RSPH6A

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).

1. 1992 Medium

   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

   PMID:1508197

   Open questions at the time

   • No localization within the spoke defined
   • No interaction partners identified
   • Function inferred only from regeneration-coupled expression
2. 1998 High

   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

   PMID:9450971

   Open questions at the time

   • Identity of the 43 and 34 kDa co-purifying partners not molecularly defined
   • Mechanism linking spoke head to waveform geometry unknown
3. 2001 Medium

   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

   PMID:11237735

   Open questions at the time

   • No subcellular localization shown
   • No functional or knockout data
   • No interaction partners
4. 2006 Medium

   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

   PMID:16936294

   Open questions at the time

   • Direct Ca2+/CaM binding by the mammalian RSPH6A not tested
   • Functional consequence of CaM binding not established
5. 2011 High

   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

   PMID:21391306

   Open questions at the time

   • Stoichiometry of the full head not resolved
   • Mammalian subcomplex composition assumed by orthology
6. 2018 High

   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

   PMID:30185526

   Open questions at the time

   • Mechanism of manchette removal defect unexplained
   • Direct RSPH6A-RSPH9 interaction not biochemically shown
   • Why axoneme initiates but fails to stabilize unclear
7. 2018 High

   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

   PMID:30070024

   Open questions at the time

   • RSP3-dependence of mammalian RSPH6A transport not directly tested
   • Adaptor coupling spoke head to IFT not identified
8. 2019 High

   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

   PMID:30239614

   Open questions at the time

   • Kinase responsible not identified
   • Functional consequence of the phosphorylation not established
9. 2020 High

   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

   PMID:32694165

   Open questions at the time

   • Resolution (15 A) insufficient for atomic model
   • Mammalian RSPH6A structure not determined
10. 2025 Medium

    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

    PMID:39849482

    Open questions at the time

    • Direct interactions among RS1 head components not biochemically mapped
    • Whether RSPH6A loss reciprocally affects IQUB not tested
11. 2024 Low

    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

    PMID:38884051

    Open questions at the time

    • Single patient without functional validation of the variant
    • No mechanistic follow-up linking variant to spoke defect

• How RSPH6A directly engages RSPH9 and other RS1 head partners in mammalian sperm, and how capacitation-induced phosphorylation alters spoke function, remain unresolved.
• 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