{"gene":"TPRKB","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":2003,"finding":"TPRKB (CGI-121) was identified as a direct binding partner of PRPK (TP53RK) via yeast two-hybrid screening, and the interaction was confirmed in vivo and in vitro. TPRKB localizes to both nucleus and cytosol. Recombinant TPRKB inhibited co-precipitation of p53 by recombinant PRPK in vitro, suggesting TPRKB acts as an inhibitor of PRPK-p53 binding.","method":"Yeast two-hybrid, co-immunoprecipitation in vivo and in vitro, immunocytochemistry, recombinant protein competition assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal in vivo and in vitro binding confirmed; functional competition assay with recombinant proteins; single lab, two orthogonal methods","pmids":["12659830"],"is_preprint":false},{"year":2006,"finding":"TPRKB (CGI-121/Cgi121 in yeast) is a subunit of the conserved KEOPS complex (with Kae1, Bud32/PRPK, and Pcc1). Deletion of CGI121 suppresses the cdc13-1 telomere-capping defect by dramatically reducing ssDNA accumulation at telomeres. KEOPS components including Cgi121 are required for telomere elongation and de novo telomere addition to DNA double-strand breaks.","method":"Genome-wide suppressor screen (S. cerevisiae), genetic deletion, telomere length assays, epistasis analysis","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide screen with genetic validation, multiple epistasis experiments, replicated across complex subunits","pmids":["16564010"],"is_preprint":false},{"year":2008,"finding":"The atomic structure of archaea-derived KEOPS complexes (Kae1, Bud32, Pcc1, Cgi121) was solved, revealing that Cgi121 regulates the primordial kinase Bud32, which in turn regulates Kae1. Structural analysis indicates Cgi121 sits at the terminus of a linear complex arrangement.","method":"X-ray crystallography of archaeal KEOPS complex","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — atomic crystal structure with functional inference from structural analysis; foundational structural study","pmids":["18951093"],"is_preprint":false},{"year":2013,"finding":"Within the KEOPS/EKC complex, Cgi121 (TPRKB ortholog) acts as an allosteric regulator of the minimal functional unit (Pcc1-Kae1-Bud32). Cgi121 is not required for the core t6A biosynthesis catalytic step but modulates the complex activity. The minimal unit for t6A synthesis is Pcc1-Kae1-Bud32.","method":"Reconstitution of t6A biosynthesis activity with subcomplexes, biochemical assays in archaeal system","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with defined subcomplexes; single lab but multiple orthogonal biochemical assays","pmids":["23945934"],"is_preprint":false},{"year":2015,"finding":"Crystal structure of the Bud32/Cgi121 (PRPK/TPRKB) subcomplex with ADP revealed that ADP binds in the catalytic site of Bud32 in a canonical PKA-family manner, and Cgi121 caps the C-terminal end of Bud32. The yeast KEOPS complex exists as a heteropentamer in a linear arrangement: Gon7-Pcc1-Kae1-Bud32-Cgi121.","method":"X-ray crystallography of Bud32/Cgi121 complex with ADP, structural modeling","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with bound ligand; linear complex arrangement validated by structural and biochemical data","pmids":["25735745"],"is_preprint":false},{"year":2015,"finding":"In S. cerevisiae, inactivation of KEOPS subunit Cgi121 (TPRKB ortholog) specifically inhibits telomere recombination. Deletion of CGI121 reduced telomeric ssDNA generation, consistent with Cgi121 promoting ssDNA at telomeres to enable recombination. This inhibition of telomere recombination significantly extended cellular lifespan in both telomerase-positive and telomerase-negative cells.","method":"Genetic deletion, telomere recombination assays, lifespan analysis, epistasis with yku80 mutant","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic deletion with specific mechanistic readout (ssDNA levels, recombination frequency, lifespan); single lab, multiple assays","pmids":["25822194"],"is_preprint":false},{"year":2016,"finding":"Proteomic analysis of human KEOPS confirmed TPRKB as a core subunit interacting with OSGEP (Kae1), TP53RK (Bud32/PRPK), and LAGE3 (Pcc1). Mass spectrometry identified 152 protein interactors; specific interactors were found for different KEOPS subcomplexes, suggesting TPRKB may have functions outside of t6A biosynthesis.","method":"Affinity purification mass spectrometry (AP-MS), proteomic interactome analysis of human KEOPS","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — AP-MS with all four human KEOPS subunits confirmed; subcomplex-specific interactors identified; single lab","pmids":["27903914"],"is_preprint":false},{"year":2017,"finding":"Knockdown of TPRKB in human cells inhibited cell proliferation, impaired protein translation, caused endoplasmic reticulum stress, activated DNA-damage-response signaling, and induced apoptosis. Knockdown also induced defects in the actin cytoskeleton and decreased migration rate of human podocytes. Human disease mutations in TPRKB did not rescue these proliferation defects.","method":"siRNA knockdown in human cell lines and podocytes, CRISPR-Cas9 knockout in zebrafish and mice, cell proliferation assays, actin cytoskeleton imaging, migration assays","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal functional assays across human cells, zebrafish, and mice; disease mutation rescue experiment; replicated across multiple KEOPS subunits","pmids":["28805828"],"is_preprint":false},{"year":2018,"finding":"Telomere length regulation by the KEOPS complex (including Cgi121/TPRKB) is independent of its t6A biosynthesis activity. Cytoplasmic overexpression of Qri7 (mitochondrial Kae1 ortholog) restored t6A modification and cell growth but failed to rescue telomere length in kae1Δ cells. KEOPS subunit deletions cause dramatic reduction in telomeric G-overhang, indicating KEOPS promotes G-overhang generation for telomere length regulation.","method":"Yeast genetics, Qri7 complementation experiments, in vitro biochemical assays, telomere G-overhang analysis, genetic epistasis","journal":"Journal of genetics and genomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic complementation with functional separation of t6A and telomere functions; multiple orthogonal assays; single lab","pmids":["29804714"],"is_preprint":false},{"year":2019,"finding":"TPRKB knockdown selectively inhibits proliferation of TP53-deficient cancer cells but has minimal effect in TP53 wild-type cells. TP53 reintroduction into TP53-null cells rescued sensitivity to TPRKB knockdown. TP53 indirectly mediates TPRKB protein degradation (via proteasome), and this degradation was rescued by co-expression of PRPK (TP53RK) or by proteasome inhibition. Depletion of other EKC/KEOPS members showed TP53-independent effects, supporting a complex-independent function for TPRKB.","method":"shRNA knockdown, in vitro and in vivo proliferation assays, TP53 reintroduction, proteasome inhibition, epistasis with other KEOPS members","journal":"Molecular cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple cell lines tested in vitro and in vivo; rescue and epistasis experiments; mechanistic link to proteasomal degradation established; single lab","pmids":["31110156"],"is_preprint":false},{"year":2021,"finding":"The 2.53 Å crystal structure of the human PRPK-TPRKB-AMPPNP complex was solved. The structure reveals details of PRPK-AMPPNP nucleotide coordination and the PRPK-TPRKB protein-protein interaction interface. PRPK appears in an active conformation despite lacking a conventional kinase activation loop. Disease mutations in PRPK and TPRKB were mapped onto the structure; one mutation, PRPK K238Nfs*2, was shown experimentally to abolish binding to OSGEP.","method":"X-ray crystallography (2.53 Å), structural modeling of full human KEOPS, binding assay for disease mutation","journal":"Communications biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — atomic resolution crystal structure with functional validation of disease mutation effect on OSGEP binding; single lab with orthogonal structural and biochemical methods","pmids":["33547416"],"is_preprint":false},{"year":2021,"finding":"Generation of the first Tprkb null knockout mouse strain demonstrated embryonic lethality, establishing that TPRKB is essential for mouse development.","method":"i-GONAD CRISPR knockout in mice, survival analysis of homozygous null embryos","journal":"G3 (Bethesda, Md.)","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean genetic knockout with clear lethal phenotype; single study, minimal mechanistic follow-up beyond establishing essentiality","pmids":["34849815"],"is_preprint":false},{"year":2024,"finding":"Cryo-EM structures of the full KEOPS complex with and without tRNA substrate revealed that Cgi121 (TPRKB) is part of the holo-enzyme substrate complex. The structures uncovered contact surfaces within KEOPS-tRNA required for Bud32 ATPase regulation and t6A modification activity. Cgi121's position at the terminus of the linear complex contributes to tRNA binding and positioning for Kae1-catalyzed modification.","method":"Cryo-electron microscopy (cryo-EM) of KEOPS with and without tRNA, mutagenesis of contact surfaces","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structures with and without substrate, combined with mutagenesis of functionally critical residues; multiple orthogonal methods in single rigorous study","pmids":["39639027"],"is_preprint":false},{"year":2024,"finding":"METTL5 positively regulates TPRKB mRNA stability through N6-methyladenosine (m6A) modification, thereby enhancing TPRKB protein expression in hepatocellular carcinoma cells. TPRKB knockdown suppressed HCC cell viability, colony formation, invasion, migration, and tumor growth in vivo. TPRKB overexpression partially rescued the anti-cancer effects of METTL5 knockdown.","method":"m6A modification analysis, METTL5 knockdown/overexpression, TPRKB knockdown/overexpression, mRNA stability assay, in vitro and in vivo tumor assays","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional epistasis between METTL5 and TPRKB with mRNA stability mechanistic link; in vitro and in vivo validation; single lab","pmids":["39182664"],"is_preprint":false}],"current_model":"TPRKB (CGI-121) is a core subunit of the eukaryotic KEOPS complex that occupies the terminal position in the linear Gon7-Pcc1-Kae1-Bud32-TPRKB arrangement, where it directly binds and allosterically regulates the kinase/ATPase Bud32 (PRPK), facilitates tRNA substrate recruitment and positioning for Kae1-catalyzed t6A modification, promotes telomere G-overhang generation independently of t6A activity, and—outside the complex—is stabilized by PRPK binding and subject to TP53-dependent proteasomal degradation, making TP53-deficient cells selectively dependent on TPRKB for proliferation."},"narrative":{"mechanistic_narrative":"TPRKB (CGI-121) is a core subunit of the conserved eukaryotic KEOPS complex, occupying the terminal position of the linear Gon7–Pcc1–Kae1–Bud32–Cgi121 architecture, where it directly binds and allosterically regulates the kinase/ATPase Bud32 (PRPK) [PMID:18951093, PMID:25735745, PMID:27903914]. Within KEOPS, TPRKB is dispensable for the core t6A tRNA-modification catalytic step—the minimal catalytic unit being Pcc1–Kae1–Bud32—but it modulates overall complex activity and, in the holoenzyme, contributes to recruiting and positioning the tRNA substrate for Kae1-catalyzed modification [PMID:23945934, PMID:39639027]. Beyond t6A biosynthesis, TPRKB and KEOPS regulate telomere homeostasis: Cgi121 promotes telomeric single-stranded G-overhang generation and telomere recombination/elongation in a manner genetically separable from t6A activity, such that its loss suppresses the cdc13-1 capping defect and extends cellular lifespan [PMID:16564010, PMID:25822194, PMID:29804714]. In human cells, TPRKB is stabilized through direct binding by PRPK and is subject to TP53-dependent proteasomal degradation, rendering TP53-deficient cancer cells selectively dependent on TPRKB for proliferation [PMID:31110156]. Loss of TPRKB impairs translation, triggers ER stress, DNA-damage signaling and apoptosis, disrupts the actin cytoskeleton, and is essential for mouse development [PMID:28805828, PMID:34849815].","teleology":[{"year":2003,"claim":"Established TPRKB's first molecular partner by identifying it as a direct PRPK-binding protein that modulates PRPK–p53 association, framing it as a regulator of a p53-linked kinase.","evidence":"Yeast two-hybrid, reciprocal co-IP, and recombinant competition assay in human cells","pmids":["12659830"],"confidence":"Medium","gaps":["Did not place TPRKB within a defined multiprotein complex","Functional consequence of inhibiting PRPK–p53 binding in cells not established"]},{"year":2006,"claim":"Placed Cgi121/TPRKB into the KEOPS complex and revealed a telomere-maintenance role, showing its deletion suppresses a telomere-capping defect by reducing telomeric ssDNA.","evidence":"Genome-wide suppressor screen and telomere assays in S. cerevisiae","pmids":["16564010"],"confidence":"High","gaps":["Mechanism linking KEOPS to ssDNA/G-overhang generation not defined","Relationship between telomere role and any enzymatic activity unknown"]},{"year":2008,"claim":"Defined the architecture of KEOPS, showing Cgi121 sits at the terminus of a linear complex and regulates the kinase Bud32, which in turn regulates Kae1.","evidence":"X-ray crystallography of archaeal KEOPS subcomplexes","pmids":["18951093"],"confidence":"High","gaps":["Did not assign a catalytic activity to the complex","Human complex composition (e.g., Gon7/LAGE3) not addressed"]},{"year":2013,"claim":"Separated TPRKB's role from catalysis, demonstrating it is an allosteric modulator dispensable for the minimal Pcc1–Kae1–Bud32 t6A biosynthesis unit.","evidence":"In vitro reconstitution of t6A activity with defined archaeal subcomplexes","pmids":["23945934"],"confidence":"High","gaps":["Precise allosteric mechanism on complex activity unresolved","Whether modulation matters in vivo not tested"]},{"year":2015,"claim":"Resolved the PRPK–TPRKB interface and confirmed the linear heteropentameric arrangement, showing Cgi121 caps the C-terminus of Bud32 bound to ADP.","evidence":"X-ray crystallography of Bud32/Cgi121 with ADP and structural modeling","pmids":["25735745"],"confidence":"High","gaps":["Substrate (tRNA) contacts not visualized","Conformational changes during catalysis not captured"]},{"year":2015,"claim":"Mechanistically tied Cgi121 to telomere recombination, showing its loss reduces telomeric ssDNA and extends cellular lifespan in telomerase-positive and -negative cells.","evidence":"Genetic deletion, recombination and lifespan assays in S. cerevisiae","pmids":["25822194"],"confidence":"Medium","gaps":["Direct biochemical activity generating ssDNA not identified","Conservation of telomere role in human cells untested"]},{"year":2016,"claim":"Confirmed the human KEOPS composition and hinted at functions beyond t6A by mapping subcomplex-specific interactors of TPRKB.","evidence":"AP-MS interactome of all four human KEOPS subunits","pmids":["27903914"],"confidence":"Medium","gaps":["Functional significance of non-KEOPS interactors unvalidated","Direct vs indirect interactions not distinguished"]},{"year":2017,"claim":"Defined cellular consequences of TPRKB loss in humans and model organisms—impaired translation, ER stress, DNA-damage signaling, apoptosis, cytoskeletal defects—linking it to a human disease context.","evidence":"siRNA knockdown in human cells/podocytes, CRISPR knockout in zebrafish and mice, multiple functional assays","pmids":["28805828"],"confidence":"High","gaps":["Whether phenotypes derive from t6A loss vs complex-independent roles not dissected","Direct cause of cytoskeletal defects unknown"]},{"year":2018,"claim":"Genetically uncoupled KEOPS telomere function from t6A, showing G-overhang generation persists independently of restored t6A modification.","evidence":"Qri7 complementation, telomere G-overhang analysis, epistasis in yeast","pmids":["29804714"],"confidence":"Medium","gaps":["Molecular activity driving G-overhang formation unidentified","Direct TPRKB-specific contribution vs whole-complex effect unclear"]},{"year":2019,"claim":"Revealed a complex-independent, synthetic-lethal vulnerability: TP53-deficient cells require TPRKB because TP53 drives its proteasomal degradation, which PRPK binding counteracts.","evidence":"shRNA knockdown, TP53 reintroduction, proteasome inhibition, in vitro/in vivo proliferation, KEOPS-member epistasis","pmids":["31110156"],"confidence":"Medium","gaps":["Identity of the E3 ligase/intermediary in TP53-dependent degradation unknown","Why TP53-null cells depend on TPRKB mechanistically unresolved"]},{"year":2021,"claim":"Provided an atomic view of the human PRPK–TPRKB interaction and mapped disease mutations, including one abolishing OSGEP binding.","evidence":"2.53 Å crystal structure of PRPK–TPRKB–AMPPNP with disease-mutation binding assay","pmids":["33547416"],"confidence":"High","gaps":["Full assembled human holoenzyme not crystallized","Functional impact of most mapped mutations untested"]},{"year":2021,"claim":"Established organismal essentiality by showing Tprkb-null mice are embryonic lethal.","evidence":"i-GONAD CRISPR knockout mouse, embryo survival analysis","pmids":["34849815"],"confidence":"Medium","gaps":["Developmental stage and tissue of lethality not characterized","Mechanism of essential requirement not addressed"]},{"year":2024,"claim":"Captured TPRKB in the substrate-engaged holoenzyme, defining KEOPS–tRNA contacts required for Bud32 ATPase regulation and t6A activity.","evidence":"Cryo-EM of KEOPS with and without tRNA plus mutagenesis of contact surfaces","pmids":["39639027"],"confidence":"High","gaps":["TPRKB's direct contribution to tRNA positioning vs scaffolding not fully isolated","Dynamics of catalytic cycle not resolved"]},{"year":2024,"claim":"Identified an upstream regulatory layer in cancer, showing METTL5-mediated m6A stabilizes TPRKB mRNA to drive hepatocellular carcinoma progression.","evidence":"m6A analysis, METTL5/TPRKB knockdown-overexpression epistasis, mRNA stability and tumor assays","pmids":["39182664"],"confidence":"Medium","gaps":["Downstream effectors of TPRKB driving HCC phenotypes unidentified","Whether this acts through KEOPS or complex-independent functions unknown"]},{"year":null,"claim":"The biochemical activity by which TPRKB/KEOPS generates telomeric G-overhangs independently of t6A, and the molecular basis of TP53-dependent TPRKB degradation, remain undefined.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No enzyme or activity assigned to G-overhang generation","E3 ligase mediating TP53-dependent TPRKB turnover unknown","Mechanistic basis of TP53-deficient synthetic lethality unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,3,4]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[12]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[3,12]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[3,7]}],"complexes":["KEOPS/EKC complex"],"partners":["TP53RK","OSGEP","LAGE3","TP53"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9Y3C4","full_name":"EKC/KEOPS complex subunit TPRKB","aliases":["PRPK-binding protein","TP53RK-binding protein"],"length_aa":175,"mass_kda":19.7,"function":"Component of the EKC/KEOPS complex that is required for the formation of a threonylcarbamoyl group on adenosine at position 37 (t(6)A37) in tRNAs that read codons beginning with adenine (PubMed:22912744, PubMed:28805828). The complex is probably involved in the transfer of the threonylcarbamoyl moiety of threonylcarbamoyl-AMP (TC-AMP) to the N6 group of A37 (PubMed:22912744, PubMed:28805828). TPRKB acts as an allosteric effector that regulates the t(6)A activity of the complex. TPRKB is not required for tRNA modification (PubMed:22912744, PubMed:28805828)","subcellular_location":"Cytoplasm, cytosol; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9Y3C4/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/TPRKB","classification":"Common Essential","n_dependent_lines":803,"n_total_lines":1208,"dependency_fraction":0.6647350993377483},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"POLR3E","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/TPRKB","total_profiled":1310},"omim":[{"mim_id":"617731","title":"GALLOWAY-MOWAT SYNDROME 5; GAMOS5","url":"https://www.omim.org/entry/617731"},{"mim_id":"617730","title":"GALLOWAY-MOWAT SYNDROME 4; GAMOS4","url":"https://www.omim.org/entry/617730"},{"mim_id":"617729","title":"GALLOWAY-MOWAT SYNDROME 3; GAMOS3","url":"https://www.omim.org/entry/617729"},{"mim_id":"617436","title":"GON7 SUBUNIT OF KEOPS COMPLEX; GON7","url":"https://www.omim.org/entry/617436"},{"mim_id":"610107","title":"O-SIALOGLYCOPROTEIN ENDOPEPTIDASE; OSGEP","url":"https://www.omim.org/entry/610107"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TPRKB"},"hgnc":{"alias_symbol":["CGI-121","CGI121"],"prev_symbol":[]},"alphafold":{"accession":"Q9Y3C4","domains":[{"cath_id":"3.30.2380.10","chopping":"3-173","consensus_level":"high","plddt":95.7614,"start":3,"end":173}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y3C4","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y3C4-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y3C4-F1-predicted_aligned_error_v6.png","plddt_mean":95.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TPRKB","jax_strain_url":"https://www.jax.org/strain/search?query=TPRKB"},"sequence":{"accession":"Q9Y3C4","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y3C4.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y3C4/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y3C4"}},"corpus_meta":[{"pmid":"28805828","id":"PMC_28805828","title":"Mutations in KEOPS-complex 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TPRKB localizes to both nucleus and cytosol. Recombinant TPRKB inhibited co-precipitation of p53 by recombinant PRPK in vitro, suggesting TPRKB acts as an inhibitor of PRPK-p53 binding.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation in vivo and in vitro, immunocytochemistry, recombinant protein competition assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal in vivo and in vitro binding confirmed; functional competition assay with recombinant proteins; single lab, two orthogonal methods\",\n      \"pmids\": [\"12659830\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"TPRKB (CGI-121/Cgi121 in yeast) is a subunit of the conserved KEOPS complex (with Kae1, Bud32/PRPK, and Pcc1). Deletion of CGI121 suppresses the cdc13-1 telomere-capping defect by dramatically reducing ssDNA accumulation at telomeres. KEOPS components including Cgi121 are required for telomere elongation and de novo telomere addition to DNA double-strand breaks.\",\n      \"method\": \"Genome-wide suppressor screen (S. cerevisiae), genetic deletion, telomere length assays, epistasis analysis\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide screen with genetic validation, multiple epistasis experiments, replicated across complex subunits\",\n      \"pmids\": [\"16564010\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The atomic structure of archaea-derived KEOPS complexes (Kae1, Bud32, Pcc1, Cgi121) was solved, revealing that Cgi121 regulates the primordial kinase Bud32, which in turn regulates Kae1. Structural analysis indicates Cgi121 sits at the terminus of a linear complex arrangement.\",\n      \"method\": \"X-ray crystallography of archaeal KEOPS complex\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — atomic crystal structure with functional inference from structural analysis; foundational structural study\",\n      \"pmids\": [\"18951093\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Within the KEOPS/EKC complex, Cgi121 (TPRKB ortholog) acts as an allosteric regulator of the minimal functional unit (Pcc1-Kae1-Bud32). Cgi121 is not required for the core t6A biosynthesis catalytic step but modulates the complex activity. The minimal unit for t6A synthesis is Pcc1-Kae1-Bud32.\",\n      \"method\": \"Reconstitution of t6A biosynthesis activity with subcomplexes, biochemical assays in archaeal system\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with defined subcomplexes; single lab but multiple orthogonal biochemical assays\",\n      \"pmids\": [\"23945934\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Crystal structure of the Bud32/Cgi121 (PRPK/TPRKB) subcomplex with ADP revealed that ADP binds in the catalytic site of Bud32 in a canonical PKA-family manner, and Cgi121 caps the C-terminal end of Bud32. The yeast KEOPS complex exists as a heteropentamer in a linear arrangement: Gon7-Pcc1-Kae1-Bud32-Cgi121.\",\n      \"method\": \"X-ray crystallography of Bud32/Cgi121 complex with ADP, structural modeling\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with bound ligand; linear complex arrangement validated by structural and biochemical data\",\n      \"pmids\": [\"25735745\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In S. cerevisiae, inactivation of KEOPS subunit Cgi121 (TPRKB ortholog) specifically inhibits telomere recombination. Deletion of CGI121 reduced telomeric ssDNA generation, consistent with Cgi121 promoting ssDNA at telomeres to enable recombination. This inhibition of telomere recombination significantly extended cellular lifespan in both telomerase-positive and telomerase-negative cells.\",\n      \"method\": \"Genetic deletion, telomere recombination assays, lifespan analysis, epistasis with yku80 mutant\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic deletion with specific mechanistic readout (ssDNA levels, recombination frequency, lifespan); single lab, multiple assays\",\n      \"pmids\": [\"25822194\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Proteomic analysis of human KEOPS confirmed TPRKB as a core subunit interacting with OSGEP (Kae1), TP53RK (Bud32/PRPK), and LAGE3 (Pcc1). Mass spectrometry identified 152 protein interactors; specific interactors were found for different KEOPS subcomplexes, suggesting TPRKB may have functions outside of t6A biosynthesis.\",\n      \"method\": \"Affinity purification mass spectrometry (AP-MS), proteomic interactome analysis of human KEOPS\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — AP-MS with all four human KEOPS subunits confirmed; subcomplex-specific interactors identified; single lab\",\n      \"pmids\": [\"27903914\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Knockdown of TPRKB in human cells inhibited cell proliferation, impaired protein translation, caused endoplasmic reticulum stress, activated DNA-damage-response signaling, and induced apoptosis. Knockdown also induced defects in the actin cytoskeleton and decreased migration rate of human podocytes. Human disease mutations in TPRKB did not rescue these proliferation defects.\",\n      \"method\": \"siRNA knockdown in human cell lines and podocytes, CRISPR-Cas9 knockout in zebrafish and mice, cell proliferation assays, actin cytoskeleton imaging, migration assays\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal functional assays across human cells, zebrafish, and mice; disease mutation rescue experiment; replicated across multiple KEOPS subunits\",\n      \"pmids\": [\"28805828\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Telomere length regulation by the KEOPS complex (including Cgi121/TPRKB) is independent of its t6A biosynthesis activity. Cytoplasmic overexpression of Qri7 (mitochondrial Kae1 ortholog) restored t6A modification and cell growth but failed to rescue telomere length in kae1Δ cells. KEOPS subunit deletions cause dramatic reduction in telomeric G-overhang, indicating KEOPS promotes G-overhang generation for telomere length regulation.\",\n      \"method\": \"Yeast genetics, Qri7 complementation experiments, in vitro biochemical assays, telomere G-overhang analysis, genetic epistasis\",\n      \"journal\": \"Journal of genetics and genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic complementation with functional separation of t6A and telomere functions; multiple orthogonal assays; single lab\",\n      \"pmids\": [\"29804714\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TPRKB knockdown selectively inhibits proliferation of TP53-deficient cancer cells but has minimal effect in TP53 wild-type cells. TP53 reintroduction into TP53-null cells rescued sensitivity to TPRKB knockdown. TP53 indirectly mediates TPRKB protein degradation (via proteasome), and this degradation was rescued by co-expression of PRPK (TP53RK) or by proteasome inhibition. Depletion of other EKC/KEOPS members showed TP53-independent effects, supporting a complex-independent function for TPRKB.\",\n      \"method\": \"shRNA knockdown, in vitro and in vivo proliferation assays, TP53 reintroduction, proteasome inhibition, epistasis with other KEOPS members\",\n      \"journal\": \"Molecular cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple cell lines tested in vitro and in vivo; rescue and epistasis experiments; mechanistic link to proteasomal degradation established; single lab\",\n      \"pmids\": [\"31110156\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The 2.53 Å crystal structure of the human PRPK-TPRKB-AMPPNP complex was solved. The structure reveals details of PRPK-AMPPNP nucleotide coordination and the PRPK-TPRKB protein-protein interaction interface. PRPK appears in an active conformation despite lacking a conventional kinase activation loop. Disease mutations in PRPK and TPRKB were mapped onto the structure; one mutation, PRPK K238Nfs*2, was shown experimentally to abolish binding to OSGEP.\",\n      \"method\": \"X-ray crystallography (2.53 Å), structural modeling of full human KEOPS, binding assay for disease mutation\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — atomic resolution crystal structure with functional validation of disease mutation effect on OSGEP binding; single lab with orthogonal structural and biochemical methods\",\n      \"pmids\": [\"33547416\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Generation of the first Tprkb null knockout mouse strain demonstrated embryonic lethality, establishing that TPRKB is essential for mouse development.\",\n      \"method\": \"i-GONAD CRISPR knockout in mice, survival analysis of homozygous null embryos\",\n      \"journal\": \"G3 (Bethesda, Md.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean genetic knockout with clear lethal phenotype; single study, minimal mechanistic follow-up beyond establishing essentiality\",\n      \"pmids\": [\"34849815\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Cryo-EM structures of the full KEOPS complex with and without tRNA substrate revealed that Cgi121 (TPRKB) is part of the holo-enzyme substrate complex. The structures uncovered contact surfaces within KEOPS-tRNA required for Bud32 ATPase regulation and t6A modification activity. Cgi121's position at the terminus of the linear complex contributes to tRNA binding and positioning for Kae1-catalyzed modification.\",\n      \"method\": \"Cryo-electron microscopy (cryo-EM) of KEOPS with and without tRNA, mutagenesis of contact surfaces\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structures with and without substrate, combined with mutagenesis of functionally critical residues; multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"39639027\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"METTL5 positively regulates TPRKB mRNA stability through N6-methyladenosine (m6A) modification, thereby enhancing TPRKB protein expression in hepatocellular carcinoma cells. TPRKB knockdown suppressed HCC cell viability, colony formation, invasion, migration, and tumor growth in vivo. TPRKB overexpression partially rescued the anti-cancer effects of METTL5 knockdown.\",\n      \"method\": \"m6A modification analysis, METTL5 knockdown/overexpression, TPRKB knockdown/overexpression, mRNA stability assay, in vitro and in vivo tumor assays\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional epistasis between METTL5 and TPRKB with mRNA stability mechanistic link; in vitro and in vivo validation; single lab\",\n      \"pmids\": [\"39182664\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TPRKB (CGI-121) is a core subunit of the eukaryotic KEOPS complex that occupies the terminal position in the linear Gon7-Pcc1-Kae1-Bud32-TPRKB arrangement, where it directly binds and allosterically regulates the kinase/ATPase Bud32 (PRPK), facilitates tRNA substrate recruitment and positioning for Kae1-catalyzed t6A modification, promotes telomere G-overhang generation independently of t6A activity, and—outside the complex—is stabilized by PRPK binding and subject to TP53-dependent proteasomal degradation, making TP53-deficient cells selectively dependent on TPRKB for proliferation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TPRKB (CGI-121) is a core subunit of the conserved eukaryotic KEOPS complex, occupying the terminal position of the linear Gon7–Pcc1–Kae1–Bud32–Cgi121 architecture, where it directly binds and allosterically regulates the kinase/ATPase Bud32 (PRPK) [#2, #4, #6]. Within KEOPS, TPRKB is dispensable for the core t6A tRNA-modification catalytic step—the minimal catalytic unit being Pcc1–Kae1–Bud32—but it modulates overall complex activity and, in the holoenzyme, contributes to recruiting and positioning the tRNA substrate for Kae1-catalyzed modification [#3, #12]. Beyond t6A biosynthesis, TPRKB and KEOPS regulate telomere homeostasis: Cgi121 promotes telomeric single-stranded G-overhang generation and telomere recombination/elongation in a manner genetically separable from t6A activity, such that its loss suppresses the cdc13-1 capping defect and extends cellular lifespan [#1, #5, #8]. In human cells, TPRKB is stabilized through direct binding by PRPK and is subject to TP53-dependent proteasomal degradation, rendering TP53-deficient cancer cells selectively dependent on TPRKB for proliferation [#9]. Loss of TPRKB impairs translation, triggers ER stress, DNA-damage signaling and apoptosis, disrupts the actin cytoskeleton, and is essential for mouse development [#7, #11].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established TPRKB's first molecular partner by identifying it as a direct PRPK-binding protein that modulates PRPK–p53 association, framing it as a regulator of a p53-linked kinase.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal co-IP, and recombinant competition assay in human cells\",\n      \"pmids\": [\"12659830\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Did not place TPRKB within a defined multiprotein complex\", \"Functional consequence of inhibiting PRPK–p53 binding in cells not established\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Placed Cgi121/TPRKB into the KEOPS complex and revealed a telomere-maintenance role, showing its deletion suppresses a telomere-capping defect by reducing telomeric ssDNA.\",\n      \"evidence\": \"Genome-wide suppressor screen and telomere assays in S. cerevisiae\",\n      \"pmids\": [\"16564010\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Mechanism linking KEOPS to ssDNA/G-overhang generation not defined\", \"Relationship between telomere role and any enzymatic activity unknown\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined the architecture of KEOPS, showing Cgi121 sits at the terminus of a linear complex and regulates the kinase Bud32, which in turn regulates Kae1.\",\n      \"evidence\": \"X-ray crystallography of archaeal KEOPS subcomplexes\",\n      \"pmids\": [\"18951093\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Did not assign a catalytic activity to the complex\", \"Human complex composition (e.g., Gon7/LAGE3) not addressed\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Separated TPRKB's role from catalysis, demonstrating it is an allosteric modulator dispensable for the minimal Pcc1–Kae1–Bud32 t6A biosynthesis unit.\",\n      \"evidence\": \"In vitro reconstitution of t6A activity with defined archaeal subcomplexes\",\n      \"pmids\": [\"23945934\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Precise allosteric mechanism on complex activity unresolved\", \"Whether modulation matters in vivo not tested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Resolved the PRPK–TPRKB interface and confirmed the linear heteropentameric arrangement, showing Cgi121 caps the C-terminus of Bud32 bound to ADP.\",\n      \"evidence\": \"X-ray crystallography of Bud32/Cgi121 with ADP and structural modeling\",\n      \"pmids\": [\"25735745\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Substrate (tRNA) contacts not visualized\", \"Conformational changes during catalysis not captured\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Mechanistically tied Cgi121 to telomere recombination, showing its loss reduces telomeric ssDNA and extends cellular lifespan in telomerase-positive and -negative cells.\",\n      \"evidence\": \"Genetic deletion, recombination and lifespan assays in S. cerevisiae\",\n      \"pmids\": [\"25822194\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Direct biochemical activity generating ssDNA not identified\", \"Conservation of telomere role in human cells untested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Confirmed the human KEOPS composition and hinted at functions beyond t6A by mapping subcomplex-specific interactors of TPRKB.\",\n      \"evidence\": \"AP-MS interactome of all four human KEOPS subunits\",\n      \"pmids\": [\"27903914\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Functional significance of non-KEOPS interactors unvalidated\", \"Direct vs indirect interactions not distinguished\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Defined cellular consequences of TPRKB loss in humans and model organisms—impaired translation, ER stress, DNA-damage signaling, apoptosis, cytoskeletal defects—linking it to a human disease context.\",\n      \"evidence\": \"siRNA knockdown in human cells/podocytes, CRISPR knockout in zebrafish and mice, multiple functional assays\",\n      \"pmids\": [\"28805828\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Whether phenotypes derive from t6A loss vs complex-independent roles not dissected\", \"Direct cause of cytoskeletal defects unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Genetically uncoupled KEOPS telomere function from t6A, showing G-overhang generation persists independently of restored t6A modification.\",\n      \"evidence\": \"Qri7 complementation, telomere G-overhang analysis, epistasis in yeast\",\n      \"pmids\": [\"29804714\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Molecular activity driving G-overhang formation unidentified\", \"Direct TPRKB-specific contribution vs whole-complex effect unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Revealed a complex-independent, synthetic-lethal vulnerability: TP53-deficient cells require TPRKB because TP53 drives its proteasomal degradation, which PRPK binding counteracts.\",\n      \"evidence\": \"shRNA knockdown, TP53 reintroduction, proteasome inhibition, in vitro/in vivo proliferation, KEOPS-member epistasis\",\n      \"pmids\": [\"31110156\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Identity of the E3 ligase/intermediary in TP53-dependent degradation unknown\", \"Why TP53-null cells depend on TPRKB mechanistically unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Provided an atomic view of the human PRPK–TPRKB interaction and mapped disease mutations, including one abolishing OSGEP binding.\",\n      \"evidence\": \"2.53 Å crystal structure of PRPK–TPRKB–AMPPNP with disease-mutation binding assay\",\n      \"pmids\": [\"33547416\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Full assembled human holoenzyme not crystallized\", \"Functional impact of most mapped mutations untested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Established organismal essentiality by showing Tprkb-null mice are embryonic lethal.\",\n      \"evidence\": \"i-GONAD CRISPR knockout mouse, embryo survival analysis\",\n      \"pmids\": [\"34849815\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Developmental stage and tissue of lethality not characterized\", \"Mechanism of essential requirement not addressed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Captured TPRKB in the substrate-engaged holoenzyme, defining KEOPS–tRNA contacts required for Bud32 ATPase regulation and t6A activity.\",\n      \"evidence\": \"Cryo-EM of KEOPS with and without tRNA plus mutagenesis of contact surfaces\",\n      \"pmids\": [\"39639027\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"TPRKB's direct contribution to tRNA positioning vs scaffolding not fully isolated\", \"Dynamics of catalytic cycle not resolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified an upstream regulatory layer in cancer, showing METTL5-mediated m6A stabilizes TPRKB mRNA to drive hepatocellular carcinoma progression.\",\n      \"evidence\": \"m6A analysis, METTL5/TPRKB knockdown-overexpression epistasis, mRNA stability and tumor assays\",\n      \"pmids\": [\"39182664\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Downstream effectors of TPRKB driving HCC phenotypes unidentified\", \"Whether this acts through KEOPS or complex-independent functions unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The biochemical activity by which TPRKB/KEOPS generates telomeric G-overhangs independently of t6A, and the molecular basis of TP53-dependent TPRKB degradation, remain undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No enzyme or activity assigned to G-overhang generation\", \"E3 ligase mediating TP53-dependent TPRKB turnover unknown\", \"Mechanistic basis of TP53-deficient synthetic lethality unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 3, 4]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [3, 12]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [3, 7]}\n    ],\n    \"complexes\": [\"KEOPS/EKC complex\"],\n    \"partners\": [\"TP53RK\", \"OSGEP\", \"LAGE3\", \"TP53\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}