{"gene":"TPR","run_date":"2026-04-28T21:42:59","timeline":{"discoveries":[{"year":1994,"finding":"Tpr (translocated promoter region) is a large coiled-coil protein (~265 kDa) localized to the cytoplasmic surface of the nuclear pore complex (NPC) in rat liver, identified by monoclonal antibody RL30; its amino-terminal ~150-250 amino acids appear in oncogenic fusions with Met, Trk, and Raf kinase domains.","method":"Immunogold electron microscopy, immunofluorescence, peptide sequencing, in vitro translation","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — direct localization by immunoEM with functional context, replicated in multiple cell types","pmids":["7798308"],"is_preprint":false},{"year":1997,"finding":"Human protein p270/Tpr is exclusively localized to the intranuclear filaments attached to the nucleoplasmic annulus of the NPC (not the cytoplasmic surface), forming filament bundles extending up to 350 nm into the nuclear interior; this was demonstrated with multiple antibodies against different Tpr epitopes across mammalian and amphibian cells.","method":"Immunoelectron microscopy with multiple antibodies to distinct Tpr epitopes, cDNA cloning, immunobiochemistry","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal antibodies and cell types; direct EM localization","pmids":["9024684"],"is_preprint":false},{"year":1998,"finding":"Tpr's NH2-terminal coiled-coil domain mediates association with the nucleoplasmic face of the NPC, while an acidic COOH-terminal domain contains a nuclear localization sequence; ectopic expression of full-length Tpr or NPC-associating domains causes dramatic nuclear accumulation of poly(A)+ RNA, implicating Tpr in mRNA export.","method":"Expression of full-length and deletion mutant Tpr in mammalian cells, immunofluorescence, poly(A)+ RNA in situ hybridization","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — domain mapping with defined cellular phenotype (poly(A)+ RNA accumulation), multiple constructs tested","pmids":["9864356"],"is_preprint":false},{"year":2001,"finding":"TPR directly binds Nup98 in vitro, and via Nup98 also associates with Nup96; TPR and Nup98 colocalize in a filamentous intranuclear network extending from NPCs to perinucleolar regions, suggesting an intranuclear phase of transport involving these nucleoporins.","method":"In vitro binding of translated proteins, double-immunofluorescence microscopy, overexpression of myc-tagged fusion proteins, double-immunoelectron microscopy","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro binding assay combined with EM and immunofluorescence colocalization","pmids":["11248057"],"is_preprint":false},{"year":2002,"finding":"Tpr is concentrated within the nuclear basket of the NPC (similar to Nup153 and Nup98); injection of anti-Tpr antibodies into mitotic or interphase cells inhibits leucine-rich NES-dependent nuclear protein export without affecting basic NLS-mediated nuclear import, establishing Tpr's role in nuclear protein export.","method":"Light and EM immunolocalization, GFP-Tpr live-cell imaging, antibody microinjection into mitotic and interphase cells, nuclear import/export assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — antibody injection with specific import/export readouts, combined light and EM localization","pmids":["11839768"],"is_preprint":false},{"year":2002,"finding":"Antibody-mediated depletion of Tpr from nuclei causes poly(A)+ RNA to accumulate in SC35-positive nuclear speckle clusters (larger and fewer in number), without affecting SV40 T-NLS import or Rev NES-dependent export, indicating Tpr is specifically required for intranuclear dynamics and processing/transport of RNA Pol II transcripts.","method":"Cytoplasmic microinjection of anti-Tpr antibodies to reconstitute Tpr-less nuclei, immunofluorescence, poly(A)+ RNA FISH","journal":"Genes to cells","confidence":"High","confidence_rationale":"Tier 2 — specific loss-of-function with defined molecular phenotype and controls for import/export","pmids":["11952838"],"is_preprint":false},{"year":2008,"finding":"Tpr directly binds Mad1 and Mad2; Tpr depletion in HeLa cells disrupts NPC localization of Mad1 and Mad2 during interphase, reduces Mad1-bound Mad2 levels, and decreases Mad1 at kinetochores during prometaphase, impairing APC(Cdc20) inhibition and the spindle assembly checkpoint.","method":"Mass spectrometry of affinity-purified Mad2-associated factors, direct binding assays, siRNA knockdown, immunofluorescence","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 — MS identification, direct binding, and KD with specific checkpoint phenotype","pmids":["18981471"],"is_preprint":false},{"year":2009,"finding":"Tpr binds strongly and specifically to importin alpha, importin beta, and CRM1-containing trimeric export complexes via distinct sites; nuclear import of Tpr is dependent on cytosolic factors and energy and is efficiently mediated by the importin alpha/beta pathway.","method":"Solid-phase binding assays with recombinant proteins, in vitro nuclear import assay","journal":"BMC cell biology","confidence":"High","confidence_rationale":"Tier 1–2 — reconstituted in vitro binding and import assay with recombinant proteins","pmids":["19835572"],"is_preprint":false},{"year":2010,"finding":"Tpr associates with dynein and dynactin motor complexes; Tpr knockdown causes lagging chromosome phenotype and disrupts spindle checkpoint proteins (Mad1/Mad2) expression and localization; overexpression of Tpr enhances multinucleated cell formation; rescue and dominant-negative experiments confirm Tpr promotes proper chromosome segregation through interaction with dynein light chain.","method":"Co-immunoprecipitation, RNAi knockdown, rescue experiments, immunofluorescence, dominant-negative overexpression","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, KD with defined phenotype, rescue experiments","pmids":["20133940"],"is_preprint":false},{"year":2011,"finding":"Tpr regulates export of mRNAs with retained introns that traffic through the Nxf1/Nxt1 (CTE) pathway but not the Crm1/Rev pathway; even modest Tpr knockdown significantly increases export and translation from CTE-containing mRNA with retained introns, without affecting completely spliced mRNA.","method":"RNAi knockdown, reporter mRNA export assays (CTE vs RRE reporters), Western blotting","journal":"RNA","confidence":"High","confidence_rationale":"Tier 2 — specific pathway discrimination with multiple reporter constructs and controls","pmids":["21613532"],"is_preprint":false},{"year":2012,"finding":"Tpr knockdown in mammalian cells dramatically enhances export of CTE-containing unspliced RNA; this regulation requires Tpr's localization to the NPC (NPC-unanchored Tpr cannot perform this function) and is independent of Sam68 and Tap/Nxf1; Nup153 (required for NPC anchoring of Tpr) similarly regulates unspliced RNA export.","method":"siRNA knockdown, reporter export assays, rescue with siRNA-resistant Tpr, immunofluorescence","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 — loss-of-function with rescue, localization-function link established","pmids":["22253824"],"is_preprint":false},{"year":2013,"finding":"Tpr stabilizes Mad1 and Mad2 protein levels before mitosis by forming a TM2 (Tpr/Mad1/Mad2) complex at NPCs during interphase; Tpr is required for Mad1–c-Mad2 recruitment to NPCs; Tpr stabilizes Mad1 and Mad2 in an mRNA- and kinetochore-independent manner; Tpr may regulate SAC proteostasis through SUMO-isopeptidases SENP1/SENP2 at NPCs.","method":"Co-immunoprecipitation, siRNA depletion, protein half-life measurements, GFP-Mad2 overexpression rescue, immunofluorescence","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — Co-IP, protein stability measurements, rescue, multiple orthogonal methods","pmids":["24344181"],"is_preprint":false},{"year":2013,"finding":"Tpr is imported into the nucleus via KPNA2 (importin alpha), which binds the bipartite NLS in Tpr with nanomolar affinity; Progerin (mutant lamin A in Hutchinson-Gilford Progeria) disrupts the nuclear/cytoplasmic Ran distribution, causing defective Tpr nuclear import because large cargo transport is particularly sensitive to changes in Ran gradient.","method":"NLS swapping experiments, binding affinity measurements, cell fractionation, fluorescence microscopy in HGPS fibroblasts","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — mechanistic dissection with NLS swapping, nanomolar affinity measurement, disease model","pmids":["23649804"],"is_preprint":false},{"year":2014,"finding":"Tpr is phosphorylated at S2059 by CDK1 and at S2094 by protein kinase A; CDK1-mediated S2059 phosphorylation is required for Tpr interaction with Mad1 and for proper localization of Mad1 and Mad2; abrogation of S2059 phosphorylation causes cell cycle defects; phosphorylated S2059-Tpr distinctly localizes with chromatin during telophase.","method":"Mass spectrometry-based phosphosite identification, kinase assays, phospho-specific antibodies, siRNA rescue with phosphomutants, immunofluorescence","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1–2 — kinase identified by in vitro assay, phosphosite mutant with defined functional consequence","pmids":["24938596"],"is_preprint":false},{"year":2018,"finding":"Depletion of Tpr (but not Nup153) dramatically increases the total number of NPCs per nucleus; this negative regulation occurs via ERK (MAPK) pathway phosphorylation, where Tpr serves as a scaffold for ERK to phosphorylate Nup153, which is critical for early stages of NPC biogenesis.","method":"AID-mediated acute depletion, NPC counting, kinase inhibition, epistasis experiments, co-immunoprecipitation","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 — acute depletion distinguishing Tpr from Nup153, pathway epistasis, scaffold-kinase mechanism","pmids":["30228202"],"is_preprint":false},{"year":2020,"finding":"Acute TPR depletion using an Auxin-Induced Degron system causes rapid transcriptomic changes similar to NXF1 or GANP (TREX-2 complex) depletion; TPR depletion disrupts association of TREX-2 subunits (GANP, PCID2, ENY2) with NPCs, resulting in abnormal RNA transcription and export, establishing TPR as an integral component of the TREX-2 mRNA export pathway.","method":"Auxin-Induced Degron system, RNA-seq transcriptomics, co-immunoprecipitation, comparison of nucleoporin depletions","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — acute inducible depletion system, pathway placement by transcriptomic comparison, Co-IP","pmids":["32917881"],"is_preprint":false},{"year":2020,"finding":"TPR is specifically required for efficient nuclear export of mRNAs and lncRNAs from short, intron-poor genes; in TPR-depleted cells these mRNAs accumulate in nuclear speckles while remaining bound to Nxf1, suggesting TPR acts downstream of Nxf1 recruitment to allow mRNAs to leave nuclear speckles and dock with the nuclear pore.","method":"Fractionated RNA-seq of nucleus vs. cytosol in TPR-depleted cells, reporter constructs, immunofluorescence","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 — genome-wide RNA fractionation, reporter validation, epistasis relative to Nxf1","pmids":["33091126"],"is_preprint":false},{"year":2021,"finding":"Tpr depletion generates transcription-dependent replication stress, DNA breaks, and genomic instability; Tpr-deficient cells show slow/asymmetric replication forks and elevated DNA-RNA hybrids; proteomic approaches identify Tpr-interacting proteins MATR3 and SUGP2 (RNA processing); Tpr interacts with GANP (TREX-2 complex), physically connecting replication forks with transcription, splicing, and mRNA export machinery.","method":"DNA fiber assays, electron microscopy of replication intermediates, complementary proteomics, Co-immunoprecipitation, siRNA knockdown, R-loop detection","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods including EM, proteomics, functional assays","pmids":["34168151"],"is_preprint":false},{"year":2015,"finding":"Tpr interacts with Aurora A kinase via its central coiled-coil domain; Tpr depletion reduces Aurora A expression levels, centrosomal localization, and phosphorylation; Tpr and Aurora A mutually regulate each other and Tpr sequesters extra Aurora A to safeguard spindle bipolarity.","method":"Co-immunoprecipitation, siRNA knockdown, immunofluorescence, Aurora A inhibitor (Alisertib) treatment","journal":"Cell cycle","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP with domain mapping, KD phenotype, single lab","pmids":["25789545"],"is_preprint":false},{"year":2011,"finding":"In Tpr-depleted cells, silencing of Tpr triggers G0-G1 arrest and senescent-like phenotype dependent on p53; Tpr depletion impairs NES-dependent nuclear export of proteins; Tpr depletion affects SUMO-protease SENP2 levels/function, altering SUMOylation at the nuclear pore.","method":"RNAi knockdown, cell cycle analysis, immunofluorescence, Western blotting","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 3 — single lab, KD with defined phenotype but limited mechanistic depth","pmids":["21811608"],"is_preprint":false},{"year":2012,"finding":"Tpr depletion reduces nuclear pore numbers and intranuclear filament number; Tpr-depleted cells show increased p53 nuclear accumulation and autophagy; Tpr controls HSP70 and HSF1 mRNA export and p53 trafficking with karyopherin CRM1.","method":"siRNA knockdown, electron microscopy, immunofluorescence, Western blotting","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 3 — single lab, multiple phenotypic readouts but limited mechanistic follow-up","pmids":["23170199"],"is_preprint":false},{"year":1998,"finding":"Molecular segments of Tpr were identified: a short C-terminal region is necessary and sufficient for nuclear import of Tpr and can confer nuclear accumulation on pyruvate kinase; the N-terminal coiled-coil domain contains clusters of heptad repeats that mediate NPC association when present in the nucleus.","method":"Expression of Tpr deletion mutants and chimeric proteins in mammalian cells, immunofluorescence","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 — systematic domain dissection with chimeric proteins, single lab","pmids":["9828100"],"is_preprint":false},{"year":1987,"finding":"The TPR-MET oncogene was generated by chromosomal rearrangement (recombination event) fusing sequences from the tpr locus on chromosome 1 (following an Alu repetitive sequence) to the met locus on chromosome 7; the rearrangement places tpr-encoded sequences upstream of the MET kinase domain.","method":"Nucleotide sequencing of rearranged genomic locus and unrearranged tpr/met fragments","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 — direct sequencing of breakpoints, foundational structural characterization","pmids":["3821733"],"is_preprint":false},{"year":1988,"finding":"The TPR-MET oncogene encodes p65(tpr-met), a constitutively active protein-tyrosine kinase that autophosphorylates on tyrosine in vitro; in vivo p65(tpr-met) is phosphorylated on serine and tyrosine, while p140(met) is phosphorylated on serine and threonine.","method":"Immunocomplex kinase assay, in vitro autophosphorylation, cell-surface iodination, phosphoamino acid analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — direct in vitro kinase assay, phosphoamino acid analysis","pmids":["3277171"],"is_preprint":false},{"year":1995,"finding":"A single tyrosine residue (Tyr489) in the C-terminus of Tpr-Met is essential for efficient transformation of fibroblasts; Y489 is required for association with Grb2, phosphatidylinositol 3-kinase activation, and phospholipase Cgamma/SHPTP2 association in vivo; mutation of Y489 does not affect exogenous kinase activity toward casein.","method":"Tyrosine-to-phenylalanine mutagenesis, transformation assays, co-immunoprecipitation, kinase assays","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1–2 — site-directed mutagenesis with transformation assay and signaling partner Co-IP","pmids":["7838524"],"is_preprint":false},{"year":1996,"finding":"Cell transformation by Tpr-Met requires signaling pathways downstream of Shc and Grb2; pathways downstream of PI3-kinase, PLCgamma, and SHPTP2/Syp alone are insufficient for transformation; a Tpr-Met mutant selectively unable to associate with Grb2 was generated to dissect these pathways.","method":"Generation of Tpr-Met mutants selectively defective in adaptor binding, transformation assays, co-immunoprecipitation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — selective loss-of-function mutants with epistasis-style pathway dissection","pmids":["8662733"],"is_preprint":false},{"year":1999,"finding":"Loss of the juxtamembrane domain of MET (which occurs in the TPR-MET rearrangement) is essential for oncogenic activation; a chimeric Tpr-juxtaMet retaining the juxtamembrane domain (aa 962-1009) strongly inhibits transformation and downstream signaling/adaptor recruitment despite equivalent kinase activity.","method":"Construction of chimeric Tpr-juxtaMet, transformation assays (proliferation, soft agar, motility, invasion), co-immunoprecipitation of signaling molecules","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 — domain-swap experiment with multiple functional readouts","pmids":["10435641"],"is_preprint":false},{"year":1992,"finding":"The human TPR gene encodes a 726 amino acid protein with extensive alpha-helical regions and three heptad repeat motifs characteristic of coiled-coil conformation; alternative splicing can extend the C-terminal domain; its N-terminal sequences contribute to oncogenic fusions with MET, RAF kinase domains.","method":"cDNA cloning and sequencing, secondary structure prediction, alternative splicing analysis","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 3 — sequence-based structural characterization, foundational but primarily computational prediction","pmids":["1549355"],"is_preprint":false}],"current_model":"Human TPR (translocated promoter region) is a large (~267 kDa) coiled-coil nucleoporin constitutively localized to the intranuclear filaments (basket) of the nuclear pore complex (NPC), where it plays multiple mechanistic roles: (1) it facilitates nuclear export of NES-containing proteins and mRNAs, particularly intronless/intron-poor mRNAs, acting downstream of Nxf1 and as an integral component of the TREX-2 mRNA export pathway via interaction with GANP; (2) it retains unspliced/intron-containing mRNAs in the nucleus through the Nxf1 pathway; (3) it is required for spindle assembly checkpoint robustness by directly binding Mad1 and Mad2 and stabilizing their protein levels at NPCs during interphase, with CDK1-mediated phosphorylation of S2059 governing its interaction with Mad1 during mitosis; (4) it interacts with dynein/dynactin to coordinate proper chromosome segregation; (5) it serves as a scaffold for ERK to phosphorylate Nup153, negatively regulating NPC biogenesis; (6) its N-terminal coiled-coil domain contributes the dimerization/activation module in TPR-MET and other oncogenic kinase fusions, while the C-terminus harbors a bipartite NLS for importin alpha/beta-mediated nuclear import; and (7) it is imported into the nucleus via KPNA2 (importin alpha), a process sensitive to the nuclear Ran gradient."},"narrative":{"teleology":[{"year":1987,"claim":"The discovery of the TPR-MET chromosomal rearrangement established that TPR sequences provide an N-terminal dimerization module fused to the MET kinase domain, founding the concept of TPR as an oncogene contributor.","evidence":"Nucleotide sequencing of rearranged and unrearranged tpr/met genomic fragments","pmids":["3821733"],"confidence":"High","gaps":["Normal TPR function was unknown","Whether TPR coiled-coil domain was sufficient for dimerization was not directly tested"]},{"year":1988,"claim":"Demonstration that TPR-MET encodes a constitutively active tyrosine kinase (p65) established that the TPR moiety drives ligand-independent kinase activation through enforced dimerization.","evidence":"Immunocomplex kinase assay and phosphoamino acid analysis of p65(tpr-met)","pmids":["3277171"],"confidence":"High","gaps":["Structural basis of TPR-mediated dimerization unresolved","Contribution of individual TPR heptad repeats to activation unknown"]},{"year":1995,"claim":"Identification of Tyr489 as the essential signaling residue in TPR-MET defined the downstream effector requirements (Grb2, PI3K, Shc) for transformation, dissecting how the fusion signals.","evidence":"Tyr-to-Phe mutagenesis with transformation assays and co-IP of signaling partners","pmids":["7838524","8662733"],"confidence":"High","gaps":["Role of TPR-derived sequences beyond dimerization in signaling not addressed","In vivo tumor models not tested"]},{"year":1997,"claim":"Correcting earlier reports, immunoelectron microscopy with multiple antibodies demonstrated that TPR exclusively localizes to the intranuclear (basket) filaments of the NPC, not the cytoplasmic face, redefining its functional context.","evidence":"Immunoelectron microscopy with antibodies to multiple distinct TPR epitopes across mammalian and amphibian cells","pmids":["9024684"],"confidence":"High","gaps":["How TPR assembles into basket filaments was unknown","Whether TPR is essential for basket structure was untested"]},{"year":1998,"claim":"Domain mapping revealed that the N-terminal coiled-coil mediates NPC association while the C-terminus contains the NLS; overexpression causing poly(A)+ RNA nuclear accumulation provided the first link between TPR and mRNA export.","evidence":"Expression of full-length and deletion mutants in mammalian cells with poly(A)+ RNA FISH","pmids":["9864356","9828100"],"confidence":"High","gaps":["Whether TPR directly contacts RNA or acts via protein partners was unknown","Export pathway specificity not determined"]},{"year":2001,"claim":"Discovery of direct TPR–Nup98 binding and their colocalization in intranuclear filaments suggested that TPR participates in an intranuclear transport network extending beyond the NPC basket.","evidence":"In vitro binding of translated proteins combined with double-immunoEM","pmids":["11248057"],"confidence":"High","gaps":["Functional consequence of TPR–Nup98 interaction on transport not tested","Whether other nucleoporins participate in intranuclear filaments unknown"]},{"year":2002,"claim":"Anti-TPR antibody injection experiments established that TPR is specifically required for leucine-rich NES-dependent nuclear protein export, while NLS-mediated import is unaffected, distinguishing TPR's directionality in transport.","evidence":"Antibody microinjection into interphase and mitotic cells with import/export reporter assays","pmids":["11839768","11952838"],"confidence":"High","gaps":["Whether TPR acts as a direct export factor or scaffold was unresolved","Pathway specificity (CRM1 vs Nxf1) not yet discriminated"]},{"year":2008,"claim":"Identification of direct TPR–Mad1–Mad2 binding and the requirement of TPR for interphase NPC localization of checkpoint proteins established an unexpected role for the nuclear basket in spindle assembly checkpoint priming.","evidence":"Mass spectrometry of Mad2-associated factors, direct binding assays, siRNA knockdown with checkpoint readouts in HeLa cells","pmids":["18981471"],"confidence":"High","gaps":["Whether TPR stabilizes Mad1/Mad2 protein levels or only localizes them was unclear","Phosphoregulation of the interaction was unknown"]},{"year":2010,"claim":"Discovery of TPR interaction with dynein/dynactin and the lagging chromosome phenotype upon TPR depletion extended its mitotic roles beyond SAC signaling to direct involvement in chromosome segregation mechanics.","evidence":"Reciprocal co-IP, RNAi knockdown with rescue and dominant-negative experiments","pmids":["20133940"],"confidence":"High","gaps":["Structural basis of TPR–dynein interaction not defined","Whether dynein interaction is direct or mediated by Mad1/Mad2 not fully resolved"]},{"year":2011,"claim":"Pathway-specific reporter assays revealed that TPR selectively retains unspliced mRNAs that use the Nxf1/CTE export route, while CRM1/Rev-dependent export is unaffected, establishing TPR as a quality-control checkpoint for intron-containing RNA at the NPC.","evidence":"RNAi knockdown with CTE vs RRE reporter mRNA export assays","pmids":["21613532"],"confidence":"High","gaps":["Whether TPR directly recognizes intron-containing features or acts via cofactors was unknown","Relationship to TREX-2 not yet established"]},{"year":2013,"claim":"Demonstration that TPR stabilizes Mad1 and Mad2 protein levels (not just localization) during interphase via TM2 complex formation resolved how TPR contributes to SAC proteostasis independently of kinetochores and mRNA.","evidence":"Co-IP, protein half-life measurements, GFP-Mad2 rescue in TPR-depleted cells","pmids":["24344181"],"confidence":"High","gaps":["Role of SUMO-isopeptidases SENP1/SENP2 in this stabilization not fully validated","Whether TPR-mediated stabilization involves ubiquitin-proteasome pathway unknown"]},{"year":2013,"claim":"Identification of KPNA2 as the importin alpha mediating TPR nuclear import, with nanomolar NLS-binding affinity, and discovery that progerin-induced Ran gradient disruption impairs TPR import linked NPC basket assembly to laminopathy pathology.","evidence":"NLS-swap experiments, binding affinity measurements, analysis in HGPS patient fibroblasts","pmids":["23649804"],"confidence":"High","gaps":["Whether reduced TPR import contributes to specific HGPS phenotypes not tested functionally","Whether other import receptors can substitute for KPNA2 unknown"]},{"year":2014,"claim":"Identification of CDK1-mediated S2059 phosphorylation as the switch governing TPR–Mad1 interaction provided the first phosphoregulatory mechanism linking cell cycle kinase activity to NPC-based SAC signaling.","evidence":"MS-based phosphosite identification, in vitro kinase assay, phosphomutant rescue in siRNA-depleted cells","pmids":["24938596"],"confidence":"High","gaps":["Whether S2059 phosphorylation is the sole regulatory switch or acts with additional modifications unknown","Structural consequences of phosphorylation on TPR–Mad1 interface not resolved"]},{"year":2018,"claim":"Acute TPR depletion causing increased NPC number revealed that TPR negatively regulates NPC biogenesis by scaffolding ERK-mediated phosphorylation of Nup153, establishing a signaling-based feedback on pore density.","evidence":"AID-mediated acute depletion, NPC counting, ERK inhibition epistasis, co-IP","pmids":["30228202"],"confidence":"High","gaps":["Which Nup153 phosphosites are ERK targets on the TPR scaffold not mapped","Whether NPC number control feeds back on gene expression was not addressed"]},{"year":2020,"claim":"Transcriptomic comparison of acute TPR, NXF1, and GANP depletions revealed overlapping gene expression changes, and co-IP showed TPR anchors the TREX-2 complex at NPCs, integrating TPR into the TREX-2 mRNA export pathway.","evidence":"Auxin-inducible degron system with RNA-seq and co-IP of TREX-2 subunits","pmids":["32917881"],"confidence":"High","gaps":["Direct versus indirect interaction between TPR and GANP not structurally resolved","Whether all TREX-2 functions require NPC-anchored TPR unknown"]},{"year":2020,"claim":"Fractionated RNA-seq after TPR depletion identified intron-poor and short genes as the specifically affected class, with their mRNAs accumulating in nuclear speckles while still bound to Nxf1, placing TPR's export function downstream of Nxf1 loading.","evidence":"Nuclear/cytoplasmic RNA fractionation-seq, reporter constructs, immunofluorescence in TPR-depleted cells","pmids":["33091126"],"confidence":"High","gaps":["Mechanism by which TPR releases Nxf1-loaded mRNPs from speckles unknown","Whether speckle retention is a cause or consequence of export failure not determined"]},{"year":2021,"claim":"Discovery that TPR depletion generates transcription-dependent replication stress, DNA breaks, and R-loops, with proteomic identification of MATR3 and SUGP2 as interactors, revealed TPR's role in coordinating transcription with replication to maintain genome stability.","evidence":"DNA fiber assays, EM of replication intermediates, R-loop detection, complementary proteomics, co-IP","pmids":["34168151"],"confidence":"High","gaps":["Whether genome instability is a direct consequence of export failure or an independent TPR function unclear","MATR3 and SUGP2 interaction directionality and stoichiometry not determined"]},{"year":null,"claim":"Key unresolved questions include the structural basis of TPR basket filament assembly, how TPR discriminates intron-poor mRNPs for export, whether its mRNA export and SAC functions are mechanistically coupled, and how TPR's multiple scaffolding roles are coordinated during the cell cycle.","evidence":"","pmids":[],"confidence":"Low","gaps":["No high-resolution structure of TPR or TPR–Mad1 complex","Mechanism of intron-poor mRNA selectivity at the molecular level unknown","Whether mRNA export and checkpoint functions share regulatory inputs untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[1,2,3,14]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[6,14,15]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[9,10,14,16]}],"localization":[{"term_id":"GO:0005635","term_label":"nuclear envelope","supporting_discovery_ids":[1,2,4]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,2,3,12]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[2,5,9,10,15,16,17]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[4,7,12,15,16]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[6,8,11,13,14]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[14,24,25]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[14,15]}],"complexes":["Nuclear pore complex (NPC basket)","TREX-2 mRNA export complex","TPR-Mad1-Mad2 (TM2) complex"],"partners":["MAD1L1","MAD2L1","NUP98","NUP153","MCM3AP","KPNA2","DYNLL1","MATR3"],"other_free_text":[]},"mechanistic_narrative":"TPR is a large coiled-coil nucleoporin that forms the intranuclear basket filaments of the nuclear pore complex, where it serves as a multifunctional scaffold integrating mRNA export, nuclear retention of unspliced transcripts, spindle assembly checkpoint signaling, and NPC biogenesis control [PMID:9024684, PMID:30228202, PMID:18981471]. TPR anchors the TREX-2 complex (GANP/PCID2/ENY2) at the NPC and is specifically required for nuclear export of mRNAs from intron-poor genes via the Nxf1 pathway, acting downstream of Nxf1 to release transcripts from nuclear speckles, while simultaneously retaining unspliced intron-containing mRNAs in the nucleus [PMID:32917881, PMID:33091126, PMID:21613532]. During interphase, TPR stabilizes Mad1 and Mad2 protein levels and recruits the Mad1–Mad2 complex to NPCs, with CDK1-mediated phosphorylation at S2059 governing the TPR–Mad1 interaction and ensuring spindle assembly checkpoint robustness; TPR also cooperates with dynein/dynactin to promote proper chromosome segregation [PMID:24344181, PMID:24938596, PMID:20133940]. TPR negatively regulates NPC number by scaffolding ERK-mediated phosphorylation of Nup153, and its N-terminal coiled-coil domain provides the dimerization/activation module in oncogenic fusions such as TPR-MET [PMID:30228202, PMID:3821733, PMID:3277171]."},"prefetch_data":{"uniprot":{"accession":"P12270","full_name":"Nucleoprotein TPR","aliases":["Megator","NPC-associated intranuclear protein","Translocated promoter region protein"],"length_aa":2363,"mass_kda":267.3,"function":"Component of the nuclear pore complex (NPC), a complex required for the trafficking across the nuclear envelope. Functions as a scaffolding element in the nuclear phase of the NPC essential for normal nucleocytoplasmic transport of proteins and mRNAs, plays a role in the establishment of nuclear-peripheral chromatin compartmentalization in interphase, and in the mitotic spindle checkpoint signaling during mitosis. Involved in the quality control and retention of unspliced mRNAs in the nucleus; in association with NUP153, regulates the nuclear export of unspliced mRNA species bearing constitutive transport element (CTE) in a NXF1- and KHDRBS1-independent manner. Negatively regulates both the association of CTE-containing mRNA with large polyribosomes and translation initiation. Does not play any role in Rev response element (RRE)-mediated export of unspliced mRNAs. Implicated in nuclear export of mRNAs transcribed from heat shock gene promoters; associates both with chromatin in the HSP70 promoter and with mRNAs transcribed from this promoter under stress-induced conditions. Modulates the nucleocytoplasmic transport of activated MAPK1/ERK2 and huntingtin/HTT and may serve as a docking site for the XPO1/CRM1-mediated nuclear export complex. According to some authors, plays a limited role in the regulation of nuclear protein export (PubMed:11952838, PubMed:22253824). Also plays a role as a structural and functional element of the perinuclear chromatin distribution; involved in the formation and/or maintenance of NPC-associated perinuclear heterochromatin exclusion zones (HEZs). Finally, acts as a spatial regulator of the spindle-assembly checkpoint (SAC) response ensuring a timely and effective recruitment of spindle checkpoint proteins like MAD1L1 and MAD2L1 to unattached kinetochore during the metaphase-anaphase transition before chromosome congression. Its N-terminus is involved in activation of oncogenic kinases","subcellular_location":"Nucleus; Nucleus membrane; Nucleus envelope; Nucleus, nuclear pore complex; Cytoplasm; Cytoplasm, cytoskeleton, spindle; Chromosome, centromere, kinetochore; Nucleus membrane","url":"https://www.uniprot.org/uniprotkb/P12270/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/TPR","classification":"Common 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ZFC3H1","url":"https://www.omim.org/entry/620956"},{"mim_id":"620875","title":"FIC DOMAIN-CONTAINING PROTEIN ADENYLYLTRANSFERASE; FICD","url":"https://www.omim.org/entry/620875"},{"mim_id":"620742","title":"INTRAFLAGELLAR TRANSPORT 70B; IFT70B","url":"https://www.omim.org/entry/620742"},{"mim_id":"620741","title":"INTRAFLAGELLAR TRANSPORT 70A; IFT70A","url":"https://www.omim.org/entry/620741"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nuclear membrane","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TPR"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P12270","domains":[{"cath_id":"-","chopping":"845-906","consensus_level":"medium","plddt":76.4682,"start":845,"end":906},{"cath_id":"1.20.5","chopping":"1367-1450","consensus_level":"medium","plddt":74.6669,"start":1367,"end":1450}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P12270","model_url":"https://alphafold.ebi.ac.uk/files/AF-P12270-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P12270-F1-predicted_aligned_error_v6.png","plddt_mean":60.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TPR","jax_strain_url":"https://www.jax.org/strain/search?query=TPR"},"sequence":{"accession":"P12270","fasta_url":"https://rest.uniprot.org/uniprotkb/P12270.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P12270/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P12270"}},"corpus_meta":[{"pmid":"1882418","id":"PMC_1882418","title":"The 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immunity","url":"https://pubmed.ncbi.nlm.nih.gov/8406803","citation_count":33,"is_preprint":false},{"pmid":"9828100","id":"PMC_9828100","title":"Molecular segments of protein Tpr that confer nuclear targeting and association with the nuclear pore complex.","date":"1998","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/9828100","citation_count":33,"is_preprint":false},{"pmid":"23898829","id":"PMC_23898829","title":"Comparison of CDC and sequence-based molecular typing of syphilis treponemes: tpr and arp loci are variable in multiple samples from the same patient.","date":"2013","source":"BMC microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/23898829","citation_count":33,"is_preprint":false},{"pmid":"34168151","id":"PMC_34168151","title":"The human nucleoporin Tpr protects cells from RNA-mediated replication stress.","date":"2021","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/34168151","citation_count":32,"is_preprint":false},{"pmid":"15223324","id":"PMC_15223324","title":"The crystal structure of Helicobacter cysteine-rich protein C at 2.0 A resolution: similar peptide-binding sites in TPR and SEL1-like repeat proteins.","date":"2004","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/15223324","citation_count":32,"is_preprint":false},{"pmid":"21811608","id":"PMC_21811608","title":"Silencing nuclear pore protein Tpr elicits a senescent-like phenotype in cancer cells.","date":"2011","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/21811608","citation_count":31,"is_preprint":false},{"pmid":"15710426","id":"PMC_15710426","title":"Overexpression of small glutamine-rich TPR-containing protein promotes apoptosis in 7721 cells.","date":"2005","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/15710426","citation_count":30,"is_preprint":false},{"pmid":"19835572","id":"PMC_19835572","title":"Karyopherin binding interactions and nuclear import mechanism of nuclear pore complex protein Tpr.","date":"2009","source":"BMC cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/19835572","citation_count":30,"is_preprint":false},{"pmid":"14729056","id":"PMC_14729056","title":"The human small glutamine-rich TPR-containing protein is required for progress through cell division.","date":"2004","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/14729056","citation_count":30,"is_preprint":false},{"pmid":"23350036","id":"PMC_23350036","title":"Novel TPR-containing subunit of TOM complex functions as cytosolic receptor for Entamoeba mitosomal transport.","date":"2013","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/23350036","citation_count":30,"is_preprint":false},{"pmid":"15719029","id":"PMC_15719029","title":"RNAi technology and lentiviral delivery as a powerful tool to suppress Tpr-Met-mediated tumorigenesis.","date":"2005","source":"Cancer gene therapy","url":"https://pubmed.ncbi.nlm.nih.gov/15719029","citation_count":29,"is_preprint":false},{"pmid":"26037924","id":"PMC_26037924","title":"The Non-canonical Tetratricopeptide Repeat (TPR) Domain of Fluorescent (FLU) Mediates Complex Formation with Glutamyl-tRNA Reductase.","date":"2015","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/26037924","citation_count":29,"is_preprint":false},{"pmid":"24563354","id":"PMC_24563354","title":"Roles of the nucleoporin Tpr in cancer and aging.","date":"2014","source":"Advances in experimental medicine and biology","url":"https://pubmed.ncbi.nlm.nih.gov/24563354","citation_count":28,"is_preprint":false},{"pmid":"24938596","id":"PMC_24938596","title":"Phosphorylation of nucleoporin Tpr governs its differential localization and is required for its mitotic function.","date":"2014","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/24938596","citation_count":28,"is_preprint":false},{"pmid":"33893288","id":"PMC_33893288","title":"Specificity of AMPylation of the human chaperone BiP is mediated by TPR motifs of FICD.","date":"2021","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/33893288","citation_count":28,"is_preprint":false},{"pmid":"25789545","id":"PMC_25789545","title":"Therapeutic potential of mitotic interaction between the nucleoporin Tpr and aurora kinase A.","date":"2015","source":"Cell cycle (Georgetown, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/25789545","citation_count":27,"is_preprint":false},{"pmid":"18320024","id":"PMC_18320024","title":"The human TPR protein TTC4 is a putative Hsp90 co-chaperone which interacts with CDC6 and shows alterations in transformed cells.","date":"2008","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/18320024","citation_count":27,"is_preprint":false},{"pmid":"17878237","id":"PMC_17878237","title":"Structurally related TPR subunits contribute differently to the function of the anaphase-promoting complex in Drosophila melanogaster.","date":"2007","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/17878237","citation_count":27,"is_preprint":false},{"pmid":"29568361","id":"PMC_29568361","title":"Colorectal cancer cells require glycogen synthase kinase-3β for sustaining mitosis via translocated promoter region (TPR)-dynein interaction.","date":"2018","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/29568361","citation_count":27,"is_preprint":false},{"pmid":"12027452","id":"PMC_12027452","title":"Structures and dynamics of Drosophila Tpr inconsistent with a static, filamentous structure.","date":"2002","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/12027452","citation_count":26,"is_preprint":false},{"pmid":"1549355","id":"PMC_1549355","title":"Nucleotide sequence analysis of human tpr cDNA clones.","date":"1992","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/1549355","citation_count":26,"is_preprint":false},{"pmid":"22504226","id":"PMC_22504226","title":"Structural and thermodynamic characterization of the interaction between two periplasmic Treponema pallidum lipoproteins that are components of a TPR-protein-associated TRAP transporter (TPAT).","date":"2012","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/22504226","citation_count":25,"is_preprint":false},{"pmid":"34725712","id":"PMC_34725712","title":"Truncation of the TPR domain of OGT alters substrate and glycosite selection.","date":"2021","source":"Analytical and bioanalytical chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/34725712","citation_count":25,"is_preprint":false},{"pmid":"32071397","id":"PMC_32071397","title":"Cancer-derived UTX TPR mutations G137V and D336G impair interaction with MLL3/4 complexes and affect UTX subcellular localization.","date":"2020","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/32071397","citation_count":25,"is_preprint":false},{"pmid":"22346747","id":"PMC_22346747","title":"The TPR domain in the host Cyp40-like cyclophilin binds to the viral replication protein and inhibits the assembly of the tombusviral replicase.","date":"2012","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/22346747","citation_count":25,"is_preprint":false},{"pmid":"15546961","id":"PMC_15546961","title":"Novel interaction partners of the TPR/MET tyrosine kinase.","date":"2004","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/15546961","citation_count":24,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":44768,"output_tokens":6500,"usd":0.115902},"stage2":{"model":"claude-opus-4-6","input_tokens":10142,"output_tokens":3987,"usd":0.225577},"total_usd":0.341479,"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\": 1994,\n      \"finding\": \"Tpr (translocated promoter region) is a large coiled-coil protein (~265 kDa) localized to the cytoplasmic surface of the nuclear pore complex (NPC) in rat liver, identified by monoclonal antibody RL30; its amino-terminal ~150-250 amino acids appear in oncogenic fusions with Met, Trk, and Raf kinase domains.\",\n      \"method\": \"Immunogold electron microscopy, immunofluorescence, peptide sequencing, in vitro translation\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct localization by immunoEM with functional context, replicated in multiple cell types\",\n      \"pmids\": [\"7798308\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Human protein p270/Tpr is exclusively localized to the intranuclear filaments attached to the nucleoplasmic annulus of the NPC (not the cytoplasmic surface), forming filament bundles extending up to 350 nm into the nuclear interior; this was demonstrated with multiple antibodies against different Tpr epitopes across mammalian and amphibian cells.\",\n      \"method\": \"Immunoelectron microscopy with multiple antibodies to distinct Tpr epitopes, cDNA cloning, immunobiochemistry\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal antibodies and cell types; direct EM localization\",\n      \"pmids\": [\"9024684\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Tpr's NH2-terminal coiled-coil domain mediates association with the nucleoplasmic face of the NPC, while an acidic COOH-terminal domain contains a nuclear localization sequence; ectopic expression of full-length Tpr or NPC-associating domains causes dramatic nuclear accumulation of poly(A)+ RNA, implicating Tpr in mRNA export.\",\n      \"method\": \"Expression of full-length and deletion mutant Tpr in mammalian cells, immunofluorescence, poly(A)+ RNA in situ hybridization\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — domain mapping with defined cellular phenotype (poly(A)+ RNA accumulation), multiple constructs tested\",\n      \"pmids\": [\"9864356\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"TPR directly binds Nup98 in vitro, and via Nup98 also associates with Nup96; TPR and Nup98 colocalize in a filamentous intranuclear network extending from NPCs to perinucleolar regions, suggesting an intranuclear phase of transport involving these nucleoporins.\",\n      \"method\": \"In vitro binding of translated proteins, double-immunofluorescence microscopy, overexpression of myc-tagged fusion proteins, double-immunoelectron microscopy\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro binding assay combined with EM and immunofluorescence colocalization\",\n      \"pmids\": [\"11248057\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Tpr is concentrated within the nuclear basket of the NPC (similar to Nup153 and Nup98); injection of anti-Tpr antibodies into mitotic or interphase cells inhibits leucine-rich NES-dependent nuclear protein export without affecting basic NLS-mediated nuclear import, establishing Tpr's role in nuclear protein export.\",\n      \"method\": \"Light and EM immunolocalization, GFP-Tpr live-cell imaging, antibody microinjection into mitotic and interphase cells, nuclear import/export assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — antibody injection with specific import/export readouts, combined light and EM localization\",\n      \"pmids\": [\"11839768\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Antibody-mediated depletion of Tpr from nuclei causes poly(A)+ RNA to accumulate in SC35-positive nuclear speckle clusters (larger and fewer in number), without affecting SV40 T-NLS import or Rev NES-dependent export, indicating Tpr is specifically required for intranuclear dynamics and processing/transport of RNA Pol II transcripts.\",\n      \"method\": \"Cytoplasmic microinjection of anti-Tpr antibodies to reconstitute Tpr-less nuclei, immunofluorescence, poly(A)+ RNA FISH\",\n      \"journal\": \"Genes to cells\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — specific loss-of-function with defined molecular phenotype and controls for import/export\",\n      \"pmids\": [\"11952838\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Tpr directly binds Mad1 and Mad2; Tpr depletion in HeLa cells disrupts NPC localization of Mad1 and Mad2 during interphase, reduces Mad1-bound Mad2 levels, and decreases Mad1 at kinetochores during prometaphase, impairing APC(Cdc20) inhibition and the spindle assembly checkpoint.\",\n      \"method\": \"Mass spectrometry of affinity-purified Mad2-associated factors, direct binding assays, siRNA knockdown, immunofluorescence\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — MS identification, direct binding, and KD with specific checkpoint phenotype\",\n      \"pmids\": [\"18981471\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Tpr binds strongly and specifically to importin alpha, importin beta, and CRM1-containing trimeric export complexes via distinct sites; nuclear import of Tpr is dependent on cytosolic factors and energy and is efficiently mediated by the importin alpha/beta pathway.\",\n      \"method\": \"Solid-phase binding assays with recombinant proteins, in vitro nuclear import assay\",\n      \"journal\": \"BMC cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — reconstituted in vitro binding and import assay with recombinant proteins\",\n      \"pmids\": [\"19835572\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Tpr associates with dynein and dynactin motor complexes; Tpr knockdown causes lagging chromosome phenotype and disrupts spindle checkpoint proteins (Mad1/Mad2) expression and localization; overexpression of Tpr enhances multinucleated cell formation; rescue and dominant-negative experiments confirm Tpr promotes proper chromosome segregation through interaction with dynein light chain.\",\n      \"method\": \"Co-immunoprecipitation, RNAi knockdown, rescue experiments, immunofluorescence, dominant-negative overexpression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, KD with defined phenotype, rescue experiments\",\n      \"pmids\": [\"20133940\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Tpr regulates export of mRNAs with retained introns that traffic through the Nxf1/Nxt1 (CTE) pathway but not the Crm1/Rev pathway; even modest Tpr knockdown significantly increases export and translation from CTE-containing mRNA with retained introns, without affecting completely spliced mRNA.\",\n      \"method\": \"RNAi knockdown, reporter mRNA export assays (CTE vs RRE reporters), Western blotting\",\n      \"journal\": \"RNA\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — specific pathway discrimination with multiple reporter constructs and controls\",\n      \"pmids\": [\"21613532\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Tpr knockdown in mammalian cells dramatically enhances export of CTE-containing unspliced RNA; this regulation requires Tpr's localization to the NPC (NPC-unanchored Tpr cannot perform this function) and is independent of Sam68 and Tap/Nxf1; Nup153 (required for NPC anchoring of Tpr) similarly regulates unspliced RNA export.\",\n      \"method\": \"siRNA knockdown, reporter export assays, rescue with siRNA-resistant Tpr, immunofluorescence\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — loss-of-function with rescue, localization-function link established\",\n      \"pmids\": [\"22253824\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Tpr stabilizes Mad1 and Mad2 protein levels before mitosis by forming a TM2 (Tpr/Mad1/Mad2) complex at NPCs during interphase; Tpr is required for Mad1–c-Mad2 recruitment to NPCs; Tpr stabilizes Mad1 and Mad2 in an mRNA- and kinetochore-independent manner; Tpr may regulate SAC proteostasis through SUMO-isopeptidases SENP1/SENP2 at NPCs.\",\n      \"method\": \"Co-immunoprecipitation, siRNA depletion, protein half-life measurements, GFP-Mad2 overexpression rescue, immunofluorescence\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, protein stability measurements, rescue, multiple orthogonal methods\",\n      \"pmids\": [\"24344181\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Tpr is imported into the nucleus via KPNA2 (importin alpha), which binds the bipartite NLS in Tpr with nanomolar affinity; Progerin (mutant lamin A in Hutchinson-Gilford Progeria) disrupts the nuclear/cytoplasmic Ran distribution, causing defective Tpr nuclear import because large cargo transport is particularly sensitive to changes in Ran gradient.\",\n      \"method\": \"NLS swapping experiments, binding affinity measurements, cell fractionation, fluorescence microscopy in HGPS fibroblasts\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic dissection with NLS swapping, nanomolar affinity measurement, disease model\",\n      \"pmids\": [\"23649804\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Tpr is phosphorylated at S2059 by CDK1 and at S2094 by protein kinase A; CDK1-mediated S2059 phosphorylation is required for Tpr interaction with Mad1 and for proper localization of Mad1 and Mad2; abrogation of S2059 phosphorylation causes cell cycle defects; phosphorylated S2059-Tpr distinctly localizes with chromatin during telophase.\",\n      \"method\": \"Mass spectrometry-based phosphosite identification, kinase assays, phospho-specific antibodies, siRNA rescue with phosphomutants, immunofluorescence\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — kinase identified by in vitro assay, phosphosite mutant with defined functional consequence\",\n      \"pmids\": [\"24938596\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Depletion of Tpr (but not Nup153) dramatically increases the total number of NPCs per nucleus; this negative regulation occurs via ERK (MAPK) pathway phosphorylation, where Tpr serves as a scaffold for ERK to phosphorylate Nup153, which is critical for early stages of NPC biogenesis.\",\n      \"method\": \"AID-mediated acute depletion, NPC counting, kinase inhibition, epistasis experiments, co-immunoprecipitation\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — acute depletion distinguishing Tpr from Nup153, pathway epistasis, scaffold-kinase mechanism\",\n      \"pmids\": [\"30228202\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Acute TPR depletion using an Auxin-Induced Degron system causes rapid transcriptomic changes similar to NXF1 or GANP (TREX-2 complex) depletion; TPR depletion disrupts association of TREX-2 subunits (GANP, PCID2, ENY2) with NPCs, resulting in abnormal RNA transcription and export, establishing TPR as an integral component of the TREX-2 mRNA export pathway.\",\n      \"method\": \"Auxin-Induced Degron system, RNA-seq transcriptomics, co-immunoprecipitation, comparison of nucleoporin depletions\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — acute inducible depletion system, pathway placement by transcriptomic comparison, Co-IP\",\n      \"pmids\": [\"32917881\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TPR is specifically required for efficient nuclear export of mRNAs and lncRNAs from short, intron-poor genes; in TPR-depleted cells these mRNAs accumulate in nuclear speckles while remaining bound to Nxf1, suggesting TPR acts downstream of Nxf1 recruitment to allow mRNAs to leave nuclear speckles and dock with the nuclear pore.\",\n      \"method\": \"Fractionated RNA-seq of nucleus vs. cytosol in TPR-depleted cells, reporter constructs, immunofluorescence\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genome-wide RNA fractionation, reporter validation, epistasis relative to Nxf1\",\n      \"pmids\": [\"33091126\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Tpr depletion generates transcription-dependent replication stress, DNA breaks, and genomic instability; Tpr-deficient cells show slow/asymmetric replication forks and elevated DNA-RNA hybrids; proteomic approaches identify Tpr-interacting proteins MATR3 and SUGP2 (RNA processing); Tpr interacts with GANP (TREX-2 complex), physically connecting replication forks with transcription, splicing, and mRNA export machinery.\",\n      \"method\": \"DNA fiber assays, electron microscopy of replication intermediates, complementary proteomics, Co-immunoprecipitation, siRNA knockdown, R-loop detection\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods including EM, proteomics, functional assays\",\n      \"pmids\": [\"34168151\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Tpr interacts with Aurora A kinase via its central coiled-coil domain; Tpr depletion reduces Aurora A expression levels, centrosomal localization, and phosphorylation; Tpr and Aurora A mutually regulate each other and Tpr sequesters extra Aurora A to safeguard spindle bipolarity.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, immunofluorescence, Aurora A inhibitor (Alisertib) treatment\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP with domain mapping, KD phenotype, single lab\",\n      \"pmids\": [\"25789545\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"In Tpr-depleted cells, silencing of Tpr triggers G0-G1 arrest and senescent-like phenotype dependent on p53; Tpr depletion impairs NES-dependent nuclear export of proteins; Tpr depletion affects SUMO-protease SENP2 levels/function, altering SUMOylation at the nuclear pore.\",\n      \"method\": \"RNAi knockdown, cell cycle analysis, immunofluorescence, Western blotting\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab, KD with defined phenotype but limited mechanistic depth\",\n      \"pmids\": [\"21811608\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Tpr depletion reduces nuclear pore numbers and intranuclear filament number; Tpr-depleted cells show increased p53 nuclear accumulation and autophagy; Tpr controls HSP70 and HSF1 mRNA export and p53 trafficking with karyopherin CRM1.\",\n      \"method\": \"siRNA knockdown, electron microscopy, immunofluorescence, Western blotting\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab, multiple phenotypic readouts but limited mechanistic follow-up\",\n      \"pmids\": [\"23170199\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Molecular segments of Tpr were identified: a short C-terminal region is necessary and sufficient for nuclear import of Tpr and can confer nuclear accumulation on pyruvate kinase; the N-terminal coiled-coil domain contains clusters of heptad repeats that mediate NPC association when present in the nucleus.\",\n      \"method\": \"Expression of Tpr deletion mutants and chimeric proteins in mammalian cells, immunofluorescence\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — systematic domain dissection with chimeric proteins, single lab\",\n      \"pmids\": [\"9828100\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1987,\n      \"finding\": \"The TPR-MET oncogene was generated by chromosomal rearrangement (recombination event) fusing sequences from the tpr locus on chromosome 1 (following an Alu repetitive sequence) to the met locus on chromosome 7; the rearrangement places tpr-encoded sequences upstream of the MET kinase domain.\",\n      \"method\": \"Nucleotide sequencing of rearranged genomic locus and unrearranged tpr/met fragments\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct sequencing of breakpoints, foundational structural characterization\",\n      \"pmids\": [\"3821733\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1988,\n      \"finding\": \"The TPR-MET oncogene encodes p65(tpr-met), a constitutively active protein-tyrosine kinase that autophosphorylates on tyrosine in vitro; in vivo p65(tpr-met) is phosphorylated on serine and tyrosine, while p140(met) is phosphorylated on serine and threonine.\",\n      \"method\": \"Immunocomplex kinase assay, in vitro autophosphorylation, cell-surface iodination, phosphoamino acid analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct in vitro kinase assay, phosphoamino acid analysis\",\n      \"pmids\": [\"3277171\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"A single tyrosine residue (Tyr489) in the C-terminus of Tpr-Met is essential for efficient transformation of fibroblasts; Y489 is required for association with Grb2, phosphatidylinositol 3-kinase activation, and phospholipase Cgamma/SHPTP2 association in vivo; mutation of Y489 does not affect exogenous kinase activity toward casein.\",\n      \"method\": \"Tyrosine-to-phenylalanine mutagenesis, transformation assays, co-immunoprecipitation, kinase assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — site-directed mutagenesis with transformation assay and signaling partner Co-IP\",\n      \"pmids\": [\"7838524\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Cell transformation by Tpr-Met requires signaling pathways downstream of Shc and Grb2; pathways downstream of PI3-kinase, PLCgamma, and SHPTP2/Syp alone are insufficient for transformation; a Tpr-Met mutant selectively unable to associate with Grb2 was generated to dissect these pathways.\",\n      \"method\": \"Generation of Tpr-Met mutants selectively defective in adaptor binding, transformation assays, co-immunoprecipitation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — selective loss-of-function mutants with epistasis-style pathway dissection\",\n      \"pmids\": [\"8662733\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Loss of the juxtamembrane domain of MET (which occurs in the TPR-MET rearrangement) is essential for oncogenic activation; a chimeric Tpr-juxtaMet retaining the juxtamembrane domain (aa 962-1009) strongly inhibits transformation and downstream signaling/adaptor recruitment despite equivalent kinase activity.\",\n      \"method\": \"Construction of chimeric Tpr-juxtaMet, transformation assays (proliferation, soft agar, motility, invasion), co-immunoprecipitation of signaling molecules\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — domain-swap experiment with multiple functional readouts\",\n      \"pmids\": [\"10435641\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"The human TPR gene encodes a 726 amino acid protein with extensive alpha-helical regions and three heptad repeat motifs characteristic of coiled-coil conformation; alternative splicing can extend the C-terminal domain; its N-terminal sequences contribute to oncogenic fusions with MET, RAF kinase domains.\",\n      \"method\": \"cDNA cloning and sequencing, secondary structure prediction, alternative splicing analysis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — sequence-based structural characterization, foundational but primarily computational prediction\",\n      \"pmids\": [\"1549355\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"Human TPR (translocated promoter region) is a large (~267 kDa) coiled-coil nucleoporin constitutively localized to the intranuclear filaments (basket) of the nuclear pore complex (NPC), where it plays multiple mechanistic roles: (1) it facilitates nuclear export of NES-containing proteins and mRNAs, particularly intronless/intron-poor mRNAs, acting downstream of Nxf1 and as an integral component of the TREX-2 mRNA export pathway via interaction with GANP; (2) it retains unspliced/intron-containing mRNAs in the nucleus through the Nxf1 pathway; (3) it is required for spindle assembly checkpoint robustness by directly binding Mad1 and Mad2 and stabilizing their protein levels at NPCs during interphase, with CDK1-mediated phosphorylation of S2059 governing its interaction with Mad1 during mitosis; (4) it interacts with dynein/dynactin to coordinate proper chromosome segregation; (5) it serves as a scaffold for ERK to phosphorylate Nup153, negatively regulating NPC biogenesis; (6) its N-terminal coiled-coil domain contributes the dimerization/activation module in TPR-MET and other oncogenic kinase fusions, while the C-terminus harbors a bipartite NLS for importin alpha/beta-mediated nuclear import; and (7) it is imported into the nucleus via KPNA2 (importin alpha), a process sensitive to the nuclear Ran gradient.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"TPR is a large coiled-coil nucleoporin that forms the intranuclear basket filaments of the nuclear pore complex, where it serves as a multifunctional scaffold integrating mRNA export, nuclear retention of unspliced transcripts, spindle assembly checkpoint signaling, and NPC biogenesis control [PMID:9024684, PMID:30228202, PMID:18981471]. TPR anchors the TREX-2 complex (GANP/PCID2/ENY2) at the NPC and is specifically required for nuclear export of mRNAs from intron-poor genes via the Nxf1 pathway, acting downstream of Nxf1 to release transcripts from nuclear speckles, while simultaneously retaining unspliced intron-containing mRNAs in the nucleus [PMID:32917881, PMID:33091126, PMID:21613532]. During interphase, TPR stabilizes Mad1 and Mad2 protein levels and recruits the Mad1–Mad2 complex to NPCs, with CDK1-mediated phosphorylation at S2059 governing the TPR–Mad1 interaction and ensuring spindle assembly checkpoint robustness; TPR also cooperates with dynein/dynactin to promote proper chromosome segregation [PMID:24344181, PMID:24938596, PMID:20133940]. TPR negatively regulates NPC number by scaffolding ERK-mediated phosphorylation of Nup153, and its N-terminal coiled-coil domain provides the dimerization/activation module in oncogenic fusions such as TPR-MET [PMID:30228202, PMID:3821733, PMID:3277171].\",\n  \"teleology\": [\n    {\n      \"year\": 1987,\n      \"claim\": \"The discovery of the TPR-MET chromosomal rearrangement established that TPR sequences provide an N-terminal dimerization module fused to the MET kinase domain, founding the concept of TPR as an oncogene contributor.\",\n      \"evidence\": \"Nucleotide sequencing of rearranged and unrearranged tpr/met genomic fragments\",\n      \"pmids\": [\"3821733\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Normal TPR function was unknown\", \"Whether TPR coiled-coil domain was sufficient for dimerization was not directly tested\"]\n    },\n    {\n      \"year\": 1988,\n      \"claim\": \"Demonstration that TPR-MET encodes a constitutively active tyrosine kinase (p65) established that the TPR moiety drives ligand-independent kinase activation through enforced dimerization.\",\n      \"evidence\": \"Immunocomplex kinase assay and phosphoamino acid analysis of p65(tpr-met)\",\n      \"pmids\": [\"3277171\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of TPR-mediated dimerization unresolved\", \"Contribution of individual TPR heptad repeats to activation unknown\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Identification of Tyr489 as the essential signaling residue in TPR-MET defined the downstream effector requirements (Grb2, PI3K, Shc) for transformation, dissecting how the fusion signals.\",\n      \"evidence\": \"Tyr-to-Phe mutagenesis with transformation assays and co-IP of signaling partners\",\n      \"pmids\": [\"7838524\", \"8662733\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Role of TPR-derived sequences beyond dimerization in signaling not addressed\", \"In vivo tumor models not tested\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Correcting earlier reports, immunoelectron microscopy with multiple antibodies demonstrated that TPR exclusively localizes to the intranuclear (basket) filaments of the NPC, not the cytoplasmic face, redefining its functional context.\",\n      \"evidence\": \"Immunoelectron microscopy with antibodies to multiple distinct TPR epitopes across mammalian and amphibian cells\",\n      \"pmids\": [\"9024684\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How TPR assembles into basket filaments was unknown\", \"Whether TPR is essential for basket structure was untested\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Domain mapping revealed that the N-terminal coiled-coil mediates NPC association while the C-terminus contains the NLS; overexpression causing poly(A)+ RNA nuclear accumulation provided the first link between TPR and mRNA export.\",\n      \"evidence\": \"Expression of full-length and deletion mutants in mammalian cells with poly(A)+ RNA FISH\",\n      \"pmids\": [\"9864356\", \"9828100\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether TPR directly contacts RNA or acts via protein partners was unknown\", \"Export pathway specificity not determined\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Discovery of direct TPR–Nup98 binding and their colocalization in intranuclear filaments suggested that TPR participates in an intranuclear transport network extending beyond the NPC basket.\",\n      \"evidence\": \"In vitro binding of translated proteins combined with double-immunoEM\",\n      \"pmids\": [\"11248057\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of TPR–Nup98 interaction on transport not tested\", \"Whether other nucleoporins participate in intranuclear filaments unknown\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Anti-TPR antibody injection experiments established that TPR is specifically required for leucine-rich NES-dependent nuclear protein export, while NLS-mediated import is unaffected, distinguishing TPR's directionality in transport.\",\n      \"evidence\": \"Antibody microinjection into interphase and mitotic cells with import/export reporter assays\",\n      \"pmids\": [\"11839768\", \"11952838\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether TPR acts as a direct export factor or scaffold was unresolved\", \"Pathway specificity (CRM1 vs Nxf1) not yet discriminated\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identification of direct TPR–Mad1–Mad2 binding and the requirement of TPR for interphase NPC localization of checkpoint proteins established an unexpected role for the nuclear basket in spindle assembly checkpoint priming.\",\n      \"evidence\": \"Mass spectrometry of Mad2-associated factors, direct binding assays, siRNA knockdown with checkpoint readouts in HeLa cells\",\n      \"pmids\": [\"18981471\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether TPR stabilizes Mad1/Mad2 protein levels or only localizes them was unclear\", \"Phosphoregulation of the interaction was unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Discovery of TPR interaction with dynein/dynactin and the lagging chromosome phenotype upon TPR depletion extended its mitotic roles beyond SAC signaling to direct involvement in chromosome segregation mechanics.\",\n      \"evidence\": \"Reciprocal co-IP, RNAi knockdown with rescue and dominant-negative experiments\",\n      \"pmids\": [\"20133940\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of TPR–dynein interaction not defined\", \"Whether dynein interaction is direct or mediated by Mad1/Mad2 not fully resolved\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Pathway-specific reporter assays revealed that TPR selectively retains unspliced mRNAs that use the Nxf1/CTE export route, while CRM1/Rev-dependent export is unaffected, establishing TPR as a quality-control checkpoint for intron-containing RNA at the NPC.\",\n      \"evidence\": \"RNAi knockdown with CTE vs RRE reporter mRNA export assays\",\n      \"pmids\": [\"21613532\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether TPR directly recognizes intron-containing features or acts via cofactors was unknown\", \"Relationship to TREX-2 not yet established\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstration that TPR stabilizes Mad1 and Mad2 protein levels (not just localization) during interphase via TM2 complex formation resolved how TPR contributes to SAC proteostasis independently of kinetochores and mRNA.\",\n      \"evidence\": \"Co-IP, protein half-life measurements, GFP-Mad2 rescue in TPR-depleted cells\",\n      \"pmids\": [\"24344181\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Role of SUMO-isopeptidases SENP1/SENP2 in this stabilization not fully validated\", \"Whether TPR-mediated stabilization involves ubiquitin-proteasome pathway unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identification of KPNA2 as the importin alpha mediating TPR nuclear import, with nanomolar NLS-binding affinity, and discovery that progerin-induced Ran gradient disruption impairs TPR import linked NPC basket assembly to laminopathy pathology.\",\n      \"evidence\": \"NLS-swap experiments, binding affinity measurements, analysis in HGPS patient fibroblasts\",\n      \"pmids\": [\"23649804\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether reduced TPR import contributes to specific HGPS phenotypes not tested functionally\", \"Whether other import receptors can substitute for KPNA2 unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identification of CDK1-mediated S2059 phosphorylation as the switch governing TPR–Mad1 interaction provided the first phosphoregulatory mechanism linking cell cycle kinase activity to NPC-based SAC signaling.\",\n      \"evidence\": \"MS-based phosphosite identification, in vitro kinase assay, phosphomutant rescue in siRNA-depleted cells\",\n      \"pmids\": [\"24938596\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether S2059 phosphorylation is the sole regulatory switch or acts with additional modifications unknown\", \"Structural consequences of phosphorylation on TPR–Mad1 interface not resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Acute TPR depletion causing increased NPC number revealed that TPR negatively regulates NPC biogenesis by scaffolding ERK-mediated phosphorylation of Nup153, establishing a signaling-based feedback on pore density.\",\n      \"evidence\": \"AID-mediated acute depletion, NPC counting, ERK inhibition epistasis, co-IP\",\n      \"pmids\": [\"30228202\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which Nup153 phosphosites are ERK targets on the TPR scaffold not mapped\", \"Whether NPC number control feeds back on gene expression was not addressed\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Transcriptomic comparison of acute TPR, NXF1, and GANP depletions revealed overlapping gene expression changes, and co-IP showed TPR anchors the TREX-2 complex at NPCs, integrating TPR into the TREX-2 mRNA export pathway.\",\n      \"evidence\": \"Auxin-inducible degron system with RNA-seq and co-IP of TREX-2 subunits\",\n      \"pmids\": [\"32917881\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct versus indirect interaction between TPR and GANP not structurally resolved\", \"Whether all TREX-2 functions require NPC-anchored TPR unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Fractionated RNA-seq after TPR depletion identified intron-poor and short genes as the specifically affected class, with their mRNAs accumulating in nuclear speckles while still bound to Nxf1, placing TPR's export function downstream of Nxf1 loading.\",\n      \"evidence\": \"Nuclear/cytoplasmic RNA fractionation-seq, reporter constructs, immunofluorescence in TPR-depleted cells\",\n      \"pmids\": [\"33091126\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which TPR releases Nxf1-loaded mRNPs from speckles unknown\", \"Whether speckle retention is a cause or consequence of export failure not determined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Discovery that TPR depletion generates transcription-dependent replication stress, DNA breaks, and R-loops, with proteomic identification of MATR3 and SUGP2 as interactors, revealed TPR's role in coordinating transcription with replication to maintain genome stability.\",\n      \"evidence\": \"DNA fiber assays, EM of replication intermediates, R-loop detection, complementary proteomics, co-IP\",\n      \"pmids\": [\"34168151\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether genome instability is a direct consequence of export failure or an independent TPR function unclear\", \"MATR3 and SUGP2 interaction directionality and stoichiometry not determined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis of TPR basket filament assembly, how TPR discriminates intron-poor mRNPs for export, whether its mRNA export and SAC functions are mechanistically coupled, and how TPR's multiple scaffolding roles are coordinated during the cell cycle.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No high-resolution structure of TPR or TPR–Mad1 complex\", \"Mechanism of intron-poor mRNA selectivity at the molecular level unknown\", \"Whether mRNA export and checkpoint functions share regulatory inputs untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [1, 2, 3, 14]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [6, 14, 15]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [9, 10, 14, 16]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005635\", \"supporting_discovery_ids\": [1, 2, 4]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 2, 3, 12]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [2, 5, 9, 10, 15, 16, 17]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [4, 7, 12, 15, 16]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [6, 8, 11, 13, 14]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [14, 24, 25]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [14, 15]}\n    ],\n    \"complexes\": [\n      \"Nuclear pore complex (NPC basket)\",\n      \"TREX-2 mRNA export complex\",\n      \"TPR-Mad1-Mad2 (TM2) complex\"\n    ],\n    \"partners\": [\n      \"MAD1L1\",\n      \"MAD2L1\",\n      \"NUP98\",\n      \"NUP153\",\n      \"MCM3AP\",\n      \"KPNA2\",\n      \"DYNLL1\",\n      \"MATR3\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}