{"gene":"RPAP3","run_date":"2026-06-10T06:43:37","timeline":{"discoveries":[{"year":2018,"finding":"The conserved C-terminal domain of RPAP3 directly binds RUVBL1/RUVBL2 hexamers, and this interaction is essential for human R2TP complex assembly; a 3.6 Å cryo-EM structure reveals direct interaction of the RPAP3 C-terminal domain with the ATPase domain of RUVBL2. The mobile TPR domains of RPAP3 map to the opposite face of the RUVBL ring, associating with PIH1D1, which mediates client protein recruitment. RPAP3 thus spans both faces of a single RUVBL ring, providing an extended scaffold that recruits clients and provides a flexible tether for HSP90.","method":"Cryo-EM (3.6 Å structure), biochemical studies, domain mapping","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure with biochemical validation, multiple orthogonal methods in a single rigorous study","pmids":["29662061"],"is_preprint":false},{"year":2018,"finding":"The RPAP3 C-terminal domain directly binds RUVBL1/RUVBL2 hexamers, and an RPAP3-like protein SPAG1 can bind PIH1D2 and RUVBL1/2 to form an R2TP-like complex termed R2SP. R2SP is enriched in testis, required for liprin-α2 expression, and facilitates assembly of liprin-α2 complexes, demonstrating a quaternary protein folding function.","method":"Structural analysis (RPAP3-C domain), systematic interaction analyses (co-IP, pulldown), functional knockdown assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — structure resolved plus interaction mapping plus functional rescue, multiple orthogonal methods","pmids":["29844425"],"is_preprint":false},{"year":2010,"finding":"In yeast, the Pih1-Tah1 (RPAP3 ortholog) heterodimer binds Hsp90 with similar affinity and stoichiometry as Tah1 alone, but the Pih1-Tah1 complex inhibits Hsp90 ATPase activity, antagonizing the stimulatory effect of Tah1 alone. Pih1 alone is unstable and degraded from its N terminus, but forms a stable heterodimer with Tah1.","method":"Analytical ultracentrifugation, microcalorimetry, noncovalent mass spectrometry, ATPase activity assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple biophysical methods, in vitro ATPase assay, replicated in same study with orthogonal approaches","pmids":["20663878"],"is_preprint":false},{"year":2008,"finding":"Yeast Tah1 (RPAP3 ortholog) specifically binds Hsp90 (yeast Hsp90, human Hsp90α and Hsp90β) via the conserved MEEVD motif at the Hsp90 C-terminus, but does not bind yeast Hsp70 (Ssa1). Ligand discrimination is achieved by favourable binding of the methionine in MEEVD and positive discrimination against the first valine in the Hsp70 VEEVD motif. Tah1 can affect Hsp90 ATPase activity.","method":"Binding assays (chaperone specificity), mutagenesis, ATPase activity assay","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding assays with mutagenesis and functional ATPase readout, single lab","pmids":["18412542"],"is_preprint":false},{"year":2011,"finding":"NMR structure of yeast Tah1 (RPAP3 ortholog) reveals two TPR motifs, a C helix, and an unstructured C-terminal region. Tah1 binds Hsp90 via the EEVD C-terminal residues through a positively charged channel with a two-carboxylate clamp. Tah1 binds the C-terminus of Pih1 through its C helix and unstructured region, stabilizing Pih1; the C-terminus of Pih1 destabilizes the protein in vitro and in vivo, and Tah1 binding allows stable complex formation.","method":"NMR structure determination, binding assays, mutagenesis, in vitro/in vivo stability assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structure with mutagenesis and in vitro/in vivo functional validation, multiple orthogonal methods","pmids":["22179618"],"is_preprint":false},{"year":2013,"finding":"High-resolution NMR solution structures of Tah1 free and in complex with the Hsp90 C-terminal peptide show that the TPR fold is similar in free and bound forms. The capping helix is essential for recognition of the Hsp90 EMEEVD motif, with Lys79, Arg83 (carboxylate clamp), and Tyr82 (π/S-CH3 interaction with Hsp90 M705) identified as key contacts. The Tah1 C-terminal unfolded region is essential for recruitment of the Pih1 C-terminal domain and folds upon binding.","method":"NMR structure (free and peptide-bound), mutagenesis, binding validation","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution NMR structure with mutagenesis and experimental validation of key residues","pmids":["24012479"],"is_preprint":false},{"year":2014,"finding":"Drosophila Spag (RPAP3 ortholog) binds Drosophila orthologs of R2TP components and Hsp90, and also interacts with and stimulates the chaperone activity of Hsp70. Spag is necessary for stabilization of snoRNP core proteins, TOR signaling activity, and likely the assembly of RNA polymerase II. Interaction with both Hsp70 and Hsp90 suggests R2TP accompanies clients from Hsp70 to Hsp90 for assembly into macromolecular complexes.","method":"Co-immunoprecipitation, RNAi knockdown/null mutants, chaperone activity assay, functional phenotype analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus functional knockouts with multiple phenotypic readouts, single lab","pmids":["24394412"],"is_preprint":false},{"year":2009,"finding":"RPAP3 interacts with Reptin (RUVBL2), a component of chromatin-remodeling complexes, as shown by immunoprecipitation and confocal microscopy. Overexpression of RPAP3 increases cell death after UV irradiation; RNAi-mediated knockdown of RPAP3 improves HeLa cell survival after UV damage and attenuates H2AX phosphorylation, while depletion of Reptin reduces survival and facilitates H2AX phosphorylation.","method":"Affinity purification/mass spectrometry, co-immunoprecipitation, confocal microscopy, RNAi knockdown, UV survival/H2AX phosphorylation assays","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — co-IP plus functional RNAi with phosphorylation readout, single lab with multiple methods","pmids":["19180575"],"is_preprint":false},{"year":2012,"finding":"RPAP3 isoform 1 (but not isoform 2) interacts with PIH1D1 and is required for PIH1D1 protein stability; RPAP3 isoform 1 knockdown downregulates PIH1D1 protein without affecting PIH1D1 mRNA. RPAP3 isoform 2 potentiates doxorubicin-induced cell death, suggesting a dominant negative effect on R2TP complex survival function.","method":"Co-immunoprecipitation, siRNA knockdown, mRNA/protein level analysis, cell death assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP plus knockdown with protein stability readout, single lab, multiple methods","pmids":["23159623"],"is_preprint":false},{"year":2010,"finding":"RPAP3 binds NEMO (NF-κB essential modulator) and inhibits NEMO ubiquitination, thereby impairing NF-κB pathway activation and enhancing doxorubicin-induced cell death in breast cancer cells.","method":"Co-immunoprecipitation, ubiquitination assay, cell death assay, NF-κB pathway analysis","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single co-IP plus functional assay, limited mechanistic follow-up","pmids":["21184742"],"is_preprint":false},{"year":2022,"finding":"The R2TP component RPAP3-TPR1 domain directly binds the TRBP-dsRBD3 domain; a 1.5 Å crystal structure identifies key residues involved. Binding of TRBP to RPAP3 and binding of TRBP to Dicer are mutually exclusive. AGO1/2, TRBP, and Dicer are sensitive to HSP90 inhibition; TRBP sensitivity is increased in the absence of RPAP3, suggesting RPAP3 modulates miRNA pathway via TRBP sequestration.","method":"Crystal structure (1.5 Å), co-immunoprecipitation, competitive binding assay, HSP90 inhibition, RPAP3 knockdown","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with key residue identification plus competitive binding and functional validation","pmids":["35150569"],"is_preprint":false},{"year":2022,"finding":"RPAP3 is phosphorylated at Ser116, Ser119, and Ser121 by kinase CK2 in HEK293 cells; the unphosphorylated form of RPAP3 binds ribosomal preassembly complexes. Phospho-null mutations at these sites enhance RPAP3 binding to proteins involved in ribosome biogenesis in AP-MS experiments, and pharmacological inhibition of CK2 similarly enhances this binding. PAQosome subunit silencing interferes with ribosomal assembly factor interactome.","method":"AP-MS (affinity purification-mass spectrometry), in vitro phosphorylation assays, phospho-null mutagenesis, CK2 inhibitor treatment, siRNA knockdown","journal":"Journal of proteome research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assay plus AP-MS plus mutagenesis, single lab, multiple orthogonal methods","pmids":["35129352"],"is_preprint":false},{"year":2021,"finding":"Yeast Tah1 (RPAP3 ortholog) interacts with Hsp70 (Ssa1) and with Ure2, improves Ure2 solubility in [URE3] strains, and inhibits Ure2 fibrillation in vitro. The N-terminal TPR domain of Tah1 is indispensable for [URE3] curing. Tah1 overproduction cures [URE3] prion and tah1 deletion increases de novo [URE3] appearance, placing Tah1 in prion propagation control.","method":"Genetic overexpression/deletion, in vitro fibrillation assay, co-immunoprecipitation, prion frequency assays, domain mutagenesis","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro fibrillation assay plus genetic epistasis plus co-IP, single lab, multiple methods","pmids":["33811921"],"is_preprint":false},{"year":2015,"finding":"Drosophila Spag (RPAP3 ortholog) antagonizes DBT (CKIε/δ ortholog) C-terminal autophosphorylation in S2 cells, as shown by Spag overexpression reducing DBT electrophoretic mobility shifts indicative of autophosphorylation.","method":"S2 cell overexpression, electrophoretic mobility shift assay, mass spectrometry of phosphorylation sites","journal":"Molecular and cellular biology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, overexpression assay with indirect phosphorylation readout, no direct in vitro reconstitution","pmids":["25939385"],"is_preprint":false},{"year":2025,"finding":"CCDC103 binds RUVBL1-RUVBL2 via a RUVBL2-binding domain (RBD) homologous to the RPAP3 C-terminal domain, but unlike RPAP3, CCDC103 lacks PIH1D1-binding motif and TPR domains. The cryo-EM structure of the RUVBL1-RUVBL2-CCDC103 complex (R2C) at 3.2 Å shows three CCDC103 molecules bound to a hetero-hexameric RUVBL1-RUVBL2 ring, with the flexible N-terminal region of CCDC103 regulating RUVBL1-RUVBL2 oligomerisation. This defines the structural distinction between R2C and R2TP (which uses RPAP3).","method":"Cryo-EM structure (3.2 Å), biochemical characterization","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — cryo-EM structure is high quality but preprint, single study; directly informs RPAP3 domain function by structural comparison","pmids":["bio_10.1101_2025.09.11.675549"],"is_preprint":true}],"current_model":"RPAP3 (yeast ortholog Tah1) functions as the central scaffold of the R2TP co-chaperone complex, bridging HSP90 (via its TPR domains, which bind the HSP90 C-terminal EEVD motif) to the RUVBL1/RUVBL2 AAA-ATPase ring (via its conserved C-terminal domain that directly contacts RUVBL2), while its middle region associates with PIH1D1 for client protein recruitment; additionally, RPAP3 interacts with Hsp70, modulates Hsp90 ATPase activity, binds TRBP to regulate the miRNA pathway, is phosphorylated by CK2 to control ribosome biogenesis client binding, and has been implicated in UV-induced DNA damage responses via H2AX phosphorylation regulation and in NF-κB pathway modulation through NEMO binding."},"narrative":{"mechanistic_narrative":"RPAP3 is the central scaffolding subunit of the R2TP co-chaperone complex, coupling the HSP90 chaperone machinery to the RUVBL1/RUVBL2 AAA-ATPase ring to drive the assembly of macromolecular complexes [PMID:29662061]. Its conserved C-terminal domain directly contacts the ATPase domain of RUVBL2 to nucleate R2TP assembly, while its mobile TPR domains project to the opposite face of the RUVBL ring and associate with PIH1D1, which mediates client recruitment; RPAP3 thus spans both faces of a single RUVBL hexamer as an extended scaffold and flexible tether for HSP90 [PMID:29662061]. The TPR domains engage HSP90 specifically through its C-terminal EEVD/MEEVD motif via a two-carboxylate clamp, discriminating against the HSP70 VEEVD motif, and this engagement modulates HSP90 ATPase activity [PMID:18412542, PMID:24012479]. Through PIH1D1 binding the same protein region stabilizes the otherwise labile PIH1D1 partner [PMID:22179618, PMID:23159623]. This architecture supports quaternary protein folding of diverse clients, exemplified by the testis-enriched RPAP3-like paralog SPAG1, which forms an analogous R2SP complex required for liprin-α2 assembly [PMID:29844425], and by client classes including snoRNP core proteins and RNA polymerase II in the orthologous machinery [PMID:24394412]. RPAP3 function is regulated by CK2 phosphorylation at Ser116/Ser119/Ser121, where the unphosphorylated form preferentially binds ribosomal preassembly complexes, linking RPAP3 to ribosome biogenesis [PMID:35129352]. Beyond core chaperone scaffolding, RPAP3 sequesters the miRNA-pathway factor TRBP through a TPR1–dsRBD3 interaction that is mutually exclusive with TRBP–Dicer binding, thereby modulating miRNA biogenesis [PMID:35150569], and it participates in the UV-induced DNA damage response together with RUVBL2 via regulation of H2AX phosphorylation [PMID:19180575].","teleology":[{"year":2008,"claim":"Established the molecular basis by which RPAP3/Tah1 selects HSP90 over HSP70, defining the chaperone connection at the heart of R2TP.","evidence":"Binding assays and mutagenesis with yeast/human HSP90 and HSP70 plus ATPase readout in yeast Tah1","pmids":["18412542"],"confidence":"Medium","gaps":["Functional consequence of HSP90 ATPase modulation for client folding not resolved","Did not address how Tah1 couples to the RUVBL ring"]},{"year":2010,"claim":"Showed that heterodimerization with PIH1 reshapes RPAP3/Tah1's effect on HSP90, converting ATPase stimulation into inhibition and revealing complex-dependent regulation.","evidence":"Analytical ultracentrifugation, microcalorimetry, native mass spectrometry, and ATPase assays on the yeast Pih1-Tah1 heterodimer","pmids":["20663878"],"confidence":"High","gaps":["Physiological clients whose folding depends on this ATPase tuning not identified","Human RPAP3-PIH1D1 ATPase regulation not directly tested"]},{"year":2009,"claim":"Linked RPAP3 to the UV DNA damage response by tying it to RUVBL2 and H2AX phosphorylation, extending its role beyond chaperone scaffolding.","evidence":"AP-MS, co-IP, confocal microscopy, and RNAi with UV survival/H2AX readouts in HeLa cells","pmids":["19180575"],"confidence":"Medium","gaps":["Direct molecular mechanism connecting RPAP3 to H2AX kinases unresolved","Whether the effect requires intact R2TP not tested"]},{"year":2011,"claim":"Provided the first atomic view of RPAP3/Tah1's TPR architecture and showed how its disordered C-terminal region stabilizes the labile PIH1 partner.","evidence":"NMR structure of yeast Tah1 with binding and in vitro/in vivo stability assays","pmids":["22179618"],"confidence":"High","gaps":["Structure of the full R2TP assembly not yet defined","Client engagement mechanism not addressed"]},{"year":2013,"claim":"Defined the residue-level carboxylate clamp and capping helix that recognize the HSP90 MEEVD motif, pinpointing the recognition determinants.","evidence":"High-resolution NMR structures of free and HSP90-peptide-bound Tah1 with mutagenesis","pmids":["24012479"],"confidence":"High","gaps":["How HSP90 tethering is coupled to RUVBL/PIH1 client handoff not shown"]},{"year":2014,"claim":"Demonstrated that the RPAP3 ortholog bridges HSP70 and HSP90 and is required for assembly of snoRNPs, TOR signaling, and RNA polymerase II, framing R2TP as a client-handoff machine.","evidence":"Co-IP, RNAi/null mutants, and chaperone activity assays in Drosophila Spag","pmids":["24394412"],"confidence":"Medium","gaps":["Direct human RPAP3-HSP70 interaction not structurally defined","Order of HSP70-to-HSP90 client transfer inferred, not demonstrated"]},{"year":2018,"claim":"Resolved how RPAP3 organizes the human R2TP complex, showing its C-terminal domain docks on RUVBL2 while TPR/PIH1D1 face the opposite ring side, and that a paralog (SPAG1) builds an analogous R2SP complex for tissue-specific client folding.","evidence":"Cryo-EM (3.6 Å), structural and biochemical mapping, and functional knockdown across two studies","pmids":["29662061","29844425"],"confidence":"High","gaps":["Conformational dynamics during active client folding not captured","Full set of human R2TP clients incomplete"]},{"year":2022,"claim":"Identified two regulatory layers: CK2 phosphorylation gating RPAP3's binding to ribosome biogenesis factors, and a structurally defined RPAP3-TPR1–TRBP interaction that competes with Dicer to modulate miRNA biogenesis.","evidence":"AP-MS, in vitro kinase and phospho-null mutagenesis; plus 1.5 Å crystal structure with competitive binding and HSP90 inhibition","pmids":["35129352","35150569"],"confidence":"High","gaps":["Physiological signals triggering CK2 control of RPAP3 not defined","Cellular consequences of TRBP sequestration for specific miRNAs not mapped"]},{"year":null,"claim":"How RPAP3's distinct activities—R2TP scaffolding, DNA damage response, miRNA regulation, and NF-κB modulation—are integrated or selected within a single cell remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No unifying model linking chaperone scaffolding to signaling and stress roles","Whether non-R2TP functions require RUVBL/PIH1D1 or HSP90 is untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,4,8]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,3,10]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[7]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,6]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[10,11]}],"complexes":["R2TP complex","R2SP complex","PAQosome"],"partners":["RUVBL2","RUVBL1","PIH1D1","HSP90","TRBP","NEMO","HSP70"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9H6T3","full_name":"RNA polymerase II-associated protein 3","aliases":[],"length_aa":665,"mass_kda":75.7,"function":"Forms an interface between the RNA polymerase II enzyme and chaperone/scaffolding protein, suggesting that it is required to connect RNA polymerase II to regulators of protein complex formation","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q9H6T3/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/RPAP3","classification":"Common Essential","n_dependent_lines":1005,"n_total_lines":1208,"dependency_fraction":0.831953642384106},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"POLR2H","stoichiometry":10.0},{"gene":"POLR2B","stoichiometry":4.0},{"gene":"POLR2E","stoichiometry":4.0},{"gene":"RPAP2","stoichiometry":4.0},{"gene":"FKBP5","stoichiometry":0.2},{"gene":"FKBP8","stoichiometry":0.2},{"gene":"HSP90AA1","stoichiometry":0.2},{"gene":"HSP90AB1","stoichiometry":0.2},{"gene":"NOP58","stoichiometry":0.2},{"gene":"PFDN6","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/RPAP3","total_profiled":1310},"omim":[{"mim_id":"611479","title":"GPN-LOOP GTPase 1; GPN1","url":"https://www.omim.org/entry/611479"},{"mim_id":"611477","title":"RNA POLYMERASE II-ASSOCIATED PROTEIN 3; RPAP3","url":"https://www.omim.org/entry/611477"},{"mim_id":"611476","title":"RNA POLYMERASE II-ASSOCIATED PROTEIN 2; RPAP2","url":"https://www.omim.org/entry/611476"},{"mim_id":"611475","title":"RNA POLYMERASE II-ASSOCIATED PROTEIN 1; RPAP1","url":"https://www.omim.org/entry/611475"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Cytosol","reliability":"Enhanced"},{"location":"Primary cilium","reliability":"Additional"},{"location":"Primary cilium tip","reliability":"Additional"},{"location":"Basal body","reliability":"Additional"},{"location":"Acrosome","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RPAP3"},"hgnc":{"alias_symbol":["FLJ21908","spag","hSpagh","Tah1"],"prev_symbol":[]},"alphafold":{"accession":"Q9H6T3","domains":[{"cath_id":"1.25.40.10","chopping":"131-234","consensus_level":"high","plddt":95.573,"start":131,"end":234},{"cath_id":"1.25.40.10","chopping":"281-406","consensus_level":"high","plddt":93.111,"start":281,"end":406},{"cath_id":"-","chopping":"550-664","consensus_level":"high","plddt":88.3728,"start":550,"end":664}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H6T3","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H6T3-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H6T3-F1-predicted_aligned_error_v6.png","plddt_mean":74.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RPAP3","jax_strain_url":"https://www.jax.org/strain/search?query=RPAP3"},"sequence":{"accession":"Q9H6T3","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9H6T3.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9H6T3/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H6T3"}},"corpus_meta":[{"pmid":"29844425","id":"PMC_29844425","title":"The RPAP3-Cterminal domain identifies R2TP-like quaternary chaperones.","date":"2018","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/29844425","citation_count":63,"is_preprint":false},{"pmid":"29662061","id":"PMC_29662061","title":"RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex.","date":"2018","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/29662061","citation_count":61,"is_preprint":false},{"pmid":"20663878","id":"PMC_20663878","title":"The Pih1-Tah1 cochaperone complex inhibits Hsp90 molecular chaperone ATPase activity.","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20663878","citation_count":61,"is_preprint":false},{"pmid":"24012479","id":"PMC_24012479","title":"High-resolution structural analysis shows how Tah1 tethers Hsp90 to the R2TP complex.","date":"2013","source":"Structure (London, England : 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peptide.","date":"2006","source":"Molecular and cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16411022","citation_count":29,"is_preprint":false},{"pmid":"7517029","id":"PMC_7517029","title":"Theileria annulata sporozoite surface antigen (SPAG-1) contains neutralizing determinants in the C terminus.","date":"1994","source":"Parasite immunology","url":"https://pubmed.ncbi.nlm.nih.gov/7517029","citation_count":26,"is_preprint":false},{"pmid":"16870344","id":"PMC_16870344","title":"Vaccination of calves with an attenuated cell line of Theileria annulata and the sporozoite antigen SPAG-1 produces a synergistic effect.","date":"2006","source":"Veterinary parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/16870344","citation_count":24,"is_preprint":false},{"pmid":"19180575","id":"PMC_19180575","title":"RPAP3 interacts with Reptin to regulate UV-induced phosphorylation of H2AX and DNA damage.","date":"2009","source":"Journal of cellular 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Complexes.","date":"2020","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/32384603","citation_count":15,"is_preprint":false},{"pmid":"35150569","id":"PMC_35150569","title":"The interaction between RPAP3 and TRBP reveals a possible involvement of the HSP90/R2TP chaperone complex in the regulation of miRNA activity.","date":"2022","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/35150569","citation_count":12,"is_preprint":false},{"pmid":"21184742","id":"PMC_21184742","title":"RPAP3 enhances cytotoxicity of doxorubicin by impairing NF-kappa B pathway.","date":"2010","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/21184742","citation_count":9,"is_preprint":false},{"pmid":"33811921","id":"PMC_33811921","title":"Tah1, A Key Component of R2TP Complex that Regulates Assembly of snoRNP, is Involved in De Novo Generation and Maintenance of Yeast Prion [URE3].","date":"2021","source":"Journal of molecular 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research","url":"https://pubmed.ncbi.nlm.nih.gov/35129352","citation_count":8,"is_preprint":false},{"pmid":"24668569","id":"PMC_24668569","title":"(1)H, (15)N and (13)C resonance assignments of the two TPR domains from the human RPAP3 protein.","date":"2014","source":"Biomolecular NMR assignments","url":"https://pubmed.ncbi.nlm.nih.gov/24668569","citation_count":8,"is_preprint":false},{"pmid":"24777385","id":"PMC_24777385","title":"Transcriptional regulation of the rat sperm-associated antigen 11e (Spag 11e) gene during endotoxin challenge.","date":"2014","source":"Molecular genetics and genomics : MGG","url":"https://pubmed.ncbi.nlm.nih.gov/24777385","citation_count":6,"is_preprint":false},{"pmid":"25939385","id":"PMC_25939385","title":"Drosophila DBT Autophosphorylation of Its C-Terminal Domain Antagonized by SPAG and Involved in UV-Induced Apoptosis.","date":"2015","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/25939385","citation_count":3,"is_preprint":false},{"pmid":"41424038","id":"PMC_41424038","title":"PIH1D1 and RPAP3, Components of the PAQosome: Emerging Roles in Cellular Physiology.","date":"2025","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/41424038","citation_count":2,"is_preprint":false},{"pmid":"41966271","id":"PMC_41966271","title":"RPAP3: Structural evolution, chaperone networks, and disease implications of a transcriptional Co-chaperone.","date":"2026","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/41966271","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.09.11.675549","title":"CCDC103-mediated assembly of the R2C complex links RUVBL1-RUVBL2 to Primary Ciliary Dyskinesia","date":"2025-09-13","source":"bioRxiv","url":"https://doi.org/10.1101/2025.09.11.675549","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":14821,"output_tokens":4289,"usd":0.054399,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12006,"output_tokens":3127,"usd":0.069102,"stage2_stop_reason":"end_turn"},"total_usd":0.123501,"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\": 2018,\n      \"finding\": \"The conserved C-terminal domain of RPAP3 directly binds RUVBL1/RUVBL2 hexamers, and this interaction is essential for human R2TP complex assembly; a 3.6 Å cryo-EM structure reveals direct interaction of the RPAP3 C-terminal domain with the ATPase domain of RUVBL2. The mobile TPR domains of RPAP3 map to the opposite face of the RUVBL ring, associating with PIH1D1, which mediates client protein recruitment. RPAP3 thus spans both faces of a single RUVBL ring, providing an extended scaffold that recruits clients and provides a flexible tether for HSP90.\",\n      \"method\": \"Cryo-EM (3.6 Å structure), biochemical studies, domain mapping\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure with biochemical validation, multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"29662061\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The RPAP3 C-terminal domain directly binds RUVBL1/RUVBL2 hexamers, and an RPAP3-like protein SPAG1 can bind PIH1D2 and RUVBL1/2 to form an R2TP-like complex termed R2SP. R2SP is enriched in testis, required for liprin-α2 expression, and facilitates assembly of liprin-α2 complexes, demonstrating a quaternary protein folding function.\",\n      \"method\": \"Structural analysis (RPAP3-C domain), systematic interaction analyses (co-IP, pulldown), functional knockdown assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — structure resolved plus interaction mapping plus functional rescue, multiple orthogonal methods\",\n      \"pmids\": [\"29844425\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"In yeast, the Pih1-Tah1 (RPAP3 ortholog) heterodimer binds Hsp90 with similar affinity and stoichiometry as Tah1 alone, but the Pih1-Tah1 complex inhibits Hsp90 ATPase activity, antagonizing the stimulatory effect of Tah1 alone. Pih1 alone is unstable and degraded from its N terminus, but forms a stable heterodimer with Tah1.\",\n      \"method\": \"Analytical ultracentrifugation, microcalorimetry, noncovalent mass spectrometry, ATPase activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple biophysical methods, in vitro ATPase assay, replicated in same study with orthogonal approaches\",\n      \"pmids\": [\"20663878\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Yeast Tah1 (RPAP3 ortholog) specifically binds Hsp90 (yeast Hsp90, human Hsp90α and Hsp90β) via the conserved MEEVD motif at the Hsp90 C-terminus, but does not bind yeast Hsp70 (Ssa1). Ligand discrimination is achieved by favourable binding of the methionine in MEEVD and positive discrimination against the first valine in the Hsp70 VEEVD motif. Tah1 can affect Hsp90 ATPase activity.\",\n      \"method\": \"Binding assays (chaperone specificity), mutagenesis, ATPase activity assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding assays with mutagenesis and functional ATPase readout, single lab\",\n      \"pmids\": [\"18412542\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"NMR structure of yeast Tah1 (RPAP3 ortholog) reveals two TPR motifs, a C helix, and an unstructured C-terminal region. Tah1 binds Hsp90 via the EEVD C-terminal residues through a positively charged channel with a two-carboxylate clamp. Tah1 binds the C-terminus of Pih1 through its C helix and unstructured region, stabilizing Pih1; the C-terminus of Pih1 destabilizes the protein in vitro and in vivo, and Tah1 binding allows stable complex formation.\",\n      \"method\": \"NMR structure determination, binding assays, mutagenesis, in vitro/in vivo stability assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structure with mutagenesis and in vitro/in vivo functional validation, multiple orthogonal methods\",\n      \"pmids\": [\"22179618\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"High-resolution NMR solution structures of Tah1 free and in complex with the Hsp90 C-terminal peptide show that the TPR fold is similar in free and bound forms. The capping helix is essential for recognition of the Hsp90 EMEEVD motif, with Lys79, Arg83 (carboxylate clamp), and Tyr82 (π/S-CH3 interaction with Hsp90 M705) identified as key contacts. The Tah1 C-terminal unfolded region is essential for recruitment of the Pih1 C-terminal domain and folds upon binding.\",\n      \"method\": \"NMR structure (free and peptide-bound), mutagenesis, binding validation\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution NMR structure with mutagenesis and experimental validation of key residues\",\n      \"pmids\": [\"24012479\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Drosophila Spag (RPAP3 ortholog) binds Drosophila orthologs of R2TP components and Hsp90, and also interacts with and stimulates the chaperone activity of Hsp70. Spag is necessary for stabilization of snoRNP core proteins, TOR signaling activity, and likely the assembly of RNA polymerase II. Interaction with both Hsp70 and Hsp90 suggests R2TP accompanies clients from Hsp70 to Hsp90 for assembly into macromolecular complexes.\",\n      \"method\": \"Co-immunoprecipitation, RNAi knockdown/null mutants, chaperone activity assay, functional phenotype analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus functional knockouts with multiple phenotypic readouts, single lab\",\n      \"pmids\": [\"24394412\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"RPAP3 interacts with Reptin (RUVBL2), a component of chromatin-remodeling complexes, as shown by immunoprecipitation and confocal microscopy. Overexpression of RPAP3 increases cell death after UV irradiation; RNAi-mediated knockdown of RPAP3 improves HeLa cell survival after UV damage and attenuates H2AX phosphorylation, while depletion of Reptin reduces survival and facilitates H2AX phosphorylation.\",\n      \"method\": \"Affinity purification/mass spectrometry, co-immunoprecipitation, confocal microscopy, RNAi knockdown, UV survival/H2AX phosphorylation assays\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — co-IP plus functional RNAi with phosphorylation readout, single lab with multiple methods\",\n      \"pmids\": [\"19180575\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"RPAP3 isoform 1 (but not isoform 2) interacts with PIH1D1 and is required for PIH1D1 protein stability; RPAP3 isoform 1 knockdown downregulates PIH1D1 protein without affecting PIH1D1 mRNA. RPAP3 isoform 2 potentiates doxorubicin-induced cell death, suggesting a dominant negative effect on R2TP complex survival function.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, mRNA/protein level analysis, cell death assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP plus knockdown with protein stability readout, single lab, multiple methods\",\n      \"pmids\": [\"23159623\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"RPAP3 binds NEMO (NF-κB essential modulator) and inhibits NEMO ubiquitination, thereby impairing NF-κB pathway activation and enhancing doxorubicin-induced cell death in breast cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, cell death assay, NF-κB pathway analysis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single co-IP plus functional assay, limited mechanistic follow-up\",\n      \"pmids\": [\"21184742\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The R2TP component RPAP3-TPR1 domain directly binds the TRBP-dsRBD3 domain; a 1.5 Å crystal structure identifies key residues involved. Binding of TRBP to RPAP3 and binding of TRBP to Dicer are mutually exclusive. AGO1/2, TRBP, and Dicer are sensitive to HSP90 inhibition; TRBP sensitivity is increased in the absence of RPAP3, suggesting RPAP3 modulates miRNA pathway via TRBP sequestration.\",\n      \"method\": \"Crystal structure (1.5 Å), co-immunoprecipitation, competitive binding assay, HSP90 inhibition, RPAP3 knockdown\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with key residue identification plus competitive binding and functional validation\",\n      \"pmids\": [\"35150569\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RPAP3 is phosphorylated at Ser116, Ser119, and Ser121 by kinase CK2 in HEK293 cells; the unphosphorylated form of RPAP3 binds ribosomal preassembly complexes. Phospho-null mutations at these sites enhance RPAP3 binding to proteins involved in ribosome biogenesis in AP-MS experiments, and pharmacological inhibition of CK2 similarly enhances this binding. PAQosome subunit silencing interferes with ribosomal assembly factor interactome.\",\n      \"method\": \"AP-MS (affinity purification-mass spectrometry), in vitro phosphorylation assays, phospho-null mutagenesis, CK2 inhibitor treatment, siRNA knockdown\",\n      \"journal\": \"Journal of proteome research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase assay plus AP-MS plus mutagenesis, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"35129352\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Yeast Tah1 (RPAP3 ortholog) interacts with Hsp70 (Ssa1) and with Ure2, improves Ure2 solubility in [URE3] strains, and inhibits Ure2 fibrillation in vitro. The N-terminal TPR domain of Tah1 is indispensable for [URE3] curing. Tah1 overproduction cures [URE3] prion and tah1 deletion increases de novo [URE3] appearance, placing Tah1 in prion propagation control.\",\n      \"method\": \"Genetic overexpression/deletion, in vitro fibrillation assay, co-immunoprecipitation, prion frequency assays, domain mutagenesis\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro fibrillation assay plus genetic epistasis plus co-IP, single lab, multiple methods\",\n      \"pmids\": [\"33811921\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Drosophila Spag (RPAP3 ortholog) antagonizes DBT (CKIε/δ ortholog) C-terminal autophosphorylation in S2 cells, as shown by Spag overexpression reducing DBT electrophoretic mobility shifts indicative of autophosphorylation.\",\n      \"method\": \"S2 cell overexpression, electrophoretic mobility shift assay, mass spectrometry of phosphorylation sites\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, overexpression assay with indirect phosphorylation readout, no direct in vitro reconstitution\",\n      \"pmids\": [\"25939385\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CCDC103 binds RUVBL1-RUVBL2 via a RUVBL2-binding domain (RBD) homologous to the RPAP3 C-terminal domain, but unlike RPAP3, CCDC103 lacks PIH1D1-binding motif and TPR domains. The cryo-EM structure of the RUVBL1-RUVBL2-CCDC103 complex (R2C) at 3.2 Å shows three CCDC103 molecules bound to a hetero-hexameric RUVBL1-RUVBL2 ring, with the flexible N-terminal region of CCDC103 regulating RUVBL1-RUVBL2 oligomerisation. This defines the structural distinction between R2C and R2TP (which uses RPAP3).\",\n      \"method\": \"Cryo-EM structure (3.2 Å), biochemical characterization\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — cryo-EM structure is high quality but preprint, single study; directly informs RPAP3 domain function by structural comparison\",\n      \"pmids\": [\"bio_10.1101_2025.09.11.675549\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"RPAP3 (yeast ortholog Tah1) functions as the central scaffold of the R2TP co-chaperone complex, bridging HSP90 (via its TPR domains, which bind the HSP90 C-terminal EEVD motif) to the RUVBL1/RUVBL2 AAA-ATPase ring (via its conserved C-terminal domain that directly contacts RUVBL2), while its middle region associates with PIH1D1 for client protein recruitment; additionally, RPAP3 interacts with Hsp70, modulates Hsp90 ATPase activity, binds TRBP to regulate the miRNA pathway, is phosphorylated by CK2 to control ribosome biogenesis client binding, and has been implicated in UV-induced DNA damage responses via H2AX phosphorylation regulation and in NF-κB pathway modulation through NEMO binding.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RPAP3 is the central scaffolding subunit of the R2TP co-chaperone complex, coupling the HSP90 chaperone machinery to the RUVBL1/RUVBL2 AAA-ATPase ring to drive the assembly of macromolecular complexes [#0]. Its conserved C-terminal domain directly contacts the ATPase domain of RUVBL2 to nucleate R2TP assembly, while its mobile TPR domains project to the opposite face of the RUVBL ring and associate with PIH1D1, which mediates client recruitment; RPAP3 thus spans both faces of a single RUVBL hexamer as an extended scaffold and flexible tether for HSP90 [#0]. The TPR domains engage HSP90 specifically through its C-terminal EEVD/MEEVD motif via a two-carboxylate clamp, discriminating against the HSP70 VEEVD motif, and this engagement modulates HSP90 ATPase activity [#3, #5]. Through PIH1D1 binding the same protein region stabilizes the otherwise labile PIH1D1 partner [#4, #8]. This architecture supports quaternary protein folding of diverse clients, exemplified by the testis-enriched RPAP3-like paralog SPAG1, which forms an analogous R2SP complex required for liprin-\\u03b12 assembly [#1], and by client classes including snoRNP core proteins and RNA polymerase II in the orthologous machinery [#6]. RPAP3 function is regulated by CK2 phosphorylation at Ser116/Ser119/Ser121, where the unphosphorylated form preferentially binds ribosomal preassembly complexes, linking RPAP3 to ribosome biogenesis [#11]. Beyond core chaperone scaffolding, RPAP3 sequesters the miRNA-pathway factor TRBP through a TPR1\\u2013dsRBD3 interaction that is mutually exclusive with TRBP\\u2013Dicer binding, thereby modulating miRNA biogenesis [#10], and it participates in the UV-induced DNA damage response together with RUVBL2 via regulation of H2AX phosphorylation [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Established the molecular basis by which RPAP3/Tah1 selects HSP90 over HSP70, defining the chaperone connection at the heart of R2TP.\",\n      \"evidence\": \"Binding assays and mutagenesis with yeast/human HSP90 and HSP70 plus ATPase readout in yeast Tah1\",\n      \"pmids\": [\"18412542\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of HSP90 ATPase modulation for client folding not resolved\", \"Did not address how Tah1 couples to the RUVBL ring\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showed that heterodimerization with PIH1 reshapes RPAP3/Tah1's effect on HSP90, converting ATPase stimulation into inhibition and revealing complex-dependent regulation.\",\n      \"evidence\": \"Analytical ultracentrifugation, microcalorimetry, native mass spectrometry, and ATPase assays on the yeast Pih1-Tah1 heterodimer\",\n      \"pmids\": [\"20663878\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological clients whose folding depends on this ATPase tuning not identified\", \"Human RPAP3-PIH1D1 ATPase regulation not directly tested\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Linked RPAP3 to the UV DNA damage response by tying it to RUVBL2 and H2AX phosphorylation, extending its role beyond chaperone scaffolding.\",\n      \"evidence\": \"AP-MS, co-IP, confocal microscopy, and RNAi with UV survival/H2AX readouts in HeLa cells\",\n      \"pmids\": [\"19180575\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular mechanism connecting RPAP3 to H2AX kinases unresolved\", \"Whether the effect requires intact R2TP not tested\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Provided the first atomic view of RPAP3/Tah1's TPR architecture and showed how its disordered C-terminal region stabilizes the labile PIH1 partner.\",\n      \"evidence\": \"NMR structure of yeast Tah1 with binding and in vitro/in vivo stability assays\",\n      \"pmids\": [\"22179618\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of the full R2TP assembly not yet defined\", \"Client engagement mechanism not addressed\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined the residue-level carboxylate clamp and capping helix that recognize the HSP90 MEEVD motif, pinpointing the recognition determinants.\",\n      \"evidence\": \"High-resolution NMR structures of free and HSP90-peptide-bound Tah1 with mutagenesis\",\n      \"pmids\": [\"24012479\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How HSP90 tethering is coupled to RUVBL/PIH1 client handoff not shown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrated that the RPAP3 ortholog bridges HSP70 and HSP90 and is required for assembly of snoRNPs, TOR signaling, and RNA polymerase II, framing R2TP as a client-handoff machine.\",\n      \"evidence\": \"Co-IP, RNAi/null mutants, and chaperone activity assays in Drosophila Spag\",\n      \"pmids\": [\"24394412\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct human RPAP3-HSP70 interaction not structurally defined\", \"Order of HSP70-to-HSP90 client transfer inferred, not demonstrated\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Resolved how RPAP3 organizes the human R2TP complex, showing its C-terminal domain docks on RUVBL2 while TPR/PIH1D1 face the opposite ring side, and that a paralog (SPAG1) builds an analogous R2SP complex for tissue-specific client folding.\",\n      \"evidence\": \"Cryo-EM (3.6 \\u00c5), structural and biochemical mapping, and functional knockdown across two studies\",\n      \"pmids\": [\"29662061\", \"29844425\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conformational dynamics during active client folding not captured\", \"Full set of human R2TP clients incomplete\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified two regulatory layers: CK2 phosphorylation gating RPAP3's binding to ribosome biogenesis factors, and a structurally defined RPAP3-TPR1\\u2013TRBP interaction that competes with Dicer to modulate miRNA biogenesis.\",\n      \"evidence\": \"AP-MS, in vitro kinase and phospho-null mutagenesis; plus 1.5 \\u00c5 crystal structure with competitive binding and HSP90 inhibition\",\n      \"pmids\": [\"35129352\", \"35150569\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological signals triggering CK2 control of RPAP3 not defined\", \"Cellular consequences of TRBP sequestration for specific miRNAs not mapped\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RPAP3's distinct activities\\u2014R2TP scaffolding, DNA damage response, miRNA regulation, and NF-\\u03baB modulation\\u2014are integrated or selected within a single cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unifying model linking chaperone scaffolding to signaling and stress roles\", \"Whether non-R2TP functions require RUVBL/PIH1D1 or HSP90 is untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 4, 8]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 3, 10]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 6]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [10, 11]}\n    ],\n    \"complexes\": [\"R2TP complex\", \"R2SP complex\", \"PAQosome\"],\n    \"partners\": [\"RUVBL2\", \"RUVBL1\", \"PIH1D1\", \"HSP90\", \"TRBP\", \"NEMO\", \"HSP70\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":7,"faith_total":7,"faith_pct":100.0}}