":["bio_10.1101_2025.05.07.652672"],"confidence":"Medium","gaps":["Preprint, not peer-reviewed","Direct Rpb3-K9–Hrp1 contact not structurally confirmed","Relevance to human termination untested"]},{"year":null,"claim":"How the eukaryote-specific factor-recruitment surface of RPB3 mechanistically integrates with the core enzyme to modulate initiation, elongation, and termination remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of factor-bound RPB3 within the holoenzyme","Human relevance of yeast termination findings untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,1]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[4,7,8,11]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,4]}],"complexes":["RNA polymerase II"],"partners":["POLR2J","POLR2B","SNAIL","MYOGENIN","ATF4","IGFBP3","GPN2","RBA50"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P19387","full_name":"DNA-directed RNA polymerase II subunit RPB3","aliases":["DNA-directed RNA polymerase II 33 kDa polypeptide","RPB33","DNA-directed RNA polymerase II subunit C","RPB31"],"length_aa":275,"mass_kda":31.4,"function":"Core component of RNA polymerase II (Pol II), a DNA-dependent RNA polymerase which synthesizes mRNA precursors and many functional non-coding RNAs using the four ribonucleoside triphosphates as substrates","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/P19387/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/POLR2C","classification":"Common Essential","n_dependent_lines":1208,"n_total_lines":1208,"dependency_fraction":1.0},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000102978","cell_line_id":"CID000698","localizations":[{"compartment":"nucleoplasm","grade":3},{"compartment":"nuclear_punctae","grade":2},{"compartment":"chromatin","grade":1},{"compartment":"cytoplasmic","grade":1}],"interactors":[{"gene":"MED19","stoichiometry":10.0},{"gene":"POLR2A","stoichiometry":10.0},{"gene":"POLR2B","stoichiometry":10.0},{"gene":"POLR2I","stoichiometry":10.0},{"gene":"POLR2K","stoichiometry":10.0},{"gene":"POLR2E","stoichiometry":10.0},{"gene":"POLR2J3;POLR2J;POLR2J2","stoichiometry":10.0},{"gene":"POLR2D","stoichiometry":10.0},{"gene":"GPN2","stoichiometry":10.0},{"gene":"GPN1","stoichiometry":10.0}],"url":"https://opencell.sf.czbiohub.org/target/CID000698","total_profiled":1310},"omim":[{"mim_id":"621545","title":"GPN-LOOP GTPase 3; GPN3","url":"https://www.omim.org/entry/621545"},{"mim_id":"621544","title":"GPN-LOOP GTPase 2; GPN2","url":"https://www.omim.org/entry/621544"},{"mim_id":"609881","title":"RNA POLYMERASE II, SUBUNIT J2; POLR2J2","url":"https://www.omim.org/entry/609881"},{"mim_id":"604150","title":"POLYMERASE II, RNA, SUBUNIT J; POLR2J","url":"https://www.omim.org/entry/604150"},{"mim_id":"180663","title":"POLYMERASE II, RNA, SUBUNIT C; POLR2C","url":"https://www.omim.org/entry/180663"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/POLR2C"},"hgnc":{"alias_symbol":["RPB3"],"prev_symbol":[]},"alphafold":{"accession":"P19387","domains":[{"cath_id":"3.30.1360.10","chopping":"7-40_178-197_218-272","consensus_level":"high","plddt":95.3525,"start":7,"end":272},{"cath_id":"2.170.120.12","chopping":"45-132_140-172","consensus_level":"high","plddt":92.266,"start":45,"end":172}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P19387","model_url":"https://alphafold.ebi.ac.uk/files/AF-P19387-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P19387-F1-predicted_aligned_error_v6.png","plddt_mean":92.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=POLR2C","jax_strain_url":"https://www.jax.org/strain/search?query=POLR2C"},"sequence":{"accession":"P19387","fasta_url":"https://rest.uniprot.org/uniprotkb/P19387.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P19387/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P19387"}},"corpus_meta":[{"pmid":"2685562","id":"PMC_2685562","title":"RNA polymerase II subunit RPB3 is an essential component of the mRNA transcription apparatus.","date":"1989","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/2685562","citation_count":89,"is_preprint":false},{"pmid":"2112240","id":"PMC_2112240","title":"A conjugation-specific gene (cnjC) from Tetrahymena encodes a protein homologous to yeast RNA polymerase subunits (RPB3, RPC40) and similar to a portion of the prokaryotic RNA polymerase alpha subunit (rpoA).","date":"1990","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/2112240","citation_count":51,"is_preprint":false},{"pmid":"16455777","id":"PMC_16455777","title":"Ribonucleic acid polymerase II binding subunit 3 (Rpb3), a potential nuclear target of insulin-like growth factor binding protein-3.","date":"2006","source":"Endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/16455777","citation_count":46,"is_preprint":false},{"pmid":"10673505","id":"PMC_10673505","title":"Activation mutants in yeast RNA polymerase II subunit RPB3 provide evidence for a structurally conserved surface required for activation in eukaryotes and bacteria.","date":"2000","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/10673505","citation_count":37,"is_preprint":false},{"pmid":"12860379","id":"PMC_12860379","title":"Functional interaction of the subunit 3 of RNA polymerase II (RPB3) with transcription factor-4 (ATF4).","date":"2003","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/12860379","citation_count":33,"is_preprint":false},{"pmid":"12207009","id":"PMC_12207009","title":"The alpha-like RNA polymerase II core subunit 3 (RPB3) is involved in tissue-specific transcription and muscle differentiation via interaction with the myogenic factor myogenin.","date":"2002","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/12207009","citation_count":28,"is_preprint":false},{"pmid":"29367954","id":"PMC_29367954","title":"POLR2C Mutations Are Associated With Primary Ovarian Insufficiency in Women.","date":"2017","source":"Journal of the Endocrine Society","url":"https://pubmed.ncbi.nlm.nih.gov/29367954","citation_count":26,"is_preprint":false},{"pmid":"29661922","id":"PMC_29661922","title":"Gpn2 and Rba50 Directly Participate in the Assembly of the Rpb3 Subcomplex in the Biogenesis of RNA Polymerase II.","date":"2018","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/29661922","citation_count":24,"is_preprint":false},{"pmid":"9556554","id":"PMC_9556554","title":"Rpb3, stoichiometry and sequence determinants of the assembly into yeast RNA polymerase II in vivo.","date":"1998","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9556554","citation_count":20,"is_preprint":false},{"pmid":"9738888","id":"PMC_9738888","title":"Mapping of Rpb3 and Rpb5 contact sites on two large subunits, Rpb1 and Rpb2, of the RNA polymerase II from fission yeast.","date":"1998","source":"Molecular & general genetics : MGG","url":"https://pubmed.ncbi.nlm.nih.gov/9738888","citation_count":16,"is_preprint":false},{"pmid":"15987790","id":"PMC_15987790","title":"Distinct regions of RPB11 are required for heterodimerization with RPB3 in human and yeast RNA polymerase II.","date":"2005","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/15987790","citation_count":14,"is_preprint":false},{"pmid":"25211001","id":"PMC_25211001","title":"Rpb3 promotes hepatocellular carcinoma through its N-terminus.","date":"2014","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/25211001","citation_count":10,"is_preprint":false},{"pmid":"10503538","id":"PMC_10503538","title":"Isolation and characterization of temperature-sensitive mutations in the gene (rpb3) for subunit 3 of RNA polymerase II in the fission yeast Schizosaccharomyces pombe.","date":"1999","source":"Molecular & general genetics : MGG","url":"https://pubmed.ncbi.nlm.nih.gov/10503538","citation_count":7,"is_preprint":false},{"pmid":"11453250","id":"PMC_11453250","title":"Functional analysis of RNA polymerase II Rpb3 mutants of the fission yeast Schizosaccharomyces pombe.","date":"2001","source":"Current genetics","url":"https://pubmed.ncbi.nlm.nih.gov/11453250","citation_count":5,"is_preprint":false},{"pmid":"1713667","id":"PMC_1713667","title":"PCR detection of the MspI polymorphism in the human IRBP gene (RPB3).","date":"1991","source":"Nucleic acids 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Sinica","url":"https://pubmed.ncbi.nlm.nih.gov/12552808","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.11.20.622938","title":"RNA Polymerase II subunits overexpressions induce genome instability and deregulate transcription","date":"2024-11-20","source":"bioRxiv","url":"https://doi.org/10.1101/2024.11.20.622938","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.05.07.652672","title":"Substitutions in RNA-binding protein Hrp1 map a potential interaction surface with the yeast RNA polymerase II elongation complex","date":"2025-05-07","source":"bioRxiv","url":"https://doi.org/10.1101/2025.05.07.652672","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.01.02.25319880","title":"Integrating GWAS and Transcriptomic Data Using PrediXcan and Multimodal Deep Learning Reveals Genetic Basis and Drug Repositioning Opportunities for Alzheimer’s Disease","date":"2025-01-02","source":"bioRxiv","url":"https://doi.org/10.1101/2025.01.02.25319880","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11363,"output_tokens":3486,"usd":0.043189,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11103,"output_tokens":2686,"usd":0.061332,"stage2_stop_reason":"end_turn"},"total_usd":0.104521,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1989,\n      \"finding\": \"RPB3 (POLR2C) is an essential single-copy gene encoding the third-largest subunit of RNA polymerase II; a temperature-sensitive rpb3 mutant prevented RNA polymerase II assembly, depleted functional enzyme, and reduced mRNA levels, demonstrating RPB3 is required for RNAPII assembly and mRNA transcription.\",\n      \"method\": \"Temperature-sensitive mutant analysis, immunological depletion of RNAPII, mRNA level measurement\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean loss-of-function with defined molecular phenotype (failure of RNAPII assembly), replicated across multiple assays in a foundational study\",\n      \"pmids\": [\"2685562\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Rpb3 (POLR2C) is present at a stoichiometry of one copy per RNA polymerase II molecule; deletion of either alpha-homology region (amino acids 29–55 or 226–267) abolishes Rpb3 assembly into RNAPII in vivo.\",\n      \"method\": \"Immunoaffinity and nickel-chelate chromatography of His6-tagged and untagged Rpb3-containing RNAPII; deletion mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct biochemical fractionation with mutagenesis, multiple orthogonal approaches establishing stoichiometry and assembly determinants\",\n      \"pmids\": [\"9556554\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Rpb3 (POLR2C) contacts Rpb2 (the beta-homologue) at the conserved region H of Rpb2, as mapped by two-hybrid screening using fission yeast subunit fragments.\",\n      \"method\": \"Yeast two-hybrid system with truncation fragments of Rpb1 and Rpb2\",\n      \"journal\": \"Molecular & general genetics : MGG\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — two-hybrid mapping in multiple fragment combinations, single lab, no in vitro biochemical confirmation\",\n      \"pmids\": [\"9738888\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Temperature-sensitive and cold-sensitive mutations in fission yeast rpb3 map to four conserved regions (A–D); cold-sensitive mutations in region A disrupt RNAPII assembly, and the Ts phenotype is suppressed by overexpression of Rpb11 (the pairing partner of Rpb3), establishing that Rpb3 mutations primarily affect the Rpb3–Rpb11 subassembly.\",\n      \"method\": \"Random mutagenesis, temperature/cold-sensitive growth assays, overexpression suppression analysis\",\n      \"journal\": \"Molecular & general genetics : MGG\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis (suppression by Rpb11 overexpression) with multiple mutant alleles, single lab\",\n      \"pmids\": [\"10503538\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Amino acid substitutions C92R and A159G in yeast RPB3 (POLR2C) specifically impair activator-dependent transcription without affecting basal transcription; homology modeling on the bacterial alpha-NTD crystal structure places residues 92–95 and 159–162 adjacent in 3D space, corresponding to the CAP activator-interaction surface of the bacterial alpha subunit, indicating a structurally conserved activation surface in RPB3.\",\n      \"method\": \"Alanine-scanning and targeted mutagenesis, activator-dependent transcription assays, homology modeling on bacterial alpha-NTD crystal structure\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — structure-based mutagenesis with functional transcription assays, multiple mutant alleles and synthetic enhancement analysis, single lab\",\n      \"pmids\": [\"10673505\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"In vitro functional analysis of fission yeast Rpb3 mutants shows that mutations in terminal conserved regions A and D impair RNAPII assembly, while mutations in central eukaryote-specific regions B and C reduce activator (GAL4-VP16)-dependent transcription without equivalent assembly defects, functionally partitioning assembly from activated transcription roles within Rpb3.\",\n      \"method\": \"In vitro GAL4-VP16 activator-dependent transcription system using S. pombe cell extracts from rpb3 mutants, heat treatment assays\",\n      \"journal\": \"Current genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro transcription assays with defined mutants, functional domain mapping, single lab\",\n      \"pmids\": [\"11453250\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The interaction site of Rpb2 with Rpb3 in fission yeast RNAPII was mapped to the C-terminal region of Rpb2 (amino acids 902–989, encoded by base 2701–2966 of Rpb2 cDNA) using two-hybrid analysis.\",\n      \"method\": \"Yeast two-hybrid system with Rpb2 deletion fragments; beta-galactosidase activity assay\",\n      \"journal\": \"Wei sheng wu xue bao = Acta microbiologica Sinica\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single two-hybrid experiment, no biochemical validation, single lab\",\n      \"pmids\": [\"12552808\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Human RPB3 (POLR2C) interacts with the myogenic transcription factor myogenin via a specific RPB3 region not homologous to the prokaryotic alpha subunit; this interaction involves the basic HLH region of myogenin but not other HLH factors (MyoD, Myf5, MRF4); coimmunoprecipitation confirmed that myogenin contacts the RNAPII complex through RPB3; a dominant-negative RPB3 fragment (Sud) counteracts myogenin transactivation and muscle differentiation.\",\n      \"method\": \"Yeast two-hybrid screening, coimmunoprecipitation, dominant-negative overexpression, muscle differentiation assays\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — reciprocal Co-IP plus yeast two-hybrid plus functional dominant-negative assay, single lab\",\n      \"pmids\": [\"12207009\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Human RPB3 (POLR2C) interacts with the transcription factor ATF4 via an RPB3-specific region (Sud) not homologous to the prokaryotic alpha subunit; RPB3 enhances ATF4 transactivation activity, and the Sud dominant-negative fragment markedly inhibits ATF4 transactivation.\",\n      \"method\": \"Yeast two-hybrid, transactivation assays with dominant-negative RPB3 fragment\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — yeast two-hybrid plus functional transactivation assay, single lab, no reciprocal Co-IP\",\n      \"pmids\": [\"12860379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"RPB3 (POLR2C) forms a heterodimer with RPB11 that initiates RNAPII assembly; in yeast, the C-terminal region of RPB11 is critical for heterodimerization, whereas in humans the conserved N-terminal alpha-motifs dominate the RPB3–RPB11 interface, indicating that the heterodimerization interface has diverged during evolution.\",\n      \"method\": \"In vitro heterodimerization assays comparing human and yeast RPB3/RPB11 truncation mutants\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct biochemical heterodimerization assays with defined mutants comparing two species, single lab\",\n      \"pmids\": [\"15987790\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"IGFBP-3 interacts with Rpb3 (POLR2C) in rat myoblasts; the interaction requires the MBD/NLS epitope of IGFBP-3 (a NLS mutant does not associate with Rpb3); interaction was confirmed by coimmunoprecipitation with specific antisera.\",\n      \"method\": \"Yeast two-hybrid library screening with IGFBP-3 deletion mutants; coimmunoprecipitation with specific antisera\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — reciprocal Co-IP plus two-hybrid domain mapping, single lab\",\n      \"pmids\": [\"16455777\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Rpb3 (POLR2C) binds directly to the transcription factor Snail via its N-terminus; this interaction downregulates E-cadherin and induces epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma cells; the N-terminal fragment of Rpb3 acts as a dominant negative blocking Rpb3–Snail binding and inhibiting proliferation and migration in Rpb3-high-expression HCC cells.\",\n      \"method\": \"Co-IP/direct binding assay, E-cadherin expression assay, dominant-negative N-terminus overexpression, cell proliferation/migration assays, xenograft tumor growth\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — direct binding demonstrated, functional rescue with dominant-negative fragment, multiple cellular assays, single lab\",\n      \"pmids\": [\"25211001\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Gpn2 and Rba50 directly participate in assembly of the Rpb3 (POLR2C) subcomplex during RNAPII biogenesis: Gpn2 interacts with Rpb12, Rba50 interacts with Rpb3, Gpn2 and Rba50 interact with each other, and loss of function of either disrupts Rpb3 subcomplex assembly and subsequent RNAPII biogenesis.\",\n      \"method\": \"Co-IP, pulldown assays, loss-of-function analysis of Gpn2/Rba50 with RNAPII assembly readout\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal interaction mapping plus functional loss-of-function with assembly readout, single lab, multiple orthogonal pulldowns\",\n      \"pmids\": [\"29661922\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"A K9E substitution at residue 9 of Rpb3 (POLR2C) causes readthrough of NNS-dependent terminators and cold-sensitive growth in yeast; genetic suppression by an R317G substitution in the RRM2 of Hrp1 (Nab4/CF1B) indicates that Rpb3-K9 forms a salt-bridge interaction surface that regulates binding of the anti-termination factor Hrp1 to the RNAPII elongation complex.\",\n      \"method\": \"Allele-specific mutagenesis, genome-wide suppressor selection, targeted suppressor selection in HRP1, transcriptome readthrough assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — allele-specific genetic epistasis with suppressor screen, multiple orthogonal alleles tested, preprint not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.05.07.652672\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"POLR2C (RPB3) is the third-largest, alpha-like core subunit of RNA polymerase II that is essential for RNAPII assembly (via conserved alpha-homology regions and heterodimerization with RPB11), participates in a structurally conserved surface required for activator-dependent transcription, directly contacts the beta-homologue Rpb2, recruits specific transcription factors (myogenin, ATF4, Snail) to the polymerase through a eukaryote-specific domain, is incorporated into the Rpb3 subcomplex with the assistance of assembly chaperones Gpn2 and Rba50, and its elongation complex surface regulates binding of the anti-termination factor Hrp1 to control NNS-dependent termination.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"POLR2C (RPB3) is the third-largest, alpha-like core subunit of RNA polymerase II and is essential for assembly of the holoenzyme and for mRNA transcription [#0]. It is present at one copy per polymerase and nucleates RNAPII assembly by heterodimerizing with RPB11 through conserved alpha-homology regions, deletion of which abolishes its incorporation into the enzyme [#1, #9]; within the assembled polymerase it directly contacts the beta-homologue Rpb2 [#2]. Structure-based mutagenesis partitions RPB3 into terminal conserved regions required for assembly and central eukaryote-specific regions required for activator-dependent transcription, the latter mapping to a structurally conserved surface homologous to the bacterial alpha CAP-interaction site [#4, #5]. Through this eukaryote-specific region RPB3 serves as a docking point that recruits sequence-specific transcription factors—including myogenin, ATF4, and Snail—to the polymerase, thereby coupling RNAPII to myogenic differentiation, stress-responsive transactivation, and Snail-driven E-cadherin repression and epithelial-mesenchymal transition [#7, #8, #11]. Its incorporation into the nascent Rpb3 subcomplex is chaperoned by Gpn2 and Rba50 [#12].\",\n  \"teleology\": [\n    {\n      \"year\": 1989,\n      \"claim\": \"Established that RPB3 is an essential gene whose product is required for the very assembly of RNA polymerase II, defining it as a core structural subunit rather than an accessory factor.\",\n      \"evidence\": \"Temperature-sensitive rpb3 mutant with immunological RNAPII depletion and mRNA measurement in yeast\",\n      \"pmids\": [\"2685562\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve which RPB3 domains drive assembly\", \"No structural placement within the enzyme\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Defined the stoichiometry and assembly determinants, showing one RPB3 copy per polymerase and that conserved alpha-homology regions are obligatory for incorporation.\",\n      \"evidence\": \"Immunoaffinity/nickel-chelate purification of tagged RNAPII plus deletion mutagenesis; two-hybrid mapping of the Rpb2 contact\",\n      \"pmids\": [\"9556554\", \"9738888\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Rpb2 contact mapped only by two-hybrid, no biochemical confirmation\", \"Order of subunit addition not established\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Localized assembly-critical mutations to conserved region A and showed RPB11 overexpression suppresses the phenotype, pinpointing the RPB3–RPB11 subassembly as the key step.\",\n      \"evidence\": \"Random mutagenesis and overexpression suppression analysis in fission yeast\",\n      \"pmids\": [\"10503538\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab genetic analysis\", \"Structural basis of the RPB3–RPB11 interface not resolved here\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Functionally separated RPB3's assembly role from its transcription-activation role, mapping assembly to terminal conserved regions and activator-dependent transcription to central eukaryote-specific regions B and C.\",\n      \"evidence\": \"In vitro GAL4-VP16 activator-dependent transcription with rpb3 mutant extracts; earlier structure-based C92R/A159G mutagenesis defining the conserved activation surface\",\n      \"pmids\": [\"11453250\", \"10673505\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct activator contacts at the surface not biochemically demonstrated\", \"Single lab\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Showed the RPB3–RPB11 heterodimer initiates assembly and that the heterodimerization interface diverged between yeast and humans, refining the molecular basis of polymerase nucleation.\",\n      \"evidence\": \"In vitro heterodimerization assays with human and yeast truncation mutants\",\n      \"pmids\": [\"15987790\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of the human interface\", \"Functional consequence of interface divergence untested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Extended RPB3's role to factor recruitment, establishing that its eukaryote-specific surface (Sud/N-terminus) directly binds sequence-specific transcription factors to couple RNAPII to differentiation, stress, and EMT programs.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal Co-IP, and dominant-negative fragment assays for myogenin, ATF4, and Snail across muscle differentiation and HCC models\",\n      \"pmids\": [\"12207009\", \"12860379\", \"25211001\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Interactions shown largely in single-lab systems\", \"How factor binding at RPB3 mechanistically alters transcription is unresolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified dedicated assembly chaperones, showing Gpn2 and Rba50 cooperatively build the Rpb3 subcomplex during RNAPII biogenesis.\",\n      \"evidence\": \"Co-IP, pulldown, and loss-of-function with RNAPII assembly readout\",\n      \"pmids\": [\"29661922\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural mechanism of chaperone-assisted assembly unknown\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed a role for the RPB3 elongation-complex surface in transcription termination, where residue K9 forms a salt-bridge surface regulating Hrp1 binding and NNS-dependent terminator readthrough.\",\n      \"evidence\": \"Allele-specific mutagenesis and suppressor selection in HRP1 with transcriptome readthrough assays in yeast (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.05.07.652672\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not peer-reviewed\", \"Direct Rpb3-K9–Hrp1 contact not structurally confirmed\", \"Relevance to human termination untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the eukaryote-specific factor-recruitment surface of RPB3 mechanistically integrates with the core enzyme to modulate initiation, elongation, and termination remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of factor-bound RPB3 within the holoenzyme\", \"Human relevance of yeast termination findings untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [4, 7, 8, 11]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 4]}\n    ],\n    \"complexes\": [\"RNA polymerase II\"],\n    \"partners\": [\"POLR2J\", \"POLR2B\", \"Snail\", \"myogenin\", \"ATF4\", \"IGFBP3\", \"Gpn2\", \"Rba50\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":5,"faith_total":5,"faith_pct":100.0}}