{"gene":"NUP35","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2006,"finding":"Crystal structure of mouse Nup35 RRM domain solved at 2.7 Å resolution, revealing atypical RNP1/RNP2 motifs and a novel homodimerization mode where the beta-sheet surface drives dimerization via hydrophobic interactions (Met236 sandwiched by Phe178 and Trp209), confirmed by analytical ultracentrifugation.","method":"X-ray crystallography + analytical ultracentrifugation","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional validation by analytical ultracentrifugation, direct mutagenesis-level structural analysis of interface residues","pmids":["16962612"],"is_preprint":false},{"year":2008,"finding":"In C. elegans, depletion of Nup35/NPP-19 inhibits NE localization of Nup155/NPP-8, blocks NPC assembly, impairs nuclear lamina formation, and disrupts nucleo-cytoplasmic transport; recruitment of Nup107/NPP-5, LEM-2, and nuclear membranes to chromatin is Nup35-independent, indicating Nup35 is specifically required for NPC assembly but not initial nuclear membrane targeting.","method":"C. elegans loss-of-function mutation (npp-19(tm2886)) + fluorescence microscopy of NPC and NE markers + transport assay","journal":"Developmental biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean in vivo loss-of-function with multiple orthogonal readouts (NE marker localization, NPC assembly, transport, nuclear morphology) in a single study","pmids":["19146848"],"is_preprint":false},{"year":2011,"finding":"Nup35 is required for the Ran-dependent translocation mechanism of inner nuclear membrane proteins; depletion of Nup35 specifically blocks Ran-dependent (but not ATP-dependent) FRAP recovery of inner nuclear membrane proteins, and adding FG repeats to membrane proteins accelerates their FRAP recovery in a Nup35-dependent manner, consistent with Nup35 facilitating transport through NPC peripheral channels.","method":"FRAP after siRNA depletion of Nup35 + Ran/ATP depletion experiments + FG-repeat addition to membrane proteins","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple complementary FRAP conditions with depletion of Nup35 and mechanistic dissection across multiple proteins in a single rigorous study","pmids":["21444689"],"is_preprint":false},{"year":2011,"finding":"FG repeats on membrane proteins facilitate their transport to the inner nuclear membrane via NPC peripheral channels, and this process requires Nup35, which itself contains FG repeats, suggesting FG–FG interactions enable integral membrane proteins to act as their own transport receptors.","method":"FRAP with FG-repeat addition constructs + Nup35 depletion","journal":"Communicative & integrative biology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — mechanistic interpretation supported by FRAP data from the companion study (PMID 21444689), but this paper is primarily a perspective/commentary reinforcing those results","pmids":["22046461"],"is_preprint":false},{"year":2013,"finding":"Nup53 (the Nup35 ortholog) interacts with Ndc1 (integral pore membrane protein) and Nup155 during NPC assembly; the Ndc1-binding site on Nup53 overlaps with its membrane-bending region, suggesting Ndc1 interaction modulates membrane deformation activity; Nup53–Nup155 interaction is the main determinant for Nup155 recruitment to the assembling pore.","method":"Co-immunoprecipitation, interaction mapping, in vitro membrane-bending assay, siRNA depletion with fluorescence microscopy","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal interaction mapping with functional membrane-bending assays and siRNA depletion readouts, multiple orthogonal methods","pmids":["24363447"],"is_preprint":false},{"year":2015,"finding":"Nup35 regulates NHE1 (Na⁺-H⁺ exchanger-1) expression in cardiomyocytes by controlling nucleo-cytoplasmic trafficking of nhe1 mRNA; the N-terminal domain of Nup35 mediates this by targeting the 5'-UTR (-412 to -213 nt) of nhe1 mRNA for nuclear export; Nup35 ablation depresses NHE1 expression and weakens resistance to acid challenge.","method":"siRNA knockdown + mRNA nuclear export assay + domain deletion mapping + pH challenge assay + in vivo/in vitro ischemia models","journal":"Journal of molecular cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple functional readouts (mRNA export, protein expression, pH homeostasis) with domain mapping in a single laboratory study","pmids":["26260029"],"is_preprint":false},{"year":2016,"finding":"A point mutation in the RRM domain of Nup35 in mice causes a degenerative myopathy specifically affecting colonic smooth muscle, leading to megacolon; no defects in enteric neurons or interstitial cells of Cajal were found, placing the pathology in smooth muscle cell function downstream of intact Nup35 RRM domain.","method":"ENU-induced point mutation mouse model + histopathology + immunohistochemistry","journal":"The American journal of pathology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean in vivo genetic model with specific phenotypic characterization, but no direct molecular mechanism identified beyond RRM domain requirement","pmids":["27427419"],"is_preprint":false},{"year":2016,"finding":"Stoichiometry of Nup35 in the nuclear pore complex was estimated at approximately 23 copies per NPC, determined by single-molecule GFP fluorescence quantification after endogenous knockdown and replacement with GFP-Nup35.","method":"Single-molecule GFP fluorescence standard + spinning-disk confocal microscopy + knockdown/replacement strategy","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct quantitative measurement in living cells with rigorous single-molecule calibration, single lab, single method","pmids":["27613095"],"is_preprint":false},{"year":2018,"finding":"Nup35 localizes to microtubules and the meiotic spindle (but not nuclear membrane) after meiosis resumption in mouse oocytes, is putatively phosphorylated after GVBD, and is required for spindle assembly and chromosome alignment; Nup35 knockdown causes first polar body extrusion failure, defective kinetochore-microtubule attachment, spindle assembly checkpoint activation, and mislocalization of p-ERK1/2 from spindle poles.","method":"Immunofluorescence + siRNA knockdown + spindle assembly checkpoint analysis + kinetochore-microtubule attachment assay","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal readouts (spindle morphology, chromosome alignment, SAC markers, p-ERK1/2 localization) in a single study, single lab","pmids":["30195030"],"is_preprint":false},{"year":2022,"finding":"Auxin-mediated rapid depletion of NUP35 in human cells does not directly control gene transcription, in contrast to NUP93 which directly activates transcription; NUP35 depletion showed no significant effect on RNA Pol II loading or BRD4 recruitment at active enhancers.","method":"Auxin-inducible degron rapid depletion + Cut&Run + in situ Hi-C","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — rigorous acute depletion system with multiple genomic readouts; negative finding for NUP35 transcriptional control established by direct experiment","pmids":["36323253"],"is_preprint":false},{"year":2023,"finding":"HIV-1 capsid (CA) makes direct contact with Nup35 via FG-motif-containing regions of Nup35; this interaction supports HIV-1 nuclear entry and is dependent on cyclophilin A (CypA) interaction with CA, as CypA-binding CA mutants show altered NPC requirements.","method":"Genetic knockdown of Nup35 + HIV-1 nuclear entry assay + CA mutant analysis + direct binding assay","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding demonstrated with functional nuclear entry readout and CA mutation validation, single study","pmids":["37355754"],"is_preprint":false},{"year":2025,"finding":"In cardiomyocytes, Nup35 directly binds Wif1 mRNA (identified by RNA immunoprecipitation sequencing) and regulates its nuclear export; Nup35 deficiency increases nuclear retention of Wif1 pre-mRNA and decreases Wif1 protein, activating Wnt signaling and causing pathological cardiac fibrosis, hypertrophy, and dysfunction; AAV9-mediated Wif1 mRNA delivery rescues the Nup35-KO cardiac phenotype.","method":"Cardiac-specific Nup35 KO and OE mice + RNA immunoprecipitation sequencing + AAV9 rescue experiment + angiotensin II/TAC models","journal":"Cardiovascular research","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo KO and OE with rescue experiment, direct RNA-binding demonstrated by RIP-seq, multiple orthogonal functional readouts across two independent cardiac stress models","pmids":["41145234"],"is_preprint":false},{"year":2026,"finding":"UBA1 (ubiquitin-activating enzyme) promotes ubiquitination and proteasomal degradation of NUP35 in macrophages during sepsis; NUP35 degradation impairs IκBα nuclear import, thereby activating NF-κB signaling and driving macrophage inflammatory activation; UBA1 knockout or pharmacologic inhibition preserves NUP35 levels, restores IκBα nuclear import, and attenuates renal injury.","method":"Myeloid-specific Uba1 KO mice + cecal ligation puncture model + ubiquitinome/proteomic analysis + IκBα nuclear import assay + co-culture studies","journal":"Antioxidants & redox signaling","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic KO with multiple orthogonal mechanistic readouts (ubiquitinome, nuclear import assay, signaling pathway), pharmacologic validation, and rescue experiments","pmids":["42049653"],"is_preprint":false}],"current_model":"NUP35 is a nucleoporin with an RRM domain that homodimerizes and functions as a structural NPC component (~23 copies/pore) required for NPC assembly (via Nup155 recruitment), Ran-dependent inner nuclear membrane protein transport through NPC peripheral channels via FG-repeat interactions, and selective mRNA export (including nhe1 and Wif1 mRNAs); it also associates with the meiotic spindle and regulates chromosome alignment, directly interacts with HIV-1 capsid FG motifs to facilitate viral nuclear entry, and is subject to UBA1-mediated ubiquitination and degradation that, when occurring in macrophages, activates NF-κB signaling by impairing IκBα nuclear import."},"narrative":{"mechanistic_narrative":"NUP35 is a structural nucleoporin that scaffolds nuclear pore complex (NPC) assembly and supports selective nucleocytoplasmic transport [PMID:19146848, PMID:24363447]. Its RRM domain adopts an atypical fold with non-canonical RNP1/RNP2 motifs and drives homodimerization through a beta-sheet hydrophobic interface centered on Met236 packed against Phe178 and Trp209 [PMID:16962612], and the protein is present at roughly 23 copies per pore [PMID:27613095]. During NPC biogenesis NUP35 (Nup53/NPP-19 in orthologs) is specifically required to recruit Nup155 to the assembling pore — its interaction with Nup155 being the principal recruitment determinant — while its binding to the integral pore membrane protein Ndc1 overlaps with a membrane-bending region that modulates membrane deformation; loss of NUP35 blocks NPC assembly, lamina formation, and transport without affecting initial nuclear membrane targeting [PMID:19146848, PMID:24363447]. NUP35 contains FG repeats and facilitates Ran-dependent translocation of inner nuclear membrane proteins through NPC peripheral channels via FG–FG interactions [PMID:21444689, PMID:22046461]. Beyond structural roles, NUP35 acts as an mRNA export factor: its N-terminal domain directs nuclear export of nhe1 mRNA in cardiomyocytes by targeting its 5'-UTR [PMID:26260029], and it directly binds Wif1 mRNA, so that NUP35 deficiency causes nuclear retention of Wif1, activation of Wnt signaling, and pathological cardiac fibrosis and hypertrophy that is rescued by AAV9-delivered Wif1 mRNA [PMID:41145234]. NUP35 additionally localizes to the meiotic spindle and microtubules in oocytes, where it is required for spindle assembly and chromosome alignment [PMID:30195030]. It is exploited by HIV-1, whose capsid contacts NUP35 via FG-motif regions to support viral nuclear entry [PMID:37355754], and its UBA1-mediated ubiquitination and proteasomal degradation in macrophages impairs IκBα nuclear import to activate NF-κB inflammatory signaling [PMID:42049653]. A point mutation in the NUP35 RRM domain causes a colonic smooth-muscle degenerative myopathy and megacolon in mice [PMID:27427419].","teleology":[{"year":2006,"claim":"Establishing the structural basis of NUP35 oligomerization showed how this nucleoporin builds higher-order assemblies, defining its RRM as a dimerization module rather than a conventional RNA-binding fold.","evidence":"X-ray crystallography of mouse Nup35 RRM with analytical ultracentrifugation","pmids":["16962612"],"confidence":"High","gaps":["Does not address whether the RRM also binds RNA in vivo","No structure of full-length protein or RRM in context of the NPC scaffold"]},{"year":2008,"claim":"In vivo loss-of-function placed NUP35 at a specific step of NPC biogenesis — required for Nup155 recruitment and pore assembly but dispensable for initial membrane-to-chromatin targeting — resolving where in the assembly hierarchy it acts.","evidence":"C. elegans npp-19 loss-of-function mutant with fluorescence imaging of NPC/NE markers and transport assays","pmids":["19146848"],"confidence":"High","gaps":["Molecular detail of the Nup35-Nup155 interaction not defined here","Did not test mRNA export functions"]},{"year":2011,"claim":"FRAP-based dissection revealed a transport function distinct from scaffolding: NUP35 mediates Ran-dependent translocation of inner nuclear membrane proteins through NPC peripheral channels via its FG repeats.","evidence":"FRAP after siRNA depletion with Ran/ATP depletion and FG-repeat addition constructs; companion perspective reinforcing FG-FG mechanism","pmids":["21444689","22046461"],"confidence":"High","gaps":["Direct FG-FG contacts not structurally resolved","Full set of membrane-protein cargoes not enumerated"]},{"year":2013,"claim":"Reciprocal interaction mapping defined the molecular interfaces — Nup53/Nup35 binding Ndc1 and Nup155 — and linked the Ndc1-binding site to membrane-bending activity, mechanistically explaining how it couples membrane deformation to pore assembly.","evidence":"Co-IP, interaction mapping, in vitro membrane-bending assay, and siRNA depletion microscopy","pmids":["24363447"],"confidence":"High","gaps":["Structural basis of membrane bending not solved","Regulation of the Ndc1/Nup155 binding switch unknown"]},{"year":2015,"claim":"Identification of nhe1 mRNA export control assigned NUP35 a selective mRNA-trafficking function with physiological consequence, showing its N-terminal domain targets a defined 5'-UTR region.","evidence":"siRNA knockdown, mRNA nuclear export assay, domain-deletion mapping, and ischemia/pH-challenge models","pmids":["26260029"],"confidence":"Medium","gaps":["Whether export selectivity is direct RNA binding or indirect not resolved here","Single laboratory; export machinery partners unidentified"]},{"year":2016,"claim":"An RRM point-mutation mouse tied NUP35 function to a tissue-specific disease, localizing the pathology to colonic smooth muscle and implicating RRM integrity in that cell type.","evidence":"ENU-induced point-mutation mouse model with histopathology and immunohistochemistry","pmids":["27427419"],"confidence":"Medium","gaps":["No molecular mechanism linking RRM mutation to smooth-muscle defect","Tissue specificity unexplained"]},{"year":2016,"claim":"Quantitative imaging fixed the stoichiometry of NUP35 at the pore (~23 copies), constraining models of its scaffolding role.","evidence":"Single-molecule GFP fluorescence calibration with knockdown/replacement in living cells","pmids":["27613095"],"confidence":"Medium","gaps":["Single method/lab","Spatial arrangement of copies within the pore not determined"]},{"year":2018,"claim":"Discovery of meiotic spindle/microtubule localization extended NUP35 function beyond the interphase NPC, implicating it in spindle assembly and chromosome alignment in oocytes.","evidence":"Immunofluorescence, siRNA knockdown, SAC and kinetochore-microtubule attachment analyses in mouse oocytes","pmids":["30195030"],"confidence":"Medium","gaps":["Direct microtubule-binding interface not defined","Role of putative post-GVBD phosphorylation unconfirmed"]},{"year":2022,"claim":"Acute degron depletion distinguished NUP35 from transcription-regulating nucleoporins, showing it does not directly control gene transcription, RNA Pol II loading, or BRD4 recruitment.","evidence":"Auxin-inducible degron depletion with Cut&Run and in situ Hi-C","pmids":["36323253"],"confidence":"Medium","gaps":["Does not exclude indirect transcriptional effects via mRNA export","Single cell system"]},{"year":2023,"claim":"Demonstrating direct HIV-1 capsid contact via NUP35 FG-motif regions identified the protein as a host factor for viral nuclear entry, gated by capsid-cyclophilin A interaction.","evidence":"Nup35 knockdown, HIV-1 nuclear entry assays, capsid mutant analysis, and direct binding assay","pmids":["37355754"],"confidence":"Medium","gaps":["Structural detail of CA-NUP35 interface unresolved","Single study"]},{"year":2025,"claim":"RIP-seq plus in vivo KO/OE with rescue established NUP35 as a direct Wif1 mRNA export factor whose loss activates Wnt signaling and drives cardiac fibrosis, providing a causal mRNA-to-disease axis.","evidence":"Cardiac-specific KO/OE mice, RIP-seq, AAV9 Wif1 mRNA rescue, and angiotensin II/TAC stress models","pmids":["41145234"],"confidence":"High","gaps":["Whether RRM directly contacts Wif1 mRNA not structurally shown","Generality of mRNA-target selection rules unknown"]},{"year":2026,"claim":"Identifying UBA1-driven ubiquitination and degradation of NUP35 connected its turnover to inflammatory signaling, showing NUP35 loss impairs IκBα nuclear import to activate NF-κB in macrophages.","evidence":"Myeloid-specific Uba1 KO mice, sepsis (CLP) model, ubiquitinome/proteomics, IκBα nuclear import assay, and pharmacologic inhibition","pmids":["42049653"],"confidence":"High","gaps":["Direct E3 ligase mediating NUP35 ubiquitination not identified","Mechanism by which NUP35 supports IκBα import not structurally defined"]},{"year":null,"claim":"How NUP35 selects specific mRNA cargoes (nhe1, Wif1) and whether its RRM directly binds these transcripts versus acting through partner export factors remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of NUP35 bound to RNA","Export-machinery partners for selective mRNA trafficking unidentified","Reconciliation of structural nucleoporin role with mRNA-export and spindle roles incomplete"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[1,4,7]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[5,11]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2,4]}],"localization":[{"term_id":"GO:0005635","term_label":"nuclear envelope","supporting_discovery_ids":[1,4]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[1,2]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[5,11]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[1,4]}],"complexes":["nuclear pore complex"],"partners":["NUP155","NDC1","UBA1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8NFH5","full_name":"Nucleoporin NUP35","aliases":["35 kDa nucleoporin","Mitotic phosphoprotein 44","MP-44","Nuclear pore complex protein Nup53","Nucleoporin NUP53"],"length_aa":326,"mass_kda":34.8,"function":"Functions as a component of the nuclear pore complex (NPC). NPC components, collectively referred to as nucleoporins (NUPs), can play the role of both NPC structural components and of docking or interaction partners for transiently associated nuclear transport factors. May play a role in the association of MAD1 with the NPC","subcellular_location":"Nucleus, nuclear pore complex; Nucleus membrane","url":"https://www.uniprot.org/uniprotkb/Q8NFH5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/NUP35","classification":"Common Essential","n_dependent_lines":589,"n_total_lines":1208,"dependency_fraction":0.48758278145695366},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000163002","cell_line_id":"CID000092","localizations":[{"compartment":"nuclear_membrane","grade":3},{"compartment":"big_aggregates","grade":2}],"interactors":[{"gene":"MAPRE1","stoichiometry":0.2},{"gene":"NUMA1","stoichiometry":0.2},{"gene":"RAB23","stoichiometry":0.2},{"gene":"RAN","stoichiometry":0.2},{"gene":"RANBP1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000092","total_profiled":1310},"omim":[{"mim_id":"614352","title":"NUCLEOPORIN, 205-KD; NUP205","url":"https://www.omim.org/entry/614352"},{"mim_id":"614351","title":"NUCLEOPORIN, 93-KD; NUP93","url":"https://www.omim.org/entry/614351"},{"mim_id":"613220","title":"TRANSMEMBRANE PROTEIN 18; TMEM18","url":"https://www.omim.org/entry/613220"},{"mim_id":"610115","title":"NDC1 TRANSMEMBRANE NUCLEOPORIN; NDC1","url":"https://www.omim.org/entry/610115"},{"mim_id":"608140","title":"NUCLEOPORIN, 35-KD; NUP35","url":"https://www.omim.org/entry/608140"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Nuclear membrane","reliability":"Approved"},{"location":"Plasma membrane","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/NUP35"},"hgnc":{"alias_symbol":["MP44"],"prev_symbol":[]},"alphafold":{"accession":"Q8NFH5","domains":[{"cath_id":"3.30.70.330","chopping":"174-247","consensus_level":"high","plddt":95.8586,"start":174,"end":247}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8NFH5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8NFH5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8NFH5-F1-predicted_aligned_error_v6.png","plddt_mean":63.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NUP35","jax_strain_url":"https://www.jax.org/strain/search?query=NUP35"},"sequence":{"accession":"Q8NFH5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8NFH5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8NFH5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8NFH5"}},"corpus_meta":[{"pmid":"21444689","id":"PMC_21444689","title":"System analysis shows distinct 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interaction.","date":"2025","source":"Plant cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/40332586","citation_count":1,"is_preprint":false},{"pmid":"42049653","id":"PMC_42049653","title":"Macrophage Ubiquitin-Activating Enzyme 1 Promotes Sepsis-Associated Acute Kidney Injury by Relieving Nucleoporin 35-Mediated Suppression of Nuclear Factor-κB Signaling.","date":"2026","source":"Antioxidants & redox signaling","url":"https://pubmed.ncbi.nlm.nih.gov/42049653","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11827,"output_tokens":3315,"usd":0.042603,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10965,"output_tokens":4128,"usd":0.079012,"stage2_stop_reason":"end_turn"},"total_usd":0.121615,"stage1_batch_id":"msgbatch_013a8chtv3AWyTqv4z4sEF4C","stage2_batch_id":"msgbatch_01AHsuW63vinsvuTgzmQ3n62","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2006,\n      \"finding\": \"Crystal structure of mouse Nup35 RRM domain solved at 2.7 Å resolution, revealing atypical RNP1/RNP2 motifs and a novel homodimerization mode where the beta-sheet surface drives dimerization via hydrophobic interactions (Met236 sandwiched by Phe178 and Trp209), confirmed by analytical ultracentrifugation.\",\n      \"method\": \"X-ray crystallography + analytical ultracentrifugation\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional validation by analytical ultracentrifugation, direct mutagenesis-level structural analysis of interface residues\",\n      \"pmids\": [\"16962612\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"In C. elegans, depletion of Nup35/NPP-19 inhibits NE localization of Nup155/NPP-8, blocks NPC assembly, impairs nuclear lamina formation, and disrupts nucleo-cytoplasmic transport; recruitment of Nup107/NPP-5, LEM-2, and nuclear membranes to chromatin is Nup35-independent, indicating Nup35 is specifically required for NPC assembly but not initial nuclear membrane targeting.\",\n      \"method\": \"C. elegans loss-of-function mutation (npp-19(tm2886)) + fluorescence microscopy of NPC and NE markers + transport assay\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean in vivo loss-of-function with multiple orthogonal readouts (NE marker localization, NPC assembly, transport, nuclear morphology) in a single study\",\n      \"pmids\": [\"19146848\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Nup35 is required for the Ran-dependent translocation mechanism of inner nuclear membrane proteins; depletion of Nup35 specifically blocks Ran-dependent (but not ATP-dependent) FRAP recovery of inner nuclear membrane proteins, and adding FG repeats to membrane proteins accelerates their FRAP recovery in a Nup35-dependent manner, consistent with Nup35 facilitating transport through NPC peripheral channels.\",\n      \"method\": \"FRAP after siRNA depletion of Nup35 + Ran/ATP depletion experiments + FG-repeat addition to membrane proteins\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple complementary FRAP conditions with depletion of Nup35 and mechanistic dissection across multiple proteins in a single rigorous study\",\n      \"pmids\": [\"21444689\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"FG repeats on membrane proteins facilitate their transport to the inner nuclear membrane via NPC peripheral channels, and this process requires Nup35, which itself contains FG repeats, suggesting FG–FG interactions enable integral membrane proteins to act as their own transport receptors.\",\n      \"method\": \"FRAP with FG-repeat addition constructs + Nup35 depletion\",\n      \"journal\": \"Communicative & integrative biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — mechanistic interpretation supported by FRAP data from the companion study (PMID 21444689), but this paper is primarily a perspective/commentary reinforcing those results\",\n      \"pmids\": [\"22046461\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Nup53 (the Nup35 ortholog) interacts with Ndc1 (integral pore membrane protein) and Nup155 during NPC assembly; the Ndc1-binding site on Nup53 overlaps with its membrane-bending region, suggesting Ndc1 interaction modulates membrane deformation activity; Nup53–Nup155 interaction is the main determinant for Nup155 recruitment to the assembling pore.\",\n      \"method\": \"Co-immunoprecipitation, interaction mapping, in vitro membrane-bending assay, siRNA depletion with fluorescence microscopy\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal interaction mapping with functional membrane-bending assays and siRNA depletion readouts, multiple orthogonal methods\",\n      \"pmids\": [\"24363447\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Nup35 regulates NHE1 (Na⁺-H⁺ exchanger-1) expression in cardiomyocytes by controlling nucleo-cytoplasmic trafficking of nhe1 mRNA; the N-terminal domain of Nup35 mediates this by targeting the 5'-UTR (-412 to -213 nt) of nhe1 mRNA for nuclear export; Nup35 ablation depresses NHE1 expression and weakens resistance to acid challenge.\",\n      \"method\": \"siRNA knockdown + mRNA nuclear export assay + domain deletion mapping + pH challenge assay + in vivo/in vitro ischemia models\",\n      \"journal\": \"Journal of molecular cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple functional readouts (mRNA export, protein expression, pH homeostasis) with domain mapping in a single laboratory study\",\n      \"pmids\": [\"26260029\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"A point mutation in the RRM domain of Nup35 in mice causes a degenerative myopathy specifically affecting colonic smooth muscle, leading to megacolon; no defects in enteric neurons or interstitial cells of Cajal were found, placing the pathology in smooth muscle cell function downstream of intact Nup35 RRM domain.\",\n      \"method\": \"ENU-induced point mutation mouse model + histopathology + immunohistochemistry\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean in vivo genetic model with specific phenotypic characterization, but no direct molecular mechanism identified beyond RRM domain requirement\",\n      \"pmids\": [\"27427419\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Stoichiometry of Nup35 in the nuclear pore complex was estimated at approximately 23 copies per NPC, determined by single-molecule GFP fluorescence quantification after endogenous knockdown and replacement with GFP-Nup35.\",\n      \"method\": \"Single-molecule GFP fluorescence standard + spinning-disk confocal microscopy + knockdown/replacement strategy\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct quantitative measurement in living cells with rigorous single-molecule calibration, single lab, single method\",\n      \"pmids\": [\"27613095\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Nup35 localizes to microtubules and the meiotic spindle (but not nuclear membrane) after meiosis resumption in mouse oocytes, is putatively phosphorylated after GVBD, and is required for spindle assembly and chromosome alignment; Nup35 knockdown causes first polar body extrusion failure, defective kinetochore-microtubule attachment, spindle assembly checkpoint activation, and mislocalization of p-ERK1/2 from spindle poles.\",\n      \"method\": \"Immunofluorescence + siRNA knockdown + spindle assembly checkpoint analysis + kinetochore-microtubule attachment assay\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal readouts (spindle morphology, chromosome alignment, SAC markers, p-ERK1/2 localization) in a single study, single lab\",\n      \"pmids\": [\"30195030\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Auxin-mediated rapid depletion of NUP35 in human cells does not directly control gene transcription, in contrast to NUP93 which directly activates transcription; NUP35 depletion showed no significant effect on RNA Pol II loading or BRD4 recruitment at active enhancers.\",\n      \"method\": \"Auxin-inducible degron rapid depletion + Cut&Run + in situ Hi-C\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — rigorous acute depletion system with multiple genomic readouts; negative finding for NUP35 transcriptional control established by direct experiment\",\n      \"pmids\": [\"36323253\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HIV-1 capsid (CA) makes direct contact with Nup35 via FG-motif-containing regions of Nup35; this interaction supports HIV-1 nuclear entry and is dependent on cyclophilin A (CypA) interaction with CA, as CypA-binding CA mutants show altered NPC requirements.\",\n      \"method\": \"Genetic knockdown of Nup35 + HIV-1 nuclear entry assay + CA mutant analysis + direct binding assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding demonstrated with functional nuclear entry readout and CA mutation validation, single study\",\n      \"pmids\": [\"37355754\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In cardiomyocytes, Nup35 directly binds Wif1 mRNA (identified by RNA immunoprecipitation sequencing) and regulates its nuclear export; Nup35 deficiency increases nuclear retention of Wif1 pre-mRNA and decreases Wif1 protein, activating Wnt signaling and causing pathological cardiac fibrosis, hypertrophy, and dysfunction; AAV9-mediated Wif1 mRNA delivery rescues the Nup35-KO cardiac phenotype.\",\n      \"method\": \"Cardiac-specific Nup35 KO and OE mice + RNA immunoprecipitation sequencing + AAV9 rescue experiment + angiotensin II/TAC models\",\n      \"journal\": \"Cardiovascular research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo KO and OE with rescue experiment, direct RNA-binding demonstrated by RIP-seq, multiple orthogonal functional readouts across two independent cardiac stress models\",\n      \"pmids\": [\"41145234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"UBA1 (ubiquitin-activating enzyme) promotes ubiquitination and proteasomal degradation of NUP35 in macrophages during sepsis; NUP35 degradation impairs IκBα nuclear import, thereby activating NF-κB signaling and driving macrophage inflammatory activation; UBA1 knockout or pharmacologic inhibition preserves NUP35 levels, restores IκBα nuclear import, and attenuates renal injury.\",\n      \"method\": \"Myeloid-specific Uba1 KO mice + cecal ligation puncture model + ubiquitinome/proteomic analysis + IκBα nuclear import assay + co-culture studies\",\n      \"journal\": \"Antioxidants & redox signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic KO with multiple orthogonal mechanistic readouts (ubiquitinome, nuclear import assay, signaling pathway), pharmacologic validation, and rescue experiments\",\n      \"pmids\": [\"42049653\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NUP35 is a nucleoporin with an RRM domain that homodimerizes and functions as a structural NPC component (~23 copies/pore) required for NPC assembly (via Nup155 recruitment), Ran-dependent inner nuclear membrane protein transport through NPC peripheral channels via FG-repeat interactions, and selective mRNA export (including nhe1 and Wif1 mRNAs); it also associates with the meiotic spindle and regulates chromosome alignment, directly interacts with HIV-1 capsid FG motifs to facilitate viral nuclear entry, and is subject to UBA1-mediated ubiquitination and degradation that, when occurring in macrophages, activates NF-κB signaling by impairing IκBα nuclear import.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NUP35 is a structural nucleoporin that scaffolds nuclear pore complex (NPC) assembly and supports selective nucleocytoplasmic transport [#1, #4]. Its RRM domain adopts an atypical fold with non-canonical RNP1/RNP2 motifs and drives homodimerization through a beta-sheet hydrophobic interface centered on Met236 packed against Phe178 and Trp209 [#0], and the protein is present at roughly 23 copies per pore [#7]. During NPC biogenesis NUP35 (Nup53/NPP-19 in orthologs) is specifically required to recruit Nup155 to the assembling pore — its interaction with Nup155 being the principal recruitment determinant — while its binding to the integral pore membrane protein Ndc1 overlaps with a membrane-bending region that modulates membrane deformation; loss of NUP35 blocks NPC assembly, lamina formation, and transport without affecting initial nuclear membrane targeting [#1, #4]. NUP35 contains FG repeats and facilitates Ran-dependent translocation of inner nuclear membrane proteins through NPC peripheral channels via FG–FG interactions [#2, #3]. Beyond structural roles, NUP35 acts as an mRNA export factor: its N-terminal domain directs nuclear export of nhe1 mRNA in cardiomyocytes by targeting its 5'-UTR [#5], and it directly binds Wif1 mRNA, so that NUP35 deficiency causes nuclear retention of Wif1, activation of Wnt signaling, and pathological cardiac fibrosis and hypertrophy that is rescued by AAV9-delivered Wif1 mRNA [#11]. NUP35 additionally localizes to the meiotic spindle and microtubules in oocytes, where it is required for spindle assembly and chromosome alignment [#8]. It is exploited by HIV-1, whose capsid contacts NUP35 via FG-motif regions to support viral nuclear entry [#10], and its UBA1-mediated ubiquitination and proteasomal degradation in macrophages impairs IκBα nuclear import to activate NF-κB inflammatory signaling [#12]. A point mutation in the NUP35 RRM domain causes a colonic smooth-muscle degenerative myopathy and megacolon in mice [#6].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Establishing the structural basis of NUP35 oligomerization showed how this nucleoporin builds higher-order assemblies, defining its RRM as a dimerization module rather than a conventional RNA-binding fold.\",\n      \"evidence\": \"X-ray crystallography of mouse Nup35 RRM with analytical ultracentrifugation\",\n      \"pmids\": [\"16962612\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not address whether the RRM also binds RNA in vivo\", \"No structure of full-length protein or RRM in context of the NPC scaffold\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"In vivo loss-of-function placed NUP35 at a specific step of NPC biogenesis — required for Nup155 recruitment and pore assembly but dispensable for initial membrane-to-chromatin targeting — resolving where in the assembly hierarchy it acts.\",\n      \"evidence\": \"C. elegans npp-19 loss-of-function mutant with fluorescence imaging of NPC/NE markers and transport assays\",\n      \"pmids\": [\"19146848\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular detail of the Nup35-Nup155 interaction not defined here\", \"Did not test mRNA export functions\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"FRAP-based dissection revealed a transport function distinct from scaffolding: NUP35 mediates Ran-dependent translocation of inner nuclear membrane proteins through NPC peripheral channels via its FG repeats.\",\n      \"evidence\": \"FRAP after siRNA depletion with Ran/ATP depletion and FG-repeat addition constructs; companion perspective reinforcing FG-FG mechanism\",\n      \"pmids\": [\"21444689\", \"22046461\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct FG-FG contacts not structurally resolved\", \"Full set of membrane-protein cargoes not enumerated\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Reciprocal interaction mapping defined the molecular interfaces — Nup53/Nup35 binding Ndc1 and Nup155 — and linked the Ndc1-binding site to membrane-bending activity, mechanistically explaining how it couples membrane deformation to pore assembly.\",\n      \"evidence\": \"Co-IP, interaction mapping, in vitro membrane-bending assay, and siRNA depletion microscopy\",\n      \"pmids\": [\"24363447\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of membrane bending not solved\", \"Regulation of the Ndc1/Nup155 binding switch unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identification of nhe1 mRNA export control assigned NUP35 a selective mRNA-trafficking function with physiological consequence, showing its N-terminal domain targets a defined 5'-UTR region.\",\n      \"evidence\": \"siRNA knockdown, mRNA nuclear export assay, domain-deletion mapping, and ischemia/pH-challenge models\",\n      \"pmids\": [\"26260029\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether export selectivity is direct RNA binding or indirect not resolved here\", \"Single laboratory; export machinery partners unidentified\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"An RRM point-mutation mouse tied NUP35 function to a tissue-specific disease, localizing the pathology to colonic smooth muscle and implicating RRM integrity in that cell type.\",\n      \"evidence\": \"ENU-induced point-mutation mouse model with histopathology and immunohistochemistry\",\n      \"pmids\": [\"27427419\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No molecular mechanism linking RRM mutation to smooth-muscle defect\", \"Tissue specificity unexplained\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Quantitative imaging fixed the stoichiometry of NUP35 at the pore (~23 copies), constraining models of its scaffolding role.\",\n      \"evidence\": \"Single-molecule GFP fluorescence calibration with knockdown/replacement in living cells\",\n      \"pmids\": [\"27613095\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single method/lab\", \"Spatial arrangement of copies within the pore not determined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Discovery of meiotic spindle/microtubule localization extended NUP35 function beyond the interphase NPC, implicating it in spindle assembly and chromosome alignment in oocytes.\",\n      \"evidence\": \"Immunofluorescence, siRNA knockdown, SAC and kinetochore-microtubule attachment analyses in mouse oocytes\",\n      \"pmids\": [\"30195030\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct microtubule-binding interface not defined\", \"Role of putative post-GVBD phosphorylation unconfirmed\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Acute degron depletion distinguished NUP35 from transcription-regulating nucleoporins, showing it does not directly control gene transcription, RNA Pol II loading, or BRD4 recruitment.\",\n      \"evidence\": \"Auxin-inducible degron depletion with Cut&Run and in situ Hi-C\",\n      \"pmids\": [\"36323253\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not exclude indirect transcriptional effects via mRNA export\", \"Single cell system\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrating direct HIV-1 capsid contact via NUP35 FG-motif regions identified the protein as a host factor for viral nuclear entry, gated by capsid-cyclophilin A interaction.\",\n      \"evidence\": \"Nup35 knockdown, HIV-1 nuclear entry assays, capsid mutant analysis, and direct binding assay\",\n      \"pmids\": [\"37355754\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural detail of CA-NUP35 interface unresolved\", \"Single study\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"RIP-seq plus in vivo KO/OE with rescue established NUP35 as a direct Wif1 mRNA export factor whose loss activates Wnt signaling and drives cardiac fibrosis, providing a causal mRNA-to-disease axis.\",\n      \"evidence\": \"Cardiac-specific KO/OE mice, RIP-seq, AAV9 Wif1 mRNA rescue, and angiotensin II/TAC stress models\",\n      \"pmids\": [\"41145234\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether RRM directly contacts Wif1 mRNA not structurally shown\", \"Generality of mRNA-target selection rules unknown\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identifying UBA1-driven ubiquitination and degradation of NUP35 connected its turnover to inflammatory signaling, showing NUP35 loss impairs IκBα nuclear import to activate NF-κB in macrophages.\",\n      \"evidence\": \"Myeloid-specific Uba1 KO mice, sepsis (CLP) model, ubiquitinome/proteomics, IκBα nuclear import assay, and pharmacologic inhibition\",\n      \"pmids\": [\"42049653\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct E3 ligase mediating NUP35 ubiquitination not identified\", \"Mechanism by which NUP35 supports IκBα import not structurally defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How NUP35 selects specific mRNA cargoes (nhe1, Wif1) and whether its RRM directly binds these transcripts versus acting through partner export factors remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of NUP35 bound to RNA\", \"Export-machinery partners for selective mRNA trafficking unidentified\", \"Reconciliation of structural nucleoporin role with mRNA-export and spindle roles incomplete\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [1, 4, 7]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [5, 11]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005643\", \"supporting_discovery_ids\": [1, 7]},\n      {\"term_id\": \"GO:0005635\", \"supporting_discovery_ids\": [1, 4]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [5, 11]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [1, 4]}\n    ],\n    \"complexes\": [\"nuclear pore complex\"],\n    \"partners\": [\"NUP155\", \"NDC1\", \"UBA1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}