{"gene":"DNAI3","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":2021,"finding":"WDR63 (DNAI3) interacts with WDR78 primarily via its WD40-repeat domain and is required for inner dynein arm (IDA) assembly in sperm flagella; loss-of-function in mice (CRISPR-Cas9 knockout) causes disorganized '9+2' axoneme and absent IDAs, resulting in male infertility.","method":"Co-immunoprecipitation (WDR63–WDR78 interaction), CRISPR-Cas9 knockout mice, transmission electron microscopy of axoneme ultrastructure","journal":"Cell discovery","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP identifying interaction domain, KO mouse with defined ultrastructural phenotype, replicated in human patient cohort","pmids":["34782613"],"is_preprint":false},{"year":2019,"finding":"WDR63 is the vertebrate orthologue of Chlamydomonas IC140, an intermediate chain of Dynein-f; it associates with Dynein-f subunits WDR78 (IC138 orthologue) and DNAH2 (heavy chain), and is expressed specifically in motile cilia-bearing cells. Unlike in Chlamydomonas, vertebrate Dynein-f (including WDR63) is essential for ciliary beat — depletion causes paralysis of mouse ependymal cilia.","method":"Co-immunoprecipitation (WDR63 with Dnah2 and Wdr78), RNAi knockdown in mouse ependymal cells, zebrafish morpholino knockdown, immunofluorescence localization","journal":"Journal of molecular cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, RNAi in mammalian cells, in vivo zebrafish morphants, replicated across two vertebrate models","pmids":["30060180"],"is_preprint":false},{"year":2020,"finding":"WDR63 is transcriptionally upregulated by p53 and acts downstream of p53 to suppress cell migration, invasion, and metastasis. Mechanistically, WDR63 directly interacts with the Arp2/3 complex and inhibits Arp2/3-mediated branched actin polymerization.","method":"Chromatin immunoprecipitation / reporter assays (p53 transcriptional activation), Co-immunoprecipitation (WDR63–Arp2/3), in vitro actin polymerization assay, migration/invasion assays, mouse xenograft models","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro actin polymerization reconstitution plus Co-IP and functional migration/invasion assays in one study, single lab","pmids":["32128961"],"is_preprint":false},{"year":2025,"finding":"WDR63 interacts with TCTEX1D2 as part of the inner dynein arm complex; Co-IP in testis confirmed WDR63 (along with WDR78) as a binding partner of TCTEX1D2, placing WDR63 in a network bridging cytoplasmic dynein 2 and axonemal inner dynein arm during sperm flagellum formation.","method":"Co-immunoprecipitation in mouse testes (TCTEX1D2 pulled down WDR63 and WDR78), Tctex1d2 knockout mice with flagellar phenotype analysis","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single Co-IP in testes from one study; mechanistic placement is indirect (via TCTEX1D2 KO rather than WDR63 manipulation)","pmids":["39827215"],"is_preprint":false},{"year":2018,"finding":"An in-frame deletion spanning WDR63 exons 14–17 (removing the 3rd and 4th WD-repeat domains) acts as a dominant-negative or gain-of-function variant; CRISPR/Cas9-mediated somatic recapitulation of this deletion in zebrafish caused brain and body malformations including sac-like brain protrusions, supporting a role for WDR63 in neural tube/encephalocele formation.","method":"CRISPR/Cas9 somatic deletion in zebrafish mimicking human deletion, overexpression of deleted vs. wild-type WDR63 RNA in zebrafish, whole-genome sequencing of human fetus","journal":"Human mutation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo zebrafish CRISPR and RNA overexpression experiments with quantified phenotype, single lab","pmids":["29285825"],"is_preprint":false},{"year":2026,"finding":"WDR63 promotes K63-linked polyubiquitination of p53 while reducing its K48-linked polyubiquitination, thereby stabilising p53 protein and activating downstream p53 signalling in alveolar epithelial cells and lung fibroblasts, driving pulmonary fibrosis.","method":"Mass spectrometry and co-immunoprecipitation to identify p53 as a WDR63 substrate, ubiquitination assays (K63 vs K48 linkage-specific), WDR63 overexpression/silencing in cell lines, bleomycin-induced mouse fibrosis model","journal":"Respirology (Carlton, Vic.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — linkage-specific ubiquitination assays plus Co-IP/MS plus in vivo mouse model, single lab","pmids":["41640377"],"is_preprint":false},{"year":2026,"finding":"WDR63 interacts and co-localizes with vimentin (VIM) in stem cells from apical papilla; WDR63 overexpression enhances VIM expression and WDR63–VIM binding, upregulates DRP1, and promotes mitochondrial fission, thereby enhancing chondrogenic differentiation.","method":"Co-immunoprecipitation and co-localization (WDR63–VIM), Western blot for DRP1 and chondrogenic markers, gain/loss-of-function in SCAPs, rabbit osteochondral defect transplantation model","journal":"Stem cell research & therapy","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP and co-localization experiment from one lab; mitochondrial fission link inferred from DRP1 protein level changes without direct reconstitution","pmids":["41796360"],"is_preprint":false},{"year":2014,"finding":"WDR63 gain-of-function enhances alkaline phosphatase activity, mineralization, and expression of osteogenic markers BSP, OSX, and RUNX2 in stem cells from apical papilla (SCAPs), and promotes osteogenesis in vivo (nude mouse transplants), identifying WDR63 as a positive regulator of SCAP osteogenic differentiation.","method":"Gain-of-function and loss-of-function (overexpression/knockdown) in SCAPs, alkaline phosphatase assay, alizarin red/calcium quantification, gene expression analysis, in vivo nude mouse transplantation; H3K4Me3 ChIP-on-chip identified WDR63 promoter activation","journal":"Journal of endodontics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — gain/loss-of-function with functional readouts but no direct molecular mechanism identified; single lab, no binding partner established","pmids":["25498833"],"is_preprint":false},{"year":2023,"finding":"WDR63 knockout (CRISPR) in human airway epithelial cells impairs rAAV2.5T vector transduction without affecting vector internalization or nuclear entry, indicating WDR63 acts at an intracellular trafficking or uncoating/conversion step post-nuclear entry.","method":"Genome-wide CRISPR screen followed by validation with individual CRISPR knockouts of WDR63 in polarized human airway epithelium; vector transduction, internalization, and nuclear entry assays","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO validated with functional assay dissecting distinct steps of AAV transduction; single lab but two independent papers (peer-reviewed + preprint) report same result","pmids":["37966249","37808760"],"is_preprint":false}],"current_model":"WDR63 (DNAI3) is a WD40-repeat protein with multiple distinct cellular roles: it is an intermediate chain of the vertebrate inner dynein arm (Dynein-f) complex, where it interacts with WDR78 via its WD40 domain and is essential for axonemal assembly and ciliary/flagellar beating; downstream of p53 it binds and inhibits the Arp2/3 complex to suppress actin polymerization and cell migration; and it promotes p53 stability through K63-linked polyubiquitination to drive pulmonary fibrosis, while also influencing stem cell differentiation and AAV intracellular transduction in airway epithelium."},"narrative":{"mechanistic_narrative":"WDR63 (DNAI3) is a WD40-repeat protein that functions as an intermediate chain of the vertebrate inner dynein arm (IDA), the Dynein-f complex that powers ciliary and flagellar motility [PMID:30060180]. As the vertebrate orthologue of Chlamydomonas IC140, it associates with the IDA subunits WDR78 (IC138 orthologue) and the heavy chain DNAH2, and is expressed specifically in motile cilia-bearing cells; unlike its algal counterpart, vertebrate Dynein-f is essential for ciliary beat, as its depletion paralyzes mouse ependymal cilia [PMID:30060180]. WDR63 binds WDR78 primarily through its WD40-repeat domain and is required for IDA assembly in sperm flagella, where loss of function produces disorganized '9+2' axonemes lacking IDAs and male infertility [PMID:34782613], and it further bridges to cytoplasmic dynein 2 trafficking machinery through TCTEX1D2 during flagellum formation [PMID:39827215]. Beyond the axoneme, WDR63 is a transcriptional target of p53 that acts downstream of p53 to suppress cell migration, invasion, and metastasis by directly binding the Arp2/3 complex and inhibiting branched actin polymerization [PMID:32128961]. WDR63 also participates in human disease beyond cilia: an in-frame deletion removing WD-repeat domains acts as a dominant-negative/gain-of-function allele causing neural tube and encephalocele malformations modeled in zebrafish [PMID:29285825], and WDR63 promotes K63-linked while reducing K48-linked polyubiquitination of p53 to stabilize it and drive pulmonary fibrosis [PMID:41640377]. A CRISPR knockout in human airway epithelium additionally implicates WDR63 in an intracellular post-nuclear-entry step of rAAV vector transduction [PMID:37966249, PMID:37808760].","teleology":[{"year":2014,"claim":"Before any molecular partner was known, WDR63 was first linked to a cellular process through a functional screen identifying it as a positive regulator of stem cell osteogenic differentiation.","evidence":"Gain/loss-of-function in SCAPs with mineralization and osteogenic marker readouts, in vivo nude mouse transplantation, H3K4Me3 ChIP-on-chip","pmids":["25498833"],"confidence":"Low","gaps":["No molecular mechanism or binding partner identified","Single lab, functional readouts only","No connection drawn to ciliary or dynein function"]},{"year":2018,"claim":"A human in-frame deletion plus zebrafish modeling established that WDR63 disruption causes neural tube/encephalocele malformations and that loss of specific WD-repeat domains can act as a dominant-negative allele.","evidence":"CRISPR/Cas9 somatic deletion and RNA overexpression in zebrafish, whole-genome sequencing of human fetus","pmids":["29285825"],"confidence":"Medium","gaps":["Molecular mechanism of malformation unresolved","Whether developmental role reflects ciliary defects not established","Single lab"]},{"year":2019,"claim":"WDR63 was defined as the vertebrate orthologue of IC140 and an intermediate chain of the inner dynein arm Dynein-f, answering what protein complex it belongs to and showing the complex is essential for vertebrate ciliary beat.","evidence":"Reciprocal Co-IP with Dnah2 and Wdr78, RNAi in mouse ependymal cells, zebrafish morpholino knockdown, immunofluorescence","pmids":["30060180"],"confidence":"High","gaps":["Structural arrangement within the dynein arm not resolved","Mechanism by which Dynein-f drives beat not detailed"]},{"year":2020,"claim":"An unexpected non-ciliary role was established: WDR63 is a p53 transcriptional target that inhibits Arp2/3-mediated branched actin polymerization to suppress migration and metastasis.","evidence":"ChIP/reporter assays, Co-IP with Arp2/3, in vitro actin polymerization reconstitution, migration/invasion assays, mouse xenografts","pmids":["32128961"],"confidence":"High","gaps":["Structural basis of Arp2/3 inhibition not defined","Single lab","Relationship between cytoskeletal/actin role and axonemal role unclear"]},{"year":2021,"claim":"The WDR63–WDR78 interaction was mapped to the WD40 domain and shown to be required for inner dynein arm assembly in sperm flagella, defining the structural basis of axonemal function and a male infertility phenotype.","evidence":"Reciprocal Co-IP identifying interaction domain, CRISPR-Cas9 knockout mice, transmission electron microscopy of axoneme ultrastructure, human patient cohort","pmids":["34782613"],"confidence":"High","gaps":["Order of assembly steps within the IDA not resolved","How WD40 domain engages WDR78 structurally not determined"]},{"year":2023,"claim":"A genome-wide screen placed WDR63 in an intracellular post-nuclear-entry step of rAAV transduction, separating its role from vector internalization and nuclear import.","evidence":"Genome-wide CRISPR screen with individual KO validation in polarized human airway epithelium; transduction, internalization, and nuclear entry assays","pmids":["37966249","37808760"],"confidence":"Medium","gaps":["Precise trafficking/uncoating step not identified","Mechanistic link to WDR63's dynein or actin functions not established"]},{"year":2025,"claim":"WDR63 was positioned in a network bridging cytoplasmic dynein 2 and the axonemal inner dynein arm via TCTEX1D2 during flagellum formation.","evidence":"Co-IP in mouse testes (TCTEX1D2 pulled down WDR63 and WDR78), Tctex1d2 knockout mice","pmids":["39827215"],"confidence":"Medium","gaps":["Single Co-IP; placement inferred via TCTEX1D2 KO rather than WDR63 manipulation","Functional dependency of WDR63 on TCTEX1D2 not directly tested"]},{"year":2026,"claim":"WDR63 was shown to control p53 protein stability through ubiquitin chain editing—promoting K63-linked while reducing K48-linked polyubiquitination—linking it to pulmonary fibrosis.","evidence":"MS/Co-IP identifying p53 as substrate, linkage-specific ubiquitination assays, overexpression/silencing, bleomycin-induced mouse fibrosis model","pmids":["41640377"],"confidence":"Medium","gaps":["Whether WDR63 has intrinsic E3 ligase activity or acts as an adaptor not established","Single lab","Reconciliation with the 2020 finding that p53 induces WDR63 (feedback architecture) not resolved"]},{"year":2026,"claim":"WDR63 was linked to vimentin binding, DRP1 upregulation, and mitochondrial fission promoting chondrogenic differentiation in stem cells.","evidence":"Co-IP and co-localization with VIM, Western blot for DRP1/chondrogenic markers, gain/loss-of-function in SCAPs, rabbit osteochondral defect model","pmids":["41796360"],"confidence":"Low","gaps":["Single Co-IP/co-localization from one lab; mitochondrial fission link inferred from DRP1 levels without direct reconstitution","Direct VIM binding not validated reciprocally","Mechanistic connection to other WDR63 functions absent"]},{"year":null,"claim":"How a single WD40 protein integrates its axonemal dynein-assembly role with its cytosolic actin/Arp2/3, p53-ubiquitin, and trafficking activities—and whether these are independent moonlighting functions or one mechanism—remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model unifying WD40-mediated interactions across complexes","Tissue-specific partner switching not characterized","Whether non-ciliary roles depend on the dynein-arm interaction unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,5]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,1]}],"localization":[{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[1]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[2]}],"pathway":[{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[0,1]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,5]}],"complexes":["inner dynein arm (Dynein-f)","Arp2/3 complex"],"partners":["WDR78","DNAH2","TCTEX1D2","TP53","VIM"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8IWG1","full_name":"Dynein axonemal intermediate chain 3","aliases":["Testis development protein NYD-SP29","WD repeat-containing protein 63"],"length_aa":891,"mass_kda":102.9,"function":"Acts as a negative regulator of cell migration, invasion, and metastasis downstream of p53/TP53, through inhibition of Arp2/3 complex-mediated actin polymerization (PubMed:32128961). Via its association with the multisubunit axonemal dynein complex, is potentially involved in the regulation of cilia function (By similarity). May play a role in osteogenesis of dental tissue-derived mesenchymal stem cells (By similarity)","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q8IWG1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/DNAI3","classification":"Not Classified","n_dependent_lines":7,"n_total_lines":1208,"dependency_fraction":0.005794701986754967},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/DNAI3","total_profiled":1310},"omim":[],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Connecting piece","reliability":"Approved"},{"location":"Perinuclear theca","reliability":"Additional"},{"location":"Calyx","reliability":"Additional"},{"location":"Mid piece","reliability":"Additional"}],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"adrenal gland","ntpm":20.6},{"tissue":"choroid plexus","ntpm":40.0},{"tissue":"fallopian tube","ntpm":13.6},{"tissue":"testis","ntpm":17.6}],"url":"https://www.proteinatlas.org/search/DNAI3"},"hgnc":{"alias_symbol":["DIC3","FLJ30067","NYD-SP29"],"prev_symbol":["WDR63"]},"alphafold":{"accession":"Q8IWG1","domains":[{"cath_id":"-","chopping":"31-128","consensus_level":"high","plddt":82.1319,"start":31,"end":128},{"cath_id":"2.130.10.10","chopping":"322-434_462-528_550-591_623-801","consensus_level":"medium","plddt":87.2367,"start":322,"end":801}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8IWG1","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8IWG1-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8IWG1-F1-predicted_aligned_error_v6.png","plddt_mean":77.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=DNAI3","jax_strain_url":"https://www.jax.org/strain/search?query=DNAI3"},"sequence":{"accession":"Q8IWG1","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8IWG1.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8IWG1/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8IWG1"}},"corpus_meta":[{"pmid":"34782613","id":"PMC_34782613","title":"Bi-allelic variants in human WDR63 cause male infertility via abnormal inner dynein arms assembly.","date":"2021","source":"Cell discovery","url":"https://pubmed.ncbi.nlm.nih.gov/34782613","citation_count":37,"is_preprint":false},{"pmid":"30060180","id":"PMC_30060180","title":"Vertebrate Dynein-f depends on Wdr78 for axonemal localization and is essential for ciliary beat.","date":"2019","source":"Journal of molecular cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/30060180","citation_count":34,"is_preprint":false},{"pmid":"32128961","id":"PMC_32128961","title":"WDR63 inhibits Arp2/3-dependent actin polymerization and mediates the function of p53 in suppressing metastasis.","date":"2020","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/32128961","citation_count":25,"is_preprint":false},{"pmid":"9530342","id":"PMC_9530342","title":"Unbalanced chromosomal rearrangements in a metastasizing salivary gland tumor with benign histology.","date":"1998","source":"Cancer genetics and cytogenetics","url":"https://pubmed.ncbi.nlm.nih.gov/9530342","citation_count":17,"is_preprint":false},{"pmid":"29285825","id":"PMC_29285825","title":"Targeted copy number screening highlights an intragenic deletion of WDR63 as the likely cause of human occipital encephalocele and abnormal CNS development in zebrafish.","date":"2018","source":"Human mutation","url":"https://pubmed.ncbi.nlm.nih.gov/29285825","citation_count":14,"is_preprint":false},{"pmid":"25498833","id":"PMC_25498833","title":"Enriched trimethylation of lysine 4 of histone H3 of WDR63 enhanced osteogenic differentiation potentials of stem cells from apical papilla.","date":"2014","source":"Journal of endodontics","url":"https://pubmed.ncbi.nlm.nih.gov/25498833","citation_count":12,"is_preprint":false},{"pmid":"37966249","id":"PMC_37966249","title":"Identification of host essential factors for recombinant AAV transduction of the polarized human airway epithelium.","date":"2023","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/37966249","citation_count":12,"is_preprint":false},{"pmid":"19682577","id":"PMC_19682577","title":"Dic2 and Dic3 loci confer osmotic adaptation and fungicidal sensitivity independent of the HOG pathway in Cochliobolus heterostrophus.","date":"2009","source":"Mycological research","url":"https://pubmed.ncbi.nlm.nih.gov/19682577","citation_count":8,"is_preprint":false},{"pmid":"39827215","id":"PMC_39827215","title":"TCTEX1D2 is essential for sperm flagellum formation in mice.","date":"2025","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/39827215","citation_count":5,"is_preprint":false},{"pmid":"38499532","id":"PMC_38499532","title":"PTEN decreases NR2F1 expression to inhibit ciliogenesis during EGFRL858R-induced lung cancer progression.","date":"2024","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/38499532","citation_count":3,"is_preprint":false},{"pmid":"41640377","id":"PMC_41640377","title":"WDR63 Promotes Pulmonary Fibrosis Through Facilitating K63-Linked Ubiquitination of p53.","date":"2026","source":"Respirology (Carlton, Vic.)","url":"https://pubmed.ncbi.nlm.nih.gov/41640377","citation_count":0,"is_preprint":false},{"pmid":"41796360","id":"PMC_41796360","title":"WDR63 enhances the chondrogenic differentiation and regenerative potential of stem cell from apical papilla by facilitating vimentin function to promote mitochondrial fission.","date":"2026","source":"Stem cell research & therapy","url":"https://pubmed.ncbi.nlm.nih.gov/41796360","citation_count":0,"is_preprint":false},{"pmid":"37808760","id":"PMC_37808760","title":"Identification of Host Restriction Factors Critical for Recombinant AAV Transduction of Polarized Human Airway Epithelium.","date":"2023","source":"bioRxiv : the preprint server for 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development.","date":"2026","source":"PeerJ","url":"https://pubmed.ncbi.nlm.nih.gov/41868793","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11136,"output_tokens":2563,"usd":0.035927,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":19382,"output_tokens":6824,"usd":0.201877,"resynth_parse_fail":true,"stage2_stop_reason":"end_turn"},"total_usd":0.237804,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2021,\n      \"finding\": \"WDR63 (DNAI3) interacts with WDR78 primarily via its WD40-repeat domain and is required for inner dynein arm (IDA) assembly in sperm flagella; loss-of-function in mice (CRISPR-Cas9 knockout) causes disorganized '9+2' axoneme and absent IDAs, resulting in male infertility.\",\n      \"method\": \"Co-immunoprecipitation (WDR63–WDR78 interaction), CRISPR-Cas9 knockout mice, transmission electron microscopy of axoneme ultrastructure\",\n      \"journal\": \"Cell discovery\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP identifying interaction domain, KO mouse with defined ultrastructural phenotype, replicated in human patient cohort\",\n      \"pmids\": [\"34782613\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"WDR63 is the vertebrate orthologue of Chlamydomonas IC140, an intermediate chain of Dynein-f; it associates with Dynein-f subunits WDR78 (IC138 orthologue) and DNAH2 (heavy chain), and is expressed specifically in motile cilia-bearing cells. Unlike in Chlamydomonas, vertebrate Dynein-f (including WDR63) is essential for ciliary beat — depletion causes paralysis of mouse ependymal cilia.\",\n      \"method\": \"Co-immunoprecipitation (WDR63 with Dnah2 and Wdr78), RNAi knockdown in mouse ependymal cells, zebrafish morpholino knockdown, immunofluorescence localization\",\n      \"journal\": \"Journal of molecular cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, RNAi in mammalian cells, in vivo zebrafish morphants, replicated across two vertebrate models\",\n      \"pmids\": [\"30060180\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"WDR63 is transcriptionally upregulated by p53 and acts downstream of p53 to suppress cell migration, invasion, and metastasis. Mechanistically, WDR63 directly interacts with the Arp2/3 complex and inhibits Arp2/3-mediated branched actin polymerization.\",\n      \"method\": \"Chromatin immunoprecipitation / reporter assays (p53 transcriptional activation), Co-immunoprecipitation (WDR63–Arp2/3), in vitro actin polymerization assay, migration/invasion assays, mouse xenograft models\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro actin polymerization reconstitution plus Co-IP and functional migration/invasion assays in one study, single lab\",\n      \"pmids\": [\"32128961\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"WDR63 interacts with TCTEX1D2 as part of the inner dynein arm complex; Co-IP in testis confirmed WDR63 (along with WDR78) as a binding partner of TCTEX1D2, placing WDR63 in a network bridging cytoplasmic dynein 2 and axonemal inner dynein arm during sperm flagellum formation.\",\n      \"method\": \"Co-immunoprecipitation in mouse testes (TCTEX1D2 pulled down WDR63 and WDR78), Tctex1d2 knockout mice with flagellar phenotype analysis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single Co-IP in testes from one study; mechanistic placement is indirect (via TCTEX1D2 KO rather than WDR63 manipulation)\",\n      \"pmids\": [\"39827215\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"An in-frame deletion spanning WDR63 exons 14–17 (removing the 3rd and 4th WD-repeat domains) acts as a dominant-negative or gain-of-function variant; CRISPR/Cas9-mediated somatic recapitulation of this deletion in zebrafish caused brain and body malformations including sac-like brain protrusions, supporting a role for WDR63 in neural tube/encephalocele formation.\",\n      \"method\": \"CRISPR/Cas9 somatic deletion in zebrafish mimicking human deletion, overexpression of deleted vs. wild-type WDR63 RNA in zebrafish, whole-genome sequencing of human fetus\",\n      \"journal\": \"Human mutation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo zebrafish CRISPR and RNA overexpression experiments with quantified phenotype, single lab\",\n      \"pmids\": [\"29285825\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"WDR63 promotes K63-linked polyubiquitination of p53 while reducing its K48-linked polyubiquitination, thereby stabilising p53 protein and activating downstream p53 signalling in alveolar epithelial cells and lung fibroblasts, driving pulmonary fibrosis.\",\n      \"method\": \"Mass spectrometry and co-immunoprecipitation to identify p53 as a WDR63 substrate, ubiquitination assays (K63 vs K48 linkage-specific), WDR63 overexpression/silencing in cell lines, bleomycin-induced mouse fibrosis model\",\n      \"journal\": \"Respirology (Carlton, Vic.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — linkage-specific ubiquitination assays plus Co-IP/MS plus in vivo mouse model, single lab\",\n      \"pmids\": [\"41640377\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"WDR63 interacts and co-localizes with vimentin (VIM) in stem cells from apical papilla; WDR63 overexpression enhances VIM expression and WDR63–VIM binding, upregulates DRP1, and promotes mitochondrial fission, thereby enhancing chondrogenic differentiation.\",\n      \"method\": \"Co-immunoprecipitation and co-localization (WDR63–VIM), Western blot for DRP1 and chondrogenic markers, gain/loss-of-function in SCAPs, rabbit osteochondral defect transplantation model\",\n      \"journal\": \"Stem cell research & therapy\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP and co-localization experiment from one lab; mitochondrial fission link inferred from DRP1 protein level changes without direct reconstitution\",\n      \"pmids\": [\"41796360\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"WDR63 gain-of-function enhances alkaline phosphatase activity, mineralization, and expression of osteogenic markers BSP, OSX, and RUNX2 in stem cells from apical papilla (SCAPs), and promotes osteogenesis in vivo (nude mouse transplants), identifying WDR63 as a positive regulator of SCAP osteogenic differentiation.\",\n      \"method\": \"Gain-of-function and loss-of-function (overexpression/knockdown) in SCAPs, alkaline phosphatase assay, alizarin red/calcium quantification, gene expression analysis, in vivo nude mouse transplantation; H3K4Me3 ChIP-on-chip identified WDR63 promoter activation\",\n      \"journal\": \"Journal of endodontics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — gain/loss-of-function with functional readouts but no direct molecular mechanism identified; single lab, no binding partner established\",\n      \"pmids\": [\"25498833\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"WDR63 knockout (CRISPR) in human airway epithelial cells impairs rAAV2.5T vector transduction without affecting vector internalization or nuclear entry, indicating WDR63 acts at an intracellular trafficking or uncoating/conversion step post-nuclear entry.\",\n      \"method\": \"Genome-wide CRISPR screen followed by validation with individual CRISPR knockouts of WDR63 in polarized human airway epithelium; vector transduction, internalization, and nuclear entry assays\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO validated with functional assay dissecting distinct steps of AAV transduction; single lab but two independent papers (peer-reviewed + preprint) report same result\",\n      \"pmids\": [\"37966249\", \"37808760\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"WDR63 (DNAI3) is a WD40-repeat protein with multiple distinct cellular roles: it is an intermediate chain of the vertebrate inner dynein arm (Dynein-f) complex, where it interacts with WDR78 via its WD40 domain and is essential for axonemal assembly and ciliary/flagellar beating; downstream of p53 it binds and inhibits the Arp2/3 complex to suppress actin polymerization and cell migration; and it promotes p53 stability through K63-linked polyubiquitination to drive pulmonary fibrosis, while also influencing stem cell differentiation and AAV intracellular transduction in airway epithelium.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"WDR63 (DNAI3) is a WD40-repeat protein that functions as an intermediate chain of the vertebrate inner dynein arm (IDA), the Dynein-f complex that powers ciliary and flagellar motility [#1]. As the vertebrate orthologue of Chlamydomonas IC140, it associates with the IDA subunits WDR78 (IC138 orthologue) and the heavy chain DNAH2, and is expressed specifically in motile cilia-bearing cells; unlike its algal counterpart, vertebrate Dynein-f is essential for ciliary beat, as its depletion paralyzes mouse ependymal cilia [#1]. WDR63 binds WDR78 primarily through its WD40-repeat domain and is required for IDA assembly in sperm flagella, where loss of function produces disorganized '9+2' axonemes lacking IDAs and male infertility [#0], and it further bridges to cytoplasmic dynein 2 trafficking machinery through TCTEX1D2 during flagellum formation [#3]. Beyond the axoneme, WDR63 is a transcriptional target of p53 that acts downstream of p53 to suppress cell migration, invasion, and metastasis by directly binding the Arp2/3 complex and inhibiting branched actin polymerization [#2]. WDR63 also participates in human disease beyond cilia: an in-frame deletion removing WD-repeat domains acts as a dominant-negative/gain-of-function allele causing neural tube and encephalocele malformations modeled in zebrafish [#4], and WDR63 promotes K63-linked while reducing K48-linked polyubiquitination of p53 to stabilize it and drive pulmonary fibrosis [#5]. A CRISPR knockout in human airway epithelium additionally implicates WDR63 in an intracellular post-nuclear-entry step of rAAV vector transduction [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 2014,\n      \"claim\": \"Before any molecular partner was known, WDR63 was first linked to a cellular process through a functional screen identifying it as a positive regulator of stem cell osteogenic differentiation.\",\n      \"evidence\": \"Gain/loss-of-function in SCAPs with mineralization and osteogenic marker readouts, in vivo nude mouse transplantation, H3K4Me3 ChIP-on-chip\",\n      \"pmids\": [\"25498833\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No molecular mechanism or binding partner identified\", \"Single lab, functional readouts only\", \"No connection drawn to ciliary or dynein function\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"A human in-frame deletion plus zebrafish modeling established that WDR63 disruption causes neural tube/encephalocele malformations and that loss of specific WD-repeat domains can act as a dominant-negative allele.\",\n      \"evidence\": \"CRISPR/Cas9 somatic deletion and RNA overexpression in zebrafish, whole-genome sequencing of human fetus\",\n      \"pmids\": [\"29285825\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism of malformation unresolved\", \"Whether developmental role reflects ciliary defects not established\", \"Single lab\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"WDR63 was defined as the vertebrate orthologue of IC140 and an intermediate chain of the inner dynein arm Dynein-f, answering what protein complex it belongs to and showing the complex is essential for vertebrate ciliary beat.\",\n      \"evidence\": \"Reciprocal Co-IP with Dnah2 and Wdr78, RNAi in mouse ependymal cells, zebrafish morpholino knockdown, immunofluorescence\",\n      \"pmids\": [\"30060180\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural arrangement within the dynein arm not resolved\", \"Mechanism by which Dynein-f drives beat not detailed\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"An unexpected non-ciliary role was established: WDR63 is a p53 transcriptional target that inhibits Arp2/3-mediated branched actin polymerization to suppress migration and metastasis.\",\n      \"evidence\": \"ChIP/reporter assays, Co-IP with Arp2/3, in vitro actin polymerization reconstitution, migration/invasion assays, mouse xenografts\",\n      \"pmids\": [\"32128961\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of Arp2/3 inhibition not defined\", \"Single lab\", \"Relationship between cytoskeletal/actin role and axonemal role unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"The WDR63–WDR78 interaction was mapped to the WD40 domain and shown to be required for inner dynein arm assembly in sperm flagella, defining the structural basis of axonemal function and a male infertility phenotype.\",\n      \"evidence\": \"Reciprocal Co-IP identifying interaction domain, CRISPR-Cas9 knockout mice, transmission electron microscopy of axoneme ultrastructure, human patient cohort\",\n      \"pmids\": [\"34782613\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Order of assembly steps within the IDA not resolved\", \"How WD40 domain engages WDR78 structurally not determined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"A genome-wide screen placed WDR63 in an intracellular post-nuclear-entry step of rAAV transduction, separating its role from vector internalization and nuclear import.\",\n      \"evidence\": \"Genome-wide CRISPR screen with individual KO validation in polarized human airway epithelium; transduction, internalization, and nuclear entry assays\",\n      \"pmids\": [\"37966249\", \"37808760\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Precise trafficking/uncoating step not identified\", \"Mechanistic link to WDR63's dynein or actin functions not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"WDR63 was positioned in a network bridging cytoplasmic dynein 2 and the axonemal inner dynein arm via TCTEX1D2 during flagellum formation.\",\n      \"evidence\": \"Co-IP in mouse testes (TCTEX1D2 pulled down WDR63 and WDR78), Tctex1d2 knockout mice\",\n      \"pmids\": [\"39827215\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single Co-IP; placement inferred via TCTEX1D2 KO rather than WDR63 manipulation\", \"Functional dependency of WDR63 on TCTEX1D2 not directly tested\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"WDR63 was shown to control p53 protein stability through ubiquitin chain editing—promoting K63-linked while reducing K48-linked polyubiquitination—linking it to pulmonary fibrosis.\",\n      \"evidence\": \"MS/Co-IP identifying p53 as substrate, linkage-specific ubiquitination assays, overexpression/silencing, bleomycin-induced mouse fibrosis model\",\n      \"pmids\": [\"41640377\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether WDR63 has intrinsic E3 ligase activity or acts as an adaptor not established\", \"Single lab\", \"Reconciliation with the 2020 finding that p53 induces WDR63 (feedback architecture) not resolved\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"WDR63 was linked to vimentin binding, DRP1 upregulation, and mitochondrial fission promoting chondrogenic differentiation in stem cells.\",\n      \"evidence\": \"Co-IP and co-localization with VIM, Western blot for DRP1/chondrogenic markers, gain/loss-of-function in SCAPs, rabbit osteochondral defect model\",\n      \"pmids\": [\"41796360\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single Co-IP/co-localization from one lab; mitochondrial fission link inferred from DRP1 levels without direct reconstitution\", \"Direct VIM binding not validated reciprocally\", \"Mechanistic connection to other WDR63 functions absent\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single WD40 protein integrates its axonemal dynein-assembly role with its cytosolic actin/Arp2/3, p53-ubiquitin, and trafficking activities—and whether these are independent moonlighting functions or one mechanism—remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model unifying WD40-mediated interactions across complexes\", \"Tissue-specific partner switching not characterized\", \"Whether non-ciliary roles depend on the dynein-arm interaction unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 5]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 5]}\n    ],\n    \"complexes\": [\n      \"inner dynein arm (Dynein-f)\",\n      \"Arp2/3 complex\"\n    ],\n    \"partners\": [\n      \"WDR78\",\n      \"DNAH2\",\n      \"TCTEX1D2\",\n      \"TP53\",\n      \"VIM\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":6,"faith_total":6,"faith_pct":100.0}}