{"gene":"PUS1","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2004,"finding":"A missense mutation in PUS1 affecting a highly conserved amino acid in the predicted catalytic center was identified as the cause of MLASA, implying that PUS1 encodes a pseudouridine synthase whose catalytic activity is essential for normal mitochondrial function.","method":"Linkage analysis, homozygosity mapping, Sanger sequencing of candidate region in affected families","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic linkage plus sequencing across multiple families; direct enzymatic activity not tested in this paper but mutation localizes to catalytic center","pmids":["15108122"],"is_preprint":false},{"year":2005,"finding":"The MLASA-associated PUS1 missense mutation abolishes pseudouridylation of both mitochondrial and cytoplasmic tRNAs at positions normally modified by Pus1p, and eliminates Pus1p enzymatic activity in cell extracts. Immunohistochemical staining demonstrated nuclear, cytoplasmic, and mitochondrial distribution of PUS1 protein.","method":"Pseudouridine assay of tRNAs from patient-derived lymphoblastoid cell lines; in vitro enzyme activity assay of patient cell extracts; immunohistochemical staining","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (tRNA modification assay, cell-extract enzyme assay, immunostaining) in patient-derived cells; directly establishes loss of enzymatic function","pmids":["15772074"],"is_preprint":false},{"year":2006,"finding":"The nuclear isoform of PUS1 contains an N-terminal extension absent in the mature mitochondrial isoform, establishing dual-compartment localization with structurally distinct isoforms. Loss-of-function PUS1 nonsense mutation (E220X) is associated with low mtDNA translation products in fibroblasts and combined respiratory chain complex defects.","method":"Sequencing of PUS1 isoforms; respiratory chain complex assays in muscle and fibroblast homogenates; mtDNA translation product measurement","journal":"Journal of medical genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — isoform structure defined by sequencing plus functional readouts (respiratory chain activity, translation), single lab","pmids":["17056637"],"is_preprint":false},{"year":1998,"finding":"Yeast Pus1p (ortholog of human PUS1) contains one zinc atom per 63-kDa monomer that is essential for its native conformation and tRNA-binding ability; zinc removal by chelation inactivates the enzyme and abolishes tRNA binding with concomitant conformational change, establishing a structural (not catalytic) role for zinc.","method":"Atomic absorption spectroscopy; EDTA/1,10-phenanthroline chelation; analytical ultracentrifugation; CD, infrared, and fluorescence spectroscopy; tRNA binding assays","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal biophysical methods with reconstitution of activity after zinc re-addition; rigorously establishes structural zinc requirement","pmids":["9585540"],"is_preprint":false},{"year":1999,"finding":"Yeast Pus1p (ortholog of human PUS1) is a multisite-specific pseudouridine synthase that catalyzes pseudouridylation at positions 27/28 of multiple tRNAs; kinetic parameters (Km ~420–740 nM, kcat ~0.4–0.5 min⁻¹) were established. Binding of Pus1p to tRNA is not the rate-limiting step. A G26·A44 base pair near the target uridine increases association rate ~100-fold, showing this structural element is a key recognition determinant.","method":"In vitro pseudouridylation assay with recombinant His-tagged Pus1p; kinetic characterization; surface plasmon resonance/real-time binding analysis; tRNA variant analysis","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified enzyme, rigorous kinetics, multiple tRNA variants tested with real-time binding measurements","pmids":["10356324"],"is_preprint":false},{"year":1999,"finding":"Yeast Pus1p (ortholog of human PUS1) forms oligomers/aggregates at low concentration in the absence of tRNA; tRNA binding prevents aggregation. The stoichiometry of the Pus1p/tRNA complex is 1:1, and the tRNA binding pocket contains a hydrophobic region responsible for aggregation.","method":"Analytical ultracentrifugation; light scattering; tRNA binding assays; detergent competition","journal":"Biochimie","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biophysical characterization with purified recombinant protein, multiple methods, single lab","pmids":["10492022"],"is_preprint":false},{"year":2013,"finding":"Crystal structures of the catalytic core domain of human PUS1 (hPus1) at 1.8 Å resolution reveal a fold with a central antiparallel β-sheet flanked by helices, a flexible hinge allowing opening/closing around an electropositive active-site cleft, and a Mes molecule mimicking the target uridine. Two unique C-terminal α-helices form the walls of the RNA binding surface and block tRNA from binding in the same orientation as in the bacterial homologue TruA, consistent with different target selectivities.","method":"X-ray crystallography (two crystal forms, 1.8 Å); molecular docking of tRNA","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structures with two crystal forms, active-site ligand mimic identified, structural basis for substrate selectivity modeled","pmids":["23707380"],"is_preprint":false},{"year":2023,"finding":"Crystal structure of yeast PUS1 bound to an mRNA-derived RNA duplex at 2.4 Å resolution reveals that PUS1 recognizes and binds both strands of a helical RNA duplex, guiding the target uridine-containing strand to the active site; this establishes the structural basis for mRNA pseudouridylation and shows divergence from tRNA recognition modes.","method":"X-ray crystallography at 2.4 Å resolution; structure-guided substrate identification","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution co-crystal structure with RNA substrate, mechanistic interpretation of active-site engagement","pmids":["37939088"],"is_preprint":false},{"year":2024,"finding":"PUS1 promotes prostate cancer bone metastasis through a non-enzymatic mechanism: PUS1 protects EIF3b from ubiquitin-mediated proteasomal degradation, and EIF3b acts as a downstream effector of PUS1-driven metastasis. FOXA1 transcriptionally activates PUS1 by binding its promoter. Knockdown of PUS1 inhibited metastasis independently of its pseudouridine synthase enzymatic activity.","method":"PUS1 knockdown (enzymatic-dead mutant rescue); co-immunoprecipitation/protein stability assays; EIF3b overexpression rescue; FOXA1 chromatin immunoprecipitation/promoter reporter assay","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — enzymatic dead mutant establishes non-enzymatic mechanism, rescue experiments, promoter binding, single lab with multiple orthogonal approaches","pmids":["39247811"],"is_preprint":false},{"year":2025,"finding":"PUS1 modulates pre-mRNA splicing; PUS1 depletion induces elevated intron retention leading to formation of endogenous double-stranded RNA (dsRNA), which activates innate antiviral immune signaling and inhibits global translation. This effect on translation is not directly mediated via pseudouridine modification of mRNA or tRNA. PUS1 isoform 2 protein is selectively upregulated in RCC.","method":"RNA-seq (intron retention analysis); dsRNA immunofluorescence; translation assays; PUS1 knockdown in RCC cells; innate immune response assays; isoform-specific protein analysis","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple cellular assays with mechanistic follow-up (splicing, dsRNA, innate immune activation), single lab","pmids":["42003926"],"is_preprint":false},{"year":2025,"finding":"PUS1 promotes cell migration in clear cell renal cell carcinoma by stabilizing SMOX mRNA via pseudouridylation; the transcription factor USF1 regulates PUS1 expression by binding to its promoter. PUS1 silencing reduces ccRCC cell migration while overexpression enhances it.","method":"PUS1 knockdown/overexpression; SMOX mRNA stability assays; pseudouridylation assay; USF1 chromatin immunoprecipitation/promoter binding","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional assays with mRNA stabilization linked to pseudouridylation and upstream transcriptional regulation, single lab with multiple methods","pmids":["39993614"],"is_preprint":false},{"year":2025,"finding":"PUS1 is a key determinant of RNA trafficking into extracellular vesicles; pseudouridine modification introduced by PUS1 on select RNAs is necessary and sufficient for their extracellular export. Myosin light chain 6 (MYL6) was identified as a pseudouridine-binding protein required for secretion of pseudouridine-modified RNAs.","method":"Genome-wide CRISPR screen; proteomics; high-sensitivity transcriptomics; pseudouridine detection in extracellular RNAs; synthetic pseudouridine-modified RNA export assays; MYL6 pulldown/binding assays","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR screen plus multiple orthogonal validation methods (proteomics, transcriptomics, synthetic RNA assays, protein binding), preprint not yet peer-reviewed","pmids":["bio_10.1101_2025.10.28.685156"],"is_preprint":true}],"current_model":"PUS1 encodes a pseudouridine synthase that localizes to the nucleus, cytoplasm, and mitochondria (via distinct isoforms), pseudouridylates specific positions in cytoplasmic and mitochondrial tRNAs (and mRNAs/ncRNAs), requires a structural zinc atom for proper folding and tRNA binding, and uses an electropositive active-site cleft with unique C-terminal helices to recognize RNA substrates; loss of its catalytic activity causes defective mitochondrial translation and oxidative phosphorylation (MLASA disease), while beyond its canonical tRNA modification role it also stabilizes select mRNAs via pseudouridylation, regulates pre-mRNA splicing to prevent dsRNA-driven innate immune activation, acts non-enzymatically to protect EIF3b from ubiquitin-mediated degradation, and marks RNAs with pseudouridine as a signal for loading into extracellular vesicles via the pseudouridine-reader protein MYL6."},"narrative":{"mechanistic_narrative":"PUS1 encodes a multisite-specific pseudouridine synthase that installs pseudouridine in tRNAs across the cytoplasmic, nuclear, and mitochondrial compartments, with structurally distinct isoforms directing dual-compartment localization [PMID:15772074, PMID:17056637]. The catalytic mechanism is built on an antiparallel β-sheet fold with a flexible hinge that opens and closes around an electropositive active-site cleft, and two unique C-terminal α-helices form the RNA-binding surface that distinguishes its substrate selectivity from bacterial homologues [PMID:23707380]; substrate recognition extends beyond tRNA to mRNA-derived RNA duplexes, where PUS1 engages both strands to guide the target uridine into the active site [PMID:37939088]. Catalytic function depends on a structural zinc atom required for native conformation and tRNA binding, and on substrate features such as a G26·A44 base pair that accelerates tRNA association [PMID:9585540, PMID:10356324]. Loss of PUS1 catalytic activity through a missense mutation in the catalytic center causes MLASA, abolishing pseudouridylation of mitochondrial and cytoplasmic tRNAs and producing defective mitochondrial translation and combined respiratory chain defects [PMID:15108122, PMID:15772074, PMID:17056637]. Beyond canonical tRNA modification, PUS1 stabilizes select mRNAs via pseudouridylation to drive cell migration [PMID:39993614], suppresses intron retention and dsRNA-driven innate immune activation through effects on pre-mRNA splicing [PMID:42003926], acts non-enzymatically to protect EIF3b from proteasomal degradation in metastasis [PMID:39247811], and marks RNAs with pseudouridine for export into extracellular vesicles via the reader protein MYL6 [PMID:bio_10.1101_2025.10.28.685156]. Its expression is transcriptionally controlled by FOXA1 and USF1 in cancer contexts [PMID:39247811, PMID:39993614].","teleology":[{"year":2004,"claim":"Established that PUS1 encodes a pseudouridine synthase whose catalytic activity is essential for mitochondrial function, by linking a catalytic-center mutation to human disease.","evidence":"Linkage analysis, homozygosity mapping, and Sanger sequencing in MLASA families","pmids":["15108122"],"confidence":"Medium","gaps":["Enzymatic activity not directly tested in this study","Mechanistic link between tRNA modification loss and respiratory failure not yet established"]},{"year":2005,"claim":"Demonstrated directly that the disease mutation abolishes enzymatic activity and tRNA pseudouridylation, and that PUS1 protein distributes across nucleus, cytoplasm, and mitochondria.","evidence":"Pseudouridine and cell-extract enzyme assays in patient-derived lymphoblastoid cells plus immunohistochemistry","pmids":["15772074"],"confidence":"High","gaps":["Mechanism connecting loss of tRNA modification to OXPHOS defect not resolved","Targeting signals directing isoforms to compartments not defined"]},{"year":2006,"claim":"Defined the structural basis for dual-compartment targeting, showing the nuclear isoform carries an N-terminal extension absent from the mitochondrial isoform, and tied loss-of-function to reduced mtDNA translation.","evidence":"Isoform sequencing plus respiratory chain assays and mtDNA translation product measurement in patient muscle and fibroblasts","pmids":["17056637"],"confidence":"Medium","gaps":["Single-lab functional readouts","Quantitative contribution of each isoform to compartmental modification not measured"]},{"year":1999,"claim":"Resolved the enzyme's catalytic and recognition logic, defining it as a multisite-specific synthase acting at tRNA positions 27/28 with a defined kinetic regime and a key recognition determinant.","evidence":"In vitro pseudouridylation with recombinant Pus1p, kinetics, real-time binding, and tRNA variant analysis (yeast ortholog)","pmids":["10356324","10492022"],"confidence":"High","gaps":["Yeast ortholog rather than human enzyme","Catalytic step kinetics distinct from binding not fully dissected"]},{"year":1998,"claim":"Established that a bound zinc atom plays a structural, not catalytic, role essential for folding and tRNA binding.","evidence":"Atomic absorption, chelation/reconstitution, ultracentrifugation, and spectroscopy with recombinant yeast Pus1p","pmids":["9585540"],"confidence":"High","gaps":["Yeast ortholog; conservation of zinc role in human PUS1 inferred","Residues coordinating zinc not mapped to human sequence here"]},{"year":2013,"claim":"Provided the high-resolution architecture of the human catalytic core, explaining substrate selectivity via unique C-terminal helices and a hinged electropositive cleft.","evidence":"X-ray crystallography of hPus1 catalytic core at 1.8 Å with active-site ligand mimic and tRNA docking","pmids":["23707380"],"confidence":"High","gaps":["No co-crystal with bound tRNA","Conformational dynamics of the hinge during catalysis inferred from static structures"]},{"year":2023,"claim":"Extended substrate scope structurally to mRNA, showing PUS1 binds both strands of an RNA duplex to position the target uridine, diverging from tRNA recognition.","evidence":"X-ray co-crystal structure of yeast PUS1 with an mRNA-derived RNA duplex at 2.4 Å","pmids":["37939088"],"confidence":"High","gaps":["Yeast enzyme; human mRNA recognition mode inferred","In vivo prevalence of duplex mRNA substrates not quantified"]},{"year":2024,"claim":"Revealed a non-enzymatic function in which PUS1 stabilizes EIF3b to drive metastasis, decoupling a disease-relevant role from catalytic activity.","evidence":"Enzymatic-dead rescue, Co-IP/protein stability assays, EIF3b rescue, and FOXA1 ChIP/promoter reporter in prostate cancer cells","pmids":["39247811"],"confidence":"Medium","gaps":["Single lab","Direct PUS1–EIF3b binding interface not mapped","Generality beyond prostate cancer bone metastasis unknown"]},{"year":2025,"claim":"Identified roles in mRNA stabilization and splicing regulation, showing pseudouridylation stabilizes SMOX mRNA while depletion drives intron retention, dsRNA accumulation, and innate immune activation.","evidence":"Knockdown/overexpression, mRNA stability and pseudouridylation assays, RNA-seq intron retention, dsRNA immunofluorescence, and immune/translation assays in RCC; USF1 ChIP","pmids":["39993614","42003926"],"confidence":"Medium","gaps":["Single-lab studies","Whether splicing effect is enzymatic or scaffolding not fully resolved","Direct targets of pseudouridylation in splicing not enumerated"]},{"year":2025,"claim":"Implicated PUS1-deposited pseudouridine as a sorting signal for RNA export into extracellular vesicles, read by MYL6.","evidence":"Genome-wide CRISPR screen, proteomics, synthetic modified-RNA export assays, and MYL6 binding assays (preprint)","pmids":["bio_10.1101_2025.10.28.685156"],"confidence":"Medium","gaps":["Preprint, not peer-reviewed","MYL6 pseudouridine-reader mechanism not structurally defined","Physiological scope of the export pathway unknown"]},{"year":null,"claim":"How PUS1's many enzymatic and non-enzymatic functions are partitioned across compartments, isoforms, and tissues remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking tRNA, mRNA, splicing, EIF3b, and EV-sorting activities","Isoform-specific function assignment incomplete","Human in vivo substrate atlas not defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[1,4,7,10]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[3,4,6,7]},{"term_id":"GO:0016853","term_label":"isomerase activity","supporting_discovery_ids":[1,4]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,2]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[1,2]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[1,4,9,10]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[0,1,8]}],"complexes":[],"partners":["EIF3B","MYL6","FOXA1","USF1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9Y606","full_name":"Pseudouridylate synthase 1 homolog","aliases":["tRNA pseudouridine synthase 1","tRNA pseudouridine(38-40) synthase","tRNA pseudouridylate synthase I","tRNA-uridine isomerase I"],"length_aa":427,"mass_kda":47.5,"function":"Pseudouridylate synthase that catalyzes pseudouridylation of tRNAs and mRNAs (PubMed:15772074, PubMed:24722331). Acts on positions 27/28 in the anticodon stem and also positions 34 and 36 in the anticodon of an intron containing tRNA (PubMed:24722331). Also catalyzes pseudouridylation of mRNAs: mediates pseudouridylation of mRNAs with the consensus sequence 5'-UGUAG-3' (PubMed:31477916, PubMed:35051350). Acts as a regulator of pre-mRNA splicing by mediating pseudouridylation of pre-mRNAs at locations associated with alternatively spliced regions (PubMed:35051350). Pseudouridylation of pre-mRNAs near splice sites directly regulates mRNA splicing and mRNA 3'-end processing (PubMed:35051350). Involved in regulation of nuclear receptor activity through pseudouridylation of SRA1 mRNA (PubMed:24722331)","subcellular_location":"Nucleus; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q9Y606/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PUS1","classification":"Not Classified","n_dependent_lines":54,"n_total_lines":1208,"dependency_fraction":0.04470198675496689},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/PUS1","total_profiled":1310},"omim":[{"mim_id":"613561","title":"MYOPATHY, LACTIC ACIDOSIS, AND SIDEROBLASTIC ANEMIA 2; MLASA2","url":"https://www.omim.org/entry/613561"},{"mim_id":"608109","title":"PSEUDOURIDINE SYNTHASE 1; PUS1","url":"https://www.omim.org/entry/608109"},{"mim_id":"600462","title":"MYOPATHY, LACTIC ACIDOSIS, AND SIDEROBLASTIC ANEMIA 1; MLASA1","url":"https://www.omim.org/entry/600462"},{"mim_id":"516060","title":"ATP SYNTHASE 6; MTATP6","url":"https://www.omim.org/entry/516060"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Mitochondria","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PUS1"},"hgnc":{"alias_symbol":["MLASA1"],"prev_symbol":[]},"alphafold":{"accession":"Q9Y606","domains":[{"cath_id":"3.30.70.580","chopping":"84-195","consensus_level":"high","plddt":94.296,"start":84,"end":195},{"cath_id":"3.30.70.660","chopping":"199-327","consensus_level":"high","plddt":97.2254,"start":199,"end":327}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y606","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y606-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y606-F1-predicted_aligned_error_v6.png","plddt_mean":80.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PUS1","jax_strain_url":"https://www.jax.org/strain/search?query=PUS1"},"sequence":{"accession":"Q9Y606","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y606.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y606/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y606"}},"corpus_meta":[{"pmid":"15108122","id":"PMC_15108122","title":"Missense mutation in pseudouridine synthase 1 (PUS1) causes mitochondrial myopathy and sideroblastic anemia (MLASA).","date":"2004","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/15108122","citation_count":245,"is_preprint":false},{"pmid":"15772074","id":"PMC_15772074","title":"Mitochondrial myopathy and sideroblastic anemia (MLASA): missense mutation in the pseudouridine synthase 1 (PUS1) gene is associated with the loss of tRNA pseudouridylation.","date":"2005","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15772074","citation_count":120,"is_preprint":false},{"pmid":"17056637","id":"PMC_17056637","title":"Nonsense mutation in pseudouridylate synthase 1 (PUS1) in two brothers affected by myopathy, lactic acidosis and sideroblastic anaemia (MLASA).","date":"2006","source":"Journal of medical 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adulthood.","date":"2014","source":"European journal of human genetics : EJHG","url":"https://pubmed.ncbi.nlm.nih.gov/25227147","citation_count":26,"is_preprint":false},{"pmid":"32287105","id":"PMC_32287105","title":"A Novel PUS1 Mutation in 2 Siblings with MLASA Syndrome: A Review of the Literature.","date":"2021","source":"Journal of pediatric hematology/oncology","url":"https://pubmed.ncbi.nlm.nih.gov/32287105","citation_count":17,"is_preprint":false},{"pmid":"36457503","id":"PMC_36457503","title":"PUS1 is a novel biomarker for predicting poor outcomes and triple-negative status in breast cancer.","date":"2022","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/36457503","citation_count":14,"is_preprint":false},{"pmid":"21686963","id":"PMC_21686963","title":"Nonsense mutation in pseudouridylate synthase 1 (PUS1) in two brothers affected by myopathy, lactic acidosis and sideroblastic anaemia (MLASA).","date":"2009","source":"BMJ case 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number.","date":"2025","source":"Annals of hematology","url":"https://pubmed.ncbi.nlm.nih.gov/39961824","citation_count":1,"is_preprint":false},{"pmid":"42003926","id":"PMC_42003926","title":"PUS1 Drives Renal Cancer Progression by Preventing Formation of Endogenous Double-stranded RNAs.","date":"2026","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/42003926","citation_count":0,"is_preprint":false},{"pmid":"41361485","id":"PMC_41361485","title":"Respiratory complex I deficiency caused by a novel multi-exonic PUS1 deletion.","date":"2025","source":"Journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/41361485","citation_count":0,"is_preprint":false},{"pmid":"18600706","id":"PMC_18600706","title":"Process development with nitrogenase-producing Escherichia coli C-M74 (pUS1) CellS.","date":"1991","source":"Biotechnology and bioengineering","url":"https://pubmed.ncbi.nlm.nih.gov/18600706","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.10.28.685156","title":"Pseudouridine selects RNAs for extracellular transport","date":"2025-10-30","source":"bioRxiv","url":"https://doi.org/10.1101/2025.10.28.685156","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":12956,"output_tokens":3215,"usd":0.043547,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10822,"output_tokens":3536,"usd":0.071255,"stage2_stop_reason":"end_turn"},"total_usd":0.114802,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2004,\n      \"finding\": \"A missense mutation in PUS1 affecting a highly conserved amino acid in the predicted catalytic center was identified as the cause of MLASA, implying that PUS1 encodes a pseudouridine synthase whose catalytic activity is essential for normal mitochondrial function.\",\n      \"method\": \"Linkage analysis, homozygosity mapping, Sanger sequencing of candidate region in affected families\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic linkage plus sequencing across multiple families; direct enzymatic activity not tested in this paper but mutation localizes to catalytic center\",\n      \"pmids\": [\"15108122\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The MLASA-associated PUS1 missense mutation abolishes pseudouridylation of both mitochondrial and cytoplasmic tRNAs at positions normally modified by Pus1p, and eliminates Pus1p enzymatic activity in cell extracts. Immunohistochemical staining demonstrated nuclear, cytoplasmic, and mitochondrial distribution of PUS1 protein.\",\n      \"method\": \"Pseudouridine assay of tRNAs from patient-derived lymphoblastoid cell lines; in vitro enzyme activity assay of patient cell extracts; immunohistochemical staining\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (tRNA modification assay, cell-extract enzyme assay, immunostaining) in patient-derived cells; directly establishes loss of enzymatic function\",\n      \"pmids\": [\"15772074\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The nuclear isoform of PUS1 contains an N-terminal extension absent in the mature mitochondrial isoform, establishing dual-compartment localization with structurally distinct isoforms. Loss-of-function PUS1 nonsense mutation (E220X) is associated with low mtDNA translation products in fibroblasts and combined respiratory chain complex defects.\",\n      \"method\": \"Sequencing of PUS1 isoforms; respiratory chain complex assays in muscle and fibroblast homogenates; mtDNA translation product measurement\",\n      \"journal\": \"Journal of medical genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — isoform structure defined by sequencing plus functional readouts (respiratory chain activity, translation), single lab\",\n      \"pmids\": [\"17056637\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Yeast Pus1p (ortholog of human PUS1) contains one zinc atom per 63-kDa monomer that is essential for its native conformation and tRNA-binding ability; zinc removal by chelation inactivates the enzyme and abolishes tRNA binding with concomitant conformational change, establishing a structural (not catalytic) role for zinc.\",\n      \"method\": \"Atomic absorption spectroscopy; EDTA/1,10-phenanthroline chelation; analytical ultracentrifugation; CD, infrared, and fluorescence spectroscopy; tRNA binding assays\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal biophysical methods with reconstitution of activity after zinc re-addition; rigorously establishes structural zinc requirement\",\n      \"pmids\": [\"9585540\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Yeast Pus1p (ortholog of human PUS1) is a multisite-specific pseudouridine synthase that catalyzes pseudouridylation at positions 27/28 of multiple tRNAs; kinetic parameters (Km ~420–740 nM, kcat ~0.4–0.5 min⁻¹) were established. Binding of Pus1p to tRNA is not the rate-limiting step. A G26·A44 base pair near the target uridine increases association rate ~100-fold, showing this structural element is a key recognition determinant.\",\n      \"method\": \"In vitro pseudouridylation assay with recombinant His-tagged Pus1p; kinetic characterization; surface plasmon resonance/real-time binding analysis; tRNA variant analysis\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified enzyme, rigorous kinetics, multiple tRNA variants tested with real-time binding measurements\",\n      \"pmids\": [\"10356324\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Yeast Pus1p (ortholog of human PUS1) forms oligomers/aggregates at low concentration in the absence of tRNA; tRNA binding prevents aggregation. The stoichiometry of the Pus1p/tRNA complex is 1:1, and the tRNA binding pocket contains a hydrophobic region responsible for aggregation.\",\n      \"method\": \"Analytical ultracentrifugation; light scattering; tRNA binding assays; detergent competition\",\n      \"journal\": \"Biochimie\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biophysical characterization with purified recombinant protein, multiple methods, single lab\",\n      \"pmids\": [\"10492022\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Crystal structures of the catalytic core domain of human PUS1 (hPus1) at 1.8 Å resolution reveal a fold with a central antiparallel β-sheet flanked by helices, a flexible hinge allowing opening/closing around an electropositive active-site cleft, and a Mes molecule mimicking the target uridine. Two unique C-terminal α-helices form the walls of the RNA binding surface and block tRNA from binding in the same orientation as in the bacterial homologue TruA, consistent with different target selectivities.\",\n      \"method\": \"X-ray crystallography (two crystal forms, 1.8 Å); molecular docking of tRNA\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structures with two crystal forms, active-site ligand mimic identified, structural basis for substrate selectivity modeled\",\n      \"pmids\": [\"23707380\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Crystal structure of yeast PUS1 bound to an mRNA-derived RNA duplex at 2.4 Å resolution reveals that PUS1 recognizes and binds both strands of a helical RNA duplex, guiding the target uridine-containing strand to the active site; this establishes the structural basis for mRNA pseudouridylation and shows divergence from tRNA recognition modes.\",\n      \"method\": \"X-ray crystallography at 2.4 Å resolution; structure-guided substrate identification\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution co-crystal structure with RNA substrate, mechanistic interpretation of active-site engagement\",\n      \"pmids\": [\"37939088\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PUS1 promotes prostate cancer bone metastasis through a non-enzymatic mechanism: PUS1 protects EIF3b from ubiquitin-mediated proteasomal degradation, and EIF3b acts as a downstream effector of PUS1-driven metastasis. FOXA1 transcriptionally activates PUS1 by binding its promoter. Knockdown of PUS1 inhibited metastasis independently of its pseudouridine synthase enzymatic activity.\",\n      \"method\": \"PUS1 knockdown (enzymatic-dead mutant rescue); co-immunoprecipitation/protein stability assays; EIF3b overexpression rescue; FOXA1 chromatin immunoprecipitation/promoter reporter assay\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — enzymatic dead mutant establishes non-enzymatic mechanism, rescue experiments, promoter binding, single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"39247811\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PUS1 modulates pre-mRNA splicing; PUS1 depletion induces elevated intron retention leading to formation of endogenous double-stranded RNA (dsRNA), which activates innate antiviral immune signaling and inhibits global translation. This effect on translation is not directly mediated via pseudouridine modification of mRNA or tRNA. PUS1 isoform 2 protein is selectively upregulated in RCC.\",\n      \"method\": \"RNA-seq (intron retention analysis); dsRNA immunofluorescence; translation assays; PUS1 knockdown in RCC cells; innate immune response assays; isoform-specific protein analysis\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple cellular assays with mechanistic follow-up (splicing, dsRNA, innate immune activation), single lab\",\n      \"pmids\": [\"42003926\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PUS1 promotes cell migration in clear cell renal cell carcinoma by stabilizing SMOX mRNA via pseudouridylation; the transcription factor USF1 regulates PUS1 expression by binding to its promoter. PUS1 silencing reduces ccRCC cell migration while overexpression enhances it.\",\n      \"method\": \"PUS1 knockdown/overexpression; SMOX mRNA stability assays; pseudouridylation assay; USF1 chromatin immunoprecipitation/promoter binding\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional assays with mRNA stabilization linked to pseudouridylation and upstream transcriptional regulation, single lab with multiple methods\",\n      \"pmids\": [\"39993614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PUS1 is a key determinant of RNA trafficking into extracellular vesicles; pseudouridine modification introduced by PUS1 on select RNAs is necessary and sufficient for their extracellular export. Myosin light chain 6 (MYL6) was identified as a pseudouridine-binding protein required for secretion of pseudouridine-modified RNAs.\",\n      \"method\": \"Genome-wide CRISPR screen; proteomics; high-sensitivity transcriptomics; pseudouridine detection in extracellular RNAs; synthetic pseudouridine-modified RNA export assays; MYL6 pulldown/binding assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR screen plus multiple orthogonal validation methods (proteomics, transcriptomics, synthetic RNA assays, protein binding), preprint not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.10.28.685156\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"PUS1 encodes a pseudouridine synthase that localizes to the nucleus, cytoplasm, and mitochondria (via distinct isoforms), pseudouridylates specific positions in cytoplasmic and mitochondrial tRNAs (and mRNAs/ncRNAs), requires a structural zinc atom for proper folding and tRNA binding, and uses an electropositive active-site cleft with unique C-terminal helices to recognize RNA substrates; loss of its catalytic activity causes defective mitochondrial translation and oxidative phosphorylation (MLASA disease), while beyond its canonical tRNA modification role it also stabilizes select mRNAs via pseudouridylation, regulates pre-mRNA splicing to prevent dsRNA-driven innate immune activation, acts non-enzymatically to protect EIF3b from ubiquitin-mediated degradation, and marks RNAs with pseudouridine as a signal for loading into extracellular vesicles via the pseudouridine-reader protein MYL6.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PUS1 encodes a multisite-specific pseudouridine synthase that installs pseudouridine in tRNAs across the cytoplasmic, nuclear, and mitochondrial compartments, with structurally distinct isoforms directing dual-compartment localization [#1, #2]. The catalytic mechanism is built on an antiparallel \\u03b2-sheet fold with a flexible hinge that opens and closes around an electropositive active-site cleft, and two unique C-terminal \\u03b1-helices form the RNA-binding surface that distinguishes its substrate selectivity from bacterial homologues [#6]; substrate recognition extends beyond tRNA to mRNA-derived RNA duplexes, where PUS1 engages both strands to guide the target uridine into the active site [#7]. Catalytic function depends on a structural zinc atom required for native conformation and tRNA binding, and on substrate features such as a G26\\u00b7A44 base pair that accelerates tRNA association [#3, #4]. Loss of PUS1 catalytic activity through a missense mutation in the catalytic center causes MLASA, abolishing pseudouridylation of mitochondrial and cytoplasmic tRNAs and producing defective mitochondrial translation and combined respiratory chain defects [#0, #1, #2]. Beyond canonical tRNA modification, PUS1 stabilizes select mRNAs via pseudouridylation to drive cell migration [#10], suppresses intron retention and dsRNA-driven innate immune activation through effects on pre-mRNA splicing [#9], acts non-enzymatically to protect EIF3b from proteasomal degradation in metastasis [#8], and marks RNAs with pseudouridine for export into extracellular vesicles via the reader protein MYL6 [#11]. Its expression is transcriptionally controlled by FOXA1 and USF1 in cancer contexts [#8, #10].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Established that PUS1 encodes a pseudouridine synthase whose catalytic activity is essential for mitochondrial function, by linking a catalytic-center mutation to human disease.\",\n      \"evidence\": \"Linkage analysis, homozygosity mapping, and Sanger sequencing in MLASA families\",\n      \"pmids\": [\"15108122\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Enzymatic activity not directly tested in this study\", \"Mechanistic link between tRNA modification loss and respiratory failure not yet established\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Demonstrated directly that the disease mutation abolishes enzymatic activity and tRNA pseudouridylation, and that PUS1 protein distributes across nucleus, cytoplasm, and mitochondria.\",\n      \"evidence\": \"Pseudouridine and cell-extract enzyme assays in patient-derived lymphoblastoid cells plus immunohistochemistry\",\n      \"pmids\": [\"15772074\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism connecting loss of tRNA modification to OXPHOS defect not resolved\", \"Targeting signals directing isoforms to compartments not defined\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defined the structural basis for dual-compartment targeting, showing the nuclear isoform carries an N-terminal extension absent from the mitochondrial isoform, and tied loss-of-function to reduced mtDNA translation.\",\n      \"evidence\": \"Isoform sequencing plus respiratory chain assays and mtDNA translation product measurement in patient muscle and fibroblasts\",\n      \"pmids\": [\"17056637\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab functional readouts\", \"Quantitative contribution of each isoform to compartmental modification not measured\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Resolved the enzyme's catalytic and recognition logic, defining it as a multisite-specific synthase acting at tRNA positions 27/28 with a defined kinetic regime and a key recognition determinant.\",\n      \"evidence\": \"In vitro pseudouridylation with recombinant Pus1p, kinetics, real-time binding, and tRNA variant analysis (yeast ortholog)\",\n      \"pmids\": [\"10356324\", \"10492022\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Yeast ortholog rather than human enzyme\", \"Catalytic step kinetics distinct from binding not fully dissected\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Established that a bound zinc atom plays a structural, not catalytic, role essential for folding and tRNA binding.\",\n      \"evidence\": \"Atomic absorption, chelation/reconstitution, ultracentrifugation, and spectroscopy with recombinant yeast Pus1p\",\n      \"pmids\": [\"9585540\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Yeast ortholog; conservation of zinc role in human PUS1 inferred\", \"Residues coordinating zinc not mapped to human sequence here\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Provided the high-resolution architecture of the human catalytic core, explaining substrate selectivity via unique C-terminal helices and a hinged electropositive cleft.\",\n      \"evidence\": \"X-ray crystallography of hPus1 catalytic core at 1.8 \\u00c5 with active-site ligand mimic and tRNA docking\",\n      \"pmids\": [\"23707380\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No co-crystal with bound tRNA\", \"Conformational dynamics of the hinge during catalysis inferred from static structures\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Extended substrate scope structurally to mRNA, showing PUS1 binds both strands of an RNA duplex to position the target uridine, diverging from tRNA recognition.\",\n      \"evidence\": \"X-ray co-crystal structure of yeast PUS1 with an mRNA-derived RNA duplex at 2.4 \\u00c5\",\n      \"pmids\": [\"37939088\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Yeast enzyme; human mRNA recognition mode inferred\", \"In vivo prevalence of duplex mRNA substrates not quantified\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealed a non-enzymatic function in which PUS1 stabilizes EIF3b to drive metastasis, decoupling a disease-relevant role from catalytic activity.\",\n      \"evidence\": \"Enzymatic-dead rescue, Co-IP/protein stability assays, EIF3b rescue, and FOXA1 ChIP/promoter reporter in prostate cancer cells\",\n      \"pmids\": [\"39247811\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Direct PUS1\\u2013EIF3b binding interface not mapped\", \"Generality beyond prostate cancer bone metastasis unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified roles in mRNA stabilization and splicing regulation, showing pseudouridylation stabilizes SMOX mRNA while depletion drives intron retention, dsRNA accumulation, and innate immune activation.\",\n      \"evidence\": \"Knockdown/overexpression, mRNA stability and pseudouridylation assays, RNA-seq intron retention, dsRNA immunofluorescence, and immune/translation assays in RCC; USF1 ChIP\",\n      \"pmids\": [\"39993614\", \"42003926\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab studies\", \"Whether splicing effect is enzymatic or scaffolding not fully resolved\", \"Direct targets of pseudouridylation in splicing not enumerated\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Implicated PUS1-deposited pseudouridine as a sorting signal for RNA export into extracellular vesicles, read by MYL6.\",\n      \"evidence\": \"Genome-wide CRISPR screen, proteomics, synthetic modified-RNA export assays, and MYL6 binding assays (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.10.28.685156\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not peer-reviewed\", \"MYL6 pseudouridine-reader mechanism not structurally defined\", \"Physiological scope of the export pathway unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How PUS1's many enzymatic and non-enzymatic functions are partitioned across compartments, isoforms, and tissues remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model linking tRNA, mRNA, splicing, EIF3b, and EV-sorting activities\", \"Isoform-specific function assignment incomplete\", \"Human in vivo substrate atlas not defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [1, 4, 7, 10]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [3, 4, 6, 7]},\n      {\"term_id\": \"GO:0016853\", \"supporting_discovery_ids\": [1, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [1, 2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [1, 4, 9, 10]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [0, 1, 8]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"EIF3b\", \"MYL6\", \"FOXA1\", \"USF1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}