{"gene":"DNAAF6","run_date":"2026-04-28T17:46:02","timeline":{"discoveries":[{"year":2014,"finding":"Pih1d3 (mouse ortholog of DNAAF6) localizes to the cytoplasm of spermatogenic cells and is required for cytoplasmic preassembly of axonemal dynein complexes; Pih1d3-null mice show absent outer and inner dynein arms in sperm flagella, and Pih1d3 was found to interact with an intermediate chain of outer dynein arm (ODA) as well as with Hsp70 and Hsp90, suggesting it stabilizes and promotes dynein complex formation.","method":"Knockout mouse model, transmission electron microscopy, co-immunoprecipitation with ODA intermediate chain and Hsp70/Hsp90, immunofluorescence localization","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — clean KO with defined cellular phenotype plus reciprocal Co-IP showing chaperone interactions, single lab with multiple orthogonal methods","pmids":["24421334"],"is_preprint":false},{"year":2016,"finding":"PIH1D3 (DNAAF6) is involved in cytoplasmic preassembly of both outer dynein arms (ODA) and inner dynein arms (IDA) of cilia and sperm flagella; loss-of-function mutations cause absent ODAs and reduced-to-absent IDAs. PIH1D3 interacts and co-precipitates with cytoplasmic ODA/IDA assembly factors DNAAF2 and DNAAF4.","method":"Patient mutations (hemizygous loss-of-function), transmission electron microscopy of cilia, co-immunoprecipitation showing interaction with DNAAF2 and DNAAF4","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 2 — human genetic evidence with TEM phenotype plus reciprocal Co-IP with DNAAF2/DNAAF4, moderate evidence from single lab with orthogonal methods","pmids":["28041644"],"is_preprint":false},{"year":2017,"finding":"PIH1D3 (DNAAF6) is part of a complementary conserved R2TP-like HSP90 co-chaperone complex in the cytoplasm; large genomic deletions and point mutations cause X-linked PCD by disrupting early axonemal dynein assembly, affecting a subset of inner arm dyneins in particular.","method":"Genomic deletion/mutation analysis in patients, functional studies of dynein arm assembly, contextual placement within DNAAF2-DNAAF4-HSP90 (R2TP-like) complex","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — human genetic evidence with mechanistic pathway placement in R2TP-like complex, replicated across multiple patient families","pmids":["28176794"],"is_preprint":false},{"year":2020,"finding":"Novel hemizygous DNAAF6 variants (frameshift and missense) lead to DNAAF6 protein degradation (validated in HEK293T cells) and cause absence of outer and inner dynein arms in sperm flagella, confirming DNAAF6 is required for dynein arm assembly.","method":"Whole-exome sequencing, protein degradation assay in HEK293T cells, transmission electron microscopy and immunostaining of patient spermatozoa","journal":"Journal of assisted reproduction and genetics","confidence":"Medium","confidence_rationale":"Tier 2 — patient variants with functional validation (protein degradation) and TEM, single lab","pmids":["32170493"],"is_preprint":false},{"year":2022,"finding":"Drosophila Dnaaf6 (ortholog of human DNAAF6) associates with Dnaaf4 in an R2TP-like complex specifically in cells with motile cilia (chordotonal neurons and sperm); loss of Dnaaf6 impairs outer dynein arm and a subset of inner dynein arm assembly, causing loss of motile sperm and defective chordotonal neuron function.","method":"Drosophila genetics (knockout flies), protein interaction studies (co-association evidence), motility and functional assays for chordotonal neurons and sperm","journal":"Frontiers in genetics","confidence":"Medium","confidence_rationale":"Tier 2 — genetic loss-of-function with defined cellular phenotype plus protein complex association, single lab","pmids":["35873488"],"is_preprint":false},{"year":2023,"finding":"In PIH1D3-knockout rats, PIH1D3 (DNAAF6) interacts with proteins required for pre-assembly and uploading of both outer dynein arms (ODA) and inner dynein arms (IDA) in cilia; KO rats show situs inversus, hydrocephalus, defects in spermatocyte survival, and mucociliary clearance failure, and PIH1D3 deficiency leads to dysfunctional pre-assembly and loading of dynein arms.","method":"TALEN-mediated knockout rat model, biochemical co-immunoprecipitation of protein partners, histological and behavioral analysis, electron microscopy","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 — clean KO with multiple phenotypic readouts and Co-IP of interacting partners, single lab","pmids":["37900281"],"is_preprint":false},{"year":2024,"finding":"DNAAF6 interacts with LARP6 (La ribonucleoprotein 6) in biomolecular condensates called dynein axonemal particles in multiciliated cells (MCCs) of Xenopus embryos; DNAAF6 is required for high α-tubulin protein expression near the apical side of MCCs during ciliogenesis, and a DNAAF6 mutant that abolishes LARP6 binding cannot restore apical α-tubulin expression, placing LARP6-DNAAF6 interaction as necessary for this function.","method":"Xenopus morpholino knockdown, co-localization imaging of DNAAF6 and LARP6 in condensates, mutagenesis of LARP6-binding site, immunofluorescence for α-tubulin","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 — interaction in condensates plus mutagenesis rescue experiment, single lab with multiple orthogonal approaches","pmids":["38762183"],"is_preprint":false},{"year":2024,"finding":"Skewed X-chromosome inactivation (XCI) in heterozygous female carriers of pathogenic DNAAF6 mutations determines the proportion of cilia lacking dynein arms; approximately one-third (20-50%) of normal ciliated airway cells was sufficient to avoid severe PCD, directly linking DNAAF6 expression level to ciliary dynein arm assembly.","method":"DNA-methylation XCI studies, immunofluorescence and high-speed videomicroscopy of airway ciliated cells from carrier mothers, correlation of XCI pattern with clinical/ciliary phenotype","journal":"Journal of medical genetics","confidence":"Medium","confidence_rationale":"Tier 2 — direct functional demonstration linking gene dosage to dynein arm assembly phenotype, multiple methods in single cohort","pmids":["38408845"],"is_preprint":false}],"current_model":"DNAAF6 (PIH1D3) is a PIH1-domain-containing cytoplasmic protein that functions as part of an R2TP-like HSP90 co-chaperone complex (with DNAAF2 and DNAAF4) to pre-assemble axonemal outer and inner dynein arm complexes in the cytoplasm before their transport into cilia and flagella; it also interacts with Hsp70/Hsp90 chaperones and with LARP6 in biomolecular condensates, and loss of DNAAF6 causes failure of dynein arm assembly resulting in primary ciliary dyskinesia and male infertility."},"narrative":{"teleology":[{"year":2014,"claim":"The first functional characterization of Pih1d3 established that it is a cytoplasmic chaperone cofactor required for axonemal dynein arm preassembly, resolving the question of where and how dynein complexes are stabilized before ciliary transport.","evidence":"Pih1d3-knockout mice analyzed by TEM, immunofluorescence, and co-immunoprecipitation with ODA intermediate chain, Hsp70, and Hsp90","pmids":["24421334"],"confidence":"High","gaps":["Identity of the broader chaperone complex containing Pih1d3 was not defined","Relevance to human disease was not established","Mechanism by which Pih1d3 stabilizes dynein subunits was unclear"]},{"year":2016,"claim":"Human genetic studies demonstrated that DNAAF6 loss-of-function mutations cause primary ciliary dyskinesia and identified DNAAF2 and DNAAF4 as direct interaction partners, establishing DNAAF6 as part of a defined cytoplasmic dynein assembly module.","evidence":"Hemizygous patient mutations with TEM of cilia, co-immunoprecipitation of DNAAF6 with DNAAF2 and DNAAF4","pmids":["28041644"],"confidence":"High","gaps":["Whether the DNAAF2–DNAAF4–DNAAF6 module constitutes a formal R2TP-like complex was not resolved","Specific contribution of DNAAF6 versus its partners to IDA vs ODA assembly was unclear"]},{"year":2017,"claim":"Placement of DNAAF6 within a conserved R2TP-like HSP90 co-chaperone complex resolved the mechanistic framework linking PIH1-domain proteins to HSP90-mediated dynein arm assembly, and confirmed X-linked inheritance of PCD caused by DNAAF6 mutations.","evidence":"Genomic deletion and mutation analysis across multiple PCD families with functional pathway contextualization","pmids":["28176794"],"confidence":"High","gaps":["Structural basis of the R2TP-like complex containing DNAAF6 was not determined","The full catalog of dynein clients handled by this specific R2TP-like module was incomplete"]},{"year":2020,"claim":"Demonstration that pathogenic DNAAF6 missense variants lead to protein degradation provided mechanistic insight into how hypomorphic alleles cause dynein arm loss, extending the mutation spectrum.","evidence":"HEK293T overexpression-based protein stability assay and TEM/immunostaining of patient sperm","pmids":["32170493"],"confidence":"Medium","gaps":["Degradation pathway (proteasomal vs autophagic) for unstable variants was not identified","Whether partial protein stability allows residual dynein assembly was untested"]},{"year":2022,"claim":"Conservation of the Dnaaf6–Dnaaf4 R2TP-like complex in Drosophila confirmed that this module operates specifically in cells bearing motile cilia, ruling out a general chaperone function.","evidence":"Drosophila Dnaaf6 knockout with motility assays in chordotonal neurons and sperm, protein co-association studies","pmids":["35873488"],"confidence":"Medium","gaps":["Direct biochemical reconstitution of the Drosophila complex was not performed","Precise step at which Dnaaf6 acts (folding, multimerization, or handoff to IFT) was unresolved"]},{"year":2023,"claim":"A knockout rat model broadened the phenotypic spectrum of DNAAF6 deficiency to include situs inversus, hydrocephalus, and spermatocyte death, and identified additional interacting partners involved in dynein uploading to axonemes.","evidence":"TALEN-mediated Pih1d3 knockout rat with Co-IP, histology, electron microscopy, and behavioral analysis","pmids":["37900281"],"confidence":"Medium","gaps":["Identity of the specific uploading/transport factors that interact with DNAAF6 was not fully resolved","Whether spermatocyte death is a direct consequence of unassembled dynein or secondary stress was unclear"]},{"year":2024,"claim":"Discovery that DNAAF6 interacts with LARP6 in biomolecular condensates (dynein axonemal particles) and that this interaction is required for apical α-tubulin expression revealed an unexpected role in translational or post-translational tubulin regulation during ciliogenesis.","evidence":"Xenopus morpholino knockdown, LARP6-binding-deficient DNAAF6 mutant rescue, co-localization imaging in multiciliated cells","pmids":["38762183"],"confidence":"Medium","gaps":["Whether DNAAF6–LARP6 condensates regulate mRNA translation or protein stability of tubulin is unresolved","Relevance of LARP6 interaction to the canonical dynein preassembly function of DNAAF6 is unclear","Whether this condensate function is conserved in mammalian multiciliated cells is untested"]},{"year":2024,"claim":"Quantification of the relationship between X-inactivation skewing and ciliary phenotype in heterozygous DNAAF6 carriers established that ~20–50% of DNAAF6-expressing cells suffice to prevent clinical PCD, defining a threshold for cell-autonomous dynein assembly.","evidence":"DNA methylation-based XCI analysis, immunofluorescence, and high-speed videomicroscopy of carrier airway epithelial cells","pmids":["38408845"],"confidence":"Medium","gaps":["Whether dynein complexes can be transferred between DNAAF6-positive and DNAAF6-negative cells was not addressed","Threshold may differ across ciliated tissues (airway vs ependymal vs reproductive)"]},{"year":null,"claim":"The structural basis for DNAAF6 within the R2TP-like complex, the precise step it catalyzes during dynein folding and multimerization, and how its LARP6-dependent condensate function integrates with its canonical chaperone role remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No high-resolution structure of DNAAF6 or the DNAAF2–DNAAF4–DNAAF6–HSP90 complex exists","The relationship between the dynein preassembly and tubulin regulation functions is undefined","Whether DNAAF6 has additional non-ciliary roles has not been systematically tested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0044183","term_label":"protein folding chaperone","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,2,4]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,1,2]}],"pathway":[{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[0,1,2,4,5]}],"complexes":["R2TP-like co-chaperone complex (DNAAF2–DNAAF4–DNAAF6–HSP90)"],"partners":["DNAAF2","DNAAF4","HSPA1A","HSP90AA1","LARP6"],"other_free_text":[]},"mechanistic_narrative":"DNAAF6 (PIH1D3) is a cytoplasmic PIH1-domain protein that functions as a component of an R2TP-like HSP90 co-chaperone complex—together with DNAAF2 and DNAAF4—to mediate the preassembly of axonemal outer dynein arm (ODA) and inner dynein arm (IDA) complexes before their transport into cilia and flagella [PMID:24421334, PMID:28041644, PMID:28176794]. DNAAF6 physically interacts with Hsp70, Hsp90, and dynein intermediate chains during this cytoplasmic assembly process, and additionally associates with LARP6 in biomolecular condensates (dynein axonemal particles) in multiciliated cells, where the DNAAF6–LARP6 interaction is required for apical α-tubulin expression during ciliogenesis [PMID:24421334, PMID:38762183]. Loss-of-function mutations in DNAAF6 cause X-linked primary ciliary dyskinesia characterized by absent ODA and reduced/absent IDA, situs inversus, hydrocephalus, and male infertility, with the severity in heterozygous female carriers determined by the degree of skewed X-chromosome inactivation [PMID:28041644, PMID:28176794, PMID:38408845]."},"prefetch_data":{"uniprot":{"accession":"Q9NQM4","full_name":"Dynein axonemal assembly factor 6","aliases":["PIH1 domain-containing protein 3","Sarcoma antigen NY-SAR-97"],"length_aa":214,"mass_kda":24.1,"function":"Plays a role in cytoplasmic pre-assembly of axonemal dynein","subcellular_location":"Cytoplasm; Golgi apparatus, trans-Golgi network","url":"https://www.uniprot.org/uniprotkb/Q9NQM4/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/DNAAF6","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/DNAAF6","total_profiled":1310},"omim":[{"mim_id":"300991","title":"CILIARY DYSKINESIA, PRIMARY, 36, X-LINKED; CILD36","url":"https://www.omim.org/entry/300991"},{"mim_id":"300933","title":"DYNEIN, AXONEMAL, ASSEMBLY FACTOR 6; DNAAF6","url":"https://www.omim.org/entry/300933"},{"mim_id":"244400","title":"CILIARY DYSKINESIA, PRIMARY, 1; CILD1","url":"https://www.omim.org/entry/244400"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Golgi apparatus","reliability":"Approved"},{"location":"Primary cilium","reliability":"Approved"},{"location":"Acrosome","reliability":"Approved"},{"location":"Nucleoplasm","reliability":"Additional"},{"location":"Primary cilium tip","reliability":"Additional"},{"location":"Basal body","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"},{"location":"Equatorial segment","reliability":"Additional"}],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"choroid plexus","ntpm":3.8},{"tissue":"fallopian tube","ntpm":10.2},{"tissue":"testis","ntpm":11.1}],"url":"https://www.proteinatlas.org/search/DNAAF6"},"hgnc":{"alias_symbol":["MGC35261","NYSAR97","TWISTER"],"prev_symbol":["CXorf41","PIH1D3"]},"alphafold":{"accession":"Q9NQM4","domains":[{"cath_id":"2.60.40.790","chopping":"113-212","consensus_level":"high","plddt":87.0452,"start":113,"end":212}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NQM4","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NQM4-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NQM4-F1-predicted_aligned_error_v6.png","plddt_mean":73.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=DNAAF6","jax_strain_url":"https://www.jax.org/strain/search?query=DNAAF6"},"sequence":{"accession":"Q9NQM4","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NQM4.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NQM4/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NQM4"}},"corpus_meta":[{"pmid":"23704092","id":"PMC_23704092","title":"Safety and efficacy of imatinib cessation for CML patients with stable undetectable minimal residual disease: results from the TWISTER study.","date":"2013","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/23704092","citation_count":598,"is_preprint":false},{"pmid":"12064933","id":"PMC_12064933","title":"Analysis of alpha-helical coiled coils with the program TWISTER reveals a structural mechanism for stutter compensation.","date":"2002","source":"Journal of structural biology","url":"https://pubmed.ncbi.nlm.nih.gov/12064933","citation_count":221,"is_preprint":false},{"pmid":"28176794","id":"PMC_28176794","title":"X-linked primary ciliary dyskinesia due to mutations in the cytoplasmic axonemal dynein assembly factor PIH1D3.","date":"2017","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/28176794","citation_count":120,"is_preprint":false},{"pmid":"28041644","id":"PMC_28041644","title":"Mutations in PIH1D3 Cause X-Linked Primary Ciliary Dyskinesia with Outer and Inner Dynein Arm Defects.","date":"2016","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/28041644","citation_count":111,"is_preprint":false},{"pmid":"25038788","id":"PMC_25038788","title":"Crystal structure and mechanistic investigation of the twister ribozyme.","date":"2014","source":"Nature chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/25038788","citation_count":100,"is_preprint":false},{"pmid":"27670347","id":"PMC_27670347","title":"Twister ribozymes as highly versatile expression platforms for artificial riboswitches.","date":"2016","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/27670347","citation_count":68,"is_preprint":false},{"pmid":"25157168","id":"PMC_25157168","title":"Structural basis for the fast self-cleavage reaction catalyzed by the twister ribozyme.","date":"2014","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/25157168","citation_count":59,"is_preprint":false},{"pmid":"27461281","id":"PMC_27461281","title":"High-Throughput Mutational Analysis of a Twister Ribozyme.","date":"2016","source":"Angewandte Chemie (International ed. in English)","url":"https://pubmed.ncbi.nlm.nih.gov/27461281","citation_count":50,"is_preprint":false},{"pmid":"24421334","id":"PMC_24421334","title":"Pih1d3 is required for cytoplasmic preassembly of axonemal dynein in mouse sperm.","date":"2014","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/24421334","citation_count":49,"is_preprint":false},{"pmid":"26859432","id":"PMC_26859432","title":"Ribozyme Catalysis with a Twist: Active State of the Twister Ribozyme in Solution Predicted from Molecular Simulation.","date":"2016","source":"Journal of the American Chemical Society","url":"https://pubmed.ncbi.nlm.nih.gov/26859432","citation_count":48,"is_preprint":false},{"pmid":"27863022","id":"PMC_27863022","title":"Unwinding the twister ribozyme: from structure to mechanism.","date":"2016","source":"Wiley interdisciplinary reviews. RNA","url":"https://pubmed.ncbi.nlm.nih.gov/27863022","citation_count":35,"is_preprint":false},{"pmid":"20703089","id":"PMC_20703089","title":"Nucleostemin: Another nucleolar \"Twister\" of the p53-MDM2 loop.","date":"2010","source":"Cell cycle (Georgetown, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/20703089","citation_count":35,"is_preprint":false},{"pmid":"11178734","id":"PMC_11178734","title":"Twister mutant mice are defective for otogelin, a component specific to inner ear acellular membranes.","date":"2000","source":"Mammalian genome : official journal of the International Mammalian Genome Society","url":"https://pubmed.ncbi.nlm.nih.gov/11178734","citation_count":35,"is_preprint":false},{"pmid":"28598157","id":"PMC_28598157","title":"Pseudoknot Formation Seeds the Twister Ribozyme Cleavage Reaction Coordinate.","date":"2017","source":"Journal of the American Chemical Society","url":"https://pubmed.ncbi.nlm.nih.gov/28598157","citation_count":32,"is_preprint":false},{"pmid":"31328021","id":"PMC_31328021","title":"Cleaning Up Mechanistic Debris Generated by Twister Ribozymes Using Computational RNA Enzymology.","date":"2019","source":"ACS catalysis","url":"https://pubmed.ncbi.nlm.nih.gov/31328021","citation_count":25,"is_preprint":false},{"pmid":"28191925","id":"PMC_28191925","title":"Mechanistic Debris Generated by Twister Ribozymes.","date":"2017","source":"ACS chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/28191925","citation_count":21,"is_preprint":false},{"pmid":"32430319","id":"PMC_32430319","title":"Light-controlled twister ribozyme with single-molecule detection resolves RNA function in time and space.","date":"2020","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/32430319","citation_count":17,"is_preprint":false},{"pmid":"32170493","id":"PMC_32170493","title":"Novel DNAAF6 variants identified by whole-exome sequencing cause male infertility and primary ciliary dyskinesia.","date":"2020","source":"Journal of assisted reproduction and genetics","url":"https://pubmed.ncbi.nlm.nih.gov/32170493","citation_count":16,"is_preprint":false},{"pmid":"30102530","id":"PMC_30102530","title":"Cellular Small Molecules Contribute to Twister Ribozyme Catalysis.","date":"2018","source":"Journal of the American Chemical Society","url":"https://pubmed.ncbi.nlm.nih.gov/30102530","citation_count":12,"is_preprint":false},{"pmid":"11472843","id":"PMC_11472843","title":"The Drosophila gene twister, an orthologue of the yeast helicase SKI2, is differentially expressed during development.","date":"2001","source":"Mechanisms of development","url":"https://pubmed.ncbi.nlm.nih.gov/11472843","citation_count":11,"is_preprint":false},{"pmid":"33812756","id":"PMC_33812756","title":"A pediatric case of primary ciliary dyskinesia caused by novel copy number variation in PIH1D3.","date":"2021","source":"Auris, nasus, larynx","url":"https://pubmed.ncbi.nlm.nih.gov/33812756","citation_count":9,"is_preprint":false},{"pmid":"32053774","id":"PMC_32053774","title":"Mg2+ Impacts the Twister Ribozyme through Push-Pull Stabilization of Nonsequential Phosphate Pairs.","date":"2020","source":"Biophysical journal","url":"https://pubmed.ncbi.nlm.nih.gov/32053774","citation_count":9,"is_preprint":false},{"pmid":"32769092","id":"PMC_32769092","title":"Contributions and competition of Mg2+ and K+ in folding and stabilization of the Twister ribozyme.","date":"2020","source":"RNA (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/32769092","citation_count":9,"is_preprint":false},{"pmid":"35873488","id":"PMC_35873488","title":"Strongly Truncated Dnaaf4 Plays a Conserved Role in Drosophila Ciliary Dynein Assembly as Part of an R2TP-Like Co-Chaperone Complex With Dnaaf6.","date":"2022","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/35873488","citation_count":5,"is_preprint":false},{"pmid":"39665326","id":"PMC_39665326","title":"Grand canonical Monte Carlo and deep learning assisted enhanced sampling to characterize the distribution of Mg2+ and influence of the Drude polarizable force field on the stability of folded states of the twister ribozyme.","date":"2024","source":"The Journal of chemical physics","url":"https://pubmed.ncbi.nlm.nih.gov/39665326","citation_count":5,"is_preprint":false},{"pmid":"38134329","id":"PMC_38134329","title":"Flanking Sequence Cotranscriptionally Regulates Twister Ribozyme Activity.","date":"2023","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/38134329","citation_count":4,"is_preprint":false},{"pmid":"38408845","id":"PMC_38408845","title":"Skewed X-chromosome inactivation drives the proportion of DNAAF6-defective airway motile cilia and variable expressivity in primary ciliary dyskinesia.","date":"2024","source":"Journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/38408845","citation_count":4,"is_preprint":false},{"pmid":"37900281","id":"PMC_37900281","title":"PIH1D3-knockout rats exhibit full ciliopathy features and dysfunctional pre-assembly and loading of dynein arms in motile cilia.","date":"2023","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/37900281","citation_count":3,"is_preprint":false},{"pmid":"37305323","id":"PMC_37305323","title":"Influence of Mg2+ Distribution on the Stability of Folded States of the Twister Ribozyme Revealed Using Grand Canonical Monte Carlo and Generative Deep Learning Enhanced Sampling.","date":"2023","source":"ACS omega","url":"https://pubmed.ncbi.nlm.nih.gov/37305323","citation_count":3,"is_preprint":false},{"pmid":"38762183","id":"PMC_38762183","title":"The binding of LARP6 and DNAAF6 in biomolecular condensates influences ciliogenesis of multiciliated cells.","date":"2024","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/38762183","citation_count":2,"is_preprint":false},{"pmid":"32790122","id":"PMC_32790122","title":"Aminoglycoside antibiotics can inhibit or activate twister ribozyme cleavage.","date":"2020","source":"The FEBS journal","url":"https://pubmed.ncbi.nlm.nih.gov/32790122","citation_count":2,"is_preprint":false},{"pmid":"39636683","id":"PMC_39636683","title":"Minimal twister sister-like self-cleaving ribozymes in the human genome revealed by deep mutational scanning.","date":"2024","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/39636683","citation_count":2,"is_preprint":false},{"pmid":"40952004","id":"PMC_40952004","title":"Discovery of novel hammerhead, twister, and DVRz-associated circular RNAs in Vitaceae, Solanaceae, and Rosaceae.","date":"2025","source":"mSystems","url":"https://pubmed.ncbi.nlm.nih.gov/40952004","citation_count":1,"is_preprint":false},{"pmid":"32820521","id":"PMC_32820521","title":"[Analysis of PIH1D3 variant in a Chinese pedigree affected with primary ciliary dyskinesia].","date":"2020","source":"Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/32820521","citation_count":1,"is_preprint":false},{"pmid":"26552830","id":"PMC_26552830","title":"Screening of Genetic Switches Based on the Twister Ribozyme Motif.","date":"2016","source":"Methods in molecular biology (Clifton, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/26552830","citation_count":1,"is_preprint":false},{"pmid":"41480157","id":"PMC_41480157","title":"Case Report: Minigene assays reveal a novel DNAAF6 intronic variant as the key etiology for primary ciliary dyskinesia.","date":"2025","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/41480157","citation_count":0,"is_preprint":false},{"pmid":"41049184","id":"PMC_41049184","title":"Deciphering the complex interplay of waterlogging and anthracnose twister disease in onion (Allium cepa L.).","date":"2025","source":"Frontiers in plant science","url":"https://pubmed.ncbi.nlm.nih.gov/41049184","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.08.26.672456","title":"The Effect of SARS-COV-2 Protein Fragments on the Dimerization of α-Synuclein","date":"2025-08-26","source":"bioRxiv","url":"https://doi.org/10.1101/2025.08.26.672456","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.08.05.668614","title":"Unique unbiased median solution for even sample sizes","date":"2025-08-07","source":"bioRxiv","url":"https://doi.org/10.1101/2025.08.05.668614","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.10.24.25338751","title":"Speaking in Tones: The role of lexical tones in Chinese-speaking Primary Progressive Aphasia","date":"2025-10-27","source":"bioRxiv","url":"https://doi.org/10.1101/2025.10.24.25338751","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.04.15.648882","title":"Spatial activation of Kinesin-1 by Ensconsin shapes microtubule networks via ncMTOCs recruitment","date":"2025-04-16","source":"bioRxiv","url":"https://doi.org/10.1101/2025.04.15.648882","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.07.11.603121","title":"Direct testing of natural twister ribozymes from over a thousand organisms reveals a broad tolerance for structural imperfections","date":"2024-07-11","source":"bioRxiv","url":"https://doi.org/10.1101/2024.07.11.603121","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.05.16.594327","title":"Self-cleaving ribozymes conserved in RNA viruses unveil a new role in protein translation","date":"2024-05-16","source":"bioRxiv","url":"https://doi.org/10.1101/2024.05.16.594327","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":20207,"output_tokens":2201,"usd":0.046818},"stage2":{"model":"claude-opus-4-6","input_tokens":5485,"output_tokens":2511,"usd":0.1353},"total_usd":0.182118,"stage1_batch_id":"msgbatch_0118RScg3WGaGkaZRtWZbtce","stage2_batch_id":"msgbatch_01Cpfzvtbj42aqp5oYZ5Te2s","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2014,\n      \"finding\": \"Pih1d3 (mouse ortholog of DNAAF6) localizes to the cytoplasm of spermatogenic cells and is required for cytoplasmic preassembly of axonemal dynein complexes; Pih1d3-null mice show absent outer and inner dynein arms in sperm flagella, and Pih1d3 was found to interact with an intermediate chain of outer dynein arm (ODA) as well as with Hsp70 and Hsp90, suggesting it stabilizes and promotes dynein complex formation.\",\n      \"method\": \"Knockout mouse model, transmission electron microscopy, co-immunoprecipitation with ODA intermediate chain and Hsp70/Hsp90, immunofluorescence localization\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined cellular phenotype plus reciprocal Co-IP showing chaperone interactions, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"24421334\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PIH1D3 (DNAAF6) is involved in cytoplasmic preassembly of both outer dynein arms (ODA) and inner dynein arms (IDA) of cilia and sperm flagella; loss-of-function mutations cause absent ODAs and reduced-to-absent IDAs. PIH1D3 interacts and co-precipitates with cytoplasmic ODA/IDA assembly factors DNAAF2 and DNAAF4.\",\n      \"method\": \"Patient mutations (hemizygous loss-of-function), transmission electron microscopy of cilia, co-immunoprecipitation showing interaction with DNAAF2 and DNAAF4\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — human genetic evidence with TEM phenotype plus reciprocal Co-IP with DNAAF2/DNAAF4, moderate evidence from single lab with orthogonal methods\",\n      \"pmids\": [\"28041644\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PIH1D3 (DNAAF6) is part of a complementary conserved R2TP-like HSP90 co-chaperone complex in the cytoplasm; large genomic deletions and point mutations cause X-linked PCD by disrupting early axonemal dynein assembly, affecting a subset of inner arm dyneins in particular.\",\n      \"method\": \"Genomic deletion/mutation analysis in patients, functional studies of dynein arm assembly, contextual placement within DNAAF2-DNAAF4-HSP90 (R2TP-like) complex\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — human genetic evidence with mechanistic pathway placement in R2TP-like complex, replicated across multiple patient families\",\n      \"pmids\": [\"28176794\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Novel hemizygous DNAAF6 variants (frameshift and missense) lead to DNAAF6 protein degradation (validated in HEK293T cells) and cause absence of outer and inner dynein arms in sperm flagella, confirming DNAAF6 is required for dynein arm assembly.\",\n      \"method\": \"Whole-exome sequencing, protein degradation assay in HEK293T cells, transmission electron microscopy and immunostaining of patient spermatozoa\",\n      \"journal\": \"Journal of assisted reproduction and genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — patient variants with functional validation (protein degradation) and TEM, single lab\",\n      \"pmids\": [\"32170493\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Drosophila Dnaaf6 (ortholog of human DNAAF6) associates with Dnaaf4 in an R2TP-like complex specifically in cells with motile cilia (chordotonal neurons and sperm); loss of Dnaaf6 impairs outer dynein arm and a subset of inner dynein arm assembly, causing loss of motile sperm and defective chordotonal neuron function.\",\n      \"method\": \"Drosophila genetics (knockout flies), protein interaction studies (co-association evidence), motility and functional assays for chordotonal neurons and sperm\",\n      \"journal\": \"Frontiers in genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic loss-of-function with defined cellular phenotype plus protein complex association, single lab\",\n      \"pmids\": [\"35873488\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In PIH1D3-knockout rats, PIH1D3 (DNAAF6) interacts with proteins required for pre-assembly and uploading of both outer dynein arms (ODA) and inner dynein arms (IDA) in cilia; KO rats show situs inversus, hydrocephalus, defects in spermatocyte survival, and mucociliary clearance failure, and PIH1D3 deficiency leads to dysfunctional pre-assembly and loading of dynein arms.\",\n      \"method\": \"TALEN-mediated knockout rat model, biochemical co-immunoprecipitation of protein partners, histological and behavioral analysis, electron microscopy\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with multiple phenotypic readouts and Co-IP of interacting partners, single lab\",\n      \"pmids\": [\"37900281\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"DNAAF6 interacts with LARP6 (La ribonucleoprotein 6) in biomolecular condensates called dynein axonemal particles in multiciliated cells (MCCs) of Xenopus embryos; DNAAF6 is required for high α-tubulin protein expression near the apical side of MCCs during ciliogenesis, and a DNAAF6 mutant that abolishes LARP6 binding cannot restore apical α-tubulin expression, placing LARP6-DNAAF6 interaction as necessary for this function.\",\n      \"method\": \"Xenopus morpholino knockdown, co-localization imaging of DNAAF6 and LARP6 in condensates, mutagenesis of LARP6-binding site, immunofluorescence for α-tubulin\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — interaction in condensates plus mutagenesis rescue experiment, single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"38762183\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Skewed X-chromosome inactivation (XCI) in heterozygous female carriers of pathogenic DNAAF6 mutations determines the proportion of cilia lacking dynein arms; approximately one-third (20-50%) of normal ciliated airway cells was sufficient to avoid severe PCD, directly linking DNAAF6 expression level to ciliary dynein arm assembly.\",\n      \"method\": \"DNA-methylation XCI studies, immunofluorescence and high-speed videomicroscopy of airway ciliated cells from carrier mothers, correlation of XCI pattern with clinical/ciliary phenotype\",\n      \"journal\": \"Journal of medical genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct functional demonstration linking gene dosage to dynein arm assembly phenotype, multiple methods in single cohort\",\n      \"pmids\": [\"38408845\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"DNAAF6 (PIH1D3) is a PIH1-domain-containing cytoplasmic protein that functions as part of an R2TP-like HSP90 co-chaperone complex (with DNAAF2 and DNAAF4) to pre-assemble axonemal outer and inner dynein arm complexes in the cytoplasm before their transport into cilia and flagella; it also interacts with Hsp70/Hsp90 chaperones and with LARP6 in biomolecular condensates, and loss of DNAAF6 causes failure of dynein arm assembly resulting in primary ciliary dyskinesia and male infertility.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"DNAAF6 (PIH1D3) is a cytoplasmic PIH1-domain protein that functions as a component of an R2TP-like HSP90 co-chaperone complex—together with DNAAF2 and DNAAF4—to mediate the preassembly of axonemal outer dynein arm (ODA) and inner dynein arm (IDA) complexes before their transport into cilia and flagella [PMID:24421334, PMID:28041644, PMID:28176794]. DNAAF6 physically interacts with Hsp70, Hsp90, and dynein intermediate chains during this cytoplasmic assembly process, and additionally associates with LARP6 in biomolecular condensates (dynein axonemal particles) in multiciliated cells, where the DNAAF6–LARP6 interaction is required for apical α-tubulin expression during ciliogenesis [PMID:24421334, PMID:38762183]. Loss-of-function mutations in DNAAF6 cause X-linked primary ciliary dyskinesia characterized by absent ODA and reduced/absent IDA, situs inversus, hydrocephalus, and male infertility, with the severity in heterozygous female carriers determined by the degree of skewed X-chromosome inactivation [PMID:28041644, PMID:28176794, PMID:38408845].\",\n  \"teleology\": [\n    {\n      \"year\": 2014,\n      \"claim\": \"The first functional characterization of Pih1d3 established that it is a cytoplasmic chaperone cofactor required for axonemal dynein arm preassembly, resolving the question of where and how dynein complexes are stabilized before ciliary transport.\",\n      \"evidence\": \"Pih1d3-knockout mice analyzed by TEM, immunofluorescence, and co-immunoprecipitation with ODA intermediate chain, Hsp70, and Hsp90\",\n      \"pmids\": [\"24421334\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Identity of the broader chaperone complex containing Pih1d3 was not defined\",\n        \"Relevance to human disease was not established\",\n        \"Mechanism by which Pih1d3 stabilizes dynein subunits was unclear\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Human genetic studies demonstrated that DNAAF6 loss-of-function mutations cause primary ciliary dyskinesia and identified DNAAF2 and DNAAF4 as direct interaction partners, establishing DNAAF6 as part of a defined cytoplasmic dynein assembly module.\",\n      \"evidence\": \"Hemizygous patient mutations with TEM of cilia, co-immunoprecipitation of DNAAF6 with DNAAF2 and DNAAF4\",\n      \"pmids\": [\"28041644\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether the DNAAF2–DNAAF4–DNAAF6 module constitutes a formal R2TP-like complex was not resolved\",\n        \"Specific contribution of DNAAF6 versus its partners to IDA vs ODA assembly was unclear\"\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Placement of DNAAF6 within a conserved R2TP-like HSP90 co-chaperone complex resolved the mechanistic framework linking PIH1-domain proteins to HSP90-mediated dynein arm assembly, and confirmed X-linked inheritance of PCD caused by DNAAF6 mutations.\",\n      \"evidence\": \"Genomic deletion and mutation analysis across multiple PCD families with functional pathway contextualization\",\n      \"pmids\": [\"28176794\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of the R2TP-like complex containing DNAAF6 was not determined\",\n        \"The full catalog of dynein clients handled by this specific R2TP-like module was incomplete\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstration that pathogenic DNAAF6 missense variants lead to protein degradation provided mechanistic insight into how hypomorphic alleles cause dynein arm loss, extending the mutation spectrum.\",\n      \"evidence\": \"HEK293T overexpression-based protein stability assay and TEM/immunostaining of patient sperm\",\n      \"pmids\": [\"32170493\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Degradation pathway (proteasomal vs autophagic) for unstable variants was not identified\",\n        \"Whether partial protein stability allows residual dynein assembly was untested\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Conservation of the Dnaaf6–Dnaaf4 R2TP-like complex in Drosophila confirmed that this module operates specifically in cells bearing motile cilia, ruling out a general chaperone function.\",\n      \"evidence\": \"Drosophila Dnaaf6 knockout with motility assays in chordotonal neurons and sperm, protein co-association studies\",\n      \"pmids\": [\"35873488\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct biochemical reconstitution of the Drosophila complex was not performed\",\n        \"Precise step at which Dnaaf6 acts (folding, multimerization, or handoff to IFT) was unresolved\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"A knockout rat model broadened the phenotypic spectrum of DNAAF6 deficiency to include situs inversus, hydrocephalus, and spermatocyte death, and identified additional interacting partners involved in dynein uploading to axonemes.\",\n      \"evidence\": \"TALEN-mediated Pih1d3 knockout rat with Co-IP, histology, electron microscopy, and behavioral analysis\",\n      \"pmids\": [\"37900281\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Identity of the specific uploading/transport factors that interact with DNAAF6 was not fully resolved\",\n        \"Whether spermatocyte death is a direct consequence of unassembled dynein or secondary stress was unclear\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Discovery that DNAAF6 interacts with LARP6 in biomolecular condensates (dynein axonemal particles) and that this interaction is required for apical α-tubulin expression revealed an unexpected role in translational or post-translational tubulin regulation during ciliogenesis.\",\n      \"evidence\": \"Xenopus morpholino knockdown, LARP6-binding-deficient DNAAF6 mutant rescue, co-localization imaging in multiciliated cells\",\n      \"pmids\": [\"38762183\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether DNAAF6–LARP6 condensates regulate mRNA translation or protein stability of tubulin is unresolved\",\n        \"Relevance of LARP6 interaction to the canonical dynein preassembly function of DNAAF6 is unclear\",\n        \"Whether this condensate function is conserved in mammalian multiciliated cells is untested\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Quantification of the relationship between X-inactivation skewing and ciliary phenotype in heterozygous DNAAF6 carriers established that ~20–50% of DNAAF6-expressing cells suffice to prevent clinical PCD, defining a threshold for cell-autonomous dynein assembly.\",\n      \"evidence\": \"DNA methylation-based XCI analysis, immunofluorescence, and high-speed videomicroscopy of carrier airway epithelial cells\",\n      \"pmids\": [\"38408845\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether dynein complexes can be transferred between DNAAF6-positive and DNAAF6-negative cells was not addressed\",\n        \"Threshold may differ across ciliated tissues (airway vs ependymal vs reproductive)\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The structural basis for DNAAF6 within the R2TP-like complex, the precise step it catalyzes during dynein folding and multimerization, and how its LARP6-dependent condensate function integrates with its canonical chaperone role remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No high-resolution structure of DNAAF6 or the DNAAF2–DNAAF4–DNAAF6–HSP90 complex exists\",\n        \"The relationship between the dynein preassembly and tubulin regulation functions is undefined\",\n        \"Whether DNAAF6 has additional non-ciliary roles has not been systematically tested\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0044183\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 2, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 1, 2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [0, 1, 2, 4, 5]}\n    ],\n    \"complexes\": [\n      \"R2TP-like co-chaperone complex (DNAAF2–DNAAF4–DNAAF6–HSP90)\"\n    ],\n    \"partners\": [\n      \"DNAAF2\",\n      \"DNAAF4\",\n      \"HSPA1A\",\n      \"HSP90AA1\",\n      \"LARP6\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}