{"gene":"RSPH3","run_date":"2026-04-28T20:42:06","timeline":{"discoveries":[{"year":2001,"finding":"RSP3 (the Chlamydomonas ortholog of RSPH3) is an A-kinase anchoring protein (AKAP) located in the radial spoke stalk; the RII-binding domain was mapped to amino acids 144–180, which form an amphipathic helix, and amino acid substitutions (L to P or VL to AA) in the central residues abolish RII binding.","method":"RII blot overlay on motility mutants, truncation analysis of RSP3, in vitro mutagenesis","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 — in vitro binding assay with mutagenesis, multiple truncations, replicated in axoneme structural context","pmids":["11309423"],"is_preprint":false},{"year":1993,"finding":"RSP3 (Chlamydomonas ortholog) contains an axoneme-binding domain within amino acids 1–85 (minimal active region 42–85); deletion of this domain abolishes binding to spokeless (pf14) axonemes, and fusion of residues 1–85 to an unrelated protein confers binding activity.","method":"In vitro axoneme-binding assay with truncated/fusion RSP3 proteins; transformation of pf14 mutants with mutagenized RSP3 genes","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with deletion mutants and functional rescue in vivo","pmids":["8408197"],"is_preprint":false},{"year":2006,"finding":"Disruption of the PKA/RII-binding domain of RSP3 (Chlamydomonas ortholog) results in unregulated axonemal cAMP-dependent protein kinase (PKA) activity and abnormal/paralyzed flagellar motility; PKA inhibitors rescue paralysis, confirming RSP3 is the axonemal AKAP that spatially regulates PKA to control motility.","method":"Transformation of pf14 mutant with RII-binding domain point mutant RSP3; reactivation experiments with demembranated cells ± PKA inhibitors","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — genetic rescue combined with pharmacological epistasis and motility assays","pmids":["16571668"],"is_preprint":false},{"year":2008,"finding":"RSP3 (Chlamydomonas ortholog) forms a homodimer; the dimerization domain coincides with the N-terminal axoneme-binding domain. Each radial spoke is proposed to be built on an RSP3 dimer, enabling localization of multiple PKAs to control dynein activity.","method":"Chemical crosslinking, native gel electrophoresis, co-immunoprecipitation with epitope-tagged RSP3, truncation analysis","journal":"Cell motility and the cytoskeleton","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal biochemical methods in single study","pmids":["18157907"],"is_preprint":false},{"year":2009,"finding":"Mammalian RSPH3 binds active (phosphorylated) ERK1/2 and is a substrate for ERK1/2 phosphorylation; RSPH3 also scaffolds the PKA holoenzyme by binding RIIα and RIIβ regulatory subunits, and ERK1/2 activity/phosphorylation regulates the interaction between RSPH3 and PKA regulatory subunits.","method":"Yeast two-hybrid screen (active ERK2 bait), co-immunoprecipitation, in vitro kinase assay, RII overlay assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods: Y2H, Co-IP, in vitro phosphorylation, RII binding","pmids":["19684019"],"is_preprint":false},{"year":2012,"finding":"RSP3 (Chlamydomonas ortholog) functions as a dimeric structural scaffold that anchors four non-PKA radial spoke proteins (involved in spoke assembly and modulation) through two distinct amphipathic helices (AHs) that bind RIIa and Dpy-30 domains; one AH can bind both domain types in vitro.","method":"In vitro pull-down assays, domain-mapping with truncated RSP3, co-immunoprecipitation","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1–2 — structural scaffold model supported by multiple binding assays and domain mutagenesis","pmids":["23148234"],"is_preprint":false},{"year":2012,"finding":"LC8 dimers bind in tandem to the N-terminal region of RSP3 (Chlamydomonas ortholog) and enhance RSP3 binding to axonemes; perturbation of RSP3's LC8-binding sites causes hypophosphorylated RSP3, defective LC8–RS–axoneme associations, and asynchronous flagellar beating.","method":"Co-sedimentation, in vitro binding assay, axoneme pull-down with RSP3 N-terminal fragments, flagellar motility analysis of mutants","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — multiple biochemical and genetic methods with functional read-out","pmids":["22753897"],"is_preprint":false},{"year":2015,"finding":"Loss-of-function RSPH3 mutations in humans cause PCD with near-complete absence of radial spokes and central-complex defects; RSPH3 protein is undetectable in cilia of affected individuals, while RS-neck protein RSPH23 and RS-head proteins RSPH1 and RSPH4A remain present, placing RSPH3 as essential for RS stalk assembly and upstream of RS-head and neck assembly.","method":"Genetic identification of loss-of-function mutations, immunofluorescence of respiratory cilia, transmission electron microscopy, high-speed videomicroscopy","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 2 — loss-of-function with ultrastructural and protein localization phenotypes; epistasis by protein absence","pmids":["26073779"],"is_preprint":false},{"year":2014,"finding":"Mouse RSP3 (mammalian RSPH3 ortholog) localizes to the perinuclear region in CHO and 293T cells and accumulates in ependymal cilia; overexpression in developing cortex increases neurons reaching the upper cortical plate, and RSP3 accumulates in the leading process of migrating cortical neurons.","method":"Immunofluorescence, in utero electroporation, subcellular fractionation","journal":"Journal of molecular histology","confidence":"Medium","confidence_rationale":"Tier 2–3 — direct localization and gain-of-function with migration phenotype, but single-lab study","pmids":["25079589"],"is_preprint":false},{"year":2015,"finding":"Mouse RSP3 (mammalian RSPH3 ortholog) is a nucleocytoplasmic shuttling protein containing two nuclear localization signals and a nuclear export signal; overexpression in developing cerebral cortex promotes neurogenesis and thickening of cortical layer II/III; RSP3 localizes to primary cilia of radial glial cells where it acts as a signaling mediator.","method":"Deletion mutant analysis of NLS/NES, in utero electroporation, immunofluorescence of primary cilia","journal":"Histochemistry and cell biology","confidence":"Medium","confidence_rationale":"Tier 2–3 — domain mapping combined with in vivo gain-of-function; single-lab study","pmids":["26082196"],"is_preprint":false},{"year":2004,"finding":"In the ascidian Ciona, RSP3 co-migrates with HSP40 and a RSP4/6 homolog as a KI-extractable complex from axonemes; immunoelectron microscopy localizes HSP40 to the distal spoke stalk (junction between head and stalk), defining the RSP3-containing subcomplex as the radial spoke stalk.","method":"Biochemical fractionation (gel filtration, ion exchange), MALDI-TOF mass spectrometry, immunoelectron microscopy","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 — biochemical complex isolation with structural localization; ortholog study","pmids":["15563603"],"is_preprint":false},{"year":2019,"finding":"Drosophila dRSPH3 interacts with RSBP15 (a novel protein containing a DD_R_PKA superfamily domain) in sperm flagella; RSBP15 stabilizes dRSPH3, and loss of RSBP15 causes absence of mature sperm, defective individualization complex, and disrupted 9+2 axoneme structure; loss of dRSPH3 alone phenocopies rsbp15 knockout.","method":"Co-immunoprecipitation, co-localization by immunofluorescence, knockout phenotype analysis, transmission electron microscopy","journal":"Journal of genetics and genomics","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP and functional knockout with structural phenotype; Drosophila ortholog","pmids":["31281031"],"is_preprint":false},{"year":2019,"finding":"Drosophila RSP3 (dRSP3) interacts with Combover (Cmb), an effector of Rho kinase; knockdown of RSP3 in Drosophila causes defective sperm individualization with actin cones failing to move synchronously, phenocopying cmb mutants, and placing RSP3 as a component coordinating the individualization machinery with axonemes.","method":"Co-immunoprecipitation (Cmb binds RSP3), RNAi knockdown with actin cone motility assay","journal":"Development (Cambridge, England)","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP plus functional knockdown phenotype; Drosophila ortholog","pmids":["31391193"],"is_preprint":false},{"year":2023,"finding":"RSPH3 physically interacts with IQUB in the yeast two-hybrid system; IQUB deficiency in mice/humans results in radial spoke defects and asthenospermia; the proposed mechanism is that IQUB recruits calmodulin to inhibit RSPH3/p-ERK1/2 AKAP activity, facilitating normal radial spoke assembly.","method":"Yeast two-hybrid, co-immunoprecipitation, western blot, knockout and knockin mouse models, transmission electron microscopy","journal":"Human reproduction (Oxford, England)","confidence":"Medium","confidence_rationale":"Tier 2 — Y2H interaction confirmed by Co-IP, with KO/KI mouse phenotype, but proposed mechanism is partly inferential","pmids":["36355624"],"is_preprint":false},{"year":2023,"finding":"LRRC23 interacts with RSPH3 in vitro (co-immunoprecipitation), and LRRC23 loss-of-function in a human patient abolishes radial spoke integrity, indicating LRRC23 functions within the RS complex upstream of or alongside RSPH3.","method":"Co-immunoprecipitation, transmission electron microscopy, whole-exome sequencing","journal":"Clinical genetics","confidence":"Low","confidence_rationale":"Tier 3 — single Co-IP, limited mechanistic follow-up","pmids":["37804054"],"is_preprint":false},{"year":2025,"finding":"RSPH3 is identified as a component of the radial spoke 1 (RS1) head in mammalian sperm; mass spectrometry in Iqub-deficient sperm shows RS1 deficiency with loss of RSPH3 among other RS1 components (RSPH6A, RSPH9, DYDC1), placing RSPH3 specifically in the RS1 subtype.","method":"Protein mass spectrometry of RS fractions, western blotting, transmission electron microscopy in Iqub-/- mice","journal":"Cell communication and signaling : CCS","confidence":"Medium","confidence_rationale":"Tier 2 — proteomics combined with structural EM; direct biochemical localization to RS1","pmids":["39849482"],"is_preprint":false},{"year":2025,"finding":"In Chlamydomonas, I1 dynein deficiency (ida1) is epistatic to the RSP3 RII-binding domain mutation (388), establishing that I1 dynein acts downstream of the RSP3–PKA signaling axis in the pathway regulating ciliary motility.","method":"Genetic epistasis — double mutant 388;ida1 motility analysis compared with single mutants","journal":"microPublication biology","confidence":"Medium","confidence_rationale":"Tier 2 — clean genetic epistasis experiment defining pathway order; single study","pmids":["41487906"],"is_preprint":false},{"year":2022,"finding":"ARMC2/PF27 is required for intraflagellar transport (IFT) of radial spokes into Chlamydomonas flagella; RSP3 and ARMC2 co-migrate on anterograde IFT trains and are co-unloaded at the flagellar tip, after which RSP3 attaches to the axoneme while ARMC2 diffuses back; in armc2 mutants, RSP3 IFT is abolished and RS assembly is limited to the proximal flagellar region.","method":"Live fluorescence imaging of tagged proteins, IFT co-migration analysis, rescue genetics in armc2/pf27 mutants","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 — direct live imaging of co-transport with loss-of-function showing abolished RSP3 IFT; strong mechanistic evidence","pmids":["34982025"],"is_preprint":false}],"current_model":"RSPH3 is a conserved radial spoke stalk protein that forms a homodimer and functions as an A-kinase anchoring protein (AKAP), scaffolding PKA (via RII-binding amphipathic helices) and ERK1/2 at the base of radial spokes in motile cilia and sperm flagella; it is essential for the assembly of the entire radial spoke structure (particularly RS1 stalk and head), is transported into cilia by IFT trains via the adapter ARMC2, and in mammals also acts as a nucleocytoplasmic shuttling protein in neurons that promotes neurogenesis, with its PKA-anchoring function placing it upstream of I1 dynein in the central pair–radial spoke signaling pathway that regulates ciliary beat."},"narrative":{"teleology":[{"year":1993,"claim":"Identifying how RSP3 attaches to the axoneme answered where the radial spoke anchors and established that a discrete N-terminal domain (aa 1–85) is both necessary and sufficient for axoneme binding.","evidence":"In vitro axoneme-binding assays with truncated/fusion RSP3 proteins and transformation rescue of spokeless pf14 Chlamydomonas mutants","pmids":["8408197"],"confidence":"High","gaps":["Binding partner on the axoneme not identified","Whether binding is direct to outer doublet or requires adaptor proteins unknown"]},{"year":2001,"claim":"Demonstrating that RSP3 is an AKAP with a defined RII-binding amphipathic helix (aa 144–180) answered how PKA is positioned at the radial spoke and linked spoke-localized kinase signaling to motility regulation.","evidence":"RII blot overlay on axonemes from motility mutants, truncation and point mutagenesis of RSP3 in Chlamydomonas","pmids":["11309423"],"confidence":"High","gaps":["Whether mammalian RSPH3 AKAP function is conserved was not yet shown","Identity of PKA substrates at the spoke unknown"]},{"year":2004,"claim":"Isolation of RSP3 in a KI-extractable spoke stalk subcomplex with HSP40 and RSP4/6 in Ciona confirmed the stalk localization across species and identified co-migrating partners.","evidence":"Biochemical fractionation, MALDI-TOF mass spectrometry, and immunoelectron microscopy in ascidian sperm axonemes","pmids":["15563603"],"confidence":"Medium","gaps":["Direct stoichiometry of the subcomplex not determined","Whether HSP40 association is functionally relevant or a co-purification artifact unclear"]},{"year":2006,"claim":"Showing that disruption of the RSP3 RII-binding domain causes unregulated axonemal PKA and paralyzed motility—rescued by PKA inhibitors—established RSP3 as the functional AKAP that restrains PKA to permit normal flagellar beating.","evidence":"Transformation of pf14 with RII-binding domain point-mutant RSP3; demembranated cell reactivation ± PKA inhibitors in Chlamydomonas","pmids":["16571668"],"confidence":"High","gaps":["Downstream PKA substrates mediating the motility defect not identified","Whether other AKAPs compensate partially unknown"]},{"year":2008,"claim":"Demonstrating RSP3 homodimerization via the N-terminal axoneme-binding domain established that each radial spoke is built on a dimeric scaffold capable of positioning multiple signaling modules.","evidence":"Chemical crosslinking, native gel electrophoresis, and co-immunoprecipitation with epitope-tagged RSP3 in Chlamydomonas","pmids":["18157907"],"confidence":"High","gaps":["Dimer interface residues not resolved at atomic level","Whether dimerization is required for IFT transport unknown"]},{"year":2009,"claim":"Identification of mammalian RSPH3 as a dual-kinase scaffold—binding active ERK1/2 and PKA RIIα/RIIβ with ERK-dependent modulation of the PKA interaction—extended the AKAP model to mammals and revealed crosstalk between MAPK and PKA pathways at radial spokes.","evidence":"Yeast two-hybrid (active ERK2 bait), co-immunoprecipitation, in vitro kinase assay, RII overlay in mammalian cells","pmids":["19684019"],"confidence":"High","gaps":["Physiological consequence of ERK-PKA crosstalk on ciliary motility not tested","RSPH3 phosphorylation sites for ERK not mapped in vivo"]},{"year":2012,"claim":"Two discoveries resolved that RSP3's amphipathic helices recruit non-PKA spoke components via RIIa/Dpy-30 domains and that LC8 dimers bind the N-terminus to enhance axoneme association, collectively defining RSP3 as a multi-valent structural hub.","evidence":"In vitro pull-downs and domain mapping for spoke protein binding; co-sedimentation, axoneme pull-down, and flagellar motility analysis of LC8-binding mutants in Chlamydomonas","pmids":["23148234","22753897"],"confidence":"High","gaps":["Full inventory of proteins recruited by each amphipathic helix in vivo not established","Structural basis of LC8-mediated enhancement of axoneme binding unknown"]},{"year":2014,"claim":"Localization of mammalian RSPH3 to ependymal cilia and the perinuclear region, with overexpression promoting cortical neuron migration, suggested roles beyond canonical ciliary motility.","evidence":"Immunofluorescence, in utero electroporation, subcellular fractionation in mouse cortex and cell lines","pmids":["25079589"],"confidence":"Medium","gaps":["Loss-of-function neuronal phenotype not shown","Whether motility phenotype is cilia-dependent or cytoplasmic unclear"]},{"year":2015,"claim":"Two key advances: human RSPH3 loss-of-function mutations were shown to cause PCD with near-complete radial spoke absence, and mammalian RSPH3 was found to shuttle between nucleus and cytoplasm to promote neurogenesis, broadening its functional repertoire.","evidence":"Genetic studies in PCD families with immunofluorescence/TEM of respiratory cilia; NLS/NES deletion mapping and in utero electroporation in mouse cortex","pmids":["26073779","26082196"],"confidence":"High","gaps":["How RSPH3 nuclear shuttling relates to its AKAP function unknown","Genotype-phenotype correlation across different RSPH3 mutations not fully explored"]},{"year":2019,"claim":"Studies in Drosophila showed RSPH3 interacts with RSBP15 for stabilization and with Combover (Cmb, a Rho kinase effector) to coordinate sperm individualization, linking radial spoke function to actin-based processes in spermatogenesis.","evidence":"Co-immunoprecipitation, knockout/RNAi phenotype analysis, and TEM in Drosophila sperm","pmids":["31281031","31391193"],"confidence":"Medium","gaps":["Whether RSBP15 and Cmb interactions are conserved in mammals unknown","Direct binding domains on RSPH3 for these partners not mapped"]},{"year":2022,"claim":"Live imaging revealed that RSPH3 is co-transported with ARMC2 on anterograde IFT trains and deposited at the flagellar tip, solving how radial spoke precursors reach their assembly site.","evidence":"Live fluorescence imaging of tagged RSP3/ARMC2, IFT co-migration analysis, and rescue genetics in armc2/pf27 Chlamydomonas mutants","pmids":["34982025"],"confidence":"High","gaps":["Whether RSPH3 is transported as a preassembled spoke subcomplex or monomer/dimer unknown","Mechanism of tip unloading not defined"]},{"year":2023,"claim":"Identification of IQUB and LRRC23 as physical interactors placed RSPH3 in a calmodulin-regulated assembly pathway where IQUB recruits calmodulin to modulate RSPH3 AKAP activity for normal RS assembly.","evidence":"Yeast two-hybrid, co-immunoprecipitation, knockout/knockin mouse models with TEM and sperm motility phenotyping; WES and Co-IP for LRRC23","pmids":["36355624","37804054"],"confidence":"Medium","gaps":["Direct calmodulin binding to RSPH3 not demonstrated","LRRC23 interaction validated only by single Co-IP","Whether IQUB regulation of RSPH3 occurs during IFT or after axoneme incorporation unclear"]},{"year":2025,"claim":"Proteomics placed RSPH3 specifically in the RS1 head, and genetic epistasis showed I1 dynein acts downstream of the RSP3–PKA signaling axis, completing the pathway order from spoke-localized PKA to dynein regulation.","evidence":"Mass spectrometry of RS fractions from Iqub-/- mouse sperm; double-mutant motility analysis (388;ida1) in Chlamydomonas","pmids":["39849482","41487906"],"confidence":"Medium","gaps":["Whether RSPH3 is in the RS1 head versus stalk needs reconciliation with prior stalk assignment","PKA substrate on I1 dynein not identified","Whether RS2/RS3 also contain RSPH3 in mammals unknown"]},{"year":null,"claim":"Key unresolved questions include the atomic structure of the RSPH3 dimer and its multi-protein spoke scaffold, the identity of PKA substrates linking RSPH3 signaling to I1 dynein regulation, and whether the nuclear shuttling and neurogenesis functions depend on the same AKAP activity used in cilia.","evidence":"","pmids":[],"confidence":"Low","gaps":["No high-resolution structure of RSPH3 dimer or spoke complex available","PKA substrate identity on I1 dynein unknown","Relationship between ciliary and nuclear RSPH3 functions unexplored"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,2,4,5]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,4,16]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[1,3,5,6]}],"localization":[{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[1,2,7,8,10,17]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[1,6,10,15]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[9]}],"pathway":[{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[7,17]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,2,4,16]},{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[11,12]}],"complexes":["Radial spoke (RS1)","RSP3 homodimer"],"partners":["PRKAR2A","PRKAR2B","MAPK1","MAPK3","ARMC2","IQUB","DYNLL1","LRRC23"],"other_free_text":[]},"mechanistic_narrative":"RSPH3 is a conserved radial spoke stalk protein that functions as a dimeric A-kinase anchoring protein (AKAP) essential for radial spoke assembly, ciliary motility regulation, and flagellar beat coordination. The N-terminal domain mediates homodimerization and axoneme binding, while internal amphipathic helices scaffold PKA regulatory subunits (RIIα/RIIβ), additional spoke components via Dpy-30/RIIa domains, and active ERK1/2, integrating kinase signaling at the spoke base to control dynein-driven motility through I1 dynein as a downstream effector [PMID:11309423, PMID:18157907, PMID:19684019, PMID:23148234, PMID:41487906]. RSPH3 is transported into cilia on anterograde IFT trains via the adapter ARMC2/PF27 and is deposited at the axoneme tip, where it incorporates into the RS1 subtype of radial spokes [PMID:34982025, PMID:39849482]. Loss-of-function mutations in human RSPH3 cause primary ciliary dyskinesia (PCD) with near-complete absence of radial spokes and central-complex defects, establishing RSPH3 as essential for RS stalk integrity upstream of RS head and neck assembly [PMID:26082196, PMID:26073779]."},"prefetch_data":{"uniprot":{"accession":"Q86UC2","full_name":"Radial spoke head protein 3 homolog","aliases":["A-kinase anchor protein RSPH3","Radial spoke head-like protein 2"],"length_aa":560,"mass_kda":63.7,"function":"Functions as part of axonemal radial spoke complexes that play an important part in the motility of sperm and cilia (By similarity). Functions as a protein kinase A-anchoring protein that scaffolds the cAMP-dependent protein kinase holoenzyme. May serve as a point of convergence for MAPK and PKA signaling in cilia (PubMed:19684019)","subcellular_location":"Cytoplasm, cytoskeleton, cilium axoneme; Cytoplasm, cytoskeleton, flagellum axoneme","url":"https://www.uniprot.org/uniprotkb/Q86UC2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RSPH3","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1089,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RSPH3","total_profiled":1310},"omim":[{"mim_id":"620708","title":"LEUCINE-RICH REPEAT-CONTAINING PROTEIN 23; LRRC23","url":"https://www.omim.org/entry/620708"},{"mim_id":"620557","title":"IQ MOTIF- AND UBIQUITIN DOMAIN-CONTAINING PROTEIN; IQUB","url":"https://www.omim.org/entry/620557"},{"mim_id":"616481","title":"CILIARY DYSKINESIA, PRIMARY, 32; CILD32","url":"https://www.omim.org/entry/616481"},{"mim_id":"615876","title":"RADIAL SPOKE HEAD 3; RSPH3","url":"https://www.omim.org/entry/615876"},{"mim_id":"612010","title":"CELIAC DISEASE, SUSCEPTIBILITY TO, 12; CELIAC12","url":"https://www.omim.org/entry/612010"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"},{"location":"Flagellar centriole","reliability":"Approved"},{"location":"Mid piece","reliability":"Approved"},{"location":"Annulus","reliability":"Approved"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RSPH3"},"hgnc":{"alias_symbol":["dJ111C20.1","RSP3"],"prev_symbol":["RSHL2"]},"alphafold":{"accession":"Q86UC2","domains":[{"cath_id":"1.20.5","chopping":"311-423","consensus_level":"medium","plddt":96.1086,"start":311,"end":423},{"cath_id":"1.20.5","chopping":"426-472","consensus_level":"high","plddt":88.6147,"start":426,"end":472}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86UC2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q86UC2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q86UC2-F1-predicted_aligned_error_v6.png","plddt_mean":64.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RSPH3","jax_strain_url":"https://www.jax.org/strain/search?query=RSPH3"},"sequence":{"accession":"Q86UC2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q86UC2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q86UC2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86UC2"}},"corpus_meta":[{"pmid":"16738661","id":"PMC_16738661","title":"Hrr25-dependent 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Each radial spoke is proposed to be built on an RSP3 dimer, enabling localization of multiple PKAs to control dynein activity.\",\n      \"method\": \"Chemical crosslinking, native gel electrophoresis, co-immunoprecipitation with epitope-tagged RSP3, truncation analysis\",\n      \"journal\": \"Cell motility and the cytoskeleton\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal biochemical methods in single study\",\n      \"pmids\": [\"18157907\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Mammalian RSPH3 binds active (phosphorylated) ERK1/2 and is a substrate for ERK1/2 phosphorylation; RSPH3 also scaffolds the PKA holoenzyme by binding RIIα and RIIβ regulatory subunits, and ERK1/2 activity/phosphorylation regulates the interaction between RSPH3 and PKA regulatory subunits.\",\n      \"method\": \"Yeast two-hybrid screen (active ERK2 bait), co-immunoprecipitation, in vitro kinase assay, RII overlay assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods: Y2H, Co-IP, in vitro phosphorylation, RII binding\",\n      \"pmids\": [\"19684019\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"RSP3 (Chlamydomonas ortholog) functions as a dimeric structural scaffold that anchors four non-PKA radial spoke proteins (involved in spoke assembly and modulation) through two distinct amphipathic helices (AHs) that bind RIIa and Dpy-30 domains; one AH can bind both domain types in vitro.\",\n      \"method\": \"In vitro pull-down assays, domain-mapping with truncated RSP3, co-immunoprecipitation\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — structural scaffold model supported by multiple binding assays and domain mutagenesis\",\n      \"pmids\": [\"23148234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"LC8 dimers bind in tandem to the N-terminal region of RSP3 (Chlamydomonas ortholog) and enhance RSP3 binding to axonemes; perturbation of RSP3's LC8-binding sites causes hypophosphorylated RSP3, defective LC8–RS–axoneme associations, and asynchronous flagellar beating.\",\n      \"method\": \"Co-sedimentation, in vitro binding assay, axoneme pull-down with RSP3 N-terminal fragments, flagellar motility analysis of mutants\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple biochemical and genetic methods with functional read-out\",\n      \"pmids\": [\"22753897\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Loss-of-function RSPH3 mutations in humans cause PCD with near-complete absence of radial spokes and central-complex defects; RSPH3 protein is undetectable in cilia of affected individuals, while RS-neck protein RSPH23 and RS-head proteins RSPH1 and RSPH4A remain present, placing RSPH3 as essential for RS stalk assembly and upstream of RS-head and neck assembly.\",\n      \"method\": \"Genetic identification of loss-of-function mutations, immunofluorescence of respiratory cilia, transmission electron microscopy, high-speed videomicroscopy\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — loss-of-function with ultrastructural and protein localization phenotypes; epistasis by protein absence\",\n      \"pmids\": [\"26073779\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Mouse RSP3 (mammalian RSPH3 ortholog) localizes to the perinuclear region in CHO and 293T cells and accumulates in ependymal cilia; overexpression in developing cortex increases neurons reaching the upper cortical plate, and RSP3 accumulates in the leading process of migrating cortical neurons.\",\n      \"method\": \"Immunofluorescence, in utero electroporation, subcellular fractionation\",\n      \"journal\": \"Journal of molecular histology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — direct localization and gain-of-function with migration phenotype, but single-lab study\",\n      \"pmids\": [\"25079589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Mouse RSP3 (mammalian RSPH3 ortholog) is a nucleocytoplasmic shuttling protein containing two nuclear localization signals and a nuclear export signal; overexpression in developing cerebral cortex promotes neurogenesis and thickening of cortical layer II/III; RSP3 localizes to primary cilia of radial glial cells where it acts as a signaling mediator.\",\n      \"method\": \"Deletion mutant analysis of NLS/NES, in utero electroporation, immunofluorescence of primary cilia\",\n      \"journal\": \"Histochemistry and cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — domain mapping combined with in vivo gain-of-function; single-lab study\",\n      \"pmids\": [\"26082196\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"In the ascidian Ciona, RSP3 co-migrates with HSP40 and a RSP4/6 homolog as a KI-extractable complex from axonemes; immunoelectron microscopy localizes HSP40 to the distal spoke stalk (junction between head and stalk), defining the RSP3-containing subcomplex as the radial spoke stalk.\",\n      \"method\": \"Biochemical fractionation (gel filtration, ion exchange), MALDI-TOF mass spectrometry, immunoelectron microscopy\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — biochemical complex isolation with structural localization; ortholog study\",\n      \"pmids\": [\"15563603\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Drosophila dRSPH3 interacts with RSBP15 (a novel protein containing a DD_R_PKA superfamily domain) in sperm flagella; RSBP15 stabilizes dRSPH3, and loss of RSBP15 causes absence of mature sperm, defective individualization complex, and disrupted 9+2 axoneme structure; loss of dRSPH3 alone phenocopies rsbp15 knockout.\",\n      \"method\": \"Co-immunoprecipitation, co-localization by immunofluorescence, knockout phenotype analysis, transmission electron microscopy\",\n      \"journal\": \"Journal of genetics and genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP and functional knockout with structural phenotype; Drosophila ortholog\",\n      \"pmids\": [\"31281031\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Drosophila RSP3 (dRSP3) interacts with Combover (Cmb), an effector of Rho kinase; knockdown of RSP3 in Drosophila causes defective sperm individualization with actin cones failing to move synchronously, phenocopying cmb mutants, and placing RSP3 as a component coordinating the individualization machinery with axonemes.\",\n      \"method\": \"Co-immunoprecipitation (Cmb binds RSP3), RNAi knockdown with actin cone motility assay\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP plus functional knockdown phenotype; Drosophila ortholog\",\n      \"pmids\": [\"31391193\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RSPH3 physically interacts with IQUB in the yeast two-hybrid system; IQUB deficiency in mice/humans results in radial spoke defects and asthenospermia; the proposed mechanism is that IQUB recruits calmodulin to inhibit RSPH3/p-ERK1/2 AKAP activity, facilitating normal radial spoke assembly.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, western blot, knockout and knockin mouse models, transmission electron microscopy\",\n      \"journal\": \"Human reproduction (Oxford, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Y2H interaction confirmed by Co-IP, with KO/KI mouse phenotype, but proposed mechanism is partly inferential\",\n      \"pmids\": [\"36355624\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"LRRC23 interacts with RSPH3 in vitro (co-immunoprecipitation), and LRRC23 loss-of-function in a human patient abolishes radial spoke integrity, indicating LRRC23 functions within the RS complex upstream of or alongside RSPH3.\",\n      \"method\": \"Co-immunoprecipitation, transmission electron microscopy, whole-exome sequencing\",\n      \"journal\": \"Clinical genetics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP, limited mechanistic follow-up\",\n      \"pmids\": [\"37804054\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RSPH3 is identified as a component of the radial spoke 1 (RS1) head in mammalian sperm; mass spectrometry in Iqub-deficient sperm shows RS1 deficiency with loss of RSPH3 among other RS1 components (RSPH6A, RSPH9, DYDC1), placing RSPH3 specifically in the RS1 subtype.\",\n      \"method\": \"Protein mass spectrometry of RS fractions, western blotting, transmission electron microscopy in Iqub-/- mice\",\n      \"journal\": \"Cell communication and signaling : CCS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — proteomics combined with structural EM; direct biochemical localization to RS1\",\n      \"pmids\": [\"39849482\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In Chlamydomonas, I1 dynein deficiency (ida1) is epistatic to the RSP3 RII-binding domain mutation (388), establishing that I1 dynein acts downstream of the RSP3–PKA signaling axis in the pathway regulating ciliary motility.\",\n      \"method\": \"Genetic epistasis — double mutant 388;ida1 motility analysis compared with single mutants\",\n      \"journal\": \"microPublication biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic epistasis experiment defining pathway order; single study\",\n      \"pmids\": [\"41487906\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ARMC2/PF27 is required for intraflagellar transport (IFT) of radial spokes into Chlamydomonas flagella; RSP3 and ARMC2 co-migrate on anterograde IFT trains and are co-unloaded at the flagellar tip, after which RSP3 attaches to the axoneme while ARMC2 diffuses back; in armc2 mutants, RSP3 IFT is abolished and RS assembly is limited to the proximal flagellar region.\",\n      \"method\": \"Live fluorescence imaging of tagged proteins, IFT co-migration analysis, rescue genetics in armc2/pf27 mutants\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct live imaging of co-transport with loss-of-function showing abolished RSP3 IFT; strong mechanistic evidence\",\n      \"pmids\": [\"34982025\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RSPH3 is a conserved radial spoke stalk protein that forms a homodimer and functions as an A-kinase anchoring protein (AKAP), scaffolding PKA (via RII-binding amphipathic helices) and ERK1/2 at the base of radial spokes in motile cilia and sperm flagella; it is essential for the assembly of the entire radial spoke structure (particularly RS1 stalk and head), is transported into cilia by IFT trains via the adapter ARMC2, and in mammals also acts as a nucleocytoplasmic shuttling protein in neurons that promotes neurogenesis, with its PKA-anchoring function placing it upstream of I1 dynein in the central pair–radial spoke signaling pathway that regulates ciliary beat.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"RSPH3 is a conserved radial spoke stalk protein that functions as a dimeric A-kinase anchoring protein (AKAP) essential for radial spoke assembly, ciliary motility regulation, and flagellar beat coordination. The N-terminal domain mediates homodimerization and axoneme binding, while internal amphipathic helices scaffold PKA regulatory subunits (RIIα/RIIβ), additional spoke components via Dpy-30/RIIa domains, and active ERK1/2, integrating kinase signaling at the spoke base to control dynein-driven motility through I1 dynein as a downstream effector [PMID:11309423, PMID:18157907, PMID:19684019, PMID:23148234, PMID:41487906]. RSPH3 is transported into cilia on anterograde IFT trains via the adapter ARMC2/PF27 and is deposited at the axoneme tip, where it incorporates into the RS1 subtype of radial spokes [PMID:34982025, PMID:39849482]. Loss-of-function mutations in human RSPH3 cause primary ciliary dyskinesia (PCD) with near-complete absence of radial spokes and central-complex defects, establishing RSPH3 as essential for RS stalk integrity upstream of RS head and neck assembly [PMID:26082196, PMID:26073779].\",\n  \"teleology\": [\n    {\n      \"year\": 1993,\n      \"claim\": \"Identifying how RSP3 attaches to the axoneme answered where the radial spoke anchors and established that a discrete N-terminal domain (aa 1–85) is both necessary and sufficient for axoneme binding.\",\n      \"evidence\": \"In vitro axoneme-binding assays with truncated/fusion RSP3 proteins and transformation rescue of spokeless pf14 Chlamydomonas mutants\",\n      \"pmids\": [\"8408197\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Binding partner on the axoneme not identified\", \"Whether binding is direct to outer doublet or requires adaptor proteins unknown\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Demonstrating that RSP3 is an AKAP with a defined RII-binding amphipathic helix (aa 144–180) answered how PKA is positioned at the radial spoke and linked spoke-localized kinase signaling to motility regulation.\",\n      \"evidence\": \"RII blot overlay on axonemes from motility mutants, truncation and point mutagenesis of RSP3 in Chlamydomonas\",\n      \"pmids\": [\"11309423\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether mammalian RSPH3 AKAP function is conserved was not yet shown\", \"Identity of PKA substrates at the spoke unknown\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Isolation of RSP3 in a KI-extractable spoke stalk subcomplex with HSP40 and RSP4/6 in Ciona confirmed the stalk localization across species and identified co-migrating partners.\",\n      \"evidence\": \"Biochemical fractionation, MALDI-TOF mass spectrometry, and immunoelectron microscopy in ascidian sperm axonemes\",\n      \"pmids\": [\"15563603\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct stoichiometry of the subcomplex not determined\", \"Whether HSP40 association is functionally relevant or a co-purification artifact unclear\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Showing that disruption of the RSP3 RII-binding domain causes unregulated axonemal PKA and paralyzed motility—rescued by PKA inhibitors—established RSP3 as the functional AKAP that restrains PKA to permit normal flagellar beating.\",\n      \"evidence\": \"Transformation of pf14 with RII-binding domain point-mutant RSP3; demembranated cell reactivation ± PKA inhibitors in Chlamydomonas\",\n      \"pmids\": [\"16571668\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream PKA substrates mediating the motility defect not identified\", \"Whether other AKAPs compensate partially unknown\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrating RSP3 homodimerization via the N-terminal axoneme-binding domain established that each radial spoke is built on a dimeric scaffold capable of positioning multiple signaling modules.\",\n      \"evidence\": \"Chemical crosslinking, native gel electrophoresis, and co-immunoprecipitation with epitope-tagged RSP3 in Chlamydomonas\",\n      \"pmids\": [\"18157907\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dimer interface residues not resolved at atomic level\", \"Whether dimerization is required for IFT transport unknown\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identification of mammalian RSPH3 as a dual-kinase scaffold—binding active ERK1/2 and PKA RIIα/RIIβ with ERK-dependent modulation of the PKA interaction—extended the AKAP model to mammals and revealed crosstalk between MAPK and PKA pathways at radial spokes.\",\n      \"evidence\": \"Yeast two-hybrid (active ERK2 bait), co-immunoprecipitation, in vitro kinase assay, RII overlay in mammalian cells\",\n      \"pmids\": [\"19684019\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological consequence of ERK-PKA crosstalk on ciliary motility not tested\", \"RSPH3 phosphorylation sites for ERK not mapped in vivo\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Two discoveries resolved that RSP3's amphipathic helices recruit non-PKA spoke components via RIIa/Dpy-30 domains and that LC8 dimers bind the N-terminus to enhance axoneme association, collectively defining RSP3 as a multi-valent structural hub.\",\n      \"evidence\": \"In vitro pull-downs and domain mapping for spoke protein binding; co-sedimentation, axoneme pull-down, and flagellar motility analysis of LC8-binding mutants in Chlamydomonas\",\n      \"pmids\": [\"23148234\", \"22753897\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full inventory of proteins recruited by each amphipathic helix in vivo not established\", \"Structural basis of LC8-mediated enhancement of axoneme binding unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Localization of mammalian RSPH3 to ependymal cilia and the perinuclear region, with overexpression promoting cortical neuron migration, suggested roles beyond canonical ciliary motility.\",\n      \"evidence\": \"Immunofluorescence, in utero electroporation, subcellular fractionation in mouse cortex and cell lines\",\n      \"pmids\": [\"25079589\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Loss-of-function neuronal phenotype not shown\", \"Whether motility phenotype is cilia-dependent or cytoplasmic unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Two key advances: human RSPH3 loss-of-function mutations were shown to cause PCD with near-complete radial spoke absence, and mammalian RSPH3 was found to shuttle between nucleus and cytoplasm to promote neurogenesis, broadening its functional repertoire.\",\n      \"evidence\": \"Genetic studies in PCD families with immunofluorescence/TEM of respiratory cilia; NLS/NES deletion mapping and in utero electroporation in mouse cortex\",\n      \"pmids\": [\"26073779\", \"26082196\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How RSPH3 nuclear shuttling relates to its AKAP function unknown\", \"Genotype-phenotype correlation across different RSPH3 mutations not fully explored\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Studies in Drosophila showed RSPH3 interacts with RSBP15 for stabilization and with Combover (Cmb, a Rho kinase effector) to coordinate sperm individualization, linking radial spoke function to actin-based processes in spermatogenesis.\",\n      \"evidence\": \"Co-immunoprecipitation, knockout/RNAi phenotype analysis, and TEM in Drosophila sperm\",\n      \"pmids\": [\"31281031\", \"31391193\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether RSBP15 and Cmb interactions are conserved in mammals unknown\", \"Direct binding domains on RSPH3 for these partners not mapped\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Live imaging revealed that RSPH3 is co-transported with ARMC2 on anterograde IFT trains and deposited at the flagellar tip, solving how radial spoke precursors reach their assembly site.\",\n      \"evidence\": \"Live fluorescence imaging of tagged RSP3/ARMC2, IFT co-migration analysis, and rescue genetics in armc2/pf27 Chlamydomonas mutants\",\n      \"pmids\": [\"34982025\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether RSPH3 is transported as a preassembled spoke subcomplex or monomer/dimer unknown\", \"Mechanism of tip unloading not defined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identification of IQUB and LRRC23 as physical interactors placed RSPH3 in a calmodulin-regulated assembly pathway where IQUB recruits calmodulin to modulate RSPH3 AKAP activity for normal RS assembly.\",\n      \"evidence\": \"Yeast two-hybrid, co-immunoprecipitation, knockout/knockin mouse models with TEM and sperm motility phenotyping; WES and Co-IP for LRRC23\",\n      \"pmids\": [\"36355624\", \"37804054\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct calmodulin binding to RSPH3 not demonstrated\", \"LRRC23 interaction validated only by single Co-IP\", \"Whether IQUB regulation of RSPH3 occurs during IFT or after axoneme incorporation unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Proteomics placed RSPH3 specifically in the RS1 head, and genetic epistasis showed I1 dynein acts downstream of the RSP3–PKA signaling axis, completing the pathway order from spoke-localized PKA to dynein regulation.\",\n      \"evidence\": \"Mass spectrometry of RS fractions from Iqub-/- mouse sperm; double-mutant motility analysis (388;ida1) in Chlamydomonas\",\n      \"pmids\": [\"39849482\", \"41487906\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether RSPH3 is in the RS1 head versus stalk needs reconciliation with prior stalk assignment\", \"PKA substrate on I1 dynein not identified\", \"Whether RS2/RS3 also contain RSPH3 in mammals unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the atomic structure of the RSPH3 dimer and its multi-protein spoke scaffold, the identity of PKA substrates linking RSPH3 signaling to I1 dynein regulation, and whether the nuclear shuttling and neurogenesis functions depend on the same AKAP activity used in cilia.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No high-resolution structure of RSPH3 dimer or spoke complex available\", \"PKA substrate identity on I1 dynein unknown\", \"Relationship between ciliary and nuclear RSPH3 functions unexplored\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 2, 4, 5]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 4, 16]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [1, 3, 5, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [1, 2, 7, 8, 10, 17]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [1, 6, 10, 15]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": []},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [7, 17]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 2, 4, 16]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [11, 12]}\n    ],\n    \"complexes\": [\n      \"Radial spoke (RS1)\",\n      \"RSP3 homodimer\"\n    ],\n    \"partners\": [\n      \"PRKAR2A\",\n      \"PRKAR2B\",\n      \"MAPK1\",\n      \"MAPK3\",\n      \"ARMC2\",\n      \"IQUB\",\n      \"DYNLL1\",\n      \"LRRC23\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}