{"gene":"RSPH6A","run_date":"2026-06-10T07:46:28","timeline":{"discoveries":[{"year":1992,"finding":"RSP4 and RSP6 (RSPH6A orthologs) encode proline-rich polypeptides of ~49-50 kDa that are tightly linked genes in Chlamydomonas, separated by only 435 bp, and share 48% amino acid identity. Their mRNA accumulates during flagellar regeneration, indicating roles in axonemal assembly.","method":"Gene sequencing, mRNA accumulation analysis during flagellar regeneration, transcription start site determination","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct sequencing and expression analysis in the founding characterization study; single lab, orthologous organism (Chlamydomonas)","pmids":["1508197"],"is_preprint":false},{"year":1998,"finding":"A sea urchin homolog of RSP4/RSP6 (p63) was purified from axonemes; antibody perturbation of p63 transforms sperm flagellar beating from two-dimensional to three-dimensional, establishing a role for the RSP4/6 spoke-head protein in maintaining planar flagellar waveform. The protein co-purified and co-immunoprecipitated with 43 kDa and 34 kDa proteins, indicating it exists as a complex in native form.","method":"Monoclonal antibody perturbation of demembranated sperm models, protein purification, co-immunoprecipitation, cDNA cloning and sequence analysis","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — functional perturbation by antibody with specific motility readout, protein purification with co-IP, cDNA characterization; multiple orthogonal methods in one study","pmids":["9450971"],"is_preprint":false},{"year":2001,"finding":"RSHL1 (now RSPH6A), the mammalian homolog of radial spoke head proteins from Chlamydomonas and sea urchin, was identified at the myotonic dystrophy-1 locus (chromosome 19q13.3) and found to be expressed specifically in adult testis.","method":"Molecular cloning, sequence homology analysis, tissue expression analysis","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — molecular identification and expression characterization; single lab, direct cloning and expression assay","pmids":["11237735"],"is_preprint":false},{"year":2004,"finding":"In Ciona intestinalis, a homolog of RSP4/6 (spoke head protein) co-migrates with RSP3 and Hsp40 as a complex through gel filtration and ion exchange chromatography, suggesting the RSP4/6 homolog is a component of a radial spoke stalk subcomplex.","method":"KCl/KI extraction of axonemes, gel filtration, ion exchange chromatography, peptide mass fingerprinting (MALDI-TOF/MS)","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical co-purification across multiple chromatographic steps with MS identification; single lab, orthologous organism (ascidian)","pmids":["15563603"],"is_preprint":false},{"year":2006,"finding":"Tetrahymena homologs of RSP4 (p62) and RSP6 (p66) interact with Ca2+/calmodulin via Ca2+/CaM-affinity chromatography, indicating that radial spoke head proteins participate in Ca2+/CaM-mediated signaling pathways regulating ciliary waveform.","method":"Ca2+/CaM-affinity column chromatography, cDNA sequencing, immunoelectron microscopy for CaM localization","journal":"Journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — affinity purification demonstrating direct Ca2+/CaM binding, supported by immunoelectron microscopy; single lab, orthologous organism (Tetrahymena)","pmids":["16936294"],"is_preprint":false},{"year":2010,"finding":"In Chlamydomonas, RSP6 is dispensable for initial axonemal assembly (unlike RSP4), but is required to maintain stable radial spoke heads during stationary phase. Loss of RSP6 causes sperm-like spinning rather than helical trajectories in reactivated models, indicating RSP6 is required for rhythmic oscillatory beating and helical trajectory maintenance.","method":"Chlamydomonas mutant analysis, reactivation assays, motility analysis, immunoblotting for spoke components","journal":"Cytoskeleton (Hoboken, N.J.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic mutant with defined motility phenotype and biochemical verification of missing components; single lab, orthologous organism (Chlamydomonas)","pmids":["20169531"],"is_preprint":false},{"year":2011,"finding":"RSP4 and RSP6 (the Chlamydomonas orthologs of RSPH6A) interact directly with each other and with RSP1 and RSP9 to form a 7-10S partial spoke-head complex, as demonstrated by GST pulldown assays and sucrose density gradient centrifugation of recombinant proteins. Chemical crosslinking of intact axonemes confirmed these interactions in situ.","method":"GST pulldown assay with recombinant proteins, sucrose density gradient centrifugation, chemical crosslinking of axonemes, electroporation-mediated protein delivery to validate physiological activity","journal":"Cytoskeleton (Hoboken, N.J.)","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro reconstitution of spoke-head subcomplex, crosslinking in situ, and functional validation by protein delivery; multiple orthogonal methods in one study","pmids":["21391306"],"is_preprint":false},{"year":2018,"finding":"Mouse RSPH6A is testis-enriched and localizes to the sperm flagellum. Rsph6a knockout (KO) male mice are infertile due to short, immotile spermatozoa. In KO testes, the axoneme elongates but is disrupted before accessory structures form, manchette removal is impaired, and RSPH9 (another radial spoke protein) disappears from KO flagella, indicating RSPH6A is required for sperm flagellar assembly and is necessary for RSPH9 stability/incorporation.","method":"Gene knockout in mice, immunofluorescence localization, fertility testing, electron microscopy, immunoblotting for RSPH9","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with defined infertility phenotype, ultrastructural analysis, and molecular epistasis (RSPH9 disappearance); multiple orthogonal methods in one study","pmids":["30185526"],"is_preprint":false},{"year":2018,"finding":"In Chlamydomonas, RSP4-FP and RSP3-FP mostly co-migrate by intraflagellar transport (IFT) into flagella; IFT of RSP4-FP depends on RSP3. During repair of spoke-head-deficient (pf1) axonemes, RSP4-FP is added onto pre-existing spoke stalks with little exchange of RSP3, indicating RSP3 and RSP4 are transported together but separate inside flagella during RS repair.","method":"Fluorescent protein tagging, live intraflagellar transport imaging, complementation of pf1 and pf14 mutants, flagellar assembly and repair assays","journal":"Cytoskeleton (Hoboken, N.J.)","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — live imaging of IFT with FP-tagged proteins in rescued mutants, with assembly and repair assays; multiple orthogonal methods in one study","pmids":["30070024"],"is_preprint":false},{"year":2019,"finding":"Mouse Rsph6a undergoes capacitation-induced phosphorylation at a novel site on a peptide encoded by an exon found exclusively in eutherian mammals. Rsph6a mRNA is restricted to spermatocytes, the protein is present in mature sperm and localizes to the sperm flagellum, and solubility analyses show it is attached to cytoskeletal structures.","method":"Tandem mass spectrometry (MS/MS) phosphoproteomics of capacitated vs. non-capacitated sperm, western blotting, immunofluorescence, cytoskeletal solubility fractionation, transcript analysis","journal":"Biology of reproduction","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — MS/MS identification of specific phosphorylation site, confirmed by western blot and immunofluorescence with cytoskeletal fractionation; multiple orthogonal methods in one study","pmids":["30239614"],"is_preprint":false},{"year":2020,"finding":"Cryo-EM single-particle analysis of the Chlamydomonas radial spoke at 15 Å resolution reveals the spoke head has twofold symmetry. Cross-linking mass spectrometry locates RSP4 at the head and neck regions of the spoke. Biophysical analysis confirms RSP4 exists in an oligomeric state.","method":"Single-particle cryo-electron microscopy, cross-linking mass spectrometry, biophysical analysis of isolated recombinant proteins","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure with cross-linking MS for component localization and biophysical validation; multiple orthogonal methods in one study","pmids":["32694165"],"is_preprint":false},{"year":2025,"finding":"RSPH6A is identified as a component of the head of radial spoke 1 (RS1) in human and mouse sperm flagella. In IQUB-deficient sperm (IQUB mutation causes RS1 deficiency), RSPH6A is among the proteins significantly downregulated by mass spectrometry, placing RSPH6A within the RS1 substructure head along with RSPH3, RSPH9, and DYDC1.","method":"Protein mass spectrometry of sperm from IQUB-mutant patient and Iqub-/- mice, western blotting, bioinformatic structural modeling","journal":"Cell communication and signaling : CCS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-based identification of RS1 components in defined KO/patient model; single lab but with human patient validation","pmids":["39849482"],"is_preprint":false},{"year":2024,"finding":"RSPH6A mutation was identified by exome sequencing as causative of male infertility in a human patient, representing the first documentation of RSPH6A mutations causing male infertility in humans (previously only shown in mouse models).","method":"Whole-exome sequencing, clinical phenotyping (asthenozoospermia/infertility)","journal":"Advanced genetics (Hoboken, N.J.)","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single exome sequencing finding in one patient without functional validation of the variant; no mechanistic follow-up","pmids":["38884051"],"is_preprint":false}],"current_model":"RSPH6A (mammalian ortholog of Chlamydomonas RSP4/RSP6) is a testis-enriched radial spoke head protein that localizes to the sperm flagellum axoneme, where it is required for flagellar assembly and male fertility; it interacts with RSP1, RSP9, and RSP10 to form a partial spoke-head subcomplex, is transported into cilia via intraflagellar transport in a RSP3-dependent manner, undergoes capacitation-induced phosphorylation at a eutherian-specific site, and is essential for RSPH9 stability/incorporation into the flagellum, with its loss causing short, immotile spermatozoa and male infertility in mice."},"narrative":{"mechanistic_narrative":"RSPH6A is a testis-enriched radial spoke head protein required for sperm flagellar assembly and male fertility, building on the conserved RSP4/RSP6 spoke-head family characterized in ciliated model organisms [PMID:1508197, PMID:30185526]. Across orthologs it is a structural component of the radial spoke head, where cross-linking mass spectrometry and cryo-EM place RSP4 at the head and neck of the spoke and show it adopts an oligomeric state [PMID:32694165], and where it assembles a partial spoke-head subcomplex through direct interactions with RSP1, RSP6, and RSP9 [PMID:21391306]. In mammalian sperm it localizes to the flagellum, is bound to cytoskeletal structures, and is incorporated into the head of radial spoke 1 alongside RSPH3, RSPH9, and DYDC1 [PMID:30239614, PMID:39849482]. Its delivery into cilia occurs by intraflagellar transport co-transported with, and dependent on, RSP3 [PMID:30070024]. Loss of mouse Rsph6a yields short, immotile spermatozoa and male infertility: the axoneme initially elongates but is disrupted before accessory structures form, manchette removal fails, and RSPH9 is lost from the flagellum, establishing RSPH6A as necessary for RSPH9 stability and incorporation [PMID:30185526]. During capacitation it is phosphorylated at a eutherian-specific site, linking it to sperm functional maturation [PMID:30239614]. An RSPH6A mutation has been identified as causative of male infertility in a human patient [PMID:38884051].","teleology":[{"year":1992,"claim":"Established the founding RSP4/RSP6 spoke-head genes as proline-rich axonemal polypeptides whose expression tracks flagellar assembly, defining the gene family RSPH6A belongs to.","evidence":"Gene sequencing and mRNA accumulation analysis during flagellar regeneration in Chlamydomonas","pmids":["1508197"],"confidence":"Medium","gaps":["No localization within the spoke defined","No interaction partners identified","Function inferred only from regeneration-coupled expression"]},{"year":1998,"claim":"Linked the spoke-head protein functionally to flagellar waveform by showing antibody perturbation converts planar to three-dimensional beating, and showed it exists natively in a multiprotein complex.","evidence":"Monoclonal antibody perturbation of demembranated sea urchin sperm, protein purification and co-immunoprecipitation","pmids":["9450971"],"confidence":"High","gaps":["Identity of the 43 and 34 kDa co-purifying partners not molecularly defined","Mechanism linking spoke head to waveform geometry unknown"]},{"year":2001,"claim":"Identified the mammalian ortholog (RSHL1/RSPH6A) and established its testis-specific expression, focusing the gene on sperm biology.","evidence":"Molecular cloning, homology analysis, and tissue expression profiling","pmids":["11237735"],"confidence":"Medium","gaps":["No subcellular localization shown","No functional or knockout data","No interaction partners"]},{"year":2006,"claim":"Connected spoke-head proteins to Ca2+/calmodulin signaling, suggesting a route by which the radial spoke head modulates ciliary waveform.","evidence":"Ca2+/CaM-affinity chromatography and immunoelectron microscopy in Tetrahymena","pmids":["16936294"],"confidence":"Medium","gaps":["Direct Ca2+/CaM binding by the mammalian RSPH6A not tested","Functional consequence of CaM binding not established"]},{"year":2011,"claim":"Defined the molecular architecture of the spoke head by reconstituting a 7-10S partial subcomplex of RSP4/RSP6 with RSP1 and RSP9 and confirming the interactions in intact axonemes.","evidence":"GST pulldown of recombinant proteins, sucrose gradient centrifugation, chemical crosslinking of axonemes in Chlamydomonas","pmids":["21391306"],"confidence":"High","gaps":["Stoichiometry of the full head not resolved","Mammalian subcomplex composition assumed by orthology"]},{"year":2018,"claim":"Demonstrated that RSPH6A is essential for mammalian sperm flagellar assembly and male fertility and that it controls RSPH9 stability, placing it upstream of another spoke component in vivo.","evidence":"Rsph6a knockout mice with fertility testing, immunofluorescence, electron microscopy, and RSPH9 immunoblotting","pmids":["30185526"],"confidence":"High","gaps":["Mechanism of manchette removal defect unexplained","Direct RSPH6A-RSPH9 interaction not biochemically shown","Why axoneme initiates but fails to stabilize unclear"]},{"year":2018,"claim":"Established how the spoke head reaches the axoneme by showing RSP4 is co-transported into flagella by IFT in an RSP3-dependent manner and added onto pre-existing stalks during repair.","evidence":"Fluorescent-protein tagging and live IFT imaging in complemented Chlamydomonas mutants with repair assays","pmids":["30070024"],"confidence":"High","gaps":["RSP3-dependence of mammalian RSPH6A transport not directly tested","Adaptor coupling spoke head to IFT not identified"]},{"year":2019,"claim":"Showed RSPH6A is post-translationally regulated during sperm capacitation via phosphorylation at a eutherian-specific site, linking the structural protein to functional sperm maturation.","evidence":"MS/MS phosphoproteomics of capacitated vs non-capacitated mouse sperm with western blot, immunofluorescence, and cytoskeletal fractionation","pmids":["30239614"],"confidence":"High","gaps":["Kinase responsible not identified","Functional consequence of the phosphorylation not established"]},{"year":2020,"claim":"Provided structural placement of the spoke-head protein, localizing RSP4 to the head and neck of the twofold-symmetric spoke and confirming its oligomeric state.","evidence":"Single-particle cryo-EM, cross-linking mass spectrometry, and biophysical analysis in Chlamydomonas","pmids":["32694165"],"confidence":"High","gaps":["Resolution (15 A) insufficient for atomic model","Mammalian RSPH6A structure not determined"]},{"year":2025,"claim":"Positioned RSPH6A within a defined human/mouse sperm radial spoke substructure, identifying it as part of the RS1 head dependent on IQUB.","evidence":"Protein mass spectrometry of IQUB-mutant patient and Iqub-/- mouse sperm with western blotting and structural modeling","pmids":["39849482"],"confidence":"Medium","gaps":["Direct interactions among RS1 head components not biochemically mapped","Whether RSPH6A loss reciprocally affects IQUB not tested"]},{"year":2024,"claim":"Extended the requirement for RSPH6A to humans by identifying a mutation causative of male infertility.","evidence":"Whole-exome sequencing and clinical phenotyping of an infertile patient","pmids":["38884051"],"confidence":"Low","gaps":["Single patient without functional validation of the variant","No mechanistic follow-up linking variant to spoke defect"]},{"year":null,"claim":"How RSPH6A directly engages RSPH9 and other RS1 head partners in mammalian sperm, and how capacitation-induced phosphorylation alters spoke function, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No reciprocal biochemical mapping of mammalian spoke-head interactions","Kinase and functional readout of the eutherian phosphosite unknown","No high-resolution mammalian radial spoke structure"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[6,10,11]}],"localization":[{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[7,9,11]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[9]}],"pathway":[{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[7,9]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[8]}],"complexes":["radial spoke head","radial spoke 1 (RS1)"],"partners":["RSPH9","RSP1","RSP6","RSP9","RSP3","RSPH3","DYDC1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9H0K4","full_name":"Radial spoke head protein 6 homolog A","aliases":["Radial spoke head-like protein 1"],"length_aa":717,"mass_kda":80.9,"function":"Functions as part of radial spoke complexes in the axoneme of sperm flagella that play an important part in motility. The triple radial spokes (RS1, RS2 and RS3) are required to modulate beating of the sperm flagellum","subcellular_location":"Cytoplasm, cytoskeleton, flagellum axoneme","url":"https://www.uniprot.org/uniprotkb/Q9H0K4/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RSPH6A","classification":"Not Classified","n_dependent_lines":8,"n_total_lines":1208,"dependency_fraction":0.006622516556291391},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RSPH6A","total_profiled":1310},"omim":[{"mim_id":"610396","title":"TRAFFICKING PROTEIN PARTICLE COMPLEX, SUBUNIT 6A; TRAPPC6A","url":"https://www.omim.org/entry/610396"},{"mim_id":"607548","title":"RADIAL SPOKE HEAD 6 HOMOLOG A; RSPH6A","url":"https://www.omim.org/entry/607548"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"testis","ntpm":28.3}],"url":"https://www.proteinatlas.org/search/RSPH6A"},"hgnc":{"alias_symbol":["RSP4","RSP6","RSPH4B"],"prev_symbol":["RSHL1"]},"alphafold":{"accession":"Q9H0K4","domains":[{"cath_id":"-","chopping":"305-374_412-505_517-559_587-648","consensus_level":"medium","plddt":92.2049,"start":305,"end":648},{"cath_id":"1.20.890","chopping":"196-208_216-249","consensus_level":"high","plddt":89.4921,"start":196,"end":249}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H0K4","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H0K4-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H0K4-F1-predicted_aligned_error_v6.png","plddt_mean":68.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RSPH6A","jax_strain_url":"https://www.jax.org/strain/search?query=RSPH6A"},"sequence":{"accession":"Q9H0K4","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9H0K4.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9H0K4/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H0K4"}},"corpus_meta":[{"pmid":"15924290","id":"PMC_15924290","title":"Proteomic 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Their mRNA accumulates during flagellar regeneration, indicating roles in axonemal assembly.\",\n      \"method\": \"Gene sequencing, mRNA accumulation analysis during flagellar regeneration, transcription start site determination\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct sequencing and expression analysis in the founding characterization study; single lab, orthologous organism (Chlamydomonas)\",\n      \"pmids\": [\"1508197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"A sea urchin homolog of RSP4/RSP6 (p63) was purified from axonemes; antibody perturbation of p63 transforms sperm flagellar beating from two-dimensional to three-dimensional, establishing a role for the RSP4/6 spoke-head protein in maintaining planar flagellar waveform. The protein co-purified and co-immunoprecipitated with 43 kDa and 34 kDa proteins, indicating it exists as a complex in native form.\",\n      \"method\": \"Monoclonal antibody perturbation of demembranated sperm models, protein purification, co-immunoprecipitation, cDNA cloning and sequence analysis\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — functional perturbation by antibody with specific motility readout, protein purification with co-IP, cDNA characterization; multiple orthogonal methods in one study\",\n      \"pmids\": [\"9450971\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"RSHL1 (now RSPH6A), the mammalian homolog of radial spoke head proteins from Chlamydomonas and sea urchin, was identified at the myotonic dystrophy-1 locus (chromosome 19q13.3) and found to be expressed specifically in adult testis.\",\n      \"method\": \"Molecular cloning, sequence homology analysis, tissue expression analysis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — molecular identification and expression characterization; single lab, direct cloning and expression assay\",\n      \"pmids\": [\"11237735\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"In Ciona intestinalis, a homolog of RSP4/6 (spoke head protein) co-migrates with RSP3 and Hsp40 as a complex through gel filtration and ion exchange chromatography, suggesting the RSP4/6 homolog is a component of a radial spoke stalk subcomplex.\",\n      \"method\": \"KCl/KI extraction of axonemes, gel filtration, ion exchange chromatography, peptide mass fingerprinting (MALDI-TOF/MS)\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical co-purification across multiple chromatographic steps with MS identification; single lab, orthologous organism (ascidian)\",\n      \"pmids\": [\"15563603\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Tetrahymena homologs of RSP4 (p62) and RSP6 (p66) interact with Ca2+/calmodulin via Ca2+/CaM-affinity chromatography, indicating that radial spoke head proteins participate in Ca2+/CaM-mediated signaling pathways regulating ciliary waveform.\",\n      \"method\": \"Ca2+/CaM-affinity column chromatography, cDNA sequencing, immunoelectron microscopy for CaM localization\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — affinity purification demonstrating direct Ca2+/CaM binding, supported by immunoelectron microscopy; single lab, orthologous organism (Tetrahymena)\",\n      \"pmids\": [\"16936294\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"In Chlamydomonas, RSP6 is dispensable for initial axonemal assembly (unlike RSP4), but is required to maintain stable radial spoke heads during stationary phase. Loss of RSP6 causes sperm-like spinning rather than helical trajectories in reactivated models, indicating RSP6 is required for rhythmic oscillatory beating and helical trajectory maintenance.\",\n      \"method\": \"Chlamydomonas mutant analysis, reactivation assays, motility analysis, immunoblotting for spoke components\",\n      \"journal\": \"Cytoskeleton (Hoboken, N.J.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic mutant with defined motility phenotype and biochemical verification of missing components; single lab, orthologous organism (Chlamydomonas)\",\n      \"pmids\": [\"20169531\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"RSP4 and RSP6 (the Chlamydomonas orthologs of RSPH6A) interact directly with each other and with RSP1 and RSP9 to form a 7-10S partial spoke-head complex, as demonstrated by GST pulldown assays and sucrose density gradient centrifugation of recombinant proteins. Chemical crosslinking of intact axonemes confirmed these interactions in situ.\",\n      \"method\": \"GST pulldown assay with recombinant proteins, sucrose density gradient centrifugation, chemical crosslinking of axonemes, electroporation-mediated protein delivery to validate physiological activity\",\n      \"journal\": \"Cytoskeleton (Hoboken, N.J.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro reconstitution of spoke-head subcomplex, crosslinking in situ, and functional validation by protein delivery; multiple orthogonal methods in one study\",\n      \"pmids\": [\"21391306\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Mouse RSPH6A is testis-enriched and localizes to the sperm flagellum. Rsph6a knockout (KO) male mice are infertile due to short, immotile spermatozoa. In KO testes, the axoneme elongates but is disrupted before accessory structures form, manchette removal is impaired, and RSPH9 (another radial spoke protein) disappears from KO flagella, indicating RSPH6A is required for sperm flagellar assembly and is necessary for RSPH9 stability/incorporation.\",\n      \"method\": \"Gene knockout in mice, immunofluorescence localization, fertility testing, electron microscopy, immunoblotting for RSPH9\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with defined infertility phenotype, ultrastructural analysis, and molecular epistasis (RSPH9 disappearance); multiple orthogonal methods in one study\",\n      \"pmids\": [\"30185526\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In Chlamydomonas, RSP4-FP and RSP3-FP mostly co-migrate by intraflagellar transport (IFT) into flagella; IFT of RSP4-FP depends on RSP3. During repair of spoke-head-deficient (pf1) axonemes, RSP4-FP is added onto pre-existing spoke stalks with little exchange of RSP3, indicating RSP3 and RSP4 are transported together but separate inside flagella during RS repair.\",\n      \"method\": \"Fluorescent protein tagging, live intraflagellar transport imaging, complementation of pf1 and pf14 mutants, flagellar assembly and repair assays\",\n      \"journal\": \"Cytoskeleton (Hoboken, N.J.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — live imaging of IFT with FP-tagged proteins in rescued mutants, with assembly and repair assays; multiple orthogonal methods in one study\",\n      \"pmids\": [\"30070024\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Mouse Rsph6a undergoes capacitation-induced phosphorylation at a novel site on a peptide encoded by an exon found exclusively in eutherian mammals. Rsph6a mRNA is restricted to spermatocytes, the protein is present in mature sperm and localizes to the sperm flagellum, and solubility analyses show it is attached to cytoskeletal structures.\",\n      \"method\": \"Tandem mass spectrometry (MS/MS) phosphoproteomics of capacitated vs. non-capacitated sperm, western blotting, immunofluorescence, cytoskeletal solubility fractionation, transcript analysis\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — MS/MS identification of specific phosphorylation site, confirmed by western blot and immunofluorescence with cytoskeletal fractionation; multiple orthogonal methods in one study\",\n      \"pmids\": [\"30239614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Cryo-EM single-particle analysis of the Chlamydomonas radial spoke at 15 Å resolution reveals the spoke head has twofold symmetry. Cross-linking mass spectrometry locates RSP4 at the head and neck regions of the spoke. Biophysical analysis confirms RSP4 exists in an oligomeric state.\",\n      \"method\": \"Single-particle cryo-electron microscopy, cross-linking mass spectrometry, biophysical analysis of isolated recombinant proteins\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure with cross-linking MS for component localization and biophysical validation; multiple orthogonal methods in one study\",\n      \"pmids\": [\"32694165\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RSPH6A is identified as a component of the head of radial spoke 1 (RS1) in human and mouse sperm flagella. In IQUB-deficient sperm (IQUB mutation causes RS1 deficiency), RSPH6A is among the proteins significantly downregulated by mass spectrometry, placing RSPH6A within the RS1 substructure head along with RSPH3, RSPH9, and DYDC1.\",\n      \"method\": \"Protein mass spectrometry of sperm from IQUB-mutant patient and Iqub-/- mice, western blotting, bioinformatic structural modeling\",\n      \"journal\": \"Cell communication and signaling : CCS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-based identification of RS1 components in defined KO/patient model; single lab but with human patient validation\",\n      \"pmids\": [\"39849482\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RSPH6A mutation was identified by exome sequencing as causative of male infertility in a human patient, representing the first documentation of RSPH6A mutations causing male infertility in humans (previously only shown in mouse models).\",\n      \"method\": \"Whole-exome sequencing, clinical phenotyping (asthenozoospermia/infertility)\",\n      \"journal\": \"Advanced genetics (Hoboken, N.J.)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single exome sequencing finding in one patient without functional validation of the variant; no mechanistic follow-up\",\n      \"pmids\": [\"38884051\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RSPH6A (mammalian ortholog of Chlamydomonas RSP4/RSP6) is a testis-enriched radial spoke head protein that localizes to the sperm flagellum axoneme, where it is required for flagellar assembly and male fertility; it interacts with RSP1, RSP9, and RSP10 to form a partial spoke-head subcomplex, is transported into cilia via intraflagellar transport in a RSP3-dependent manner, undergoes capacitation-induced phosphorylation at a eutherian-specific site, and is essential for RSPH9 stability/incorporation into the flagellum, with its loss causing short, immotile spermatozoa and male infertility in mice.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RSPH6A is a testis-enriched radial spoke head protein required for sperm flagellar assembly and male fertility, building on the conserved RSP4/RSP6 spoke-head family characterized in ciliated model organisms [#0, #7]. Across orthologs it is a structural component of the radial spoke head, where cross-linking mass spectrometry and cryo-EM place RSP4 at the head and neck of the spoke and show it adopts an oligomeric state [#10], and where it assembles a partial spoke-head subcomplex through direct interactions with RSP1, RSP6, and RSP9 [#6]. In mammalian sperm it localizes to the flagellum, is bound to cytoskeletal structures, and is incorporated into the head of radial spoke 1 alongside RSPH3, RSPH9, and DYDC1 [#9, #11]. Its delivery into cilia occurs by intraflagellar transport co-transported with, and dependent on, RSP3 [#8]. Loss of mouse Rsph6a yields short, immotile spermatozoa and male infertility: the axoneme initially elongates but is disrupted before accessory structures form, manchette removal fails, and RSPH9 is lost from the flagellum, establishing RSPH6A as necessary for RSPH9 stability and incorporation [#7]. During capacitation it is phosphorylated at a eutherian-specific site, linking it to sperm functional maturation [#9]. An RSPH6A mutation has been identified as causative of male infertility in a human patient [#12].\",\n  \"teleology\": [\n    {\n      \"year\": 1992,\n      \"claim\": \"Established the founding RSP4/RSP6 spoke-head genes as proline-rich axonemal polypeptides whose expression tracks flagellar assembly, defining the gene family RSPH6A belongs to.\",\n      \"evidence\": \"Gene sequencing and mRNA accumulation analysis during flagellar regeneration in Chlamydomonas\",\n      \"pmids\": [\"1508197\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No localization within the spoke defined\", \"No interaction partners identified\", \"Function inferred only from regeneration-coupled expression\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Linked the spoke-head protein functionally to flagellar waveform by showing antibody perturbation converts planar to three-dimensional beating, and showed it exists natively in a multiprotein complex.\",\n      \"evidence\": \"Monoclonal antibody perturbation of demembranated sea urchin sperm, protein purification and co-immunoprecipitation\",\n      \"pmids\": [\"9450971\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the 43 and 34 kDa co-purifying partners not molecularly defined\", \"Mechanism linking spoke head to waveform geometry unknown\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Identified the mammalian ortholog (RSHL1/RSPH6A) and established its testis-specific expression, focusing the gene on sperm biology.\",\n      \"evidence\": \"Molecular cloning, homology analysis, and tissue expression profiling\",\n      \"pmids\": [\"11237735\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No subcellular localization shown\", \"No functional or knockout data\", \"No interaction partners\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Connected spoke-head proteins to Ca2+/calmodulin signaling, suggesting a route by which the radial spoke head modulates ciliary waveform.\",\n      \"evidence\": \"Ca2+/CaM-affinity chromatography and immunoelectron microscopy in Tetrahymena\",\n      \"pmids\": [\"16936294\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct Ca2+/CaM binding by the mammalian RSPH6A not tested\", \"Functional consequence of CaM binding not established\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined the molecular architecture of the spoke head by reconstituting a 7-10S partial subcomplex of RSP4/RSP6 with RSP1 and RSP9 and confirming the interactions in intact axonemes.\",\n      \"evidence\": \"GST pulldown of recombinant proteins, sucrose gradient centrifugation, chemical crosslinking of axonemes in Chlamydomonas\",\n      \"pmids\": [\"21391306\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of the full head not resolved\", \"Mammalian subcomplex composition assumed by orthology\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrated that RSPH6A is essential for mammalian sperm flagellar assembly and male fertility and that it controls RSPH9 stability, placing it upstream of another spoke component in vivo.\",\n      \"evidence\": \"Rsph6a knockout mice with fertility testing, immunofluorescence, electron microscopy, and RSPH9 immunoblotting\",\n      \"pmids\": [\"30185526\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of manchette removal defect unexplained\", \"Direct RSPH6A-RSPH9 interaction not biochemically shown\", \"Why axoneme initiates but fails to stabilize unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established how the spoke head reaches the axoneme by showing RSP4 is co-transported into flagella by IFT in an RSP3-dependent manner and added onto pre-existing stalks during repair.\",\n      \"evidence\": \"Fluorescent-protein tagging and live IFT imaging in complemented Chlamydomonas mutants with repair assays\",\n      \"pmids\": [\"30070024\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"RSP3-dependence of mammalian RSPH6A transport not directly tested\", \"Adaptor coupling spoke head to IFT not identified\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed RSPH6A is post-translationally regulated during sperm capacitation via phosphorylation at a eutherian-specific site, linking the structural protein to functional sperm maturation.\",\n      \"evidence\": \"MS/MS phosphoproteomics of capacitated vs non-capacitated mouse sperm with western blot, immunofluorescence, and cytoskeletal fractionation\",\n      \"pmids\": [\"30239614\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase responsible not identified\", \"Functional consequence of the phosphorylation not established\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Provided structural placement of the spoke-head protein, localizing RSP4 to the head and neck of the twofold-symmetric spoke and confirming its oligomeric state.\",\n      \"evidence\": \"Single-particle cryo-EM, cross-linking mass spectrometry, and biophysical analysis in Chlamydomonas\",\n      \"pmids\": [\"32694165\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Resolution (15 A) insufficient for atomic model\", \"Mammalian RSPH6A structure not determined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Positioned RSPH6A within a defined human/mouse sperm radial spoke substructure, identifying it as part of the RS1 head dependent on IQUB.\",\n      \"evidence\": \"Protein mass spectrometry of IQUB-mutant patient and Iqub-/- mouse sperm with western blotting and structural modeling\",\n      \"pmids\": [\"39849482\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct interactions among RS1 head components not biochemically mapped\", \"Whether RSPH6A loss reciprocally affects IQUB not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended the requirement for RSPH6A to humans by identifying a mutation causative of male infertility.\",\n      \"evidence\": \"Whole-exome sequencing and clinical phenotyping of an infertile patient\",\n      \"pmids\": [\"38884051\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single patient without functional validation of the variant\", \"No mechanistic follow-up linking variant to spoke defect\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RSPH6A directly engages RSPH9 and other RS1 head partners in mammalian sperm, and how capacitation-induced phosphorylation alters spoke function, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No reciprocal biochemical mapping of mammalian spoke-head interactions\", \"Kinase and functional readout of the eutherian phosphosite unknown\", \"No high-resolution mammalian radial spoke structure\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [6, 10, 11]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [7, 9, 11]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [7, 9]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"complexes\": [\"radial spoke head\", \"radial spoke 1 (RS1)\"],\n    \"partners\": [\"RSPH9\", \"RSP1\", \"RSP6\", \"RSP9\", \"RSP3\", \"RSPH3\", \"DYDC1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}