{"gene":"TUBGCP6","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2001,"finding":"GCP6 (TUBGCP6) is a bona fide subunit of the human γ-tubulin ring complex (γ-TuRC), identified by mass spectrometry. The complex forms a ~25 nm ring, nucleates microtubule polymerization in vitro, and GCP6 shares five conserved sequence regions with other GCPs, defining a novel protein superfamily conserved in metazoans. GCP6 localizes to the centrosome and associates with microtubules.","method":"Purification of human γ-TuRC, mass spectrometry identification of subunits, in vitro microtubule nucleation assay, sequence analysis, immunolocalization","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution of microtubule nucleation, MS identification, stoichiometry analysis; foundational study replicated across subsequent work","pmids":["11694571"],"is_preprint":false},{"year":2006,"finding":"GCP6 binds directly to keratin intermediate filaments (IFs) via its C-terminal fragment (residues 1397–1819), mediating attachment of γ-TuRC/MTOCs to IFs in polarized epithelial cells. This interaction is blocked by Cdk1-mediated phosphorylation of GCP6 at S1397. GCP6 knockdown by shRNA scatters γ-tubulin signal throughout the cytoplasm and displaces microtubule-nucleating activity from the apical domain.","method":"Yeast two-hybrid, in vitro binding of purified His-tagged GCP6 to keratins, IF pulldown, shRNA knockdown in CACO-2 cells, phosphomimetic mutant (S1397D) overexpression, immunofluorescence","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro binding with purified proteins, yeast two-hybrid validation, shRNA loss-of-function with defined cellular phenotype, phosphomimetic mutagenesis; single lab but multiple orthogonal methods","pmids":["17182859"],"is_preprint":false},{"year":2012,"finding":"GCP6 is a substrate of Plk4 kinase: GCP6 physically interacts with Plk4 and is phosphorylated by Plk4. This phosphorylation regulates centriole duplication. Depletion of GCP6 prevents γ-TuRC assembly, causes loss of centrosomal γ-tubulin, reduces centriole numbers, and produces a high frequency of monopolar spindles. GCP6 localizes to both the pericentriolar material and distal portions of centrioles.","method":"siRNA depletion, co-immunoprecipitation with Plk4, in vitro kinase assay (Plk4 phosphorylates GCP6), immunofluorescence, centriole counting","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay combined with co-IP, siRNA loss-of-function with multiple defined phenotypic readouts (γ-TuRC assembly, centriole number, spindle formation); single lab, multiple orthogonal methods","pmids":["22302995"],"is_preprint":false},{"year":2019,"finding":"WBP11, a pre-mRNA splicing factor, is required for proper splicing of TUBGCP6 pre-mRNA. Loss of WBP11 causes retention of multiple introns in TUBGCP6, leading to a rapid decline in TUBGCP6 protein levels, which in turn causes centriole duplication defects. Several other splicing factors that interact with WBP11 are also required for TUBGCP6 expression and centriole duplication.","method":"siRNA/shRNA depletion of WBP11, RNA-seq to identify intron retention events, western blot for TUBGCP6 protein levels, co-immunoprecipitation of WBP11 with other splicing factors, centriole duplication assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — RNA-seq identification of TUBGCP6 intron retention, protein-level confirmation, functional rescue/depletion experiments linking splicing to centriole duplication; single lab, multiple orthogonal methods","pmids":["31874114"],"is_preprint":false},{"year":2020,"finding":"GCP4, GCP5, and GCP6 form a salt (KCl)-resistant sub-complex within the γ-TuRC containing two copies of GCP4 and one copy each of GCP5 and GCP6. This sub-complex assembles independently of γTuSCs and, when incubated with cytoplasmic extracts containing γTuSCs, reconstitutes γTuRCs competent to nucleate microtubules. Sequence extensions and insertions specific to GCP6 (N-terminal extension and region between grip1 and grip2 motifs) are involved in γTuRC assembly or stabilization.","method":"Biochemical fractionation, salt-resistance assay, in vitro reconstitution of γTuRC from sub-complex + cytoplasmic extracts, microtubule nucleation assay, deletion/truncation analysis of GCP6 sequence insertions","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of γTuRC, biochemical sub-complex isolation, functional microtubule nucleation assay, domain analysis; single lab, multiple orthogonal methods","pmids":["32317396"],"is_preprint":false},{"year":2021,"finding":"SF3B14, a component of the spliceosomal U2 snRNP, is required for splicing of TUBGCP6 pre-mRNA. SF3B14 depletion increases the unspliced/spliced TUBGCP6 mRNA ratio and reduces TUBGCP6 protein levels, resulting in monopolar spindles and chromosome misalignment.","method":"RNAi depletion of SF3B14, RT-PCR to measure spliced vs unspliced TUBGCP6 mRNA, western blot for TUBGCP6 protein, immunofluorescence for mitotic phenotypes","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — loss-of-function with mRNA splicing ratio measurement and protein level confirmation, single lab, single study","pmids":["34954520"],"is_preprint":false},{"year":2026,"finding":"In zebrafish, loss-of-function mutation of tubgcp6 (nonsense mutation identified by positional cloning) causes mitotic arrest, disorganized spindle formation, increased p53-dependent apoptosis, and preferential symmetric differentiation over self-renewal in hematopoietic stem and progenitor cells (HSPCs), leading to HSPC exhaustion during definitive hematopoiesis.","method":"Forward genetic screen, positional cloning, time-lapse imaging, lineage tracing, immunofluorescence for spindle organization, genetic epistasis with p53","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — loss-of-function genetics in zebrafish with multiple cellular readouts (spindle, apoptosis, cell fate), time-lapse and lineage tracing; single study, model organism","pmids":["42092188"],"is_preprint":false}],"current_model":"TUBGCP6 (GCP6) is a core structural subunit of the human γ-tubulin ring complex (γ-TuRC) that, together with GCP4 and GCP5, forms a salt-resistant sub-complex that scaffolds γTuRC assembly and microtubule nucleation; it localizes to the pericentriolar material and distal centrioles where it is required for centriole duplication downstream of Plk4-mediated phosphorylation, and in polarized epithelial cells it anchors γ-TuRC/MTOCs to keratin intermediate filaments via a Cdk1-regulated interaction, while its protein levels are maintained by WBP11- and SF3B14-dependent pre-mRNA splicing."},"narrative":{"mechanistic_narrative":"TUBGCP6 (GCP6) is a core structural subunit of the human γ-tubulin ring complex (γ-TuRC) that scaffolds the microtubule-nucleating machinery at centrosomes [PMID:11694571]. Within the γ-TuRC, GCP6 partners with GCP4 and GCP5 to form a salt-resistant sub-complex containing two copies of GCP4 and one each of GCP5 and GCP6; this sub-complex assembles independently of γ-tubulin small complexes (γTuSCs) and, when combined with γTuSC-containing extracts, reconstitutes nucleation-competent γ-TuRCs, with GCP6-specific sequence extensions contributing to ring assembly and stabilization [PMID:32317396]. GCP6 is essential for centriole duplication: it localizes to the pericentriolar material and distal centrioles, is a direct substrate of the master centriole-duplication kinase Plk4, and its depletion blocks γ-TuRC assembly, depletes centrosomal γ-tubulin, reduces centriole number, and produces monopolar spindles [PMID:22302995]. In polarized epithelial cells, GCP6 anchors γ-TuRC/MTOCs to keratin intermediate filaments through its C-terminal region, an interaction switched off by Cdk1-mediated phosphorylation at S1397, thereby positioning microtubule-nucleating activity at the apical domain [PMID:17182859]. GCP6 protein levels are set post-transcriptionally by efficient splicing of its intron-rich pre-mRNA: the splicing factors WBP11 and the U2 snRNP component SF3B14 are required to prevent intron retention, and their loss collapses GCP6 levels and phenocopies centriole/spindle defects [PMID:31874114, PMID:34954520]. Loss of tubgcp6 in zebrafish causes mitotic arrest, disorganized spindles, and p53-dependent apoptosis, driving hematopoietic stem/progenitor cell exhaustion [PMID:42092188].","teleology":[{"year":2001,"claim":"Established GCP6 as a genuine subunit of the microtubule-nucleating γ-TuRC, defining the molecular machine in which it acts rather than treating it as an orphan centrosomal protein.","evidence":"Purification of human γ-TuRC with mass-spectrometry subunit identification, in vitro microtubule nucleation assay, and immunolocalization","pmids":["11694571"],"confidence":"High","gaps":["Stoichiometry and structural position of GCP6 within the ring not resolved","Functional contribution of GCP6 to nucleation versus structural scaffolding not separated"]},{"year":2006,"claim":"Showed that GCP6 does more than scaffold nucleation—it physically tethers MTOCs to the cytoskeleton, explaining how nucleating activity is spatially positioned in polarized cells and how that positioning is cell-cycle regulated.","evidence":"Yeast two-hybrid, in vitro binding of purified GCP6 to keratins, shRNA knockdown in CACO-2 cells, and S1397D phosphomimetic mutagenesis","pmids":["17182859"],"confidence":"High","gaps":["Whether the keratin interaction operates outside epithelial cell types unknown","Identity of the kinase driving S1397 phosphorylation in vivo beyond Cdk1 not established"]},{"year":2012,"claim":"Placed GCP6 downstream of Plk4, linking γ-TuRC assembly directly to the centriole duplication program and demonstrating its requirement for bipolar spindle formation.","evidence":"siRNA depletion, co-immunoprecipitation with Plk4, in vitro Plk4 kinase assay, and centriole counting by immunofluorescence","pmids":["22302995"],"confidence":"High","gaps":["Phosphosites on GCP6 targeted by Plk4 not mapped","Mechanistic link between GCP6 phosphorylation and centriole assembly not defined"]},{"year":2019,"claim":"Revealed that GCP6 abundance is rate-limiting for centriole duplication and is controlled post-transcriptionally, identifying intron retention as a regulatory chokepoint for the protein.","evidence":"WBP11 siRNA/shRNA depletion, RNA-seq detection of TUBGCP6 intron retention, western blot, and centriole duplication assays","pmids":["31874114"],"confidence":"High","gaps":["Why TUBGCP6 is especially sensitive to splicing efficiency not explained","Direct binding of WBP11 to TUBGCP6 pre-mRNA versus indirect effect not distinguished"]},{"year":2020,"claim":"Defined the GCP4/GCP5/GCP6 salt-resistant sub-complex as an assembly-competent module and reconstituted functional γ-TuRC from it, clarifying GCP6's structural role in ring assembly.","evidence":"Biochemical fractionation, salt-resistance assay, in vitro γTuRC reconstitution from sub-complex plus cytoplasmic extracts, nucleation assay, and GCP6 truncation analysis","pmids":["32317396"],"confidence":"High","gaps":["High-resolution structure of GCP6 within the assembled ring not determined","Precise function of GCP6 N-terminal extension and grip1–grip2 insertion not dissected"]},{"year":2021,"claim":"Confirmed and extended the splicing-dependent control of GCP6 by implicating the U2 snRNP component SF3B14, reinforcing that spliceosome activity gates GCP6 protein levels and mitotic fidelity.","evidence":"RNAi depletion of SF3B14, RT-PCR of spliced vs unspliced TUBGCP6 mRNA, western blot, and mitotic phenotype imaging","pmids":["34954520"],"confidence":"Medium","gaps":["Single-study, single-lab finding without reciprocal validation","Whether SF3B14 acts directly on TUBGCP6 introns or globally not resolved"]},{"year":2026,"claim":"Connected GCP6 loss to an organ-level phenotype, showing that its requirement for spindle integrity governs stem-cell self-renewal versus differentiation during hematopoiesis.","evidence":"Zebrafish forward genetic screen, positional cloning of a tubgcp6 nonsense allele, time-lapse imaging, lineage tracing, and p53 epistasis","pmids":["42092188"],"confidence":"Medium","gaps":["Whether the HSPC phenotype reflects centriole/γ-TuRC loss specifically versus general mitotic failure not isolated","Relevance to mammalian hematopoiesis not established"]},{"year":null,"claim":"How GCP6's distinct roles—γ-TuRC ring assembly, Plk4-dependent centriole duplication, and intermediate-filament anchoring—are coordinated within a single cell cycle remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of GCP6 within the assembled γ-TuRC","Plk4 phosphosites on GCP6 unmapped","Integration of splicing-based abundance control with phosphoregulation not addressed"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,4]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[1]}],"localization":[{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[0,2]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[2,6]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[0,4]}],"complexes":["γ-tubulin ring complex (γ-TuRC)","GCP4/GCP5/GCP6 salt-resistant sub-complex"],"partners":["TUBGCP4","TUBGCP5","PLK4","WBP11","SF3B14","KERATIN"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96RT7","full_name":"Gamma-tubulin complex component 6","aliases":[],"length_aa":1819,"mass_kda":200.5,"function":"Component of the gamma-tubulin ring complex (gTuRC) which mediates microtubule nucleation (PubMed:11694571, PubMed:38305685, PubMed:38609661, PubMed:39321809). The gTuRC regulates the minus-end nucleation of alpha-beta tubulin heterodimers that grow into microtubule protafilaments, a critical step in centrosome duplication and spindle formation (PubMed:38305685, PubMed:38609661, PubMed:39321809)","subcellular_location":"Cytoplasm, cytoskeleton, microtubule organizing center, centrosome","url":"https://www.uniprot.org/uniprotkb/Q96RT7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/TUBGCP6","classification":"Common Essential","n_dependent_lines":1113,"n_total_lines":1208,"dependency_fraction":0.9213576158940397},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"UBA1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/TUBGCP6","total_profiled":1310},"omim":[{"mim_id":"618083","title":"WW-BINDING PROTEIN 11; WBP11","url":"https://www.omim.org/entry/618083"},{"mim_id":"610053","title":"TUBULIN-GAMMA COMPLEX-ASSOCIATED PROTEIN 6; TUBGCP6","url":"https://www.omim.org/entry/610053"},{"mim_id":"251270","title":"MICROCEPHALY AND CHORIORETINOPATHY, AUTOSOMAL RECESSIVE, 1; MCCRP1","url":"https://www.omim.org/entry/251270"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TUBGCP6"},"hgnc":{"alias_symbol":["GCP6","KIAA1669","DJ402G11.6"],"prev_symbol":[]},"alphafold":{"accession":"Q96RT7","domains":[{"cath_id":"-","chopping":"340-482","consensus_level":"medium","plddt":84.2692,"start":340,"end":482},{"cath_id":"1.20.120.1900","chopping":"1512-1815","consensus_level":"medium","plddt":84.3413,"start":1512,"end":1815}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96RT7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96RT7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96RT7-F1-predicted_aligned_error_v6.png","plddt_mean":59.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TUBGCP6","jax_strain_url":"https://www.jax.org/strain/search?query=TUBGCP6"},"sequence":{"accession":"Q96RT7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96RT7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96RT7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96RT7"}},"corpus_meta":[{"pmid":"11694571","id":"PMC_11694571","title":"GCP5 and GCP6: two new members of the human gamma-tubulin complex.","date":"2001","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/11694571","citation_count":154,"is_preprint":false},{"pmid":"22302995","id":"PMC_22302995","title":"GCP6 is a substrate of Plk4 and required for centriole duplication.","date":"2012","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/22302995","citation_count":69,"is_preprint":false},{"pmid":"17182859","id":"PMC_17182859","title":"GCP6 binds to intermediate filaments: a novel function of keratins in the organization of microtubules in epithelial cells.","date":"2006","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/17182859","citation_count":43,"is_preprint":false},{"pmid":"31874114","id":"PMC_31874114","title":"WBP11 is required for splicing the TUBGCP6 pre-mRNA to promote centriole duplication.","date":"2019","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/31874114","citation_count":18,"is_preprint":false},{"pmid":"32317396","id":"PMC_32317396","title":"A stable sub-complex between GCP4, GCP5 and GCP6 promotes the assembly of γ-tubulin ring complexes.","date":"2020","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/32317396","citation_count":18,"is_preprint":false},{"pmid":"37031378","id":"PMC_37031378","title":"Biallelic variants in TUBGCP6 result in microcephaly and chorioretinopathy 1: Report of four cases and a literature review.","date":"2023","source":"American journal of medical genetics. Part A","url":"https://pubmed.ncbi.nlm.nih.gov/37031378","citation_count":6,"is_preprint":false},{"pmid":"34954520","id":"PMC_34954520","title":"SF3B14 is involved in the centrosome regulation through splicing of TUBGCP6 pre-mRNA.","date":"2021","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/34954520","citation_count":2,"is_preprint":false},{"pmid":"42092188","id":"PMC_42092188","title":"Mutation of Tubgcp6 induces hematopoietic stem and progenitor cell exhaustion in zebrafish.","date":"2026","source":"Communications biology","url":"https://pubmed.ncbi.nlm.nih.gov/42092188","citation_count":0,"is_preprint":false},{"pmid":"42033392","id":"PMC_42033392","title":"Novel Homozygous TUBGCP6 Variant Impairs Brain Development: Case Report and Literature Review.","date":"2026","source":"Journal of child neurology","url":"https://pubmed.ncbi.nlm.nih.gov/42033392","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":6234,"output_tokens":2272,"usd":0.026391,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9336,"output_tokens":3073,"usd":0.061753,"stage2_stop_reason":"end_turn"},"total_usd":0.088144,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2001,\n      \"finding\": \"GCP6 (TUBGCP6) is a bona fide subunit of the human γ-tubulin ring complex (γ-TuRC), identified by mass spectrometry. The complex forms a ~25 nm ring, nucleates microtubule polymerization in vitro, and GCP6 shares five conserved sequence regions with other GCPs, defining a novel protein superfamily conserved in metazoans. GCP6 localizes to the centrosome and associates with microtubules.\",\n      \"method\": \"Purification of human γ-TuRC, mass spectrometry identification of subunits, in vitro microtubule nucleation assay, sequence analysis, immunolocalization\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution of microtubule nucleation, MS identification, stoichiometry analysis; foundational study replicated across subsequent work\",\n      \"pmids\": [\"11694571\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"GCP6 binds directly to keratin intermediate filaments (IFs) via its C-terminal fragment (residues 1397–1819), mediating attachment of γ-TuRC/MTOCs to IFs in polarized epithelial cells. This interaction is blocked by Cdk1-mediated phosphorylation of GCP6 at S1397. GCP6 knockdown by shRNA scatters γ-tubulin signal throughout the cytoplasm and displaces microtubule-nucleating activity from the apical domain.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding of purified His-tagged GCP6 to keratins, IF pulldown, shRNA knockdown in CACO-2 cells, phosphomimetic mutant (S1397D) overexpression, immunofluorescence\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro binding with purified proteins, yeast two-hybrid validation, shRNA loss-of-function with defined cellular phenotype, phosphomimetic mutagenesis; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"17182859\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"GCP6 is a substrate of Plk4 kinase: GCP6 physically interacts with Plk4 and is phosphorylated by Plk4. This phosphorylation regulates centriole duplication. Depletion of GCP6 prevents γ-TuRC assembly, causes loss of centrosomal γ-tubulin, reduces centriole numbers, and produces a high frequency of monopolar spindles. GCP6 localizes to both the pericentriolar material and distal portions of centrioles.\",\n      \"method\": \"siRNA depletion, co-immunoprecipitation with Plk4, in vitro kinase assay (Plk4 phosphorylates GCP6), immunofluorescence, centriole counting\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay combined with co-IP, siRNA loss-of-function with multiple defined phenotypic readouts (γ-TuRC assembly, centriole number, spindle formation); single lab, multiple orthogonal methods\",\n      \"pmids\": [\"22302995\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"WBP11, a pre-mRNA splicing factor, is required for proper splicing of TUBGCP6 pre-mRNA. Loss of WBP11 causes retention of multiple introns in TUBGCP6, leading to a rapid decline in TUBGCP6 protein levels, which in turn causes centriole duplication defects. Several other splicing factors that interact with WBP11 are also required for TUBGCP6 expression and centriole duplication.\",\n      \"method\": \"siRNA/shRNA depletion of WBP11, RNA-seq to identify intron retention events, western blot for TUBGCP6 protein levels, co-immunoprecipitation of WBP11 with other splicing factors, centriole duplication assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNA-seq identification of TUBGCP6 intron retention, protein-level confirmation, functional rescue/depletion experiments linking splicing to centriole duplication; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"31874114\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"GCP4, GCP5, and GCP6 form a salt (KCl)-resistant sub-complex within the γ-TuRC containing two copies of GCP4 and one copy each of GCP5 and GCP6. This sub-complex assembles independently of γTuSCs and, when incubated with cytoplasmic extracts containing γTuSCs, reconstitutes γTuRCs competent to nucleate microtubules. Sequence extensions and insertions specific to GCP6 (N-terminal extension and region between grip1 and grip2 motifs) are involved in γTuRC assembly or stabilization.\",\n      \"method\": \"Biochemical fractionation, salt-resistance assay, in vitro reconstitution of γTuRC from sub-complex + cytoplasmic extracts, microtubule nucleation assay, deletion/truncation analysis of GCP6 sequence insertions\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of γTuRC, biochemical sub-complex isolation, functional microtubule nucleation assay, domain analysis; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"32317396\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SF3B14, a component of the spliceosomal U2 snRNP, is required for splicing of TUBGCP6 pre-mRNA. SF3B14 depletion increases the unspliced/spliced TUBGCP6 mRNA ratio and reduces TUBGCP6 protein levels, resulting in monopolar spindles and chromosome misalignment.\",\n      \"method\": \"RNAi depletion of SF3B14, RT-PCR to measure spliced vs unspliced TUBGCP6 mRNA, western blot for TUBGCP6 protein, immunofluorescence for mitotic phenotypes\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — loss-of-function with mRNA splicing ratio measurement and protein level confirmation, single lab, single study\",\n      \"pmids\": [\"34954520\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"In zebrafish, loss-of-function mutation of tubgcp6 (nonsense mutation identified by positional cloning) causes mitotic arrest, disorganized spindle formation, increased p53-dependent apoptosis, and preferential symmetric differentiation over self-renewal in hematopoietic stem and progenitor cells (HSPCs), leading to HSPC exhaustion during definitive hematopoiesis.\",\n      \"method\": \"Forward genetic screen, positional cloning, time-lapse imaging, lineage tracing, immunofluorescence for spindle organization, genetic epistasis with p53\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — loss-of-function genetics in zebrafish with multiple cellular readouts (spindle, apoptosis, cell fate), time-lapse and lineage tracing; single study, model organism\",\n      \"pmids\": [\"42092188\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TUBGCP6 (GCP6) is a core structural subunit of the human γ-tubulin ring complex (γ-TuRC) that, together with GCP4 and GCP5, forms a salt-resistant sub-complex that scaffolds γTuRC assembly and microtubule nucleation; it localizes to the pericentriolar material and distal centrioles where it is required for centriole duplication downstream of Plk4-mediated phosphorylation, and in polarized epithelial cells it anchors γ-TuRC/MTOCs to keratin intermediate filaments via a Cdk1-regulated interaction, while its protein levels are maintained by WBP11- and SF3B14-dependent pre-mRNA splicing.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TUBGCP6 (GCP6) is a core structural subunit of the human γ-tubulin ring complex (γ-TuRC) that scaffolds the microtubule-nucleating machinery at centrosomes [#0]. Within the γ-TuRC, GCP6 partners with GCP4 and GCP5 to form a salt-resistant sub-complex containing two copies of GCP4 and one each of GCP5 and GCP6; this sub-complex assembles independently of γ-tubulin small complexes (γTuSCs) and, when combined with γTuSC-containing extracts, reconstitutes nucleation-competent γ-TuRCs, with GCP6-specific sequence extensions contributing to ring assembly and stabilization [#4]. GCP6 is essential for centriole duplication: it localizes to the pericentriolar material and distal centrioles, is a direct substrate of the master centriole-duplication kinase Plk4, and its depletion blocks γ-TuRC assembly, depletes centrosomal γ-tubulin, reduces centriole number, and produces monopolar spindles [#2]. In polarized epithelial cells, GCP6 anchors γ-TuRC/MTOCs to keratin intermediate filaments through its C-terminal region, an interaction switched off by Cdk1-mediated phosphorylation at S1397, thereby positioning microtubule-nucleating activity at the apical domain [#1]. GCP6 protein levels are set post-transcriptionally by efficient splicing of its intron-rich pre-mRNA: the splicing factors WBP11 and the U2 snRNP component SF3B14 are required to prevent intron retention, and their loss collapses GCP6 levels and phenocopies centriole/spindle defects [#3, #5]. Loss of tubgcp6 in zebrafish causes mitotic arrest, disorganized spindles, and p53-dependent apoptosis, driving hematopoietic stem/progenitor cell exhaustion [#6].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established GCP6 as a genuine subunit of the microtubule-nucleating γ-TuRC, defining the molecular machine in which it acts rather than treating it as an orphan centrosomal protein.\",\n      \"evidence\": \"Purification of human γ-TuRC with mass-spectrometry subunit identification, in vitro microtubule nucleation assay, and immunolocalization\",\n      \"pmids\": [\"11694571\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and structural position of GCP6 within the ring not resolved\", \"Functional contribution of GCP6 to nucleation versus structural scaffolding not separated\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Showed that GCP6 does more than scaffold nucleation—it physically tethers MTOCs to the cytoskeleton, explaining how nucleating activity is spatially positioned in polarized cells and how that positioning is cell-cycle regulated.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro binding of purified GCP6 to keratins, shRNA knockdown in CACO-2 cells, and S1397D phosphomimetic mutagenesis\",\n      \"pmids\": [\"17182859\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the keratin interaction operates outside epithelial cell types unknown\", \"Identity of the kinase driving S1397 phosphorylation in vivo beyond Cdk1 not established\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Placed GCP6 downstream of Plk4, linking γ-TuRC assembly directly to the centriole duplication program and demonstrating its requirement for bipolar spindle formation.\",\n      \"evidence\": \"siRNA depletion, co-immunoprecipitation with Plk4, in vitro Plk4 kinase assay, and centriole counting by immunofluorescence\",\n      \"pmids\": [\"22302995\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphosites on GCP6 targeted by Plk4 not mapped\", \"Mechanistic link between GCP6 phosphorylation and centriole assembly not defined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Revealed that GCP6 abundance is rate-limiting for centriole duplication and is controlled post-transcriptionally, identifying intron retention as a regulatory chokepoint for the protein.\",\n      \"evidence\": \"WBP11 siRNA/shRNA depletion, RNA-seq detection of TUBGCP6 intron retention, western blot, and centriole duplication assays\",\n      \"pmids\": [\"31874114\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why TUBGCP6 is especially sensitive to splicing efficiency not explained\", \"Direct binding of WBP11 to TUBGCP6 pre-mRNA versus indirect effect not distinguished\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined the GCP4/GCP5/GCP6 salt-resistant sub-complex as an assembly-competent module and reconstituted functional γ-TuRC from it, clarifying GCP6's structural role in ring assembly.\",\n      \"evidence\": \"Biochemical fractionation, salt-resistance assay, in vitro γTuRC reconstitution from sub-complex plus cytoplasmic extracts, nucleation assay, and GCP6 truncation analysis\",\n      \"pmids\": [\"32317396\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"High-resolution structure of GCP6 within the assembled ring not determined\", \"Precise function of GCP6 N-terminal extension and grip1–grip2 insertion not dissected\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Confirmed and extended the splicing-dependent control of GCP6 by implicating the U2 snRNP component SF3B14, reinforcing that spliceosome activity gates GCP6 protein levels and mitotic fidelity.\",\n      \"evidence\": \"RNAi depletion of SF3B14, RT-PCR of spliced vs unspliced TUBGCP6 mRNA, western blot, and mitotic phenotype imaging\",\n      \"pmids\": [\"34954520\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-study, single-lab finding without reciprocal validation\", \"Whether SF3B14 acts directly on TUBGCP6 introns or globally not resolved\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Connected GCP6 loss to an organ-level phenotype, showing that its requirement for spindle integrity governs stem-cell self-renewal versus differentiation during hematopoiesis.\",\n      \"evidence\": \"Zebrafish forward genetic screen, positional cloning of a tubgcp6 nonsense allele, time-lapse imaging, lineage tracing, and p53 epistasis\",\n      \"pmids\": [\"42092188\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the HSPC phenotype reflects centriole/γ-TuRC loss specifically versus general mitotic failure not isolated\", \"Relevance to mammalian hematopoiesis not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How GCP6's distinct roles—γ-TuRC ring assembly, Plk4-dependent centriole duplication, and intermediate-filament anchoring—are coordinated within a single cell cycle remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of GCP6 within the assembled γ-TuRC\", \"Plk4 phosphosites on GCP6 unmapped\", \"Integration of splicing-based abundance control with phosphoregulation not addressed\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"GO:0005813\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [2, 6]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [0, 4]}\n    ],\n    \"complexes\": [\"γ-tubulin ring complex (γ-TuRC)\", \"GCP4/GCP5/GCP6 salt-resistant sub-complex\"],\n    \"partners\": [\"TUBGCP4\", \"TUBGCP5\", \"PLK4\", \"WBP11\", \"SF3B14\", \"keratin\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}