{"gene":"POLR3F","run_date":"2026-04-28T19:45:44","timeline":{"discoveries":[{"year":1997,"finding":"The yeast C34 subunit (ortholog of human POLR3F/hRPC39) is part of an RNA Pol III-specific subunit that directly interacts with TFIIIB70 (BRF1). Mutations in RPC34 that impair growth also impaired C34 interaction with TFIIIB70. Mutant pol III had normal catalytic activity on non-specific templates but was defective in promoter-dependent transcription initiation, demonstrating that C34 is required for Pol III recruitment to pre-initiation complexes and for open complex formation.","method":"Site-directed mutagenesis of RPC34, purification of mutant Pol III, in vitro transcription assays on poly[d(A-T)] and specific gene templates, interaction assays with TFIIIB70","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with mutagenesis and specific transcription assays; multiple orthogonal methods","pmids":["9312031"],"is_preprint":false},{"year":1997,"finding":"Human POLR3F (hRPC39) is part of a specific subcomplex with hRPC32 and hRPC62 that dissociates from the human Pol III core under partial denaturing conditions. The core Pol III lacking this subcomplex can perform elongation and termination on tailed templates but cannot support promoter-dependent transcription initiation; addition of the recombinant hRPC32-hRPC39-hRPC62 subcomplex restores specific initiation. hRPC39 physically interacts with both hTBP and hTFIIIB90, indicating it mediates Pol III recruitment to the TFIIIB-DNA complex.","method":"Immunopurification of human Pol III, sucrose gradient sedimentation, reconstitution of recombinant subcomplex, in vitro transcription assays, physical interaction assays (co-immunoprecipitation, pulldown)","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 — reconstitution of recombinant subcomplex with functional rescue, multiple orthogonal methods in a single study","pmids":["9171375"],"is_preprint":false},{"year":1999,"finding":"Yeast TFIIIB70 C-terminal conserved regions II and III are required for interaction with the C34 subunit (yeast POLR3F ortholog). Conditional mutations in region II that impaired C34 interaction were co-lethal with rpc34 mutations, establishing a functional genetic interaction. These mutations also impaired assembly of TFIIIB·TFIIIC·DNA complexes and in vitro transcription of the SUP4 tRNA gene, placing C34-TFIIIB70 interaction as essential for pre-initiation complex assembly.","method":"Extensive mutagenesis of TFIIIB70, co-lethality/epistasis analysis with rpc34 mutations, in vitro transcription assays, interaction assays","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1-2 — epistasis (double-mutant co-lethality) combined with in vitro transcription and interaction assays","pmids":["10329159"],"is_preprint":false},{"year":1999,"finding":"The hTFIIIC90 subunit of human TFIIIC physically interacts with hRPC39 (POLR3F) and hRPC62 subunits of the Pol III initiation-specific subcomplex, as shown by immunodepletion and immunoprecipitation. This interaction is proposed to facilitate TFIIIB and RNA Pol III recruitment to the pre-initiation complex.","method":"Immunodepletion, co-immunoprecipitation, in vitro interaction assays","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2-3 — reciprocal co-IP with functional context but single laboratory","pmids":["10523658"],"is_preprint":false},{"year":2001,"finding":"Plakophilin 2, a dual-location desmosomal/nuclear protein, is present in the RNA Pol III holoenzyme (but not the core complex) and co-immunoselects with other Pol III subunits and TFIIIB. Plakophilin 2 binds specifically to RPC155 (the largest Pol III subunit) in vitro, placing it in a nuclear particle that contains the full Pol III holoenzyme including POLR3F-containing subcomplex.","method":"Co-immunoselection, in vitro binding assays, colocalization by immunofluorescence","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2-3 — co-immunoselection with in vitro binding confirmation; POLR3F implicated as part of holoenzyme context","pmids":["11416169"],"is_preprint":false},{"year":2019,"finding":"Endogenous microDNA (small extrachromosomal circular DNAs) associate with RNA polymerase subunits including POLR3F, as identified by affinity purification, suggesting POLR3F participates in microDNA-mediated transcription of small regulatory RNAs independent of canonical promoters.","method":"Affinity purification of microDNA-associated proteins, identification by mass spectrometry","journal":"Nucleic acids research","confidence":"Low","confidence_rationale":"Tier 3 — single pulldown/association experiment; POLR3F identified as associated factor without direct mechanistic follow-up","pmids":["30828735"],"is_preprint":false},{"year":2020,"finding":"In citrus (plant ortholog system), MAF1 binds predominantly to the WH1 domain of the Pol III C34 subunit (CsC34, ortholog of POLR3F), and the MAF1 phosphoregulatory region (loop-3 and α-helix-2) contributes to this interaction. Phosphorylation of MAF1's phosphoregulatory region decreases its affinity for CsC34, leading to Pol III derepression. The C-terminal region of BRF1 (TFIIIB component) competes with MAF1 for CsC34 interaction, revealing a competitive mechanism regulating Pol III activity.","method":"NMR structural analysis of CsMAF1, co-immunoprecipitation, phosphorylation assays with citrus AGC1 kinase, competition binding assays","journal":"The Plant cell","confidence":"Medium","confidence_rationale":"Tier 1-2 — structural data combined with biochemical interaction and phosphorylation assays, but in plant ortholog system","pmids":["32641350"],"is_preprint":false},{"year":2021,"finding":"Cryo-EM structures of human RNA Pol III at 2.8–3.3 Å resolution in unbound and transcribing states reveal that an iron-sulfur cluster tethers the heterotrimer subcomplex (which includes POLR3F/RPC39) to the Pol III core, an element absent in yeast Pol III. The cancer-associated RPC7α isoform binds the polymerase clamp and may interfere with Pol III inhibition by tumor suppressor MAF1. Disease-related mutations are mapped onto the structure.","method":"Cryo-electron microscopy at 2.8–3.3 Å resolution","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 — high-resolution cryo-EM structure with functional interpretation of subunit connectivity and disease mutation mapping","pmids":["33558764"],"is_preprint":false},{"year":2009,"finding":"RNA polymerase III (which contains POLR3F as part of its initiation subcomplex) acts as a cytosolic DNA sensor: it transcribes AT-rich dsDNA (poly(dA-dT)) into 5'-triphosphate RNA, which then activates RIG-I and MAVS to induce IFN-β. Inhibition of RNA Pol III prevents IFN-β induction by cytosolic DNA and by intracellular bacteria (Legionella pneumophila).","method":"Biochemical purification of the responsible enzyme, siRNA knockdown of Pol III, in vitro transcription assays, reporter gene assays for IFN-β induction","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 — biochemical purification and reconstitution combined with loss-of-function validation and pathogen infection model; highly cited foundational study","pmids":["19631370"],"is_preprint":false}],"current_model":"POLR3F (hRPC39) is a subunit of an RNA Pol III-specific heterotrimer (with hRPC32 and hRPC62) that is essential for promoter-dependent transcription initiation but dispensable for elongation and termination; it physically bridges the polymerase to the TFIIIB initiation factor (via interactions with TBP and TFIIIB90/BRF1) and to TFIIIC (via hTFIIIC90), and cryo-EM structures show it is tethered to the Pol III core through an iron-sulfur cluster; MAF1 represses Pol III by competing with BRF1 for binding to the WH1 domain of the C34 subunit, an interaction regulated by phosphorylation, while the intact Pol III holoenzyme also functions as a cytosolic DNA sensor that transcribes AT-rich DNA into 5'-ppp RNA to trigger innate immune signaling via RIG-I."},"narrative":{"teleology":[{"year":1997,"claim":"Establishing that C34/hRPC39 resides in an initiation-specific subcomplex and is required for promoter-dependent transcription but not for catalytic elongation resolved a long-standing question about how Pol III distinguishes specific promoters from random templates.","evidence":"Purification of mutant yeast Pol III and reconstitution of recombinant human hRPC32–hRPC39–hRPC62 subcomplex with functional transcription assays","pmids":["9312031","9171375"],"confidence":"High","gaps":["Structural basis for how the heterotrimer engages the Pol III core was unknown","It was unclear whether hRPC39 contacts TFIIIC directly or only through TFIIIB"]},{"year":1999,"claim":"Mapping the C34–TFIIIB70(BRF1) and C34/hRPC39–hTFIIIC90 interfaces defined POLR3F as a dual bridge connecting the polymerase simultaneously to both basal transcription factors, explaining how Pol III is recruited to pre-initiation complexes.","evidence":"Mutagenesis of TFIIIB70 with co-lethality analysis in yeast; co-immunoprecipitation of hTFIIIC90 with hRPC39 and hRPC62 in human extracts","pmids":["10329159","10523658"],"confidence":"High","gaps":["No atomic-resolution map of the C34–BRF1 or C34–TFIIIC90 contact surfaces","Whether the TFIIIC interaction is maintained throughout initiation or only during recruitment was unresolved"]},{"year":2009,"claim":"Discovery that the Pol III holoenzyme (containing POLR3F) acts as a cytosolic DNA sensor expanded its known biology beyond nuclear gene transcription into innate immunity, showing it transcribes AT-rich DNA into 5′-ppp RNA to activate RIG-I signaling.","evidence":"Biochemical purification of the responsible polymerase, siRNA knockdown of Pol III subunits, IFN-β reporter assays, and Legionella infection model","pmids":["19631370"],"confidence":"High","gaps":["The specific contribution of POLR3F versus other subunits to cytosolic DNA sensing was not dissected","No structure of the Pol III–cytosolic DNA complex is available"]},{"year":2020,"claim":"Demonstration that MAF1 and BRF1 compete for the WH1 domain of C34 (POLR3F ortholog), with MAF1 affinity modulated by phosphorylation, revealed the molecular switch controlling Pol III repression and derepression.","evidence":"NMR structural analysis of citrus MAF1, co-immunoprecipitation, phosphorylation assays, and competition binding assays with plant orthologs","pmids":["32641350"],"confidence":"Medium","gaps":["Demonstrated in a plant ortholog system; direct validation of this competitive mechanism on human POLR3F is lacking","Identity and hierarchy of kinases regulating MAF1 phosphorylation at the C34 interface in mammals remain unresolved"]},{"year":2021,"claim":"High-resolution cryo-EM of human Pol III revealed that an iron–sulfur cluster, absent in yeast, anchors the POLR3F-containing heterotrimer to the polymerase core, providing the first structural explanation for how this subcomplex is physically integrated into the human enzyme.","evidence":"Cryo-EM of human Pol III at 2.8–3.3 Å resolution in unbound and transcribing states","pmids":["33558764"],"confidence":"High","gaps":["No structure captures the heterotrimer engaged with both TFIIIB and TFIIIC simultaneously","Functional role of the iron–sulfur cluster beyond structural tethering has not been tested"]},{"year":null,"claim":"It remains unknown how the POLR3F-containing heterotrimer coordinates promoter opening (open complex formation) at atomic resolution, whether the iron–sulfur cluster serves a redox-sensing or regulatory role, and how the MAF1–C34 competition is regulated in human cells under different nutrient and stress conditions.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural snapshot of the open complex with the heterotrimer engaged on melted DNA","No functional dissection of the Fe–S cluster beyond structural tethering","MAF1–C34 competitive binding not validated biochemically with human proteins"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[0,1,8]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[1,7]},{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[0,1,8]}],"localization":[{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[1,4]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,1,2,7]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[8]}],"complexes":["RNA polymerase III","Pol III initiation-specific heterotrimer (RPC32–RPC39–RPC62)"],"partners":["POLR3B","POLR3C","POLR3E","BRF1","TBP","GTF3C4","MAF1","PKP2"],"other_free_text":[]},"mechanistic_narrative":"POLR3F (RPC6/hRPC39/C34) is a subunit of the RNA polymerase III initiation-specific heterotrimer (with RPC32 and RPC62) that is essential for promoter-dependent transcription initiation but dispensable for elongation and termination. The heterotrimer dissociates from the Pol III core under partial denaturing conditions, and reconstitution of recombinant hRPC32–hRPC39–hRPC62 restores specific initiation; POLR3F directly contacts both TFIIIB components (TBP, BRF1) and TFIIIC (hTFIIIC90), thereby bridging the polymerase to its promoter-recognition machinery [PMID:9171375, PMID:9312031, PMID:10523658]. Cryo-EM structures of human Pol III reveal that an iron–sulfur cluster, absent in yeast, tethers this heterotrimer to the polymerase core, and the repressor MAF1 competes with BRF1 for binding to the WH1 domain of the C34 subunit in a phosphorylation-regulated manner [PMID:33558764, PMID:32641350]. Beyond canonical gene transcription, the intact Pol III holoenzyme containing POLR3F functions as a cytosolic DNA sensor that transcribes AT-rich dsDNA into 5′-triphosphate RNA to trigger RIG-I–dependent innate immune signaling [PMID:19631370]."},"prefetch_data":{"uniprot":{"accession":"Q9H1D9","full_name":"DNA-directed RNA polymerase III subunit RPC6","aliases":["DNA-directed RNA polymerase III subunit F","RNA polymerase III 39 kDa subunit","RPC39"],"length_aa":316,"mass_kda":35.7,"function":"DNA-dependent RNA polymerase catalyzes the transcription of DNA into RNA using the four ribonucleoside triphosphates as substrates (PubMed:20413673, PubMed:21358628, PubMed:33558764, PubMed:34675218). Specific peripheric component of RNA polymerase III (Pol III) which synthesizes small non-coding RNAs including 5S rRNA, snRNAs, tRNAs and miRNAs from at least 500 distinct genomic loci. Part of POLR3C/RPC3-POLR3F/RPC6-POLR3G/RPC7 heterotrimer that coordinates the dynamics of Pol III stalk and clamp modules during the transition from apo to elongation state (PubMed:20413673, PubMed:33558764, PubMed:33558766). Pol III plays a key role in sensing and limiting infection by intracellular bacteria and DNA viruses, including varicella zoster virus. Acts as a nuclear and cytosolic DNA sensor detecting AT-rich DNA, involved in innate immune response. Can sense non-self dsDNA that serves as template for transcription into dsRNA. The non-self RNA polymerase III transcripts, such as Epstein-Barr virus-encoded RNAs (EBERs) induce type I interferon and NF-kappa-B through the RIG-I pathway (PubMed:19609254, PubMed:19631370, PubMed:30211253). Preferentially binds double-stranded DNA (dsDNA) (PubMed:21358628)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9H1D9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/POLR3F","classification":"Common Essential","n_dependent_lines":1201,"n_total_lines":1208,"dependency_fraction":0.9942052980132451},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000132664","cell_line_id":"CID000714","localizations":[{"compartment":"nuclear_punctae","grade":3},{"compartment":"nucleoplasm","grade":2}],"interactors":[{"gene":"POLR1C","stoichiometry":10.0},{"gene":"POLR2E","stoichiometry":10.0},{"gene":"POLR2F","stoichiometry":10.0},{"gene":"POLR2H","stoichiometry":10.0},{"gene":"POLR2K","stoichiometry":10.0},{"gene":"POLR3A","stoichiometry":10.0},{"gene":"POLR3B","stoichiometry":10.0},{"gene":"POLR3C","stoichiometry":10.0},{"gene":"POLR3D","stoichiometry":10.0},{"gene":"POLR3E","stoichiometry":10.0}],"url":"https://opencell.sf.czbiohub.org/target/CID000714","total_profiled":1310},"omim":[{"mim_id":"619872","title":"IMMUNODEFICIENCY 101 (VARICELLA ZOSTER VIRUS-SPECIFIC); IMD101","url":"https://www.omim.org/entry/619872"},{"mim_id":"617456","title":"POLYMERASE III, RNA, SUBUNIT G; POLR3G","url":"https://www.omim.org/entry/617456"},{"mim_id":"617455","title":"POLYMERASE III, RNA, SUBUNIT F; POLR3F","url":"https://www.omim.org/entry/617455"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/POLR3F"},"hgnc":{"alias_symbol":["RPC39","RPC6","C34"],"prev_symbol":[]},"alphafold":{"accession":"Q9H1D9","domains":[{"cath_id":"1.10.10.10","chopping":"14-78","consensus_level":"high","plddt":81.8894,"start":14,"end":78},{"cath_id":"1.10.10.10","chopping":"86-163","consensus_level":"high","plddt":87.2083,"start":86,"end":163},{"cath_id":"1.10.10","chopping":"171-271","consensus_level":"high","plddt":91.0456,"start":171,"end":271},{"cath_id":"-","chopping":"280-314","consensus_level":"medium","plddt":90.6011,"start":280,"end":314}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H1D9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H1D9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H1D9-F1-predicted_aligned_error_v6.png","plddt_mean":84.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=POLR3F","jax_strain_url":"https://www.jax.org/strain/search?query=POLR3F"},"sequence":{"accession":"Q9H1D9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9H1D9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9H1D9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H1D9"}},"corpus_meta":[{"pmid":"15640162","id":"PMC_15640162","title":"Different 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2.","date":"2001","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/11416169","citation_count":98,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"23455924","id":"PMC_23455924","title":"A Y2H-seq approach defines the human protein methyltransferase interactome.","date":"2013","source":"Nature methods","url":"https://pubmed.ncbi.nlm.nih.gov/23455924","citation_count":93,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"10523658","id":"PMC_10523658","title":"The TFIIIC90 subunit of TFIIIC interacts with multiple components of the RNA polymerase III machinery and contains a histone-specific acetyltransferase activity.","date":"1999","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/10523658","citation_count":86,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"33558764","id":"PMC_33558764","title":"Cryo-EM structures of human RNA polymerase III in its unbound and transcribing states.","date":"2021","source":"Nature structural & molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/33558764","citation_count":80,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":22267,"output_tokens":1016,"usd":0.041021},"stage2":{"model":"claude-opus-4-6","input_tokens":4215,"output_tokens":1246,"usd":0.078338},"total_usd":0.316626,"stage1_batch_id":"msgbatch_01SUR8NDzVrNmyszWyJeCct7","stage2_batch_id":"msgbatch_01G7Yfutko3cDWzejrwH5AMx","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":33631,"output_tokens":3162,"usd":0.074162},"round2_rules_fired":"R3","round2_stage2":{"model":"claude-opus-4-6","input_tokens":6219,"output_tokens":2039,"usd":0.123105}},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1997,\n      \"finding\": \"The C34 subunit (POLR3F ortholog in yeast, encoded by RPC34) of RNA Polymerase III directly interacts with TFIIIB70 (BRF) and is required for pol III recruitment to pre-initiation complexes; specific mutations in C34 impair either pol III recruitment (reduced affinity for pre-initiation complexes) or open complex formation during transcription initiation, while leaving catalytic activity on non-specific templates unaffected.\",\n      \"method\": \"Site-directed mutagenesis of RPC34, purification of mutant pol III, in vitro transcription assays on poly[d(A-T)] and specific pol III gene templates, complementation by increased pol III concentration\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with mutagenesis and purified components, multiple orthogonal assays\",\n      \"pmids\": [\"9312031\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"The C-terminal extension of TFIIIB70 (BRF) contains conserved regions that mediate direct interaction with the C34 subunit of pol III; mutations in TFIIIB70 region II and III that abolish C34 interaction are co-lethal with rpc34 mutations, and these same mutations impair assembly of TFIIIB·TFIIIC·DNA pre-initiation complexes and in vitro transcription.\",\n      \"method\": \"Extensive mutagenesis of TFIIIB70, genetic co-lethality (synthetic lethality) with rpc34 alleles, in vitro transcription of SUP4 tRNA gene, pre-initiation complex assembly assays\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — genetic epistasis combined with in vitro biochemical assays and mutagenesis, replicated the C34–TFIIIB70 interaction of PMID:9312031\",\n      \"pmids\": [\"10329159\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In the plant ortholog system, the Pol III C34 subunit (CsC34) interacts predominantly through its WH1 domain with the MAF1 phosphoregulatory region (loop-3 and α-helix-2); phosphorylation of MAF1 (including Ser45 by a citrus AGC1 kinase) decreases MAF1 affinity for C34, relieving Pol III repression. Additionally, the C-terminal region of TFIIIB component BRF1 competes with MAF1 for C34 binding.\",\n      \"method\": \"NMR structural data of CsMAF1, pull-down and binding assays between CsMAF1 and CsC34 WH1 domain, phosphorylation assays with AGC1 kinase, competition assays with BRF1 C-terminal region\",\n      \"journal\": \"The Plant cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — structural data plus direct binding assays and kinase phosphorylation assay; plant ortholog context limits direct translation to human POLR3F but mechanism is conserved\",\n      \"pmids\": [\"32641350\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"POLR3F (yeast C34/RPC34) is a subunit of RNA Polymerase III that directly contacts the TFIIIB component TFIIIB70/BRF through its C-terminal region, functioning as a key determinant for pol III recruitment to pre-initiation complexes and for enabling open complex formation during transcription initiation; its interaction with MAF1 (mediated via the C34 WH1 domain) is regulated by phosphorylation and competes with BRF binding, providing a mechanism for transcriptional repression.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper first, then extract discoveries only from KEEP papers.\n\n**Classification:**\n\nMost papers in the retrieved corpus (papers 1-42) are about \"C34\" as an HIV fusion inhibitor peptide, TLR4 inhibitor, chemical compound, bacterial strain, etc. — NOT about POLR3F (the RNA polymerase III subunit F, also known as RPC39/hRPC39/C39).\n\nFrom the gene2pubmed-curated papers, I need to identify which describe POLR3F (human RNA Pol III subunit, hRPC39):\n- Paper [25]: KEEP — directly describes hRPC39 (POLR3F) as part of the hRPC32-hRPC39-hRPC62 subcomplex\n- Paper [30]: KEEP — cryo-EM of human RNA Pol III, resolves subunit structure including the heterotrimer\n- Paper [27]: KEEP — describes nuclear particles with RNA Pol III subunits including RPC39 context\n- Paper [29]: KEEP — hTFIIIC90 interacts with hRPC39 and hRPC62\n- Paper [24]: KEEP — microDNA associate with POLR3F\n- Papers [1-12, 14-23, 26, 28]: Large-scale interactome/proteome studies — include POLR3F only as a node, no mechanistic findings specific to POLR3F\n\nFrom retrieved papers:\n- Paper [2]: KEEP (yeast C34 = POLR3F ortholog, RPC34/yeast C34 subunit of Pol III) — directly about the yeast ortholog\n- Paper [7]: KEEP (yeast TFIIIB70 interaction with C34 subunit = yeast POLR3F ortholog)\n- Paper [29] in retrieved: KEEP (plant MAF1/C34 interaction — but this is plant, SYMBOL COLLISION — EXCLUDE)\n\nRetrieved paper [29] (PMID:32641350): Plant CsC34 — this is a plant ortholog context. The cellular context (citrus, plant Pol III) is consistent with the mammalian gene function. KEEP as ortholog.\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1997,\n      \"finding\": \"The yeast C34 subunit (ortholog of human POLR3F/hRPC39) is part of an RNA Pol III-specific subunit that directly interacts with TFIIIB70 (BRF1). Mutations in RPC34 that impair growth also impaired C34 interaction with TFIIIB70. Mutant pol III had normal catalytic activity on non-specific templates but was defective in promoter-dependent transcription initiation, demonstrating that C34 is required for Pol III recruitment to pre-initiation complexes and for open complex formation.\",\n      \"method\": \"Site-directed mutagenesis of RPC34, purification of mutant Pol III, in vitro transcription assays on poly[d(A-T)] and specific gene templates, interaction assays with TFIIIB70\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with mutagenesis and specific transcription assays; multiple orthogonal methods\",\n      \"pmids\": [\"9312031\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Human POLR3F (hRPC39) is part of a specific subcomplex with hRPC32 and hRPC62 that dissociates from the human Pol III core under partial denaturing conditions. The core Pol III lacking this subcomplex can perform elongation and termination on tailed templates but cannot support promoter-dependent transcription initiation; addition of the recombinant hRPC32-hRPC39-hRPC62 subcomplex restores specific initiation. hRPC39 physically interacts with both hTBP and hTFIIIB90, indicating it mediates Pol III recruitment to the TFIIIB-DNA complex.\",\n      \"method\": \"Immunopurification of human Pol III, sucrose gradient sedimentation, reconstitution of recombinant subcomplex, in vitro transcription assays, physical interaction assays (co-immunoprecipitation, pulldown)\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstitution of recombinant subcomplex with functional rescue, multiple orthogonal methods in a single study\",\n      \"pmids\": [\"9171375\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Yeast TFIIIB70 C-terminal conserved regions II and III are required for interaction with the C34 subunit (yeast POLR3F ortholog). Conditional mutations in region II that impaired C34 interaction were co-lethal with rpc34 mutations, establishing a functional genetic interaction. These mutations also impaired assembly of TFIIIB·TFIIIC·DNA complexes and in vitro transcription of the SUP4 tRNA gene, placing C34-TFIIIB70 interaction as essential for pre-initiation complex assembly.\",\n      \"method\": \"Extensive mutagenesis of TFIIIB70, co-lethality/epistasis analysis with rpc34 mutations, in vitro transcription assays, interaction assays\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — epistasis (double-mutant co-lethality) combined with in vitro transcription and interaction assays\",\n      \"pmids\": [\"10329159\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"The hTFIIIC90 subunit of human TFIIIC physically interacts with hRPC39 (POLR3F) and hRPC62 subunits of the Pol III initiation-specific subcomplex, as shown by immunodepletion and immunoprecipitation. This interaction is proposed to facilitate TFIIIB and RNA Pol III recruitment to the pre-initiation complex.\",\n      \"method\": \"Immunodepletion, co-immunoprecipitation, in vitro interaction assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — reciprocal co-IP with functional context but single laboratory\",\n      \"pmids\": [\"10523658\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Plakophilin 2, a dual-location desmosomal/nuclear protein, is present in the RNA Pol III holoenzyme (but not the core complex) and co-immunoselects with other Pol III subunits and TFIIIB. Plakophilin 2 binds specifically to RPC155 (the largest Pol III subunit) in vitro, placing it in a nuclear particle that contains the full Pol III holoenzyme including POLR3F-containing subcomplex.\",\n      \"method\": \"Co-immunoselection, in vitro binding assays, colocalization by immunofluorescence\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — co-immunoselection with in vitro binding confirmation; POLR3F implicated as part of holoenzyme context\",\n      \"pmids\": [\"11416169\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Endogenous microDNA (small extrachromosomal circular DNAs) associate with RNA polymerase subunits including POLR3F, as identified by affinity purification, suggesting POLR3F participates in microDNA-mediated transcription of small regulatory RNAs independent of canonical promoters.\",\n      \"method\": \"Affinity purification of microDNA-associated proteins, identification by mass spectrometry\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single pulldown/association experiment; POLR3F identified as associated factor without direct mechanistic follow-up\",\n      \"pmids\": [\"30828735\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In citrus (plant ortholog system), MAF1 binds predominantly to the WH1 domain of the Pol III C34 subunit (CsC34, ortholog of POLR3F), and the MAF1 phosphoregulatory region (loop-3 and α-helix-2) contributes to this interaction. Phosphorylation of MAF1's phosphoregulatory region decreases its affinity for CsC34, leading to Pol III derepression. The C-terminal region of BRF1 (TFIIIB component) competes with MAF1 for CsC34 interaction, revealing a competitive mechanism regulating Pol III activity.\",\n      \"method\": \"NMR structural analysis of CsMAF1, co-immunoprecipitation, phosphorylation assays with citrus AGC1 kinase, competition binding assays\",\n      \"journal\": \"The Plant cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — structural data combined with biochemical interaction and phosphorylation assays, but in plant ortholog system\",\n      \"pmids\": [\"32641350\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cryo-EM structures of human RNA Pol III at 2.8–3.3 Å resolution in unbound and transcribing states reveal that an iron-sulfur cluster tethers the heterotrimer subcomplex (which includes POLR3F/RPC39) to the Pol III core, an element absent in yeast Pol III. The cancer-associated RPC7α isoform binds the polymerase clamp and may interfere with Pol III inhibition by tumor suppressor MAF1. Disease-related mutations are mapped onto the structure.\",\n      \"method\": \"Cryo-electron microscopy at 2.8–3.3 Å resolution\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution cryo-EM structure with functional interpretation of subunit connectivity and disease mutation mapping\",\n      \"pmids\": [\"33558764\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"RNA polymerase III (which contains POLR3F as part of its initiation subcomplex) acts as a cytosolic DNA sensor: it transcribes AT-rich dsDNA (poly(dA-dT)) into 5'-triphosphate RNA, which then activates RIG-I and MAVS to induce IFN-β. Inhibition of RNA Pol III prevents IFN-β induction by cytosolic DNA and by intracellular bacteria (Legionella pneumophila).\",\n      \"method\": \"Biochemical purification of the responsible enzyme, siRNA knockdown of Pol III, in vitro transcription assays, reporter gene assays for IFN-β induction\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — biochemical purification and reconstitution combined with loss-of-function validation and pathogen infection model; highly cited foundational study\",\n      \"pmids\": [\"19631370\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"POLR3F (hRPC39) is a subunit of an RNA Pol III-specific heterotrimer (with hRPC32 and hRPC62) that is essential for promoter-dependent transcription initiation but dispensable for elongation and termination; it physically bridges the polymerase to the TFIIIB initiation factor (via interactions with TBP and TFIIIB90/BRF1) and to TFIIIC (via hTFIIIC90), and cryo-EM structures show it is tethered to the Pol III core through an iron-sulfur cluster; MAF1 represses Pol III by competing with BRF1 for binding to the WH1 domain of the C34 subunit, an interaction regulated by phosphorylation, while the intact Pol III holoenzyme also functions as a cytosolic DNA sensor that transcribes AT-rich DNA into 5'-ppp RNA to trigger innate immune signaling via RIG-I.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"POLR3F (yeast C34/RPC34) is a dedicated subunit of RNA Polymerase III that mediates enzyme recruitment to pre-initiation complexes through direct interaction between its C-terminal region and the TFIIIB component BRF (TFIIIB70); specific mutations in this subunit selectively impair either pol III recruitment or open complex formation during transcription initiation without affecting catalytic activity on non-specific templates [PMID:9312031, PMID:10329159]. The WH1 domain of POLR3F binds the transcriptional repressor MAF1, and phosphorylation of MAF1 reduces this interaction, while BRF1 competes with MAF1 for POLR3F binding, thereby coupling nutrient/stress signaling to pol III transcriptional output [PMID:32641350].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Establishing that the C34 subunit is not merely structural but functions as the key pol III surface for pre-initiation complex engagement resolved how pol III is specifically recruited to promoters: C34 directly contacts BRF/TFIIIB70, and distinct mutations separately abolish recruitment versus open complex formation.\",\n      \"evidence\": \"Site-directed mutagenesis of yeast RPC34, purified mutant pol III tested in vitro on specific and non-specific templates\",\n      \"pmids\": [\"9312031\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of the C34–BRF interface was not resolved at atomic resolution\",\n        \"Whether C34 contributes to promoter specificity beyond recruitment was not addressed\",\n        \"Role in vivo under physiological regulation was not tested\"\n      ]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Mapping the reciprocal BRF side of the interaction showed that conserved C-terminal regions II and III of TFIIIB70 are essential for C34 binding, and genetic co-lethality with rpc34 alleles confirmed the functional importance of this interface in vivo.\",\n      \"evidence\": \"Extensive mutagenesis of yeast TFIIIB70, synthetic lethality with rpc34 alleles, in vitro transcription and pre-initiation complex assembly assays\",\n      \"pmids\": [\"10329159\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No high-resolution structure of the C34–BRF complex was available\",\n        \"How the interaction is regulated remained unknown\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identifying the C34 WH1 domain as the binding surface for the repressor MAF1 — and showing that MAF1 phosphorylation weakens this contact while BRF1 competes for the same site — provided a mechanistic explanation for how pol III transcription is toggled between active and repressed states.\",\n      \"evidence\": \"NMR structure of plant MAF1, pull-down/binding assays between CsMAF1 and CsC34 WH1 domain, AGC1 kinase phosphorylation assays, BRF1 competition assays\",\n      \"pmids\": [\"32641350\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Demonstrated in plant orthologs; direct validation with human POLR3F and MAF1 is needed\",\n        \"Whether additional pol III subunits modulate the MAF1–C34 competition is unknown\",\n        \"Structural basis of the ternary competition at the WH1 domain at atomic resolution is unresolved\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A high-resolution structure of the human POLR3F–BRF1–MAF1 interface, and direct demonstration that the competitive binding mechanism operates in mammalian cells under physiological signaling, remain open questions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No atomic-resolution structure of the human POLR3F–BRF1 or POLR3F–MAF1 complex\",\n        \"In vivo validation of the phosphorylation-dependent competition model in mammalian systems is lacking\",\n        \"Whether POLR3F mutations contribute to human POLR3-related disorders has not been directly tested\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 1, 2]}\n    ],\n    \"complexes\": [\"RNA Polymerase III\"],\n    \"partners\": [\"BRF1\", \"MAF1\"],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"POLR3F (RPC6/hRPC39/C34) is a subunit of the RNA polymerase III initiation-specific heterotrimer (with RPC32 and RPC62) that is essential for promoter-dependent transcription initiation but dispensable for elongation and termination. The heterotrimer dissociates from the Pol III core under partial denaturing conditions, and reconstitution of recombinant hRPC32–hRPC39–hRPC62 restores specific initiation; POLR3F directly contacts both TFIIIB components (TBP, BRF1) and TFIIIC (hTFIIIC90), thereby bridging the polymerase to its promoter-recognition machinery [PMID:9171375, PMID:9312031, PMID:10523658]. Cryo-EM structures of human Pol III reveal that an iron–sulfur cluster, absent in yeast, tethers this heterotrimer to the polymerase core, and the repressor MAF1 competes with BRF1 for binding to the WH1 domain of the C34 subunit in a phosphorylation-regulated manner [PMID:33558764, PMID:32641350]. Beyond canonical gene transcription, the intact Pol III holoenzyme containing POLR3F functions as a cytosolic DNA sensor that transcribes AT-rich dsDNA into 5′-triphosphate RNA to trigger RIG-I–dependent innate immune signaling [PMID:19631370].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Establishing that C34/hRPC39 resides in an initiation-specific subcomplex and is required for promoter-dependent transcription but not for catalytic elongation resolved a long-standing question about how Pol III distinguishes specific promoters from random templates.\",\n      \"evidence\": \"Purification of mutant yeast Pol III and reconstitution of recombinant human hRPC32–hRPC39–hRPC62 subcomplex with functional transcription assays\",\n      \"pmids\": [\"9312031\", \"9171375\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis for how the heterotrimer engages the Pol III core was unknown\",\n        \"It was unclear whether hRPC39 contacts TFIIIC directly or only through TFIIIB\"\n      ]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Mapping the C34–TFIIIB70(BRF1) and C34/hRPC39–hTFIIIC90 interfaces defined POLR3F as a dual bridge connecting the polymerase simultaneously to both basal transcription factors, explaining how Pol III is recruited to pre-initiation complexes.\",\n      \"evidence\": \"Mutagenesis of TFIIIB70 with co-lethality analysis in yeast; co-immunoprecipitation of hTFIIIC90 with hRPC39 and hRPC62 in human extracts\",\n      \"pmids\": [\"10329159\", \"10523658\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No atomic-resolution map of the C34–BRF1 or C34–TFIIIC90 contact surfaces\",\n        \"Whether the TFIIIC interaction is maintained throughout initiation or only during recruitment was unresolved\"\n      ]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Discovery that the Pol III holoenzyme (containing POLR3F) acts as a cytosolic DNA sensor expanded its known biology beyond nuclear gene transcription into innate immunity, showing it transcribes AT-rich DNA into 5′-ppp RNA to activate RIG-I signaling.\",\n      \"evidence\": \"Biochemical purification of the responsible polymerase, siRNA knockdown of Pol III subunits, IFN-β reporter assays, and Legionella infection model\",\n      \"pmids\": [\"19631370\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The specific contribution of POLR3F versus other subunits to cytosolic DNA sensing was not dissected\",\n        \"No structure of the Pol III–cytosolic DNA complex is available\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstration that MAF1 and BRF1 compete for the WH1 domain of C34 (POLR3F ortholog), with MAF1 affinity modulated by phosphorylation, revealed the molecular switch controlling Pol III repression and derepression.\",\n      \"evidence\": \"NMR structural analysis of citrus MAF1, co-immunoprecipitation, phosphorylation assays, and competition binding assays with plant orthologs\",\n      \"pmids\": [\"32641350\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Demonstrated in a plant ortholog system; direct validation of this competitive mechanism on human POLR3F is lacking\",\n        \"Identity and hierarchy of kinases regulating MAF1 phosphorylation at the C34 interface in mammals remain unresolved\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"High-resolution cryo-EM of human Pol III revealed that an iron–sulfur cluster, absent in yeast, anchors the POLR3F-containing heterotrimer to the polymerase core, providing the first structural explanation for how this subcomplex is physically integrated into the human enzyme.\",\n      \"evidence\": \"Cryo-EM of human Pol III at 2.8–3.3 Å resolution in unbound and transcribing states\",\n      \"pmids\": [\"33558764\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No structure captures the heterotrimer engaged with both TFIIIB and TFIIIC simultaneously\",\n        \"Functional role of the iron–sulfur cluster beyond structural tethering has not been tested\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown how the POLR3F-containing heterotrimer coordinates promoter opening (open complex formation) at atomic resolution, whether the iron–sulfur cluster serves a redox-sensing or regulatory role, and how the MAF1–C34 competition is regulated in human cells under different nutrient and stress conditions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No structural snapshot of the open complex with the heterotrimer engaged on melted DNA\",\n        \"No functional dissection of the Fe–S cluster beyond structural tethering\",\n        \"MAF1–C34 competitive binding not validated biochemically with human proteins\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [0, 1, 8]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [1, 7]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [0, 1, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [1, 4]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 1, 2, 7]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"complexes\": [\n      \"RNA polymerase III\",\n      \"Pol III initiation-specific heterotrimer (RPC32–RPC39–RPC62)\"\n    ],\n    \"partners\": [\n      \"POLR3B\",\n      \"POLR3C\",\n      \"POLR3E\",\n      \"BRF1\",\n      \"TBP\",\n      \"GTF3C4\",\n      \"MAF1\",\n      \"PKP2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}