{"gene":"ROPN1L","run_date":"2026-06-10T06:43:37","timeline":{"discoveries":[{"year":2013,"finding":"ROPN1L binds AKAP3 in the sperm fibrous sheath (FS). Knockout of ROPN1L (RLKO) alone causes slightly reduced sperm motility and increased levels of ROPN1, while knockout of ROPN1 (RKO) increases ROPN1L levels, indicating mutual compensation. Double knockout (DKO) causes complete sperm immotility, structural thinning/shredding of the principal piece, and reduction in AKAP3 levels, demonstrating that ROPN1 and ROPN1L together are required for AKAP3 incorporation into the FS and for PKA-dependent signaling (including PKA phosphorylation of a 270-kDa protein and capacitation-induced tyrosine phosphorylation).","method":"Genetic knockout mice (single and double KO), sperm motility assays, Western blot, immunofluorescence, morphological analysis","journal":"Biology of reproduction","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with defined cellular phenotypes, reciprocal compensatory protein-level changes, multiple orthogonal readouts in a single rigorous study","pmids":["23303679"],"is_preprint":false},{"year":2016,"finding":"Phosphorylated FSCB binds ROPN1/ROPN1L with significantly higher affinity than non-phosphorylated FSCB (shown by immunoprecipitation). FSCB phosphorylation suppresses SUMOylation of ROPN1/ROPN1L, and pharmacological suppression of ROPN1/ROPN1L SUMOylation mimics the effect of FSCB phosphorylation on sperm motility, placing ROPN1/ROPN1L downstream of PKA-mediated FSCB phosphorylation in the capacitation signaling pathway.","method":"Immunoprecipitation assay, SUMOylation inhibition experiments, sperm motility assays","journal":"American journal of translational research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus functional rescue experiment, single lab, two orthogonal methods","pmids":["27398160"],"is_preprint":false},{"year":2021,"finding":"During bull sperm capacitation, ROPN1L undergoes reversible oxidative post-translational modifications (oxPTMs) manifested by reduction or oxidation of susceptible cysteines, linking ROPN1L to the cAMP/PKA redox signaling pathway during capacitation.","method":"Fluorescent gel-based redox proteomics, Western blot, flow cytometry, fluorescence microscopy","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — redox proteomic identification, single lab, but limited functional follow-up specific to ROPN1L","pmids":["34360666"],"is_preprint":false},{"year":2025,"finding":"ROPN1L is identified as a component of the stalk of radial spoke 1 (RS1) in sperm flagella in both humans and mice. In IQUB-deficient patients and Iqub-/- mice, RS1 is specifically absent and ROPN1L is among the proteins constituting the RS1 stalk, as determined by protein mass spectrometry and western blotting.","method":"Protein mass spectrometry, western blotting, electron microscopy, Iqub-/- mouse model","journal":"Cell communication and signaling : CCS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mass spectrometry identification with KO model validation, single lab, two orthogonal methods","pmids":["39849482"],"is_preprint":false},{"year":2025,"finding":"Loss of NSUN7 in mouse spermatids reduces the abundance of ROPN1L during spermiogenesis, as detected by proteomic profiling, linking ROPN1L protein levels to NSUN7-dependent fibrous sheath and flagellar assembly.","method":"Proteomic profiling of sorted spermatids from Nsun7 knockout mice","journal":"International journal of molecular sciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single proteomics dataset from one KO model, no direct functional follow-up on ROPN1L specifically","pmids":["41516134"],"is_preprint":false},{"year":2026,"finding":"RNAi-mediated knockdown of ropn1l in Schmidtea mediterranea (planarian flatworm) significantly reduced swim rate by 26.4% and shortened cilia by 37.1%, establishing a role for ROPN1L in ciliary structure and motility in a metazoan model.","method":"RNAi knockdown in Schmidtea mediterranea, swim rate measurement, cilia length measurement","journal":"microPublication biology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean loss-of-function with defined morphological and functional phenotype, single study, non-mammalian model","pmids":["42079374"],"is_preprint":false},{"year":2025,"finding":"scRNA-seq analysis of breast cancer identified a subpopulation of neoplastic multi-ciliated cells (MCCs) marked by ROPN1L and FOXJ1 co-expression, validated by immunohistochemistry and electron microscopy, indicating ROPN1L as a marker of MCC identity in this context.","method":"Single-cell RNA sequencing, immunohistochemistry, electron microscopy","journal":"Cells","confidence":"Low","confidence_rationale":"Tier 3 / Weak — expression/localization marker identification, no direct functional mechanistic experiment on ROPN1L protein","pmids":["39936988"],"is_preprint":false}],"current_model":"ROPN1L is an R2D2-domain protein that localizes to the sperm fibrous sheath (FS) and radial spoke 1 (RS1) stalk of the flagellar axoneme, where it binds AKAP3 to scaffold PKA signaling; it compensates functionally with its paralog ROPN1, undergoes FSCB-phosphorylation-regulated SUMOylation changes and redox modifications during capacitation, and is required for sperm motility and male fertility, while also playing a broader role in motile cilia structure and function."},"narrative":{"mechanistic_narrative":"ROPN1L is an A-kinase anchoring scaffold of the motile axoneme that organizes PKA-dependent signaling required for sperm flagellar motility and male fertility [PMID:23303679]. In the sperm fibrous sheath it binds AKAP3 and functions redundantly with its paralog ROPN1, such that single loss of either protein causes only modest motility defects with compensatory upregulation of the other, whereas combined loss abolishes motility, thins and shreds the principal piece, and depletes AKAP3, establishing that ROPN1L and ROPN1 together are required to incorporate AKAP3 into the fibrous sheath and to support PKA-dependent phosphorylation events during capacitation [PMID:23303679]. ROPN1L acts downstream of capacitation signaling: phosphorylated FSCB binds ROPN1L/ROPN1 with high affinity and suppresses their SUMOylation, and ROPN1L cysteines undergo reversible oxidative modification within the cAMP/PKA redox pathway during capacitation [PMID:27398160, PMID:34360666]. Beyond the fibrous sheath, ROPN1L is a constituent of the radial spoke 1 stalk of the flagellar axoneme [PMID:39849482], and its role in ciliary structure and motility extends to motile cilia in a metazoan model, where its knockdown shortens cilia and reduces swimming [PMID:42079374].","teleology":[{"year":2013,"claim":"Established that ROPN1L is an AKAP3-binding fibrous sheath scaffold acting redundantly with ROPN1 to enable PKA signaling and sperm motility, answering whether these paralogs are functionally required and how they relate.","evidence":"Single and double knockout mice with sperm motility assays, Western blot, immunofluorescence and morphology","pmids":["23303679"],"confidence":"High","gaps":["Does not resolve the direct molecular consequence of AKAP3 loss on specific PKA substrates","Compensation mechanism between ROPN1L and ROPN1 at the regulatory level is undefined"]},{"year":2016,"claim":"Placed ROPN1L downstream of PKA-mediated FSCB phosphorylation by showing phospho-FSCB binds ROPN1L and suppresses its SUMOylation to regulate motility.","evidence":"Co-immunoprecipitation and SUMOylation-inhibition rescue with sperm motility assays","pmids":["27398160"],"confidence":"Medium","gaps":["SUMOylation sites on ROPN1L not mapped","Single-lab finding without reciprocal validation of the FSCB-ROPN1L interaction"]},{"year":2021,"claim":"Linked ROPN1L to redox signaling by demonstrating reversible cysteine oxidation during capacitation.","evidence":"Fluorescent gel-based redox proteomics, Western blot, flow cytometry in bull sperm","pmids":["34360666"],"confidence":"Medium","gaps":["Functional consequence of ROPN1L oxidation not tested","Specific oxidized cysteines and their effect on AKAP3/PKA scaffolding unknown"]},{"year":2025,"claim":"Identified ROPN1L as a structural component of the radial spoke 1 stalk, extending its role beyond the fibrous sheath to the axonemal radial spoke.","evidence":"Protein mass spectrometry, Western blotting and electron microscopy in IQUB-deficient patients and Iqub-/- mice","pmids":["39849482"],"confidence":"Medium","gaps":["Whether ROPN1L is structurally essential for RS1 assembly versus a passenger is not resolved","Relationship between its RS1 role and its fibrous sheath/AKAP3 role is unclear"]},{"year":2025,"claim":"Connected ROPN1L abundance to spermiogenic flagellar assembly programs via NSUN7-dependent regulation.","evidence":"Proteomic profiling of sorted spermatids from Nsun7 knockout mice","pmids":["41516134"],"confidence":"Low","gaps":["Single proteomics dataset with no direct functional follow-up on ROPN1L","Whether NSUN7 regulates ROPN1L directly or indirectly is unknown"]},{"year":2025,"claim":"Identified ROPN1L as a marker of multi-ciliated cell identity in a neoplastic context, suggesting expression beyond sperm.","evidence":"Single-cell RNA sequencing, immunohistochemistry and electron microscopy in breast cancer","pmids":["39936988"],"confidence":"Low","gaps":["No functional mechanistic experiment on ROPN1L in these cells","Marker association does not establish a functional role"]},{"year":2026,"claim":"Demonstrated a conserved requirement for ROPN1L in motile cilia structure and motility in a non-mammalian metazoan.","evidence":"RNAi knockdown in Schmidtea mediterranea with swim rate and cilia length measurement","pmids":["42079374"],"confidence":"Medium","gaps":["Molecular partners of ROPN1L in planarian cilia not defined","Whether the AKAP3/PKA mechanism operates in non-sperm cilia is untested"]},{"year":null,"claim":"How ROPN1L coordinates its dual localization to the fibrous sheath and the radial spoke 1 stalk, and whether its PKA-scaffolding mechanism generalizes to somatic motile cilia, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of ROPN1L within RS1 or its AKAP3 complex","Functional importance of capacitation-induced SUMOylation and redox modifications in vivo not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[3,5]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,3]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1]},{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[0]}],"complexes":["radial spoke 1","sperm fibrous sheath"],"partners":["AKAP3","ROPN1","FSCB"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96C74","full_name":"Ropporin-1-like protein","aliases":["AKAP-associated sperm protein"],"length_aa":230,"mass_kda":26.1,"function":"Functions as part of axonemal radial spoke complexes that play an important part in the motility of sperm and cilia. Important for male fertility. With ROPN1, involved in fibrous sheath integrity and sperm motility, plays a role in PKA-dependent signaling processes required for spermatozoa capacitation","subcellular_location":"Cell projection, cilium, flagellum; Cell projection, cilium","url":"https://www.uniprot.org/uniprotkb/Q96C74/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ROPN1L","classification":"Not Classified","n_dependent_lines":22,"n_total_lines":1208,"dependency_fraction":0.018211920529801324},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ROPN1L","total_profiled":1310},"omim":[{"mim_id":"615876","title":"RADIAL SPOKE HEAD 3; RSPH3","url":"https://www.omim.org/entry/615876"},{"mim_id":"611757","title":"RHOPHILIN-ASSOCIATED TAIL PROTEIN 1; ROPN1","url":"https://www.omim.org/entry/611757"},{"mim_id":"611756","title":"RHOPHILIN ASSOCIATED TAIL PROTEIN 1-LIKE; ROPN1L","url":"https://www.omim.org/entry/611756"},{"mim_id":"123450","title":"CRI-DU-CHAT SYNDROME","url":"https://www.omim.org/entry/123450"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Primary cilium transition zone","reliability":"Supported"},{"location":"Mid piece","reliability":"Supported"},{"location":"Principal piece","reliability":"Supported"},{"location":"End piece","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"},{"location":"Vesicles","reliability":"Additional"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"testis","ntpm":304.2}],"url":"https://www.proteinatlas.org/search/ROPN1L"},"hgnc":{"alias_symbol":["ASP","FLJ25776","RSPH11","RP11-1C1.7","TCONS_00009352"],"prev_symbol":["LINC01513"]},"alphafold":{"accession":"Q96C74","domains":[{"cath_id":"-","chopping":"140-218","consensus_level":"medium","plddt":88.0706,"start":140,"end":218}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96C74","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96C74-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96C74-F1-predicted_aligned_error_v6.png","plddt_mean":85.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ROPN1L","jax_strain_url":"https://www.jax.org/strain/search?query=ROPN1L"},"sequence":{"accession":"Q96C74","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96C74.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96C74/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96C74"}},"corpus_meta":[{"pmid":"22452962","id":"PMC_22452962","title":"A genome-wide association study identifies a breast cancer risk variant in ERBB4 at 2q34: results from the Seoul Breast Cancer Study.","date":"2012","source":"Breast cancer research : BCR","url":"https://pubmed.ncbi.nlm.nih.gov/22452962","citation_count":111,"is_preprint":false},{"pmid":"24219008","id":"PMC_24219008","title":"Dynamically regulated miRNA-mRNA networks revealed by exercise.","date":"2013","source":"BMC physiology","url":"https://pubmed.ncbi.nlm.nih.gov/24219008","citation_count":99,"is_preprint":false},{"pmid":"23303679","id":"PMC_23303679","title":"Loss of R2D2 proteins ROPN1 and ROPN1L causes defects in murine sperm motility, phosphorylation, and fibrous sheath integrity.","date":"2013","source":"Biology of reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/23303679","citation_count":83,"is_preprint":false},{"pmid":"21424380","id":"PMC_21424380","title":"Potential novel candidate polymorphisms identified in genome-wide association study for breast cancer 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Cancer.","date":"2025","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/39936988","citation_count":8,"is_preprint":false},{"pmid":"38027210","id":"PMC_38027210","title":"RNA sequencing profiles reveals progressively reduced spermatogenesis with progression in adult cryptorchidism.","date":"2023","source":"Frontiers in endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/38027210","citation_count":7,"is_preprint":false},{"pmid":"32871643","id":"PMC_32871643","title":"Evaluation of Rhophilin Associated Tail Protein (ROPN1L) in the Human Liver Fluke Opisthorchis viverrini for Diagnostic Approach.","date":"2020","source":"The Korean journal of parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/32871643","citation_count":6,"is_preprint":false},{"pmid":"40231527","id":"PMC_40231527","title":"Cilia Plays a Pivotal Role in the Hypersecretion of Airway Mucus in Mice.","date":"2024","source":"Current molecular 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Part D, Genomics & proteomics","url":"https://pubmed.ncbi.nlm.nih.gov/37354751","citation_count":5,"is_preprint":false},{"pmid":"36977161","id":"PMC_36977161","title":"Production and Immunological Characterization of scFv Specific to Epitope of Opisthorchis viverrini Rhophilin-Associated Tail Protein 1-like (OvROPN1L).","date":"2023","source":"Tropical medicine and infectious disease","url":"https://pubmed.ncbi.nlm.nih.gov/36977161","citation_count":5,"is_preprint":false},{"pmid":"35003358","id":"PMC_35003358","title":"Linc01513 inhibits the malignant potential of Nasopharyngeal carcinoma by binding to PTBP1.","date":"2021","source":"Journal of Cancer","url":"https://pubmed.ncbi.nlm.nih.gov/35003358","citation_count":3,"is_preprint":false},{"pmid":"37162640","id":"PMC_37162640","title":"Effect of arginine-induced motility and capacitation on RNA population in goat spermatozoa.","date":"2023","source":"Veterinary research communications","url":"https://pubmed.ncbi.nlm.nih.gov/37162640","citation_count":3,"is_preprint":false},{"pmid":"42079374","id":"PMC_42079374","title":"Insights into Human Ciliopathies: Gene Silencing of ropn1l and tex9 in Schmidtea mediterranea Indicate Association with Ciliary Structure and Motility.","date":"2026","source":"microPublication biology","url":"https://pubmed.ncbi.nlm.nih.gov/42079374","citation_count":0,"is_preprint":false},{"pmid":"40538010","id":"PMC_40538010","title":"Machine Learning-Driven Discovery of TRIM Genes as Diagnostic Biomarkers for Idiopathic Pulmonary Fibrosis.","date":"2025","source":"Medical science monitor : international medical journal of experimental and clinical research","url":"https://pubmed.ncbi.nlm.nih.gov/40538010","citation_count":0,"is_preprint":false},{"pmid":"41516134","id":"PMC_41516134","title":"NSUN7 Regulates Sperm Flagella Formation at All Stages of Spermiogenesis.","date":"2025","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/41516134","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16646,"output_tokens":1818,"usd":0.038604,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":8605,"output_tokens":2464,"usd":0.052312,"stage2_stop_reason":"end_turn"},"total_usd":0.090916,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2013,\n      \"finding\": \"ROPN1L binds AKAP3 in the sperm fibrous sheath (FS). Knockout of ROPN1L (RLKO) alone causes slightly reduced sperm motility and increased levels of ROPN1, while knockout of ROPN1 (RKO) increases ROPN1L levels, indicating mutual compensation. Double knockout (DKO) causes complete sperm immotility, structural thinning/shredding of the principal piece, and reduction in AKAP3 levels, demonstrating that ROPN1 and ROPN1L together are required for AKAP3 incorporation into the FS and for PKA-dependent signaling (including PKA phosphorylation of a 270-kDa protein and capacitation-induced tyrosine phosphorylation).\",\n      \"method\": \"Genetic knockout mice (single and double KO), sperm motility assays, Western blot, immunofluorescence, morphological analysis\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with defined cellular phenotypes, reciprocal compensatory protein-level changes, multiple orthogonal readouts in a single rigorous study\",\n      \"pmids\": [\"23303679\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Phosphorylated FSCB binds ROPN1/ROPN1L with significantly higher affinity than non-phosphorylated FSCB (shown by immunoprecipitation). FSCB phosphorylation suppresses SUMOylation of ROPN1/ROPN1L, and pharmacological suppression of ROPN1/ROPN1L SUMOylation mimics the effect of FSCB phosphorylation on sperm motility, placing ROPN1/ROPN1L downstream of PKA-mediated FSCB phosphorylation in the capacitation signaling pathway.\",\n      \"method\": \"Immunoprecipitation assay, SUMOylation inhibition experiments, sperm motility assays\",\n      \"journal\": \"American journal of translational research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus functional rescue experiment, single lab, two orthogonal methods\",\n      \"pmids\": [\"27398160\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"During bull sperm capacitation, ROPN1L undergoes reversible oxidative post-translational modifications (oxPTMs) manifested by reduction or oxidation of susceptible cysteines, linking ROPN1L to the cAMP/PKA redox signaling pathway during capacitation.\",\n      \"method\": \"Fluorescent gel-based redox proteomics, Western blot, flow cytometry, fluorescence microscopy\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — redox proteomic identification, single lab, but limited functional follow-up specific to ROPN1L\",\n      \"pmids\": [\"34360666\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ROPN1L is identified as a component of the stalk of radial spoke 1 (RS1) in sperm flagella in both humans and mice. In IQUB-deficient patients and Iqub-/- mice, RS1 is specifically absent and ROPN1L is among the proteins constituting the RS1 stalk, as determined by protein mass spectrometry and western blotting.\",\n      \"method\": \"Protein mass spectrometry, western blotting, electron microscopy, Iqub-/- mouse model\",\n      \"journal\": \"Cell communication and signaling : CCS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mass spectrometry identification with KO model validation, single lab, two orthogonal methods\",\n      \"pmids\": [\"39849482\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Loss of NSUN7 in mouse spermatids reduces the abundance of ROPN1L during spermiogenesis, as detected by proteomic profiling, linking ROPN1L protein levels to NSUN7-dependent fibrous sheath and flagellar assembly.\",\n      \"method\": \"Proteomic profiling of sorted spermatids from Nsun7 knockout mice\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single proteomics dataset from one KO model, no direct functional follow-up on ROPN1L specifically\",\n      \"pmids\": [\"41516134\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"RNAi-mediated knockdown of ropn1l in Schmidtea mediterranea (planarian flatworm) significantly reduced swim rate by 26.4% and shortened cilia by 37.1%, establishing a role for ROPN1L in ciliary structure and motility in a metazoan model.\",\n      \"method\": \"RNAi knockdown in Schmidtea mediterranea, swim rate measurement, cilia length measurement\",\n      \"journal\": \"microPublication biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean loss-of-function with defined morphological and functional phenotype, single study, non-mammalian model\",\n      \"pmids\": [\"42079374\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"scRNA-seq analysis of breast cancer identified a subpopulation of neoplastic multi-ciliated cells (MCCs) marked by ROPN1L and FOXJ1 co-expression, validated by immunohistochemistry and electron microscopy, indicating ROPN1L as a marker of MCC identity in this context.\",\n      \"method\": \"Single-cell RNA sequencing, immunohistochemistry, electron microscopy\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — expression/localization marker identification, no direct functional mechanistic experiment on ROPN1L protein\",\n      \"pmids\": [\"39936988\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ROPN1L is an R2D2-domain protein that localizes to the sperm fibrous sheath (FS) and radial spoke 1 (RS1) stalk of the flagellar axoneme, where it binds AKAP3 to scaffold PKA signaling; it compensates functionally with its paralog ROPN1, undergoes FSCB-phosphorylation-regulated SUMOylation changes and redox modifications during capacitation, and is required for sperm motility and male fertility, while also playing a broader role in motile cilia structure and function.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ROPN1L is an A-kinase anchoring scaffold of the motile axoneme that organizes PKA-dependent signaling required for sperm flagellar motility and male fertility [#0]. In the sperm fibrous sheath it binds AKAP3 and functions redundantly with its paralog ROPN1, such that single loss of either protein causes only modest motility defects with compensatory upregulation of the other, whereas combined loss abolishes motility, thins and shreds the principal piece, and depletes AKAP3, establishing that ROPN1L and ROPN1 together are required to incorporate AKAP3 into the fibrous sheath and to support PKA-dependent phosphorylation events during capacitation [#0]. ROPN1L acts downstream of capacitation signaling: phosphorylated FSCB binds ROPN1L/ROPN1 with high affinity and suppresses their SUMOylation, and ROPN1L cysteines undergo reversible oxidative modification within the cAMP/PKA redox pathway during capacitation [#1, #2]. Beyond the fibrous sheath, ROPN1L is a constituent of the radial spoke 1 stalk of the flagellar axoneme [#3], and its role in ciliary structure and motility extends to motile cilia in a metazoan model, where its knockdown shortens cilia and reduces swimming [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 2013,\n      \"claim\": \"Established that ROPN1L is an AKAP3-binding fibrous sheath scaffold acting redundantly with ROPN1 to enable PKA signaling and sperm motility, answering whether these paralogs are functionally required and how they relate.\",\n      \"evidence\": \"Single and double knockout mice with sperm motility assays, Western blot, immunofluorescence and morphology\",\n      \"pmids\": [\"23303679\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not resolve the direct molecular consequence of AKAP3 loss on specific PKA substrates\", \"Compensation mechanism between ROPN1L and ROPN1 at the regulatory level is undefined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Placed ROPN1L downstream of PKA-mediated FSCB phosphorylation by showing phospho-FSCB binds ROPN1L and suppresses its SUMOylation to regulate motility.\",\n      \"evidence\": \"Co-immunoprecipitation and SUMOylation-inhibition rescue with sperm motility assays\",\n      \"pmids\": [\"27398160\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"SUMOylation sites on ROPN1L not mapped\", \"Single-lab finding without reciprocal validation of the FSCB-ROPN1L interaction\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Linked ROPN1L to redox signaling by demonstrating reversible cysteine oxidation during capacitation.\",\n      \"evidence\": \"Fluorescent gel-based redox proteomics, Western blot, flow cytometry in bull sperm\",\n      \"pmids\": [\"34360666\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of ROPN1L oxidation not tested\", \"Specific oxidized cysteines and their effect on AKAP3/PKA scaffolding unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified ROPN1L as a structural component of the radial spoke 1 stalk, extending its role beyond the fibrous sheath to the axonemal radial spoke.\",\n      \"evidence\": \"Protein mass spectrometry, Western blotting and electron microscopy in IQUB-deficient patients and Iqub-/- mice\",\n      \"pmids\": [\"39849482\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether ROPN1L is structurally essential for RS1 assembly versus a passenger is not resolved\", \"Relationship between its RS1 role and its fibrous sheath/AKAP3 role is unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Connected ROPN1L abundance to spermiogenic flagellar assembly programs via NSUN7-dependent regulation.\",\n      \"evidence\": \"Proteomic profiling of sorted spermatids from Nsun7 knockout mice\",\n      \"pmids\": [\"41516134\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single proteomics dataset with no direct functional follow-up on ROPN1L\", \"Whether NSUN7 regulates ROPN1L directly or indirectly is unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified ROPN1L as a marker of multi-ciliated cell identity in a neoplastic context, suggesting expression beyond sperm.\",\n      \"evidence\": \"Single-cell RNA sequencing, immunohistochemistry and electron microscopy in breast cancer\",\n      \"pmids\": [\"39936988\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No functional mechanistic experiment on ROPN1L in these cells\", \"Marker association does not establish a functional role\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Demonstrated a conserved requirement for ROPN1L in motile cilia structure and motility in a non-mammalian metazoan.\",\n      \"evidence\": \"RNAi knockdown in Schmidtea mediterranea with swim rate and cilia length measurement\",\n      \"pmids\": [\"42079374\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular partners of ROPN1L in planarian cilia not defined\", \"Whether the AKAP3/PKA mechanism operates in non-sperm cilia is untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How ROPN1L coordinates its dual localization to the fibrous sheath and the radial spoke 1 stalk, and whether its PKA-scaffolding mechanism generalizes to somatic motile cilia, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of ROPN1L within RS1 or its AKAP3 complex\", \"Functional importance of capacitation-induced SUMOylation and redox modifications in vivo not established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [3, 5]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"complexes\": [\"radial spoke 1\", \"sperm fibrous sheath\"],\n    \"partners\": [\"AKAP3\", \"ROPN1\", \"FSCB\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":4,"faith_total":4,"faith_pct":100.0}}