{"gene":"TRUB1","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":1997,"finding":"Yeast Pus4 (encoded by YNL292w/PUS4) is the pseudouridine synthase responsible for forming Ψ55 in both cytoplasmic and mitochondrial tRNAs. Recombinant Pus4 purified from E. coli catalyzes Ψ55 formation on T7 in vitro transcripts of yeast tRNA genes with strict site-specificity (no other uridines modified), and deletion of YNL292w abolishes all Ψ55 formation activity in cell-free extracts.","method":"Recombinant protein purification, in vitro pseudouridylation assay on tRNA transcripts, gene disruption, chemical mapping of pseudouridine residues in cytoplasmic and mitochondrial tRNAs","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified recombinant protein plus genetic deletion with chemical mapping, multiple orthogonal methods in a single rigorous study","pmids":["9358157"],"is_preprint":false},{"year":1998,"finding":"Yeast Pus4 catalyzes Ψ55 formation in the tRNA-like 3′-domain of mutant TYMV RNA (at position 37 in TYMV-mutant G37U, equivalent to Ψ55 in tRNA), demonstrating substrate flexibility of Pus4 beyond canonical tRNA.","method":"In vitro pseudouridylation assay using purified recombinant yeast Pus4 on in vitro-transcribed TYMV RNA variants","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro reconstitution with purified enzyme but single lab, single study","pmids":["9705510"],"is_preprint":false},{"year":1999,"finding":"Yeast PUS4 deletion does not affect pseudouridine formation in spliceosomal UsnRNAs (U1, U2, U4, U5, U6), establishing that Pus4 activity is not required for UsnRNA pseudouridylation and that its substrate specificity is restricted to tRNAs in this context.","method":"Chemical mapping of pseudouridine in UsnRNAs from pus4Δ yeast mutants","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic deletion with chemical mapping, replicated in context of broader PUS enzyme survey","pmids":["10022901"],"is_preprint":false},{"year":2000,"finding":"Overexpression of PUS4 in yeast causes accumulation of tRNA precursors and derepression of GCN4 translation (Gcd− phenotype) independently of eIF2α phosphorylation, by impeding tRNA 5′-end processing or nuclear export. Importantly, this Gcd− phenotype does not require PUS4 enzymatic activity, indicating a non-catalytic function of the protein in tRNA biogenesis.","method":"High-copy PUS4 overexpression, GCN4-lacZ reporter assay, genetic epistasis with RNase P (RPR1), LOS1, suppression analysis, enzymatic activity mutants","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal genetic epistasis experiments with activity-dead mutant establishing non-catalytic role, replicated across several genetic backgrounds","pmids":["10713174"],"is_preprint":false},{"year":2003,"finding":"Human TRUB1 was identified as the first human ortholog of bacterial TruB/Ψ55 synthase, encoding a 349-amino acid protein with a conserved TruB domain (W104–I255) containing catalytic motif II with the conserved aspartate residue involved in uridine recognition and catalytic function. Northern blot showed wide tissue expression.","method":"Gene cloning, sequence/phylogenetic analysis, domain identification, Northern blot","journal":"International journal of molecular medicine","confidence":"Low","confidence_rationale":"Tier 4 / Weak — computational/sequence-based identification with no direct enzymatic assay for human TRUB1 protein; domain inference only","pmids":["12736709"],"is_preprint":false},{"year":2006,"finding":"Yeast Pus4 is functionally redundant with La protein for tRNA structural stability: depletion of Pus4p in strains carrying a mutant tRNA(Arg)(CCG) decreases tRNA stability (while La deletion is lethal in this background), placing Pus4 in a pathway ensuring tRNA structural integrity and biogenesis.","method":"Genetic epistasis (double-mutant analysis), La deletion/Pus4 depletion in mutant tRNA strains, stability assays","journal":"RNA (New York, N.Y.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic epistasis with defined tRNA stability phenotype, single lab","pmids":["16581807"],"is_preprint":false},{"year":2007,"finding":"Yeast Pus4 (screened as an RNA-binding protein on proteome arrays) binds a CAM-containing RNA hairpin and inhibits BMV RNA encapsidation in plants and virion reassembly in vitro, demonstrating an RNA-binding antiviral activity beyond its pseudouridine synthase role.","method":"Proteome array binding screen, in planta BMV accumulation assay, in vitro virion reassembly assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteome array plus in vitro functional assay and in planta validation, single lab","pmids":["17360619"],"is_preprint":false},{"year":2014,"finding":"Yeast Pus4 modifies TEF1 mRNA at Ψ-239 in vivo, and this mRNA pseudouridylation is conserved in S. mikitae and S. pombe, establishing Pus4 as a mRNA pseudouridine synthase in addition to its tRNA role. Pus4 activity was shown to be necessary and sufficient for TEF1 Ψ-239 by genetic deletion and in vitro reconstitution.","method":"PSI-seq (transcriptome-wide pseudouridine mapping), genetic deletion of PUS4, in vitro reconstitution with purified Pus4","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution showing Pus4 is sufficient, plus genetic deletion showing necessity, plus evolutionary conservation across species","pmids":["25353621"],"is_preprint":false},{"year":2017,"finding":"Human TRUB1 is the predominant pseudouridine synthase acting on mammalian mRNA, targeting a specific sequence/structural context (computationally modeled with AUC=0.974 for substrate prediction). Genetic perturbation of TRUB1 combined with Ψ-seq and massively parallel reporter assays defined the sequence and structural determinants governing TRUB1 specificity.","method":"Ψ-seq on >2.5 billion reads, TRUB1 genetic knockdown/knockout, massively parallel reporter assays with thousands of synthetic sequence variants, computational modeling of specificity","journal":"Genome research","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (transcriptome-wide mapping, genetic perturbation, reporter assays), large-scale data, independently consistent findings","pmids":["28073919"],"is_preprint":false},{"year":2020,"finding":"Human TRUB1 (nuclear) produces Ψ55 in most elongator tRNAs in the nucleus, whereas cytoplasmic PUS10 produces Ψ55 (and Ψ54) in tRNAs that contain Ψ54Ψ55. The nuclear isoform of PUS10 (catalytically inactive) specifically binds unmodified U54U55 tRNAs and inhibits TRUB1-mediated U55→Ψ55 conversion, establishing compartmentalized and non-redundant Ψ55 synthase activities.","method":"Nearest-neighbor analysis, recombinant protein assays, subcellular fractionation, specific Ψ55 synthase knockdown cells, binding competition assays","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro enzymatic assays with recombinant proteins, subcellular fractionation, knockdown with biochemical readout, multiple orthogonal approaches in single study","pmids":["33023933"],"is_preprint":false},{"year":2020,"finding":"TruB1 directly binds the stem-loop structure of pri-let-7 miRNA (HITS-CLIP and biochemical assays showing binding to endogenous pri-let-7), selectively enhances interaction between pri-let-7 and the microprocessor DGCR8, and promotes let-7 maturation without pseudouridylating the miRNA. TruB1 suppresses cell proliferation partly via this let-7 pathway.","method":"Cell-based luciferase reporter screen, HITS-CLIP, biochemical binding assays (pulldown), let-7 maturation assays, cell proliferation assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal biochemical binding (HITS-CLIP + pulldown), functional maturation assay, cellular proliferation phenotype, multiple orthogonal methods","pmids":["32926445"],"is_preprint":false},{"year":2021,"finding":"The pseudouridine synthase Pus4/TruB can act as a prion in yeast ([BIG+] state), epigenetically increasing cell proliferation and size while shortening lifespan, with altered protein synthesis and differential synthesis of dozens of proteins including proliferation and aging regulators.","method":"Prion formation assays, cell growth/size measurements, lifespan assays, proteomic analysis of [BIG+] cells, protein synthesis measurements","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple cellular phenotype readouts with defined mechanistic outcome (prion-based epigenetic state), single lab","pmids":["34545808"],"is_preprint":false},{"year":2022,"finding":"Human TRUB1 is responsible for Ψ55 formation in mitochondrial tRNAAsn, tRNAGln, tRNAGlu, and tRNAPro but not the other 18 mitochondrial tRNAs. TRUB1 knockout (CRISPR/Cas9) abolishes Ψ55 in these four tRNAs; recombinant TRUB1 efficiently catalyzes Ψ55 in tRNAAsn and tRNAGln in vitro. TRUB1 deficiency affects tRNA base-pairing (18A/G–Ψ55), conformation, and stability, and impairs mitochondrial translation and oxidative phosphorylation system biogenesis.","method":"CRISPR/Cas9 TRUB1 knockout, CMC/reverse transcription Ψ-mapping assay, in vitro pseudouridylation assay with recombinant TRUB1, cDNA rescue experiments, mitochondrial translation assay","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with recombinant protein, CRISPR knockout with Ψ mapping, cDNA rescue, multiple orthogonal methods demonstrating substrate specificity and downstream consequences","pmids":["36018806"],"is_preprint":false},{"year":2022,"finding":"BID-seq transcriptome-wide mapping in human cancer cells revealed that TRUB1-installed Ψ sites have a transcript stabilization role; depletion of TRUB1 reduced stability of transcripts bearing TRUB1-dependent Ψ modifications.","method":"BID-seq (bisulfite-induced deletion sequencing), TRUB1 knockdown/knockout, transcript stability assays","journal":"Nature biotechnology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — transcriptome-wide mapping with genetic perturbation and functional stability readout, single lab","pmids":["36302989"],"is_preprint":false},{"year":2024,"finding":"Human TruB1 binds specifically to the terminal loop of pri-let-7a1 at nucleotides 31–41 (a small stem-loop architecture). A conserved KRKK motif in human and higher eukaryotes provides an additional electrostatic binding interface beyond what is seen in E. coli TruB–tRNA interaction. The structural basis was determined by biochemical assays and structural investigation.","method":"Biochemical binding assays, structural investigation (crystal/structural study), mutagenesis of the KRKK motif","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — structural data plus biochemical validation but single lab, single study; abstract does not specify crystal vs NMR resolution level","pmids":["38776834"],"is_preprint":false},{"year":2025,"finding":"TRUB1 and PUS10 function redundantly to catalyze the conserved Ψ55 modification in cytosolic tRNAs in human HCT116 cells; individual knockouts of TRUB1 or PUS10 each reduce but do not eliminate cytosolic tRNA Ψ55. Additionally, TRUB1 introduces Ψ modifications at distinct stages of pre-tRNA processing.","method":"Systematic PUS knockout/knockdown in HCT116 cells, BACS (2-bromoacrylamide-assisted cyclization sequencing) for Ψ mapping, comprehensive tRNA Ψ profiling","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — systematic genetic perturbations with quantitative Ψ mapping across nine PUS enzymes, defining redundancy with multiple orthogonal validations","pmids":["41136621"],"is_preprint":false},{"year":2025,"finding":"Pus4/TruB (yeast ortholog of TRUB1) both catalyzes Ψ55 formation on tRNA and remodels tRNA conformational dynamics. Wild-type Pus4 binding to unmodified tRNA populates additional conformational states that gradually approach the ensemble adopted more rapidly by pre-pseudouridylated tRNA. A catalytically incompetent Pus4 mutant binds more slowly and remodels tRNA into distinct conformational ensembles, demonstrating that catalytic and remodeling activities are separable.","method":"Single-molecule FRET, optical binding assays, catalytically inactive Pus4 mutant analysis","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — single-molecule FRET with active-site mutagenesis separating catalytic from remodeling activities, peer-reviewed publication","pmids":["41916762"],"is_preprint":false},{"year":2025,"finding":"TRUB1 knockdown in CRC cells activates the TNFα/NFκB pathway, leading to increased expression of apoptosis-related proteins and decreased Ψ modification. BIRC3 was identified as a downstream target gene regulated by TRUB1 in the NFκB pathway.","method":"TRUB1 knockdown in HCT116 cells, RNA sequencing, Western blot, immunofluorescence, in vivo tumor growth assay in nude mice","journal":"Gastroenterology report","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, pathway inferred from transcriptomics with limited direct mechanistic validation of TRUB1→BIRC3→NFκB connection","pmids":["40260225"],"is_preprint":false},{"year":2026,"finding":"TRUB1-installed Ψ at single conserved sites on mRNAs is causally associated with increased protein production, as shown by TRUB1 knockout experiments demonstrating motif-specific reduction in protein abundance. In contrast, transcripts with clustered/high-density Ψ modifications show reduced protein abundance despite elevated translation efficiency, establishing a density-dependent effect of pseudouridylation on translation output.","method":"TRUB1 knockout, nanopore direct RNA sequencing (Mod-p ID), proteomics, ribosome profiling, controlled in vitro translation assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Strong — TRUB1 KO with multimodal readouts (proteomics + ribosome profiling + in vitro translation), direct causal evidence from genetic perturbation plus functional assay","pmids":["42011786"],"is_preprint":false},{"year":2025,"finding":"Nanopore direct RNA sequencing of yeast mitochondria lacking PUS4 demonstrated that Pus4 pseudouridylates 23 of 24 mitochondrially-encoded tRNAs at Ψ55 in the T-loop in vivo.","method":"Nanopore direct RNA sequencing of mitochondrially-enriched RNA, PUS4 gene knockout comparison","journal":"bioRxiv : the preprint server for biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — nanopore sequencing with genetic knockout providing comprehensive in vivo map, but preprint and single lab","pmids":["40654949"],"is_preprint":true}],"current_model":"TRUB1 (human ortholog of yeast Pus4/bacterial TruB) is a pseudouridine synthase that installs Ψ55 in most cytosolic elongator tRNAs (in the nucleus, where it is partially restricted by catalytically inactive nuclear PUS10), in four mitochondrial tRNAs (tRNAAsn, tRNAGln, tRNAGlu, tRNAPro), and at specific mRNA sites with a predictable sequence/structural code—TRUB1-deposited mRNA Ψ enhances transcript stability and protein output at single sites but impairs translation when pseudouridine density is high; beyond pseudouridylation, TRUB1 directly binds the terminal loop of pri-let-7 via a conserved KRKK motif and recruits the DGCR8 microprocessor to promote let-7 miRNA maturation, and the enzyme additionally remodels tRNA conformational dynamics in a manner separable from its catalytic activity."},"narrative":{"mechanistic_narrative":"TRUB1 is the human ortholog of bacterial TruB/yeast Pus4, a stand-alone pseudouridine synthase that installs the conserved Ψ55 modification in the T-loop of tRNAs and extends this activity to mRNA and microRNA biogenesis [PMID:12736709, PMID:28073919]. In the nucleus it generates Ψ55 in most cytosolic elongator tRNAs, an activity that is partially restrained by a catalytically inactive nuclear PUS10 isoform that binds unmodified U54U55 tRNAs and blocks U55→Ψ55 conversion, while TRUB1 and PUS10 together act redundantly to ensure cytosolic tRNA Ψ55 [PMID:33023933, PMID:41136621]. TRUB1 also pseudouridylates four mitochondrial tRNAs (tRNAAsn, tRNAGln, tRNAGlu, tRNAPro), and its loss disrupts tRNA base-pairing, conformation, and stability, impairing mitochondrial translation and oxidative phosphorylation biogenesis [PMID:36018806]. Beyond tRNA, TRUB1 is the predominant mRNA pseudouridine synthase in mammalian cells, recognizing a defined sequence/structural context [PMID:28073919]; single-site Ψ deposition stabilizes transcripts and enhances protein output, whereas clustered high-density Ψ reduces protein abundance, defining a density-dependent effect on translation [PMID:36302989, PMID:42011786]. Independent of catalysis, TRUB1 directly binds the terminal loop of pri-let-7 through a conserved KRKK electrostatic interface and recruits the DGCR8 microprocessor to promote let-7 maturation and suppress cell proliferation [PMID:32926445, PMID:38776834]. Conformational studies of the yeast ortholog further establish that TRUB1/Pus4 remodels tRNA structural dynamics in a manner separable from its enzymatic activity [PMID:41916762].","teleology":[{"year":1997,"claim":"Established the enzymatic identity of the Ψ55 synthase, answering which protein forms the universally conserved T-loop pseudouridine in tRNA.","evidence":"Recombinant yeast Pus4 in vitro pseudouridylation of tRNA transcripts plus PUS4 gene disruption with chemical Ψ mapping","pmids":["9358157"],"confidence":"High","gaps":["Did not address human ortholog activity","Substrate scope beyond tRNA untested"]},{"year":1998,"claim":"Showed the enzyme's substrate recognition is structure-based rather than strictly tRNA-sequence-dependent, by modifying a tRNA-like viral RNA domain.","evidence":"In vitro pseudouridylation of TYMV RNA variants with purified recombinant Pus4","pmids":["9705510"],"confidence":"Medium","gaps":["Single lab, single study","In vivo relevance of viral RNA modification unknown"]},{"year":1999,"claim":"Delimited substrate specificity by showing the enzyme is not required for spliceosomal UsnRNA pseudouridylation.","evidence":"Chemical Ψ mapping in UsnRNAs from pus4Δ yeast","pmids":["10022901"],"confidence":"Medium","gaps":["Negative result does not exclude other non-tRNA substrates"]},{"year":2000,"claim":"Revealed a catalysis-independent role in tRNA biogenesis, separating the protein's structural/scaffolding function from its enzymatic activity.","evidence":"High-copy PUS4 overexpression with GCN4-lacZ reporter, genetic epistasis, and activity-dead mutants in yeast","pmids":["10713174"],"confidence":"High","gaps":["Molecular basis of tRNA processing/export interference not defined","Not tested for human TRUB1"]},{"year":2003,"claim":"Identified the human ortholog TRUB1 and its conserved catalytic TruB domain, opening study of the mammalian enzyme.","evidence":"Gene cloning, phylogenetic/domain analysis, and Northern blot tissue survey","pmids":["12736709"],"confidence":"Low","gaps":["No direct enzymatic assay for human protein","Catalytic activity inferred from domain only"]},{"year":2006,"claim":"Placed the enzyme in a tRNA structural-stability pathway functionally overlapping with La protein.","evidence":"Genetic epistasis and tRNA stability assays in mutant tRNA(Arg) yeast strains","pmids":["16581807"],"confidence":"Medium","gaps":["Mechanism of stabilization not resolved","Catalytic vs structural contribution unseparated here"]},{"year":2007,"claim":"Demonstrated a sequence-specific RNA-binding/antiviral activity distinct from pseudouridylation.","evidence":"Proteome array binding screen, in planta BMV accumulation, and in vitro virion reassembly assays","pmids":["17360619"],"confidence":"Medium","gaps":["Physiological relevance to host cells unclear","Single lab"]},{"year":2014,"claim":"Established the enzyme as a bona fide mRNA pseudouridine synthase, extending its substrate repertoire beyond structured tRNA.","evidence":"PSI-seq, PUS4 deletion, and in vitro reconstitution showing necessity and sufficiency for TEF1 Ψ-239, conserved across species","pmids":["25353621"],"confidence":"High","gaps":["Functional consequence of mRNA Ψ not addressed","Human ortholog mRNA activity not tested here"]},{"year":2017,"claim":"Identified TRUB1 as the dominant mammalian mRNA Ψ synthase and defined the sequence/structural code governing its specificity.","evidence":"Ψ-seq, TRUB1 knockdown/knockout, massively parallel reporter assays, and computational specificity modeling","pmids":["28073919"],"confidence":"High","gaps":["Downstream functional impact of mRNA Ψ not yet defined","Mitochondrial substrates not addressed"]},{"year":2020,"claim":"Resolved compartmentalized division of labor for tRNA Ψ55, showing nuclear TRUB1 is regulated by an inactive nuclear PUS10 isoform.","evidence":"Recombinant assays, subcellular fractionation, specific synthase knockdown, and binding competition assays","pmids":["33023933"],"confidence":"High","gaps":["Structural basis of PUS10 inhibition of TRUB1 unresolved","Regulation of compartment switching unknown"]},{"year":2020,"claim":"Uncovered a non-catalytic role in miRNA biogenesis: TRUB1 binds pri-let-7 and recruits DGCR8 to promote let-7 maturation and suppress proliferation.","evidence":"Luciferase reporter screen, HITS-CLIP, pulldown binding, let-7 maturation and proliferation assays","pmids":["32926445"],"confidence":"High","gaps":["Whether other miRNAs are regulated unknown","Mechanism of DGCR8 recruitment not structurally defined here"]},{"year":2021,"claim":"Showed the yeast ortholog can adopt a heritable prion state altering proteostasis, growth, and lifespan.","evidence":"Prion formation, growth/size, lifespan, proteomic, and protein synthesis assays in [BIG+] yeast","pmids":["34545808"],"confidence":"Medium","gaps":["No evidence human TRUB1 forms a prion","Link to canonical synthase activity unclear"]},{"year":2022,"claim":"Defined TRUB1 as the Ψ55 synthase for four specific mitochondrial tRNAs and linked its loss to defective mitochondrial translation.","evidence":"CRISPR knockout, CMC Ψ mapping, in vitro pseudouridylation, cDNA rescue, and mitochondrial translation assays","pmids":["36018806"],"confidence":"High","gaps":["Why only four of 22 mt-tRNAs are substrates unresolved","OXPHOS phenotype mechanism not fully traced"]},{"year":2022,"claim":"Assigned a transcript-stabilizing function to TRUB1-installed mRNA Ψ in human cancer cells.","evidence":"BID-seq transcriptome-wide Ψ mapping with TRUB1 depletion and transcript stability assays","pmids":["36302989"],"confidence":"Medium","gaps":["Stability mechanism (RNA-binding readers) not identified","Single lab"]},{"year":2024,"claim":"Provided the structural/molecular basis for pri-let-7 recognition, identifying a higher-eukaryote KRKK electrostatic interface.","evidence":"Biochemical binding, structural investigation, and KRKK motif mutagenesis on pri-let-7a1","pmids":["38776834"],"confidence":"Medium","gaps":["Resolution/method of structure not specified","Single study"]},{"year":2025,"claim":"Established TRUB1/PUS10 redundancy for cytosolic tRNA Ψ55 and timing of Ψ deposition during pre-tRNA processing.","evidence":"Systematic PUS knockout/knockdown in HCT116 cells with BACS Ψ mapping","pmids":["41136621"],"confidence":"High","gaps":["Functional consequence of redundancy buffering unclear","Regulation of processing-stage choice unknown"]},{"year":2025,"claim":"Separated catalytic from conformational-remodeling activities, showing the enzyme reshapes tRNA dynamics independent of Ψ formation.","evidence":"Single-molecule FRET and optical binding assays with catalytically inactive Pus4 mutant","pmids":["41916762"],"confidence":"High","gaps":["Functional output of remodeling in vivo unknown","Demonstrated in yeast ortholog"]},{"year":2025,"claim":"Linked TRUB1 loss to TNFα/NF-κB activation and BIRC3 regulation in colorectal cancer cells.","evidence":"TRUB1 knockdown in HCT116, RNA-seq, Western blot, and nude mouse tumor assays","pmids":["40260225"],"confidence":"Low","gaps":["TRUB1→BIRC3→NF-κB connection inferred from transcriptomics without direct mechanistic validation","Single lab"]},{"year":2026,"claim":"Established a density-dependent rule for how TRUB1-installed mRNA Ψ controls translation output.","evidence":"TRUB1 knockout with nanopore direct RNA-seq, proteomics, ribosome profiling, and in vitro translation","pmids":["42011786"],"confidence":"High","gaps":["Reader proteins distinguishing single vs clustered Ψ not identified","Mechanism of translation impairment at high density unresolved"]},{"year":null,"claim":"How TRUB1 integrates its multiple activities — catalytic Ψ deposition, non-catalytic tRNA remodeling, and microprocessor recruitment — into coordinated cellular regulation, and what reader machinery interprets its mRNA marks, remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No Ψ reader proteins identified for TRUB1 marks","Coordination between catalytic and scaffolding roles unknown","Structural model of human TRUB1 on substrate tRNA/mRNA lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140098","term_label":"catalytic activity, acting on RNA","supporting_discovery_ids":[0,7,8,9,12,15]},{"term_id":"GO:0016853","term_label":"isomerase activity","supporting_discovery_ids":[0,12]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[6,10,14]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[9]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[12]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[9,15]}],"pathway":[{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[8,9,12,13,15,18]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[10]}],"complexes":[],"partners":["DGCR8","PUS10"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8WWH5","full_name":"Pseudouridylate synthase TRUB1","aliases":["TruB pseudouridine synthase homolog 1","tRNA pseudouridine 55 synthase TRUB1","Psi55 synthase TRUB1"],"length_aa":349,"mass_kda":37.3,"function":"Pseudouridine synthase that catalyzes pseudouridylation of mRNAs and tRNAs (PubMed:28073919, PubMed:31477916, PubMed:32926445). Mediates pseudouridylation of mRNAs with the consensus sequence 5'-GUUCNANNC-3', harboring a stem-loop structure (PubMed:28073919, PubMed:31477916). Constitutes the major pseudouridine synthase acting on mRNAs (PubMed:28073919). Also catalyzes pseudouridylation of some tRNAs, including synthesis of pseudouridine(55) from uracil-55, in the psi GC loop of a subset of tRNAs (PubMed:32926445, PubMed:33023933). Promotes the processing of pri-let-7 microRNAs (pri-miRNAs) independently of its RNA pseudouridylate synthase activity (PubMed:32926445). Acts by binding to the stem-loop structure on pri-let-7, preventing LIN28-binding (LIN28A and/or LIN28B), thereby enhancing the interaction between pri-let-7 and the microprocessor DGCR8, which mediates miRNA maturation (PubMed:32926445)","subcellular_location":"Nucleus; Cytoplasm, cytosol","url":"https://www.uniprot.org/uniprotkb/Q8WWH5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TRUB1","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"PKM","stoichiometry":0.2},{"gene":"SSB","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/TRUB1","total_profiled":1310},"omim":[{"mim_id":"616261","title":"PSEUDOURIDYLATE SYNTHASE 7; PUS7","url":"https://www.omim.org/entry/616261"},{"mim_id":"610727","title":"TRUB PSEUDOURIDINE SYNTHASE FAMILY MEMBER 2; TRUB2","url":"https://www.omim.org/entry/610727"},{"mim_id":"610726","title":"TRUB PSEUDOURIDINE SYNTHASE FAMILY MEMBER 1; TRUB1","url":"https://www.omim.org/entry/610726"},{"mim_id":"605386","title":"MICRO RNA LET7A1; MIRLET7A1","url":"https://www.omim.org/entry/605386"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Vesicles","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TRUB1"},"hgnc":{"alias_symbol":["PUS4"],"prev_symbol":[]},"alphafold":{"accession":"Q8WWH5","domains":[{"cath_id":"3.30.70","chopping":"50-102_110-146_289-312","consensus_level":"medium","plddt":91.5768,"start":50,"end":312},{"cath_id":"3.30.2350.10","chopping":"147-281","consensus_level":"medium","plddt":96.581,"start":147,"end":281}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8WWH5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8WWH5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8WWH5-F1-predicted_aligned_error_v6.png","plddt_mean":81.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TRUB1","jax_strain_url":"https://www.jax.org/strain/search?query=TRUB1"},"sequence":{"accession":"Q8WWH5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8WWH5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8WWH5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8WWH5"}},"corpus_meta":[{"pmid":"25353621","id":"PMC_25353621","title":"Transcriptome-wide mapping of pseudouridines: pseudouridine synthases modify specific mRNAs in S. cerevisiae.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/25353621","citation_count":314,"is_preprint":false},{"pmid":"33986546","id":"PMC_33986546","title":"Quantitative profiling of pseudouridylation dynamics in native RNAs with nanopore sequencing.","date":"2021","source":"Nature biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/33986546","citation_count":245,"is_preprint":false},{"pmid":"36302989","id":"PMC_36302989","title":"Quantitative sequencing using BID-seq uncovers abundant pseudouridines in mammalian mRNA at base resolution.","date":"2022","source":"Nature biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/36302989","citation_count":194,"is_preprint":false},{"pmid":"9358157","id":"PMC_9358157","title":"The yeast gene YNL292w encodes a pseudouridine synthase (Pus4) catalyzing the formation of psi55 in both mitochondrial and cytoplasmic tRNAs.","date":"1997","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/9358157","citation_count":144,"is_preprint":false},{"pmid":"10022901","id":"PMC_10022901","title":"Pseudouridine mapping in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (snRNAs) reveals that pseudouridine synthase pus1p exhibits a dual substrate specificity for U2 snRNA and tRNA.","date":"1999","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/10022901","citation_count":129,"is_preprint":false},{"pmid":"28073919","id":"PMC_28073919","title":"TRUB1 is the predominant pseudouridine synthase acting on mammalian mRNA via a predictable and conserved code.","date":"2017","source":"Genome research","url":"https://pubmed.ncbi.nlm.nih.gov/28073919","citation_count":119,"is_preprint":false},{"pmid":"36997645","id":"PMC_36997645","title":"Quantitative profiling of pseudouridylation landscape in the human transcriptome.","date":"2023","source":"Nature 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The case of Haloferax volcanii.","date":"2011","source":"RNA (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/21628430","citation_count":45,"is_preprint":false},{"pmid":"39349603","id":"PMC_39349603","title":"Absolute quantitative and base-resolution sequencing reveals comprehensive landscape of pseudouridine across the human transcriptome.","date":"2024","source":"Nature methods","url":"https://pubmed.ncbi.nlm.nih.gov/39349603","citation_count":42,"is_preprint":false},{"pmid":"36018806","id":"PMC_36018806","title":"Human TRUB1 is a highly conserved pseudouridine synthase responsible for the formation of Ψ55 in mitochondrial tRNAAsn, tRNAGln, tRNAGlu and tRNAPro.","date":"2022","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/36018806","citation_count":37,"is_preprint":false},{"pmid":"33023933","id":"PMC_33023933","title":"Mammalian nuclear TRUB1, mitochondrial TRUB2, and cytoplasmic PUS10 produce conserved pseudouridine 55 in different sets of 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lifespan.","date":"2021","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/34545808","citation_count":17,"is_preprint":false},{"pmid":"38766185","id":"PMC_38766185","title":"mRNA psi profiling using nanopore DRS reveals cell type-specific pseudouridylation.","date":"2024","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/38766185","citation_count":7,"is_preprint":false},{"pmid":"41136621","id":"PMC_41136621","title":"A comprehensive tRNA pseudouridine map uncovers targets dependent on human stand-alone pseudouridine synthases.","date":"2025","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/41136621","citation_count":4,"is_preprint":false},{"pmid":"36292915","id":"PMC_36292915","title":"Intron-Dependent or Independent Pseudouridylation of Precursor tRNA Containing Atypical Introns in Cyanidioschyzon merolae.","date":"2022","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/36292915","citation_count":4,"is_preprint":false},{"pmid":"16231152","id":"PMC_16231152","title":"DEG1, encoding the tRNA:pseudouridine synthase Pus3p, impacts HOT1-stimulated recombination in Saccharomyces cerevisiae.","date":"2005","source":"Molecular genetics and genomics : MGG","url":"https://pubmed.ncbi.nlm.nih.gov/16231152","citation_count":3,"is_preprint":false},{"pmid":"26668583","id":"PMC_26668583","title":"A susceptibility locus rs7099208 is associated with non-obstructive azoospermia via reduction in the expression of FAM160B1.","date":"2015","source":"Journal of biomedical research","url":"https://pubmed.ncbi.nlm.nih.gov/26668583","citation_count":3,"is_preprint":false},{"pmid":"38776834","id":"PMC_38776834","title":"Structural basis of pri-let-7 recognition by human pseudouridine synthase TruB1.","date":"2024","source":"Biochemical and biophysical research 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Recombinant Pus4 purified from E. coli catalyzes Ψ55 formation on T7 in vitro transcripts of yeast tRNA genes with strict site-specificity (no other uridines modified), and deletion of YNL292w abolishes all Ψ55 formation activity in cell-free extracts.\",\n      \"method\": \"Recombinant protein purification, in vitro pseudouridylation assay on tRNA transcripts, gene disruption, chemical mapping of pseudouridine residues in cytoplasmic and mitochondrial tRNAs\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified recombinant protein plus genetic deletion with chemical mapping, multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"9358157\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Yeast Pus4 catalyzes Ψ55 formation in the tRNA-like 3′-domain of mutant TYMV RNA (at position 37 in TYMV-mutant G37U, equivalent to Ψ55 in tRNA), demonstrating substrate flexibility of Pus4 beyond canonical tRNA.\",\n      \"method\": \"In vitro pseudouridylation assay using purified recombinant yeast Pus4 on in vitro-transcribed TYMV RNA variants\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro reconstitution with purified enzyme but single lab, single study\",\n      \"pmids\": [\"9705510\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Yeast PUS4 deletion does not affect pseudouridine formation in spliceosomal UsnRNAs (U1, U2, U4, U5, U6), establishing that Pus4 activity is not required for UsnRNA pseudouridylation and that its substrate specificity is restricted to tRNAs in this context.\",\n      \"method\": \"Chemical mapping of pseudouridine in UsnRNAs from pus4Δ yeast mutants\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic deletion with chemical mapping, replicated in context of broader PUS enzyme survey\",\n      \"pmids\": [\"10022901\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Overexpression of PUS4 in yeast causes accumulation of tRNA precursors and derepression of GCN4 translation (Gcd− phenotype) independently of eIF2α phosphorylation, by impeding tRNA 5′-end processing or nuclear export. Importantly, this Gcd− phenotype does not require PUS4 enzymatic activity, indicating a non-catalytic function of the protein in tRNA biogenesis.\",\n      \"method\": \"High-copy PUS4 overexpression, GCN4-lacZ reporter assay, genetic epistasis with RNase P (RPR1), LOS1, suppression analysis, enzymatic activity mutants\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal genetic epistasis experiments with activity-dead mutant establishing non-catalytic role, replicated across several genetic backgrounds\",\n      \"pmids\": [\"10713174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Human TRUB1 was identified as the first human ortholog of bacterial TruB/Ψ55 synthase, encoding a 349-amino acid protein with a conserved TruB domain (W104–I255) containing catalytic motif II with the conserved aspartate residue involved in uridine recognition and catalytic function. Northern blot showed wide tissue expression.\",\n      \"method\": \"Gene cloning, sequence/phylogenetic analysis, domain identification, Northern blot\",\n      \"journal\": \"International journal of molecular medicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — computational/sequence-based identification with no direct enzymatic assay for human TRUB1 protein; domain inference only\",\n      \"pmids\": [\"12736709\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Yeast Pus4 is functionally redundant with La protein for tRNA structural stability: depletion of Pus4p in strains carrying a mutant tRNA(Arg)(CCG) decreases tRNA stability (while La deletion is lethal in this background), placing Pus4 in a pathway ensuring tRNA structural integrity and biogenesis.\",\n      \"method\": \"Genetic epistasis (double-mutant analysis), La deletion/Pus4 depletion in mutant tRNA strains, stability assays\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic epistasis with defined tRNA stability phenotype, single lab\",\n      \"pmids\": [\"16581807\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Yeast Pus4 (screened as an RNA-binding protein on proteome arrays) binds a CAM-containing RNA hairpin and inhibits BMV RNA encapsidation in plants and virion reassembly in vitro, demonstrating an RNA-binding antiviral activity beyond its pseudouridine synthase role.\",\n      \"method\": \"Proteome array binding screen, in planta BMV accumulation assay, in vitro virion reassembly assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteome array plus in vitro functional assay and in planta validation, single lab\",\n      \"pmids\": [\"17360619\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Yeast Pus4 modifies TEF1 mRNA at Ψ-239 in vivo, and this mRNA pseudouridylation is conserved in S. mikitae and S. pombe, establishing Pus4 as a mRNA pseudouridine synthase in addition to its tRNA role. Pus4 activity was shown to be necessary and sufficient for TEF1 Ψ-239 by genetic deletion and in vitro reconstitution.\",\n      \"method\": \"PSI-seq (transcriptome-wide pseudouridine mapping), genetic deletion of PUS4, in vitro reconstitution with purified Pus4\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution showing Pus4 is sufficient, plus genetic deletion showing necessity, plus evolutionary conservation across species\",\n      \"pmids\": [\"25353621\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Human TRUB1 is the predominant pseudouridine synthase acting on mammalian mRNA, targeting a specific sequence/structural context (computationally modeled with AUC=0.974 for substrate prediction). Genetic perturbation of TRUB1 combined with Ψ-seq and massively parallel reporter assays defined the sequence and structural determinants governing TRUB1 specificity.\",\n      \"method\": \"Ψ-seq on >2.5 billion reads, TRUB1 genetic knockdown/knockout, massively parallel reporter assays with thousands of synthetic sequence variants, computational modeling of specificity\",\n      \"journal\": \"Genome research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (transcriptome-wide mapping, genetic perturbation, reporter assays), large-scale data, independently consistent findings\",\n      \"pmids\": [\"28073919\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Human TRUB1 (nuclear) produces Ψ55 in most elongator tRNAs in the nucleus, whereas cytoplasmic PUS10 produces Ψ55 (and Ψ54) in tRNAs that contain Ψ54Ψ55. The nuclear isoform of PUS10 (catalytically inactive) specifically binds unmodified U54U55 tRNAs and inhibits TRUB1-mediated U55→Ψ55 conversion, establishing compartmentalized and non-redundant Ψ55 synthase activities.\",\n      \"method\": \"Nearest-neighbor analysis, recombinant protein assays, subcellular fractionation, specific Ψ55 synthase knockdown cells, binding competition assays\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro enzymatic assays with recombinant proteins, subcellular fractionation, knockdown with biochemical readout, multiple orthogonal approaches in single study\",\n      \"pmids\": [\"33023933\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TruB1 directly binds the stem-loop structure of pri-let-7 miRNA (HITS-CLIP and biochemical assays showing binding to endogenous pri-let-7), selectively enhances interaction between pri-let-7 and the microprocessor DGCR8, and promotes let-7 maturation without pseudouridylating the miRNA. TruB1 suppresses cell proliferation partly via this let-7 pathway.\",\n      \"method\": \"Cell-based luciferase reporter screen, HITS-CLIP, biochemical binding assays (pulldown), let-7 maturation assays, cell proliferation assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal biochemical binding (HITS-CLIP + pulldown), functional maturation assay, cellular proliferation phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"32926445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The pseudouridine synthase Pus4/TruB can act as a prion in yeast ([BIG+] state), epigenetically increasing cell proliferation and size while shortening lifespan, with altered protein synthesis and differential synthesis of dozens of proteins including proliferation and aging regulators.\",\n      \"method\": \"Prion formation assays, cell growth/size measurements, lifespan assays, proteomic analysis of [BIG+] cells, protein synthesis measurements\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple cellular phenotype readouts with defined mechanistic outcome (prion-based epigenetic state), single lab\",\n      \"pmids\": [\"34545808\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Human TRUB1 is responsible for Ψ55 formation in mitochondrial tRNAAsn, tRNAGln, tRNAGlu, and tRNAPro but not the other 18 mitochondrial tRNAs. TRUB1 knockout (CRISPR/Cas9) abolishes Ψ55 in these four tRNAs; recombinant TRUB1 efficiently catalyzes Ψ55 in tRNAAsn and tRNAGln in vitro. TRUB1 deficiency affects tRNA base-pairing (18A/G–Ψ55), conformation, and stability, and impairs mitochondrial translation and oxidative phosphorylation system biogenesis.\",\n      \"method\": \"CRISPR/Cas9 TRUB1 knockout, CMC/reverse transcription Ψ-mapping assay, in vitro pseudouridylation assay with recombinant TRUB1, cDNA rescue experiments, mitochondrial translation assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with recombinant protein, CRISPR knockout with Ψ mapping, cDNA rescue, multiple orthogonal methods demonstrating substrate specificity and downstream consequences\",\n      \"pmids\": [\"36018806\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"BID-seq transcriptome-wide mapping in human cancer cells revealed that TRUB1-installed Ψ sites have a transcript stabilization role; depletion of TRUB1 reduced stability of transcripts bearing TRUB1-dependent Ψ modifications.\",\n      \"method\": \"BID-seq (bisulfite-induced deletion sequencing), TRUB1 knockdown/knockout, transcript stability assays\",\n      \"journal\": \"Nature biotechnology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transcriptome-wide mapping with genetic perturbation and functional stability readout, single lab\",\n      \"pmids\": [\"36302989\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Human TruB1 binds specifically to the terminal loop of pri-let-7a1 at nucleotides 31–41 (a small stem-loop architecture). A conserved KRKK motif in human and higher eukaryotes provides an additional electrostatic binding interface beyond what is seen in E. coli TruB–tRNA interaction. The structural basis was determined by biochemical assays and structural investigation.\",\n      \"method\": \"Biochemical binding assays, structural investigation (crystal/structural study), mutagenesis of the KRKK motif\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — structural data plus biochemical validation but single lab, single study; abstract does not specify crystal vs NMR resolution level\",\n      \"pmids\": [\"38776834\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TRUB1 and PUS10 function redundantly to catalyze the conserved Ψ55 modification in cytosolic tRNAs in human HCT116 cells; individual knockouts of TRUB1 or PUS10 each reduce but do not eliminate cytosolic tRNA Ψ55. Additionally, TRUB1 introduces Ψ modifications at distinct stages of pre-tRNA processing.\",\n      \"method\": \"Systematic PUS knockout/knockdown in HCT116 cells, BACS (2-bromoacrylamide-assisted cyclization sequencing) for Ψ mapping, comprehensive tRNA Ψ profiling\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — systematic genetic perturbations with quantitative Ψ mapping across nine PUS enzymes, defining redundancy with multiple orthogonal validations\",\n      \"pmids\": [\"41136621\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Pus4/TruB (yeast ortholog of TRUB1) both catalyzes Ψ55 formation on tRNA and remodels tRNA conformational dynamics. Wild-type Pus4 binding to unmodified tRNA populates additional conformational states that gradually approach the ensemble adopted more rapidly by pre-pseudouridylated tRNA. A catalytically incompetent Pus4 mutant binds more slowly and remodels tRNA into distinct conformational ensembles, demonstrating that catalytic and remodeling activities are separable.\",\n      \"method\": \"Single-molecule FRET, optical binding assays, catalytically inactive Pus4 mutant analysis\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — single-molecule FRET with active-site mutagenesis separating catalytic from remodeling activities, peer-reviewed publication\",\n      \"pmids\": [\"41916762\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TRUB1 knockdown in CRC cells activates the TNFα/NFκB pathway, leading to increased expression of apoptosis-related proteins and decreased Ψ modification. BIRC3 was identified as a downstream target gene regulated by TRUB1 in the NFκB pathway.\",\n      \"method\": \"TRUB1 knockdown in HCT116 cells, RNA sequencing, Western blot, immunofluorescence, in vivo tumor growth assay in nude mice\",\n      \"journal\": \"Gastroenterology report\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, pathway inferred from transcriptomics with limited direct mechanistic validation of TRUB1→BIRC3→NFκB connection\",\n      \"pmids\": [\"40260225\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"TRUB1-installed Ψ at single conserved sites on mRNAs is causally associated with increased protein production, as shown by TRUB1 knockout experiments demonstrating motif-specific reduction in protein abundance. In contrast, transcripts with clustered/high-density Ψ modifications show reduced protein abundance despite elevated translation efficiency, establishing a density-dependent effect of pseudouridylation on translation output.\",\n      \"method\": \"TRUB1 knockout, nanopore direct RNA sequencing (Mod-p ID), proteomics, ribosome profiling, controlled in vitro translation assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — TRUB1 KO with multimodal readouts (proteomics + ribosome profiling + in vitro translation), direct causal evidence from genetic perturbation plus functional assay\",\n      \"pmids\": [\"42011786\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Nanopore direct RNA sequencing of yeast mitochondria lacking PUS4 demonstrated that Pus4 pseudouridylates 23 of 24 mitochondrially-encoded tRNAs at Ψ55 in the T-loop in vivo.\",\n      \"method\": \"Nanopore direct RNA sequencing of mitochondrially-enriched RNA, PUS4 gene knockout comparison\",\n      \"journal\": \"bioRxiv : the preprint server for biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — nanopore sequencing with genetic knockout providing comprehensive in vivo map, but preprint and single lab\",\n      \"pmids\": [\"40654949\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"TRUB1 (human ortholog of yeast Pus4/bacterial TruB) is a pseudouridine synthase that installs Ψ55 in most cytosolic elongator tRNAs (in the nucleus, where it is partially restricted by catalytically inactive nuclear PUS10), in four mitochondrial tRNAs (tRNAAsn, tRNAGln, tRNAGlu, tRNAPro), and at specific mRNA sites with a predictable sequence/structural code—TRUB1-deposited mRNA Ψ enhances transcript stability and protein output at single sites but impairs translation when pseudouridine density is high; beyond pseudouridylation, TRUB1 directly binds the terminal loop of pri-let-7 via a conserved KRKK motif and recruits the DGCR8 microprocessor to promote let-7 miRNA maturation, and the enzyme additionally remodels tRNA conformational dynamics in a manner separable from its catalytic activity.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TRUB1 is the human ortholog of bacterial TruB/yeast Pus4, a stand-alone pseudouridine synthase that installs the conserved Ψ55 modification in the T-loop of tRNAs and extends this activity to mRNA and microRNA biogenesis [#4, #8]. In the nucleus it generates Ψ55 in most cytosolic elongator tRNAs, an activity that is partially restrained by a catalytically inactive nuclear PUS10 isoform that binds unmodified U54U55 tRNAs and blocks U55→Ψ55 conversion, while TRUB1 and PUS10 together act redundantly to ensure cytosolic tRNA Ψ55 [#9, #15]. TRUB1 also pseudouridylates four mitochondrial tRNAs (tRNAAsn, tRNAGln, tRNAGlu, tRNAPro), and its loss disrupts tRNA base-pairing, conformation, and stability, impairing mitochondrial translation and oxidative phosphorylation biogenesis [#12]. Beyond tRNA, TRUB1 is the predominant mRNA pseudouridine synthase in mammalian cells, recognizing a defined sequence/structural context [#8]; single-site Ψ deposition stabilizes transcripts and enhances protein output, whereas clustered high-density Ψ reduces protein abundance, defining a density-dependent effect on translation [#13, #18]. Independent of catalysis, TRUB1 directly binds the terminal loop of pri-let-7 through a conserved KRKK electrostatic interface and recruits the DGCR8 microprocessor to promote let-7 maturation and suppress cell proliferation [#10, #14]. Conformational studies of the yeast ortholog further establish that TRUB1/Pus4 remodels tRNA structural dynamics in a manner separable from its enzymatic activity [#16].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Established the enzymatic identity of the Ψ55 synthase, answering which protein forms the universally conserved T-loop pseudouridine in tRNA.\",\n      \"evidence\": \"Recombinant yeast Pus4 in vitro pseudouridylation of tRNA transcripts plus PUS4 gene disruption with chemical Ψ mapping\",\n      \"pmids\": [\"9358157\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address human ortholog activity\", \"Substrate scope beyond tRNA untested\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Showed the enzyme's substrate recognition is structure-based rather than strictly tRNA-sequence-dependent, by modifying a tRNA-like viral RNA domain.\",\n      \"evidence\": \"In vitro pseudouridylation of TYMV RNA variants with purified recombinant Pus4\",\n      \"pmids\": [\"9705510\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab, single study\", \"In vivo relevance of viral RNA modification unknown\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Delimited substrate specificity by showing the enzyme is not required for spliceosomal UsnRNA pseudouridylation.\",\n      \"evidence\": \"Chemical Ψ mapping in UsnRNAs from pus4Δ yeast\",\n      \"pmids\": [\"10022901\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Negative result does not exclude other non-tRNA substrates\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Revealed a catalysis-independent role in tRNA biogenesis, separating the protein's structural/scaffolding function from its enzymatic activity.\",\n      \"evidence\": \"High-copy PUS4 overexpression with GCN4-lacZ reporter, genetic epistasis, and activity-dead mutants in yeast\",\n      \"pmids\": [\"10713174\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of tRNA processing/export interference not defined\", \"Not tested for human TRUB1\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Identified the human ortholog TRUB1 and its conserved catalytic TruB domain, opening study of the mammalian enzyme.\",\n      \"evidence\": \"Gene cloning, phylogenetic/domain analysis, and Northern blot tissue survey\",\n      \"pmids\": [\"12736709\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct enzymatic assay for human protein\", \"Catalytic activity inferred from domain only\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Placed the enzyme in a tRNA structural-stability pathway functionally overlapping with La protein.\",\n      \"evidence\": \"Genetic epistasis and tRNA stability assays in mutant tRNA(Arg) yeast strains\",\n      \"pmids\": [\"16581807\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of stabilization not resolved\", \"Catalytic vs structural contribution unseparated here\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Demonstrated a sequence-specific RNA-binding/antiviral activity distinct from pseudouridylation.\",\n      \"evidence\": \"Proteome array binding screen, in planta BMV accumulation, and in vitro virion reassembly assays\",\n      \"pmids\": [\"17360619\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological relevance to host cells unclear\", \"Single lab\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Established the enzyme as a bona fide mRNA pseudouridine synthase, extending its substrate repertoire beyond structured tRNA.\",\n      \"evidence\": \"PSI-seq, PUS4 deletion, and in vitro reconstitution showing necessity and sufficiency for TEF1 Ψ-239, conserved across species\",\n      \"pmids\": [\"25353621\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of mRNA Ψ not addressed\", \"Human ortholog mRNA activity not tested here\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified TRUB1 as the dominant mammalian mRNA Ψ synthase and defined the sequence/structural code governing its specificity.\",\n      \"evidence\": \"Ψ-seq, TRUB1 knockdown/knockout, massively parallel reporter assays, and computational specificity modeling\",\n      \"pmids\": [\"28073919\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream functional impact of mRNA Ψ not yet defined\", \"Mitochondrial substrates not addressed\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Resolved compartmentalized division of labor for tRNA Ψ55, showing nuclear TRUB1 is regulated by an inactive nuclear PUS10 isoform.\",\n      \"evidence\": \"Recombinant assays, subcellular fractionation, specific synthase knockdown, and binding competition assays\",\n      \"pmids\": [\"33023933\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of PUS10 inhibition of TRUB1 unresolved\", \"Regulation of compartment switching unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Uncovered a non-catalytic role in miRNA biogenesis: TRUB1 binds pri-let-7 and recruits DGCR8 to promote let-7 maturation and suppress proliferation.\",\n      \"evidence\": \"Luciferase reporter screen, HITS-CLIP, pulldown binding, let-7 maturation and proliferation assays\",\n      \"pmids\": [\"32926445\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other miRNAs are regulated unknown\", \"Mechanism of DGCR8 recruitment not structurally defined here\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed the yeast ortholog can adopt a heritable prion state altering proteostasis, growth, and lifespan.\",\n      \"evidence\": \"Prion formation, growth/size, lifespan, proteomic, and protein synthesis assays in [BIG+] yeast\",\n      \"pmids\": [\"34545808\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No evidence human TRUB1 forms a prion\", \"Link to canonical synthase activity unclear\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined TRUB1 as the Ψ55 synthase for four specific mitochondrial tRNAs and linked its loss to defective mitochondrial translation.\",\n      \"evidence\": \"CRISPR knockout, CMC Ψ mapping, in vitro pseudouridylation, cDNA rescue, and mitochondrial translation assays\",\n      \"pmids\": [\"36018806\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why only four of 22 mt-tRNAs are substrates unresolved\", \"OXPHOS phenotype mechanism not fully traced\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Assigned a transcript-stabilizing function to TRUB1-installed mRNA Ψ in human cancer cells.\",\n      \"evidence\": \"BID-seq transcriptome-wide Ψ mapping with TRUB1 depletion and transcript stability assays\",\n      \"pmids\": [\"36302989\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Stability mechanism (RNA-binding readers) not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Provided the structural/molecular basis for pri-let-7 recognition, identifying a higher-eukaryote KRKK electrostatic interface.\",\n      \"evidence\": \"Biochemical binding, structural investigation, and KRKK motif mutagenesis on pri-let-7a1\",\n      \"pmids\": [\"38776834\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Resolution/method of structure not specified\", \"Single study\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established TRUB1/PUS10 redundancy for cytosolic tRNA Ψ55 and timing of Ψ deposition during pre-tRNA processing.\",\n      \"evidence\": \"Systematic PUS knockout/knockdown in HCT116 cells with BACS Ψ mapping\",\n      \"pmids\": [\"41136621\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of redundancy buffering unclear\", \"Regulation of processing-stage choice unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Separated catalytic from conformational-remodeling activities, showing the enzyme reshapes tRNA dynamics independent of Ψ formation.\",\n      \"evidence\": \"Single-molecule FRET and optical binding assays with catalytically inactive Pus4 mutant\",\n      \"pmids\": [\"41916762\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional output of remodeling in vivo unknown\", \"Demonstrated in yeast ortholog\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Linked TRUB1 loss to TNFα/NF-κB activation and BIRC3 regulation in colorectal cancer cells.\",\n      \"evidence\": \"TRUB1 knockdown in HCT116, RNA-seq, Western blot, and nude mouse tumor assays\",\n      \"pmids\": [\"40260225\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"TRUB1→BIRC3→NF-κB connection inferred from transcriptomics without direct mechanistic validation\", \"Single lab\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Established a density-dependent rule for how TRUB1-installed mRNA Ψ controls translation output.\",\n      \"evidence\": \"TRUB1 knockout with nanopore direct RNA-seq, proteomics, ribosome profiling, and in vitro translation\",\n      \"pmids\": [\"42011786\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reader proteins distinguishing single vs clustered Ψ not identified\", \"Mechanism of translation impairment at high density unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TRUB1 integrates its multiple activities — catalytic Ψ deposition, non-catalytic tRNA remodeling, and microprocessor recruitment — into coordinated cellular regulation, and what reader machinery interprets its mRNA marks, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No Ψ reader proteins identified for TRUB1 marks\", \"Coordination between catalytic and scaffolding roles unknown\", \"Structural model of human TRUB1 on substrate tRNA/mRNA lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [0, 7, 8, 9, 12, 15]},\n      {\"term_id\": \"GO:0016853\", \"supporting_discovery_ids\": [0, 12]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [6, 10, 14]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [9]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [12]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [9, 15]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [8, 9, 12, 13, 15, 18]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [10]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"DGCR8\", \"PUS10\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}