{"gene":"TNPO3","run_date":"2026-04-28T21:42:59","timeline":{"discoveries":[{"year":2009,"finding":"The HIV-1 capsid protein (CA), not integrase, is the dominant viral determinant that dictates TNPO3 dependency during HIV-1 infection, as demonstrated by MLV/HIV-1 chimera viruses pinpointing CA as the genetic determinant of sensitization to TNPO3 knockdown.","method":"MLV/HIV-1 chimeric virus infectivity assays in TNPO3 knockdown cells; in vitro pulldown and surface plasmon resonance assays comparing integrase binding hierarchy vs. infection dependency","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal genetic and biochemical approaches, replicated across multiple retroviral systems","pmids":["19846519"],"is_preprint":false},{"year":2012,"finding":"Purified recombinant TNPO3 directly stimulates HIV-1 core uncoating in vitro; this stimulatory effect is reduced by RanGTP. TNPO3 and cyclophilin A (CypA) exert opposing effects on HIV-1 uncoating, with CypA inhibiting uncoating and reducing TNPO3-stimulated uncoating in vitro.","method":"In vitro HIV-1 core uncoating assay with purified recombinant TNPO3; RanGTP competition; CypA addition assay; cyclosporine treatment in TNPO3-depleted cells","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with purified proteins, multiple orthogonal methods","pmids":["23097435"],"is_preprint":false},{"year":2013,"finding":"TNPO3 promotes HIV-1 infectivity indirectly by maintaining nuclear localization of the CA-binding protein CPSF6; TNPO3 knockdown causes CPSF6 to accumulate in the cytoplasm, leading to abnormal stabilization of the HIV-1 CA core and inhibition of HIV-1 replication. Targeting CPSF6 to the nucleus via a heterologous NLS rescues HIV-1 from TNPO3 knockdown-induced inhibition.","method":"TNPO3 siRNA knockdown; CPSF6 nuclear localization signal deletion; nuclear export signal fusion; heterologous NLS rescue; fate-of-capsid assay; 2-LTR circle qPCR; massive parallel sequencing of HIV-1 cDNA","journal":"Retrovirology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal functional rescue experiments, strong mechanistic model with genetic epistasis","pmids":["23414560"],"is_preprint":false},{"year":2013,"finding":"The ability of TNPO3-depleted cells to inhibit HIV-1 infection requires CPSF6; simultaneous depletion of TNPO3 and CPSF6 rescues HIV-1 infection, and cytosolic full-length CPSF6 blocks HIV-1 infection at the nuclear import step and enhances stability of the HIV-1 core.","method":"Double siRNA knockdown of TNPO3 and CPSF6; overexpression of cytosolic CPSF6; fate-of-capsid assay; 2-LTR circle formation assay","journal":"Retrovirology","confidence":"High","confidence_rationale":"Tier 2 — epistasis with double KD, multiple readouts, independently corroborates PMID 23414560","pmids":["23622145"],"is_preprint":false},{"year":2011,"finding":"TNPO3 promotes HIV-1 infectivity at a step detectable after the preintegration complex arrives in the nucleus (after 2-LTR circle formation but before integration), and CA is the viral determinant for TNPO3 dependence, as demonstrated by a panel of 27 CA mutants with varying TNPO3 dependence.","method":"TNPO3 knockdown (lentiviral vector and siRNA) in multiple cell types; panel of 27 CA mutant single-cycle HIV-1 vectors; qPCR for viral cDNA, 2-LTR circles, and proviral DNA","journal":"Retrovirology","confidence":"High","confidence_rationale":"Tier 2 — systematic CA mutant panel, multiple cell types, replicated knockdown methods","pmids":["22145813"],"is_preprint":false},{"year":2012,"finding":"TNPO3 is a structured protein existing in monomer-dimer equilibrium in solution; it binds directly to the HIV-1 intasome (IN tetramer prebound to cognate DNA) but not to naked viral DNA or capsid cores in vitro. Interacting amino acids map to the HIV-1 IN C-terminal domain and the cargo-binding domain of TNPO3.","method":"Circular dichroism, analytical ultracentrifugation, small-angle X-ray scattering, homology modeling; in vitro biochemical binding assays; mass spectrometry-based protein footprinting; site-directed mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — multiple biophysical methods with mutagenesis mapping of interaction interface","pmids":["22872640"],"is_preprint":false},{"year":2011,"finding":"HIV-1 IN mutations (W131A, Q168L) that impair TNPO3 binding do not significantly affect 2-LTR circle formation (nuclear import), indicating the IN-TNPO3 interaction is not a major determinant of nuclear import but may act at a nuclear step prior to integration.","method":"IN mutant viruses; TNPO3 binding assays; qPCR for 2-LTR circles and proviral DNA; integration assay","journal":"Retrovirology","confidence":"Medium","confidence_rationale":"Tier 2 — functional mutant panel with defined molecular readouts, single lab","pmids":["22176773"],"is_preprint":false},{"year":2014,"finding":"HIV-1 IN double mutant R263A/K264A significantly reduces TRN-SR2/TNPO3 interaction while retaining wild-type reverse transcription activity, and results in a block in nuclear import and integration, supporting the importance of the IN-TNPO3 interaction for HIV nuclear import.","method":"Site-directed mutagenesis; TRN-SR2 binding assay; quantitative PCR for 2-LTR circles and integration; eGFP-IN fluorescence-based nuclear import assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — mutagenesis with multiple molecular readouts including live-cell imaging, single lab","pmids":["25063804"],"is_preprint":false},{"year":2013,"finding":"TNPO3 is the causative gene for LGMD1F (now LGMD D2); a heterozygous frameshift variant in TNPO3 causes limb-girdle muscular dystrophy. Mutant TNPO3 localizes around the nucleus but not inside, unlike wild-type TNPO3.","method":"Whole-exome sequencing; Sanger validation; subcellular localization of mutant TNPO3 by transfection and microscopy","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 — disease-causing mutation identified with exome sequencing and localization experiment, replicated in additional families","pmids":["23667635"],"is_preprint":false},{"year":2019,"finding":"miR-128 directly targets two sites in the TNPO3 mRNA to downregulate TNPO3 protein expression, and this reduction of TNPO3 by miR-128 contributes to inhibition of HIV-1 replication but not MLV infection; anti-miR-128 partly neutralizes the IFN-mediated block of HIV-1.","method":"miR-128 overexpression/knockdown in Jurkat cells and primary CD4+ T cells; TNPO3 mRNA/protein quantification; HIV-1 and MLV infectivity assays; TNPO3-independent HIV-1 challenge","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 3 — functional rescue with TNPO3-independent virus provides mechanistic specificity, single lab","pmids":["31341054"],"is_preprint":false},{"year":2019,"finding":"A novel TNPO3 frameshift mutation (c.2757delC) causes LGMD D2; mutant TNPO3 protein accumulates in subsarcolemmal and perinuclear areas and fails to localize to cytoplasmic annulate lamellae pore complexes in transfected cells, while at least one SR cargo (SRSF1/SRRM2) remains normally located in the nucleus.","method":"Genetic sequencing; TNPO3 construct transfection; immunofluorescence localization in patient muscle and transfected cells","journal":"Neurology. Genetics","confidence":"Medium","confidence_rationale":"Tier 3 — localization experiment in disease context with mechanistic implication, second independent family confirming mechanism","pmids":["31192305"],"is_preprint":false},{"year":2021,"finding":"TNPO3 interacts with the splicing factor SRSF1 during myogenesis; TNPO3 decreases in the cytoplasm and becomes strongly clustered in nuclei of differentiated myotubes, while SRSF1 remains primarily nuclear, indicating coordinated nuclear import activity during muscle differentiation.","method":"Confocal, structured illumination, and electron microscopy of TNPO3 and SRSF1 during myogenesis in myoblast differentiation model","journal":"Molecular and cellular biochemistry","confidence":"Low","confidence_rationale":"Tier 3 — localization study without direct functional consequence or loss-of-function readout","pmids":["33452620"],"is_preprint":false},{"year":2020,"finding":"TNPO3 directly binds RSV Gag protein and mediates its nuclear entry; this interaction does not require the canonical cargo-binding domain (CBD) of TNPO3, suggesting a unique nuclear import mechanism for retroviral Gag distinct from SR-protein import.","method":"TNPO3 CBD deletion mutants; co-immunoprecipitation; nuclear import assays in avian cells; yeast genetic screen","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 — direct binding shown with domain deletion mutants and functional import assay, mechanistically distinct finding","pmids":["32581109"],"is_preprint":false},{"year":2022,"finding":"Tnpo3 interacts with the immunoglobulin-like fold domain of transcription factor EBF1 (via glutamic acid E271) in pro-B cells; B lineage-specific inactivation of Tnpo3 in mice blocks early B cell differentiation with down-regulation of B lineage genes and up-regulation of T/NK lineage genes.","method":"Mass spectrometry of EBF1-associated proteins; co-immunoprecipitation; EBF1 E271A point mutant retroviral transduction into Ebf1-/- progenitors; Tnpo3 conditional knockout mice; RNA-seq","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 — mass spec identification, mutagenesis of binding interface, conditional KO with defined phenotype and RNA-seq","pmids":["36167471"],"is_preprint":false},{"year":2023,"finding":"Tnpo3 is required for correct splicing of the Trav11-Traj18-Trac pre-mRNA encoding the semi-invariant TCRα chain of iNKT cells; the developmental block of iNKT cells in Tnpo3-deficient mice is rescued by transgenic provision of a pre-spliced cDNA, demonstrating Tnpo3 acts at the splicing step.","method":"Tnpo3-deficient mouse model; iNKT cell development analysis; transgenic cDNA rescue experiment; pre-mRNA splicing analysis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis via transgenic rescue definitively positions Tnpo3 at the splicing step, clean KO phenotype","pmids":["37339974"],"is_preprint":false},{"year":2025,"finding":"TNPO3 mediates nuclear import of CIRBP via a non-classical RSY-NLS in which tyrosine residues play a key role in binding, independent of serine phosphorylation. Serine and tyrosine phosphorylation within CIRBP's NLS inhibits TNPO3 binding, revealing a phosphorylation-independent (and phosphorylation-regulated) nuclear import mechanism distinct from classical SR-domain recognition.","method":"X-ray crystallography of TNPO3-CIRBP complex; mutagenesis of tyrosine residues; phosphopeptide binding assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with mutagenesis and biochemical validation of phosphorylation-dependent regulation","pmids":["40360518"],"is_preprint":false}],"current_model":"TNPO3 (Transportin-3) is a nuclear import receptor of the importin-β family that canonically transports serine/arginine-rich (SR) splicing factors into the nucleus via recognition of RS/SR-repeat NLS motifs (with tyrosine-containing non-classical NLS motifs also recognized in a phosphorylation-regulated manner); it additionally facilitates HIV-1 infection primarily by keeping the CA-binding protein CPSF6 nuclear (preventing cytoplasmic CPSF6-mediated abnormal capsid stabilization), promotes HIV-1 core uncoating in vitro in a RanGTP-sensitive manner, interacts with the HIV-1 intasome through the IN C-terminal domain, and in normal biology controls B cell programming by interacting with EBF1 and regulates iNKT cell development by supporting splicing of the TCRα pre-mRNA, with loss-of-function frameshift mutations in TNPO3 causing LGMD D2 through disruption of nuclear import and myogenic pathways."},"narrative":{"teleology":[{"year":2009,"claim":"Establishing that HIV-1 capsid (CA), not integrase, is the dominant viral determinant of TNPO3 dependency resolved the initial confusion over which viral component TNPO3 functionally engages during infection.","evidence":"MLV/HIV-1 chimeric virus infectivity assays in TNPO3-knockdown cells with surface plasmon resonance binding comparisons","pmids":["19846519"],"confidence":"High","gaps":["How CA dictates TNPO3 dependency if TNPO3 does not directly bind assembled cores remained unresolved","No structural basis for the CA-TNPO3 functional link"]},{"year":2011,"claim":"Mapping the TNPO3-dependent step in HIV-1 replication to a point after nuclear entry (post-2-LTR circle formation but before integration) revealed that TNPO3 acts beyond classical nuclear import, and a systematic panel of 27 CA mutants confirmed CA as the genetic determinant.","evidence":"TNPO3 knockdown with qPCR quantification of viral cDNA, 2-LTR circles, and proviral DNA across 27 CA mutants in multiple cell types","pmids":["22145813","22176773"],"confidence":"High","gaps":["Whether the nuclear-stage effect reflects a direct TNPO3 activity or an indirect consequence via a host co-factor was unknown"]},{"year":2012,"claim":"Biophysical and biochemical characterization showed TNPO3 directly binds the HIV-1 intasome (not naked DNA or capsid cores) through the IN C-terminal domain and the TNPO3 cargo-binding domain, while a separate in vitro reconstitution demonstrated TNPO3 stimulates HIV-1 core uncoating in a RanGTP-sensitive manner, establishing two distinct biochemical activities relevant to HIV-1.","evidence":"Purified recombinant protein binding assays, mass spectrometry footprinting, mutagenesis (intasome binding); in vitro core uncoating assay with RanGTP and CypA (uncoating activity)","pmids":["22872640","23097435"],"confidence":"High","gaps":["Whether in vitro uncoating activity reflects the physiologically dominant mechanism was unclear","The relative contributions of intasome binding versus uncoating stimulation to infectivity were not deconvolved"]},{"year":2013,"claim":"The discovery that TNPO3's principal role in HIV-1 infection is maintaining nuclear localization of the CA-binding factor CPSF6 — with cytoplasmic CPSF6 being the actual restriction factor that stabilizes cores and blocks nuclear import — unified the CA-dependency and post-nuclear-entry observations into a coherent indirect mechanism.","evidence":"TNPO3/CPSF6 double knockdown rescue; heterologous NLS fusion rescue of CPSF6; fate-of-capsid assays; 2-LTR circle qPCR","pmids":["23414560","23622145"],"confidence":"High","gaps":["Whether TNPO3 has any CPSF6-independent contribution to HIV-1 replication remained debated","How CPSF6 nuclear import by TNPO3 is regulated was not resolved"]},{"year":2013,"claim":"Identification of a heterozygous frameshift mutation in TNPO3 as the cause of LGMD D2 established TNPO3 as a disease gene for muscular dystrophy and showed that mutant TNPO3 mislocalizes to perinuclear regions rather than entering the nucleus.","evidence":"Whole-exome sequencing of LGMD1F families; Sanger validation; subcellular localization of mutant TNPO3 by immunofluorescence","pmids":["23667635"],"confidence":"Medium","gaps":["The downstream molecular pathway from impaired nuclear import to muscle degeneration was not defined","Whether dominant-negative effects or haploinsufficiency underlies pathology was unclear"]},{"year":2014,"claim":"The IN R263A/K264A double mutant that selectively disrupts TNPO3 binding while preserving reverse transcription resulted in impaired nuclear import and integration, supporting a direct functional role for the IN-TNPO3 interaction in HIV-1 nuclear entry.","evidence":"Site-directed IN mutagenesis; TNPO3 binding assay; qPCR for 2-LTR circles and integration; eGFP-IN imaging","pmids":["25063804"],"confidence":"Medium","gaps":["Whether this nuclear import defect is separable from the CPSF6-mediated indirect mechanism was not tested","Single-lab finding without independent replication"]},{"year":2019,"claim":"A second LGMD D2-causing TNPO3 frameshift mutation confirmed the disease locus and showed that mutant protein accumulates at subsarcolemmal/perinuclear sites and fails to localize to annulate lamellae, while at least some SR-protein cargoes retain normal nuclear localization.","evidence":"Genetic sequencing of independent family; immunofluorescence of patient muscle and transfected cells","pmids":["31192305"],"confidence":"Medium","gaps":["Which specific cargo(es) are critically mis-imported in LGMD D2 muscle remains unknown","No functional rescue experiment in patient cells"]},{"year":2020,"claim":"Demonstration that TNPO3 binds RSV Gag and mediates its nuclear entry through a mechanism independent of the canonical cargo-binding domain broadened the repertoire of TNPO3 import mechanisms beyond SR-domain recognition.","evidence":"TNPO3 CBD deletion mutants; co-immunoprecipitation; nuclear import assays in avian cells","pmids":["32581109"],"confidence":"Medium","gaps":["The binding site on TNPO3 for Gag was not mapped at residue resolution","Whether this non-CBD mechanism extends to other cargo proteins is unknown"]},{"year":2022,"claim":"Conditional knockout of Tnpo3 in the B lineage revealed that TNPO3 is required for early B cell differentiation by interacting with EBF1 through its immunoglobulin-like domain (E271), with loss of Tnpo3 causing downregulation of B lineage genes and aberrant upregulation of T/NK genes.","evidence":"EBF1 interactome mass spectrometry; co-immunoprecipitation; EBF1 E271A point mutant retroviral complementation in Ebf1−/− progenitors; Tnpo3 conditional KO mice; RNA-seq","pmids":["36167471"],"confidence":"High","gaps":["Whether TNPO3 acts by importing EBF1 into the nucleus or by an import-independent mechanism was not fully resolved","The splicing targets relevant to the B cell differentiation block were not identified"]},{"year":2023,"claim":"Tnpo3 was shown to be essential for iNKT cell development specifically by enabling correct splicing of the Trav11-Traj18-Trac pre-mRNA, as a pre-spliced cDNA transgene fully rescued the iNKT cell defect, definitively positioning TNPO3's physiological function at the nuclear import-to-splicing axis.","evidence":"Tnpo3-deficient mouse model; iNKT cell flow cytometry; pre-spliced TCRα cDNA transgenic rescue; pre-mRNA splicing analysis","pmids":["37339974"],"confidence":"High","gaps":["Which SR-protein cargo(es) are rate-limiting for TCRα splicing downstream of TNPO3 import is unknown","Whether analogous splicing defects occur in other Tnpo3-dependent cell lineages was not tested"]},{"year":2025,"claim":"The crystal structure of TNPO3 bound to CIRBP revealed a non-classical RSY-NLS recognition mode in which tyrosine residues (not serine phosphorylation) drive binding, with phosphorylation of serine and tyrosine residues inhibiting the interaction, establishing a phosphorylation-regulated import mechanism distinct from canonical RS-domain recognition.","evidence":"X-ray crystallography of TNPO3–CIRBP complex; tyrosine mutagenesis; phosphopeptide binding assays","pmids":["40360518"],"confidence":"High","gaps":["Whether the RSY-NLS mode applies to other TNPO3 cargoes beyond CIRBP is untested","Structural basis for how phosphorylation blocks binding at atomic resolution is incomplete"]},{"year":null,"claim":"Major unresolved questions include which specific cargo(es) are critically mis-imported in LGMD D2 muscle, whether TNPO3 has CPSF6-independent contributions to HIV-1 replication in vivo, and the full repertoire of physiological cargoes recognized via the RSY-NLS versus classical RS-domain mechanisms.","evidence":"Open question arising from accumulated literature","pmids":[],"confidence":"Low","gaps":["No systematic cargo profiling in TNPO3-deficient cells has been reported","The pathogenic mechanism of LGMD D2 at the molecular cargo level is undefined","No structural basis for the TNPO3–CPSF6 interaction has been determined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[2,4,7,12,13,14,15]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,3,14]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[8,10,11,13]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2,11]},{"term_id":"GO:0005635","term_label":"nuclear envelope","supporting_discovery_ids":[10]}],"pathway":[{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[2,4,7,12,15]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[14]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[13,14]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[13,14]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[8,10]}],"complexes":[],"partners":["CPSF6","EBF1","SRSF1","CIRBP"],"other_free_text":[]},"mechanistic_narrative":"TNPO3 (Transportin-3/TRN-SR2) is a nuclear import receptor of the importin-β superfamily that transports serine/arginine-rich (SR) splicing factors and other cargoes into the nucleus, thereby coupling nuclear import to RNA splicing, lymphocyte differentiation, and myogenesis. Its cargo-binding domain recognizes RS/SR-repeat nuclear localization signals as well as non-classical RSY-type NLS motifs in which tyrosine residues are critical for binding, with serine and tyrosine phosphorylation acting as negative regulators of cargo recognition [PMID:40360518]. TNPO3 is co-opted during HIV-1 infection: its primary contribution to viral replication is maintaining the nuclear localization of the capsid-binding factor CPSF6, whose cytoplasmic accumulation upon TNPO3 depletion abnormally stabilizes the HIV-1 core and blocks infection at the nuclear import/integration step [PMID:23414560, PMID:23622145]; TNPO3 also directly stimulates HIV-1 core uncoating in vitro in a RanGTP-sensitive manner and binds the HIV-1 intasome through the integrase C-terminal domain [PMID:23097435, PMID:22872640]. In normal physiology, conditional Tnpo3 knockout in mice blocks early B cell differentiation through interaction with the transcription factor EBF1, and Tnpo3 deficiency impairs iNKT cell development by disrupting splicing of the semi-invariant TCRα pre-mRNA, a defect rescued by a pre-spliced cDNA transgene [PMID:36167471, PMID:37339974]; heterozygous frameshift mutations in TNPO3 cause limb-girdle muscular dystrophy type D2 (LGMD D2) [PMID:23667635, PMID:31192305]."},"prefetch_data":{"uniprot":{"accession":"Q9Y5L0","full_name":"Transportin-3","aliases":["Importin-12","Imp12","Transportin-SR","TRN-SR"],"length_aa":923,"mass_kda":104.2,"function":"Importin, which transports target proteins into the nucleus (PubMed:10366588, PubMed:10713112, PubMed:11517331, PubMed:12628928, PubMed:24449914). Specifically mediates the nuclear import of splicing factor serine/arginine (SR) proteins, such as RBM4, SFRS1 and SFRS2, by recognizing phosphorylated SR domains (PubMed:10366588, PubMed:10713112, PubMed:11517331, PubMed:12628928, PubMed:24449914). Also mediates the nuclear import of serine/arginine (SR) protein CPSF6, independently of CPSF6 phosphorylation (PubMed:30916345, PubMed:31465518). The nuclear import process is regulated by the small GTPase Ran that partitions between cytoplasm and nucleus in the predominantly GDP- and GTP-bound form, respectively (PubMed:23878195, PubMed:24449914). Importin associates with target cargo proteins in the cytoplasm, and the competitive binding of GTP-bound Ran induces the release of cargos in the nucleus (PubMed:23878195, PubMed:24449914) (Microbial infection) Involved in immunodeficiency virus (HIV-1) infection by importing the pre-integration complex (PIC) into the nucleus (PubMed:18722123, PubMed:21901095, PubMed:22398280, PubMed:29329553). Required for a nuclear maturation step of HIV-1 prior to integration (PubMed:21901095, PubMed:22398280)","subcellular_location":"Nucleus envelope; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q9Y5L0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/TNPO3","classification":"Common Essential","n_dependent_lines":1203,"n_total_lines":1208,"dependency_fraction":0.9958609271523179},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000064419","cell_line_id":"CID001578","localizations":[{"compartment":"big_aggregates","grade":3},{"compartment":"cytoplasmic","grade":3},{"compartment":"nucleoplasm","grade":3}],"interactors":[{"gene":"TRA2B","stoichiometry":10.0},{"gene":"LUC7L","stoichiometry":10.0},{"gene":"CIRBP","stoichiometry":10.0},{"gene":"CPSF6","stoichiometry":4.0},{"gene":"RBM27","stoichiometry":4.0},{"gene":"CACTIN","stoichiometry":4.0},{"gene":"NUDT21","stoichiometry":4.0},{"gene":"RBM14","stoichiometry":0.2},{"gene":"RBM39","stoichiometry":0.2},{"gene":"RBM26","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001578","total_profiled":1310},"omim":[{"mim_id":"614221","title":"BILIARY CIRRHOSIS, PRIMARY, 5; PBC5","url":"https://www.omim.org/entry/614221"},{"mim_id":"614220","title":"BILIARY CIRRHOSIS, PRIMARY, 4; PBC4","url":"https://www.omim.org/entry/614220"},{"mim_id":"610032","title":"TRANSPORTIN 3; TNPO3","url":"https://www.omim.org/entry/610032"},{"mim_id":"609423","title":"HUMAN IMMUNODEFICIENCY VIRUS TYPE 1, SUSCEPTIBILITY TO","url":"https://www.omim.org/entry/609423"},{"mim_id":"608423","title":"MUSCULAR DYSTROPHY, LIMB-GIRDLE, AUTOSOMAL DOMINANT 2; LGMDD2","url":"https://www.omim.org/entry/608423"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TNPO3"},"hgnc":{"alias_symbol":["TRN-SR","MTR10A","TRN-SR2","IPO12"],"prev_symbol":["LGMD1F"]},"alphafold":{"accession":"Q9Y5L0","domains":[{"cath_id":"-","chopping":"252-357","consensus_level":"medium","plddt":96.4575,"start":252,"end":357},{"cath_id":"-","chopping":"358-494","consensus_level":"medium","plddt":94.7673,"start":358,"end":494},{"cath_id":"-","chopping":"806-923","consensus_level":"medium","plddt":94.3273,"start":806,"end":923}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y5L0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y5L0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y5L0-F1-predicted_aligned_error_v6.png","plddt_mean":94.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TNPO3","jax_strain_url":"https://www.jax.org/strain/search?query=TNPO3"},"sequence":{"accession":"Q9Y5L0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y5L0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y5L0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y5L0"}},"corpus_meta":[{"pmid":"19846519","id":"PMC_19846519","title":"The requirement for cellular transportin 3 (TNPO3 or TRN-SR2) during infection maps to human immunodeficiency virus type 1 capsid and not integrase.","date":"2009","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/19846519","citation_count":153,"is_preprint":false},{"pmid":"23414560","id":"PMC_23414560","title":"TNPO3 protects HIV-1 replication from CPSF6-mediated capsid stabilization in the host cell cytoplasm.","date":"2013","source":"Retrovirology","url":"https://pubmed.ncbi.nlm.nih.gov/23414560","citation_count":122,"is_preprint":false},{"pmid":"23097435","id":"PMC_23097435","title":"The host proteins transportin SR2/TNPO3 and cyclophilin A exert opposing effects on HIV-1 uncoating.","date":"2012","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/23097435","citation_count":114,"is_preprint":false},{"pmid":"23622145","id":"PMC_23622145","title":"The ability of TNPO3-depleted cells to inhibit HIV-1 infection requires CPSF6.","date":"2013","source":"Retrovirology","url":"https://pubmed.ncbi.nlm.nih.gov/23622145","citation_count":81,"is_preprint":false},{"pmid":"22145813","id":"PMC_22145813","title":"Inhibition of HIV-1 infection by TNPO3 depletion is determined by capsid and detectable after viral cDNA enters the nucleus.","date":"2011","source":"Retrovirology","url":"https://pubmed.ncbi.nlm.nih.gov/22145813","citation_count":81,"is_preprint":false},{"pmid":"35876041","id":"PMC_35876041","title":"Circular RNA circ-TNPO3 inhibits clear cell renal cell carcinoma metastasis by binding to IGF2BP2 and destabilizing SERPINH1 mRNA.","date":"2022","source":"Clinical and translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/35876041","citation_count":67,"is_preprint":false},{"pmid":"25205108","id":"PMC_25205108","title":"The IRF5-TNPO3 association with systemic lupus erythematosus has two components that other autoimmune disorders variably share.","date":"2014","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/25205108","citation_count":66,"is_preprint":false},{"pmid":"34703650","id":"PMC_34703650","title":"Circular RNA circ-TNPO3 suppresses metastasis of GC by acting as a protein decoy for IGF2BP3 to regulate the expression of MYC and SNAIL.","date":"2021","source":"Molecular therapy. Nucleic acids","url":"https://pubmed.ncbi.nlm.nih.gov/34703650","citation_count":66,"is_preprint":false},{"pmid":"23667635","id":"PMC_23667635","title":"Next-generation sequencing identifies transportin 3 as the causative gene for LGMD1F.","date":"2013","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/23667635","citation_count":65,"is_preprint":false},{"pmid":"22872640","id":"PMC_22872640","title":"Interaction of the HIV-1 intasome with transportin 3 protein (TNPO3 or TRN-SR2).","date":"2012","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22872640","citation_count":46,"is_preprint":false},{"pmid":"22176773","id":"PMC_22176773","title":"Mutations affecting interaction of integrase with TNPO3 do not prevent HIV-1 cDNA nuclear import.","date":"2011","source":"Retrovirology","url":"https://pubmed.ncbi.nlm.nih.gov/22176773","citation_count":34,"is_preprint":false},{"pmid":"24779422","id":"PMC_24779422","title":"Mouse mammary tumor virus-based vector transduces non-dividing cells, enters the nucleus via a TNPO3-independent pathway and integrates in a less biased fashion than other retroviruses.","date":"2014","source":"Retrovirology","url":"https://pubmed.ncbi.nlm.nih.gov/24779422","citation_count":26,"is_preprint":false},{"pmid":"31341054","id":"PMC_31341054","title":"Interferon-Inducible MicroRNA miR-128 Modulates HIV-1 Replication by Targeting TNPO3 mRNA.","date":"2019","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/31341054","citation_count":25,"is_preprint":false},{"pmid":"23632945","id":"PMC_23632945","title":"Clinical phenotype, muscle MRI and muscle pathology of LGMD1F.","date":"2013","source":"Journal of neurology","url":"https://pubmed.ncbi.nlm.nih.gov/23632945","citation_count":25,"is_preprint":false},{"pmid":"25063804","id":"PMC_25063804","title":"The HIV-1 integrase mutant R263A/K264A is 2-fold defective for TRN-SR2 binding and viral nuclear import.","date":"2014","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/25063804","citation_count":24,"is_preprint":false},{"pmid":"23279333","id":"PMC_23279333","title":"Ultrastructural changes in LGMD1F.","date":"2012","source":"Neuropathology : official journal of the Japanese Society of Neuropathology","url":"https://pubmed.ncbi.nlm.nih.gov/23279333","citation_count":21,"is_preprint":false},{"pmid":"31192305","id":"PMC_31192305","title":"Novel mutation in TNPO3 causes congenital limb-girdle myopathy with slow progression.","date":"2019","source":"Neurology. Genetics","url":"https://pubmed.ncbi.nlm.nih.gov/31192305","citation_count":19,"is_preprint":false},{"pmid":"35309568","id":"PMC_35309568","title":"LGMD D2 TNPO3-Related: From Clinical Spectrum to Pathogenetic Mechanism.","date":"2022","source":"Frontiers in neurology","url":"https://pubmed.ncbi.nlm.nih.gov/35309568","citation_count":16,"is_preprint":false},{"pmid":"33452620","id":"PMC_33452620","title":"Morphological study of TNPO3 and SRSF1 interaction during myogenesis by combining confocal, structured illumination and electron microscopy analysis.","date":"2021","source":"Molecular and cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/33452620","citation_count":15,"is_preprint":false},{"pmid":"32690349","id":"PMC_32690349","title":"Transportin 3 (TNPO3) and related proteins in limb girdle muscular dystrophy D2 muscle biopsies: A morphological study and pathogenetic hypothesis.","date":"2020","source":"Neuromuscular disorders : NMD","url":"https://pubmed.ncbi.nlm.nih.gov/32690349","citation_count":14,"is_preprint":false},{"pmid":"32581109","id":"PMC_32581109","title":"TNPO3-Mediated Nuclear Entry of the Rous Sarcoma Virus Gag Protein Is Independent of the Cargo-Binding Domain.","date":"2020","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/32581109","citation_count":9,"is_preprint":false},{"pmid":"36167471","id":"PMC_36167471","title":"Tnpo3 enables EBF1 function in conditions of antagonistic Notch signaling.","date":"2022","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/36167471","citation_count":8,"is_preprint":false},{"pmid":"36789274","id":"PMC_36789274","title":"CRISPR-Cas9 editing of a TNPO3 mutation in a muscle cell model of limb-girdle muscular dystrophy type D2.","date":"2023","source":"Molecular therapy. Nucleic acids","url":"https://pubmed.ncbi.nlm.nih.gov/36789274","citation_count":7,"is_preprint":false},{"pmid":"37339974","id":"PMC_37339974","title":"Tnpo3 controls splicing of the pre-mRNA encoding the canonical TCR α chain of iNKT cells.","date":"2023","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/37339974","citation_count":3,"is_preprint":false},{"pmid":"40360518","id":"PMC_40360518","title":"Structural basis of phosphorylation-independent nuclear import of CIRBP by TNPO3.","date":"2025","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/40360518","citation_count":0,"is_preprint":false},{"pmid":"36082569","id":"PMC_36082569","title":"[Analysis of TNPO3 gene variant and clinical phenotype in a neonate with limb-girdle muscular dystrophies form 1F].","date":"2022","source":"Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/36082569","citation_count":0,"is_preprint":false},{"pmid":"41291251","id":"PMC_41291251","title":"Novel insights into the molecular mechanisms of LGMDD2: role of TNPO3 in experimental cell and zebrafish models.","date":"2025","source":"Cellular and molecular life sciences : CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/41291251","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.12.27.24319460","title":"Deleterious coding variation associated with autism is consistent across populations, as exemplified by admixed Latin American populations","date":"2025-01-06","source":"bioRxiv","url":"https://doi.org/10.1101/2024.12.27.24319460","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":17094,"output_tokens":4034,"usd":0.055896},"stage2":{"model":"claude-opus-4-6","input_tokens":7465,"output_tokens":3432,"usd":0.184688},"total_usd":0.240584,"stage1_batch_id":"msgbatch_01RPbVc9PDH9zQmYK5Hs6srm","stage2_batch_id":"msgbatch_01JVbVLzeKN2yVrsGp3zC8aM","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2009,\n      \"finding\": \"The HIV-1 capsid protein (CA), not integrase, is the dominant viral determinant that dictates TNPO3 dependency during HIV-1 infection, as demonstrated by MLV/HIV-1 chimera viruses pinpointing CA as the genetic determinant of sensitization to TNPO3 knockdown.\",\n      \"method\": \"MLV/HIV-1 chimeric virus infectivity assays in TNPO3 knockdown cells; in vitro pulldown and surface plasmon resonance assays comparing integrase binding hierarchy vs. infection dependency\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal genetic and biochemical approaches, replicated across multiple retroviral systems\",\n      \"pmids\": [\"19846519\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Purified recombinant TNPO3 directly stimulates HIV-1 core uncoating in vitro; this stimulatory effect is reduced by RanGTP. TNPO3 and cyclophilin A (CypA) exert opposing effects on HIV-1 uncoating, with CypA inhibiting uncoating and reducing TNPO3-stimulated uncoating in vitro.\",\n      \"method\": \"In vitro HIV-1 core uncoating assay with purified recombinant TNPO3; RanGTP competition; CypA addition assay; cyclosporine treatment in TNPO3-depleted cells\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with purified proteins, multiple orthogonal methods\",\n      \"pmids\": [\"23097435\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TNPO3 promotes HIV-1 infectivity indirectly by maintaining nuclear localization of the CA-binding protein CPSF6; TNPO3 knockdown causes CPSF6 to accumulate in the cytoplasm, leading to abnormal stabilization of the HIV-1 CA core and inhibition of HIV-1 replication. Targeting CPSF6 to the nucleus via a heterologous NLS rescues HIV-1 from TNPO3 knockdown-induced inhibition.\",\n      \"method\": \"TNPO3 siRNA knockdown; CPSF6 nuclear localization signal deletion; nuclear export signal fusion; heterologous NLS rescue; fate-of-capsid assay; 2-LTR circle qPCR; massive parallel sequencing of HIV-1 cDNA\",\n      \"journal\": \"Retrovirology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal functional rescue experiments, strong mechanistic model with genetic epistasis\",\n      \"pmids\": [\"23414560\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The ability of TNPO3-depleted cells to inhibit HIV-1 infection requires CPSF6; simultaneous depletion of TNPO3 and CPSF6 rescues HIV-1 infection, and cytosolic full-length CPSF6 blocks HIV-1 infection at the nuclear import step and enhances stability of the HIV-1 core.\",\n      \"method\": \"Double siRNA knockdown of TNPO3 and CPSF6; overexpression of cytosolic CPSF6; fate-of-capsid assay; 2-LTR circle formation assay\",\n      \"journal\": \"Retrovirology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — epistasis with double KD, multiple readouts, independently corroborates PMID 23414560\",\n      \"pmids\": [\"23622145\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"TNPO3 promotes HIV-1 infectivity at a step detectable after the preintegration complex arrives in the nucleus (after 2-LTR circle formation but before integration), and CA is the viral determinant for TNPO3 dependence, as demonstrated by a panel of 27 CA mutants with varying TNPO3 dependence.\",\n      \"method\": \"TNPO3 knockdown (lentiviral vector and siRNA) in multiple cell types; panel of 27 CA mutant single-cycle HIV-1 vectors; qPCR for viral cDNA, 2-LTR circles, and proviral DNA\",\n      \"journal\": \"Retrovirology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — systematic CA mutant panel, multiple cell types, replicated knockdown methods\",\n      \"pmids\": [\"22145813\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"TNPO3 is a structured protein existing in monomer-dimer equilibrium in solution; it binds directly to the HIV-1 intasome (IN tetramer prebound to cognate DNA) but not to naked viral DNA or capsid cores in vitro. Interacting amino acids map to the HIV-1 IN C-terminal domain and the cargo-binding domain of TNPO3.\",\n      \"method\": \"Circular dichroism, analytical ultracentrifugation, small-angle X-ray scattering, homology modeling; in vitro biochemical binding assays; mass spectrometry-based protein footprinting; site-directed mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple biophysical methods with mutagenesis mapping of interaction interface\",\n      \"pmids\": [\"22872640\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"HIV-1 IN mutations (W131A, Q168L) that impair TNPO3 binding do not significantly affect 2-LTR circle formation (nuclear import), indicating the IN-TNPO3 interaction is not a major determinant of nuclear import but may act at a nuclear step prior to integration.\",\n      \"method\": \"IN mutant viruses; TNPO3 binding assays; qPCR for 2-LTR circles and proviral DNA; integration assay\",\n      \"journal\": \"Retrovirology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional mutant panel with defined molecular readouts, single lab\",\n      \"pmids\": [\"22176773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HIV-1 IN double mutant R263A/K264A significantly reduces TRN-SR2/TNPO3 interaction while retaining wild-type reverse transcription activity, and results in a block in nuclear import and integration, supporting the importance of the IN-TNPO3 interaction for HIV nuclear import.\",\n      \"method\": \"Site-directed mutagenesis; TRN-SR2 binding assay; quantitative PCR for 2-LTR circles and integration; eGFP-IN fluorescence-based nuclear import assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mutagenesis with multiple molecular readouts including live-cell imaging, single lab\",\n      \"pmids\": [\"25063804\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TNPO3 is the causative gene for LGMD1F (now LGMD D2); a heterozygous frameshift variant in TNPO3 causes limb-girdle muscular dystrophy. Mutant TNPO3 localizes around the nucleus but not inside, unlike wild-type TNPO3.\",\n      \"method\": \"Whole-exome sequencing; Sanger validation; subcellular localization of mutant TNPO3 by transfection and microscopy\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — disease-causing mutation identified with exome sequencing and localization experiment, replicated in additional families\",\n      \"pmids\": [\"23667635\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"miR-128 directly targets two sites in the TNPO3 mRNA to downregulate TNPO3 protein expression, and this reduction of TNPO3 by miR-128 contributes to inhibition of HIV-1 replication but not MLV infection; anti-miR-128 partly neutralizes the IFN-mediated block of HIV-1.\",\n      \"method\": \"miR-128 overexpression/knockdown in Jurkat cells and primary CD4+ T cells; TNPO3 mRNA/protein quantification; HIV-1 and MLV infectivity assays; TNPO3-independent HIV-1 challenge\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — functional rescue with TNPO3-independent virus provides mechanistic specificity, single lab\",\n      \"pmids\": [\"31341054\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"A novel TNPO3 frameshift mutation (c.2757delC) causes LGMD D2; mutant TNPO3 protein accumulates in subsarcolemmal and perinuclear areas and fails to localize to cytoplasmic annulate lamellae pore complexes in transfected cells, while at least one SR cargo (SRSF1/SRRM2) remains normally located in the nucleus.\",\n      \"method\": \"Genetic sequencing; TNPO3 construct transfection; immunofluorescence localization in patient muscle and transfected cells\",\n      \"journal\": \"Neurology. Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — localization experiment in disease context with mechanistic implication, second independent family confirming mechanism\",\n      \"pmids\": [\"31192305\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TNPO3 interacts with the splicing factor SRSF1 during myogenesis; TNPO3 decreases in the cytoplasm and becomes strongly clustered in nuclei of differentiated myotubes, while SRSF1 remains primarily nuclear, indicating coordinated nuclear import activity during muscle differentiation.\",\n      \"method\": \"Confocal, structured illumination, and electron microscopy of TNPO3 and SRSF1 during myogenesis in myoblast differentiation model\",\n      \"journal\": \"Molecular and cellular biochemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — localization study without direct functional consequence or loss-of-function readout\",\n      \"pmids\": [\"33452620\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TNPO3 directly binds RSV Gag protein and mediates its nuclear entry; this interaction does not require the canonical cargo-binding domain (CBD) of TNPO3, suggesting a unique nuclear import mechanism for retroviral Gag distinct from SR-protein import.\",\n      \"method\": \"TNPO3 CBD deletion mutants; co-immunoprecipitation; nuclear import assays in avian cells; yeast genetic screen\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct binding shown with domain deletion mutants and functional import assay, mechanistically distinct finding\",\n      \"pmids\": [\"32581109\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Tnpo3 interacts with the immunoglobulin-like fold domain of transcription factor EBF1 (via glutamic acid E271) in pro-B cells; B lineage-specific inactivation of Tnpo3 in mice blocks early B cell differentiation with down-regulation of B lineage genes and up-regulation of T/NK lineage genes.\",\n      \"method\": \"Mass spectrometry of EBF1-associated proteins; co-immunoprecipitation; EBF1 E271A point mutant retroviral transduction into Ebf1-/- progenitors; Tnpo3 conditional knockout mice; RNA-seq\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mass spec identification, mutagenesis of binding interface, conditional KO with defined phenotype and RNA-seq\",\n      \"pmids\": [\"36167471\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Tnpo3 is required for correct splicing of the Trav11-Traj18-Trac pre-mRNA encoding the semi-invariant TCRα chain of iNKT cells; the developmental block of iNKT cells in Tnpo3-deficient mice is rescued by transgenic provision of a pre-spliced cDNA, demonstrating Tnpo3 acts at the splicing step.\",\n      \"method\": \"Tnpo3-deficient mouse model; iNKT cell development analysis; transgenic cDNA rescue experiment; pre-mRNA splicing analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis via transgenic rescue definitively positions Tnpo3 at the splicing step, clean KO phenotype\",\n      \"pmids\": [\"37339974\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TNPO3 mediates nuclear import of CIRBP via a non-classical RSY-NLS in which tyrosine residues play a key role in binding, independent of serine phosphorylation. Serine and tyrosine phosphorylation within CIRBP's NLS inhibits TNPO3 binding, revealing a phosphorylation-independent (and phosphorylation-regulated) nuclear import mechanism distinct from classical SR-domain recognition.\",\n      \"method\": \"X-ray crystallography of TNPO3-CIRBP complex; mutagenesis of tyrosine residues; phosphopeptide binding assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with mutagenesis and biochemical validation of phosphorylation-dependent regulation\",\n      \"pmids\": [\"40360518\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TNPO3 (Transportin-3) is a nuclear import receptor of the importin-β family that canonically transports serine/arginine-rich (SR) splicing factors into the nucleus via recognition of RS/SR-repeat NLS motifs (with tyrosine-containing non-classical NLS motifs also recognized in a phosphorylation-regulated manner); it additionally facilitates HIV-1 infection primarily by keeping the CA-binding protein CPSF6 nuclear (preventing cytoplasmic CPSF6-mediated abnormal capsid stabilization), promotes HIV-1 core uncoating in vitro in a RanGTP-sensitive manner, interacts with the HIV-1 intasome through the IN C-terminal domain, and in normal biology controls B cell programming by interacting with EBF1 and regulates iNKT cell development by supporting splicing of the TCRα pre-mRNA, with loss-of-function frameshift mutations in TNPO3 causing LGMD D2 through disruption of nuclear import and myogenic pathways.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"TNPO3 (Transportin-3/TRN-SR2) is a nuclear import receptor of the importin-β superfamily that transports serine/arginine-rich (SR) splicing factors and other cargoes into the nucleus, thereby coupling nuclear import to RNA splicing, lymphocyte differentiation, and myogenesis. Its cargo-binding domain recognizes RS/SR-repeat nuclear localization signals as well as non-classical RSY-type NLS motifs in which tyrosine residues are critical for binding, with serine and tyrosine phosphorylation acting as negative regulators of cargo recognition [PMID:40360518]. TNPO3 is co-opted during HIV-1 infection: its primary contribution to viral replication is maintaining the nuclear localization of the capsid-binding factor CPSF6, whose cytoplasmic accumulation upon TNPO3 depletion abnormally stabilizes the HIV-1 core and blocks infection at the nuclear import/integration step [PMID:23414560, PMID:23622145]; TNPO3 also directly stimulates HIV-1 core uncoating in vitro in a RanGTP-sensitive manner and binds the HIV-1 intasome through the integrase C-terminal domain [PMID:23097435, PMID:22872640]. In normal physiology, conditional Tnpo3 knockout in mice blocks early B cell differentiation through interaction with the transcription factor EBF1, and Tnpo3 deficiency impairs iNKT cell development by disrupting splicing of the semi-invariant TCRα pre-mRNA, a defect rescued by a pre-spliced cDNA transgene [PMID:36167471, PMID:37339974]; heterozygous frameshift mutations in TNPO3 cause limb-girdle muscular dystrophy type D2 (LGMD D2) [PMID:23667635, PMID:31192305].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Establishing that HIV-1 capsid (CA), not integrase, is the dominant viral determinant of TNPO3 dependency resolved the initial confusion over which viral component TNPO3 functionally engages during infection.\",\n      \"evidence\": \"MLV/HIV-1 chimeric virus infectivity assays in TNPO3-knockdown cells with surface plasmon resonance binding comparisons\",\n      \"pmids\": [\"19846519\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CA dictates TNPO3 dependency if TNPO3 does not directly bind assembled cores remained unresolved\", \"No structural basis for the CA-TNPO3 functional link\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Mapping the TNPO3-dependent step in HIV-1 replication to a point after nuclear entry (post-2-LTR circle formation but before integration) revealed that TNPO3 acts beyond classical nuclear import, and a systematic panel of 27 CA mutants confirmed CA as the genetic determinant.\",\n      \"evidence\": \"TNPO3 knockdown with qPCR quantification of viral cDNA, 2-LTR circles, and proviral DNA across 27 CA mutants in multiple cell types\",\n      \"pmids\": [\"22145813\", \"22176773\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the nuclear-stage effect reflects a direct TNPO3 activity or an indirect consequence via a host co-factor was unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Biophysical and biochemical characterization showed TNPO3 directly binds the HIV-1 intasome (not naked DNA or capsid cores) through the IN C-terminal domain and the TNPO3 cargo-binding domain, while a separate in vitro reconstitution demonstrated TNPO3 stimulates HIV-1 core uncoating in a RanGTP-sensitive manner, establishing two distinct biochemical activities relevant to HIV-1.\",\n      \"evidence\": \"Purified recombinant protein binding assays, mass spectrometry footprinting, mutagenesis (intasome binding); in vitro core uncoating assay with RanGTP and CypA (uncoating activity)\",\n      \"pmids\": [\"22872640\", \"23097435\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether in vitro uncoating activity reflects the physiologically dominant mechanism was unclear\", \"The relative contributions of intasome binding versus uncoating stimulation to infectivity were not deconvolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"The discovery that TNPO3's principal role in HIV-1 infection is maintaining nuclear localization of the CA-binding factor CPSF6 — with cytoplasmic CPSF6 being the actual restriction factor that stabilizes cores and blocks nuclear import — unified the CA-dependency and post-nuclear-entry observations into a coherent indirect mechanism.\",\n      \"evidence\": \"TNPO3/CPSF6 double knockdown rescue; heterologous NLS fusion rescue of CPSF6; fate-of-capsid assays; 2-LTR circle qPCR\",\n      \"pmids\": [\"23414560\", \"23622145\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether TNPO3 has any CPSF6-independent contribution to HIV-1 replication remained debated\", \"How CPSF6 nuclear import by TNPO3 is regulated was not resolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identification of a heterozygous frameshift mutation in TNPO3 as the cause of LGMD D2 established TNPO3 as a disease gene for muscular dystrophy and showed that mutant TNPO3 mislocalizes to perinuclear regions rather than entering the nucleus.\",\n      \"evidence\": \"Whole-exome sequencing of LGMD1F families; Sanger validation; subcellular localization of mutant TNPO3 by immunofluorescence\",\n      \"pmids\": [\"23667635\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The downstream molecular pathway from impaired nuclear import to muscle degeneration was not defined\", \"Whether dominant-negative effects or haploinsufficiency underlies pathology was unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"The IN R263A/K264A double mutant that selectively disrupts TNPO3 binding while preserving reverse transcription resulted in impaired nuclear import and integration, supporting a direct functional role for the IN-TNPO3 interaction in HIV-1 nuclear entry.\",\n      \"evidence\": \"Site-directed IN mutagenesis; TNPO3 binding assay; qPCR for 2-LTR circles and integration; eGFP-IN imaging\",\n      \"pmids\": [\"25063804\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether this nuclear import defect is separable from the CPSF6-mediated indirect mechanism was not tested\", \"Single-lab finding without independent replication\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"A second LGMD D2-causing TNPO3 frameshift mutation confirmed the disease locus and showed that mutant protein accumulates at subsarcolemmal/perinuclear sites and fails to localize to annulate lamellae, while at least some SR-protein cargoes retain normal nuclear localization.\",\n      \"evidence\": \"Genetic sequencing of independent family; immunofluorescence of patient muscle and transfected cells\",\n      \"pmids\": [\"31192305\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Which specific cargo(es) are critically mis-imported in LGMD D2 muscle remains unknown\", \"No functional rescue experiment in patient cells\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstration that TNPO3 binds RSV Gag and mediates its nuclear entry through a mechanism independent of the canonical cargo-binding domain broadened the repertoire of TNPO3 import mechanisms beyond SR-domain recognition.\",\n      \"evidence\": \"TNPO3 CBD deletion mutants; co-immunoprecipitation; nuclear import assays in avian cells\",\n      \"pmids\": [\"32581109\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The binding site on TNPO3 for Gag was not mapped at residue resolution\", \"Whether this non-CBD mechanism extends to other cargo proteins is unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Conditional knockout of Tnpo3 in the B lineage revealed that TNPO3 is required for early B cell differentiation by interacting with EBF1 through its immunoglobulin-like domain (E271), with loss of Tnpo3 causing downregulation of B lineage genes and aberrant upregulation of T/NK genes.\",\n      \"evidence\": \"EBF1 interactome mass spectrometry; co-immunoprecipitation; EBF1 E271A point mutant retroviral complementation in Ebf1−/− progenitors; Tnpo3 conditional KO mice; RNA-seq\",\n      \"pmids\": [\"36167471\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether TNPO3 acts by importing EBF1 into the nucleus or by an import-independent mechanism was not fully resolved\", \"The splicing targets relevant to the B cell differentiation block were not identified\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Tnpo3 was shown to be essential for iNKT cell development specifically by enabling correct splicing of the Trav11-Traj18-Trac pre-mRNA, as a pre-spliced cDNA transgene fully rescued the iNKT cell defect, definitively positioning TNPO3's physiological function at the nuclear import-to-splicing axis.\",\n      \"evidence\": \"Tnpo3-deficient mouse model; iNKT cell flow cytometry; pre-spliced TCRα cDNA transgenic rescue; pre-mRNA splicing analysis\",\n      \"pmids\": [\"37339974\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which SR-protein cargo(es) are rate-limiting for TCRα splicing downstream of TNPO3 import is unknown\", \"Whether analogous splicing defects occur in other Tnpo3-dependent cell lineages was not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"The crystal structure of TNPO3 bound to CIRBP revealed a non-classical RSY-NLS recognition mode in which tyrosine residues (not serine phosphorylation) drive binding, with phosphorylation of serine and tyrosine residues inhibiting the interaction, establishing a phosphorylation-regulated import mechanism distinct from canonical RS-domain recognition.\",\n      \"evidence\": \"X-ray crystallography of TNPO3–CIRBP complex; tyrosine mutagenesis; phosphopeptide binding assays\",\n      \"pmids\": [\"40360518\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the RSY-NLS mode applies to other TNPO3 cargoes beyond CIRBP is untested\", \"Structural basis for how phosphorylation blocks binding at atomic resolution is incomplete\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Major unresolved questions include which specific cargo(es) are critically mis-imported in LGMD D2 muscle, whether TNPO3 has CPSF6-independent contributions to HIV-1 replication in vivo, and the full repertoire of physiological cargoes recognized via the RSY-NLS versus classical RS-domain mechanisms.\",\n      \"evidence\": \"Open question arising from accumulated literature\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No systematic cargo profiling in TNPO3-deficient cells has been reported\", \"The pathogenic mechanism of LGMD D2 at the molecular cargo level is undefined\", \"No structural basis for the TNPO3–CPSF6 interaction has been determined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [2, 4, 7, 12, 13, 14, 15]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 3, 14]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [8, 10, 11, 13]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2, 11]},\n      {\"term_id\": \"GO:0005635\", \"supporting_discovery_ids\": [10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [2, 4, 7, 12, 15]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [14]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [13, 14]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [13, 14]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [8, 10]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"CPSF6\",\n      \"EBF1\",\n      \"SRSF1\",\n      \"CIRBP\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}