id: O95900 gene_symbol: TRUB2 product_type: PROTEIN status: COMPLETE taxon: id: NCBITaxon:9606 label: Homo sapiens description: TRUB2 is a nuclear-encoded pseudouridine synthase of the TruB family that is imported into the mitochondrial matrix, where it is concentrated in mitochondrial RNA granules and co-sediments with the large mitoribosomal subunit. Recombinant TRUB2 isomerizes uridine-55 of tRNA to pseudouridine, and it accounts for the tRNA Psi55 synthase activity of mitochondria, consistent with modification of the T-loop of the subset of mitochondrial tRNAs that carry Psi55. It also contributes, apparently as a minor enzyme alongside RPUSD3, to pseudouridylation of specific sites in the COX1 and COX3 mitochondrial mRNAs. TRUB2 associates with RPUSD3, RPUSD4, NGRN, RCC1L (WBSCR16), FASTKD2 and MTERF3 in a mitochondrial RNA-granule module that supports 16S rRNA stability, mitoribosome assembly and mitochondrial translation; its depletion impairs mitochondrial protein synthesis, particularly of ATP8/ATP6, and oxidative phosphorylation, and it is essential for viability in several human cell lines. alternative_products: - name: '1' id: O95900-1 - name: '2' id: O95900-2 sequence_note: VSP_056094 existing_annotations: - term: id: GO:0001522 label: pseudouridine synthesis evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: involved_in supporting_entities: - ARBA:ARBA00028294 - InterPro:IPR020103 - InterPro:IPR039048 review: summary: Electronic (ARBA/InterPro) assignment of the generic process pseudouridine synthesis from TruB-family membership. action: MODIFY reason: Correct but general. TRUB2 is a TruB-family pseudouridine synthase whose experimentally supported process roles are pseudouridylation of mitochondrial tRNA (Psi55) and, as a minor contributor, mitochondrial mRNA. Replace with the specific child terms the gene already carries. proposed_replacement_terms: - &id006 id: GO:0070902 label: mitochondrial tRNA pseudouridine synthesis - &id007 id: GO:1990481 label: mRNA pseudouridine synthesis supported_by: - &id008 reference_id: file:human/TRUB2/TRUB2-uniprot.txt record_section: ["CC:SIMILARITY"] supporting_text: Belongs to the pseudouridine synthase TruB family. - &id001 reference_id: PMID:33023933 supporting_text: We show here that all three human recombinant proteins, TRUB1, PUS10, and TRUB2, have Ψ55 synthase activities, but differ slightly in their tRNA structural requirements. - &id005 reference_id: PMID:33023933 supporting_text: Knockdown of TRUB1, TRUB2, and PUS10 reduced Ψ55 synthesis by the nuclear, mitochondrial, and cytoplasmic extracts, respectively, in both tRNATrp and tRNAAla - term: id: GO:0003723 label: RNA binding evidence_type: HDA original_reference_id: PMID:22658674 qualifier: enables review: summary: TRUB2 was recovered in high-throughput UV-crosslinking/oligo(dT) mRNA interactome capture in human cells. action: KEEP_AS_NON_CORE reason: RNA binding is inherent to a pseudouridine synthase that acts on tRNA and mRNA substrates, and interactome capture supports direct RNA contact. It is not the informative description of the gene's activity (that is the catalytic activity), and the specific RNA ligands are not defined by these screens, so it is retained as non-core. supported_by: - reference_id: PMID:22658674 supporting_text: to define the mRNA interactome of proliferating human HeLa cells - term: id: GO:0003723 label: RNA binding evidence_type: HDA original_reference_id: PMID:22681889 qualifier: enables review: summary: TRUB2 was recovered in high-throughput UV-crosslinking/oligo(dT) mRNA interactome capture in human cells. action: KEEP_AS_NON_CORE reason: RNA binding is inherent to a pseudouridine synthase that acts on tRNA and mRNA substrates, and interactome capture supports direct RNA contact. It is not the informative description of the gene's activity (that is the catalytic activity), and the specific RNA ligands are not defined by these screens, so it is retained as non-core. supported_by: - reference_id: PMID:22681889 supporting_text: to identify the mRNA-bound proteome using quantitative proteomics - term: id: GO:0003723 label: RNA binding evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables supporting_entities: - InterPro:IPR020103 review: summary: InterPro2GO assignment of RNA binding from the pseudouridine synthase catalytic domain superfamily (IPR020103). action: KEEP_AS_NON_CORE reason: Consistent with the HDA interactome-capture rows and with the enzyme's RNA substrates; true but peripheral to the catalytic function, retained as non-core. supported_by: - *id001 - term: id: GO:0005739 label: mitochondrion evidence_type: HTP original_reference_id: PMID:34800366 qualifier: located_in review: summary: TRUB2 is in the MitoCoP high-confidence human mitochondrial proteome. action: MODIFY reason: Correct but general. The protein has a predicted N-terminal mitochondrial transit peptide and is experimentally placed in the mitochondrial matrix and in matrix-located mitochondrial RNA granules, so the specific term mitochondrial matrix (already carried by the gene) is preferred. proposed_replacement_terms: - &id002 id: GO:0005759 label: mitochondrial matrix supported_by: - reference_id: PMID:34800366 supporting_text: mitochondrial high-confidence proteome of >1,100 proteins (MitoCoP) - &id003 reference_id: file:human/TRUB2/TRUB2-uniprot.txt record_section: ["CC:SUBCELLULAR LOCATION"] supporting_text: 'SUBCELLULAR LOCATION: Mitochondrion matrix' - term: id: GO:0005739 label: mitochondrion evidence_type: IDA original_reference_id: PMID:28082677 qualifier: located_in review: summary: Immunofluorescence localizes TRUB2 to mitochondria, enriched in mitochondrial RNA granules. action: MODIFY reason: Correct but general. The protein has a predicted N-terminal mitochondrial transit peptide and is experimentally placed in the mitochondrial matrix and in matrix-located mitochondrial RNA granules, so the specific term mitochondrial matrix (already carried by the gene) is preferred. proposed_replacement_terms: - *id002 supported_by: - &id012 reference_id: PMID:28082677 supporting_text: We observed a clear enrichment of RPUSD3, RPUSD4, and TRUB2 in MRGs, whereas neither PUS1 nor PUSL1 appeared to be specifically enriched in discrete foci - *id003 - term: id: GO:0005759 label: mitochondrial matrix evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: is_active_in supporting_entities: - PANTHER:PTN000324318 - UniProtKB:O95900 review: summary: Phylogenetic inference of mitochondrial matrix activity. The IBA derives from a PAINT IBD at Bilateria node PTN000324318, seeded solely by human TRUB2 (O95900); TRUB2's appearance in its own WITH/FROM reflects its experimental grounding, not circularity. action: ACCEPT reason: TRUB2 localizes to the mitochondrial matrix, concentrated in mitochondrial RNA granules where it co-sediments with the large mitoribosomal subunit and associates with the RPUSD3/RPUSD4/NGRN/RCC1L/FASTKD2 module. The PAINT placement at the Bilateria TRUB2-subfamily node is consistent with the conserved transit peptide and with human experimental data. supported_by: - &id004 reference_id: PMID:27974379 supporting_text: we identified a protein module in mitochondrial RNA granules - *id003 - &id009 reference_id: file:human/TRUB2/TRUB2-uniprot.txt record_section: ["DR:PANTHER"] supporting_text: PTHR13195:SF0; PSEUDOURIDYLATE SYNTHASE TRUB2, MITOCHONDRIAL - term: id: GO:0005759 label: mitochondrial matrix evidence_type: IDA original_reference_id: PMID:27974379 qualifier: located_in review: summary: BioID proximity labelling and immunofluorescence place TRUB2 in mitochondrial RNA granules within the matrix. action: ACCEPT reason: TRUB2 localizes to the mitochondrial matrix, concentrated in mitochondrial RNA granules where it co-sediments with the large mitoribosomal subunit and associates with the RPUSD3/RPUSD4/NGRN/RCC1L/FASTKD2 module. supported_by: - *id004 - reference_id: PMID:27974379 supporting_text: Using BioID, we reciprocally confirmed the specific interaction of TRUB2 with all these proteins - term: id: GO:0005759 label: mitochondrial matrix evidence_type: IEA original_reference_id: GO_REF:0000044 qualifier: located_in supporting_entities: - UniProtKB-SubCell:SL-0170 review: summary: UniProt subcellular-location mapping (SL-0170, mitochondrion matrix), curated from experimental papers. action: ACCEPT reason: TRUB2 localizes to the mitochondrial matrix, concentrated in mitochondrial RNA granules where it co-sediments with the large mitoribosomal subunit and associates with the RPUSD3/RPUSD4/NGRN/RCC1L/FASTKD2 module. supported_by: - *id003 - term: id: GO:0005759 label: mitochondrial matrix evidence_type: IMP original_reference_id: PMID:33023933 qualifier: is_active_in review: summary: Psi55 synthase activity of mitochondrial extracts is lost on TRUB2 knockdown, locating TRUB2 activity in mitochondria. action: ACCEPT reason: Knockdown specifically reduced Psi55 synthesis by mitochondrial extracts, supporting is_active_in the mitochondrial matrix. TRUB2 localizes to the mitochondrial matrix, concentrated in mitochondrial RNA granules where it co-sediments with the large mitoribosomal subunit and associates with the RPUSD3/RPUSD4/NGRN/RCC1L/FASTKD2 module. supported_by: - *id005 - term: id: GO:0005759 label: mitochondrial matrix evidence_type: TAS original_reference_id: Reactome:R-HSA-9936243 qualifier: located_in review: summary: Reactome places TRUB2, as part of the mitochondrial pseudouridylation module, in the mitochondrial matrix. action: ACCEPT reason: TRUB2 localizes to the mitochondrial matrix, concentrated in mitochondrial RNA granules where it co-sediments with the large mitoribosomal subunit and associates with the RPUSD3/RPUSD4/NGRN/RCC1L/FASTKD2 module. supported_by: - reference_id: Reactome:R-HSA-9936243 supporting_text: which binds 16S rRNA - *id003 - term: id: GO:0005759 label: mitochondrial matrix evidence_type: TAS original_reference_id: Reactome:R-HSA-9937369 qualifier: located_in review: summary: Reactome places TRUB2, as part of the mitochondrial pseudouridylation module, in the mitochondrial matrix. action: ACCEPT reason: TRUB2 localizes to the mitochondrial matrix, concentrated in mitochondrial RNA granules where it co-sediments with the large mitoribosomal subunit and associates with the RPUSD3/RPUSD4/NGRN/RCC1L/FASTKD2 module. supported_by: - &id010 reference_id: Reactome:R-HSA-9937369 supporting_text: Either RPUSD3 or TRUB2 in the module can catalyze the conversion of uridine-390 of MT-CO1 mRNA and uridine-697 of MT-CO3 mRNA to pseudouridine residues - *id003 - term: id: GO:0006396 label: RNA processing evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: involved_in supporting_entities: - ARBA:ARBA00028223 - InterPro:IPR002501 review: summary: Electronic assignment of the very general process RNA processing from the TruB N-terminal domain (IPR002501). action: MODIFY reason: Uninformative for an RNA-modifying enzyme. Replace with the specific pseudouridylation processes the gene already carries. proposed_replacement_terms: - *id006 - *id007 supported_by: - *id001 - *id008 - term: id: GO:0009451 label: RNA modification evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: involved_in supporting_entities: - InterPro:IPR020103 review: summary: InterPro2GO assignment of RNA modification from the pseudouridine synthase catalytic domain superfamily. action: MODIFY reason: Correct but general. The supported specific processes are mitochondrial tRNA pseudouridine synthesis and mRNA pseudouridine synthesis, both already annotated. proposed_replacement_terms: - *id006 - *id007 supported_by: - *id001 - &id011 reference_id: PMID:27974379 supporting_text: Depletion of both TRUB2 and RPUSD3 resulted in decreased pseudouridylation in the COXI - term: id: GO:0009982 label: pseudouridine synthase activity evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: enables supporting_entities: - PANTHER:PTN000324318 - UniProtKB:O95900 review: summary: Phylogenetic inference of pseudouridine synthase activity. The IBA derives from a PAINT IBD at Bilateria node PTN000324318, seeded solely by human TRUB2 (O95900); TRUB2's appearance in its own WITH/FROM reflects its experimental grounding, not circularity. action: ACCEPT reason: Recombinant human TRUB2 converts U55 to pseudouridine in tRNA transcripts in vitro and also modified one mRNA site in an in vitro mRNA pool, so pseudouridine synthase activity is directly established. The tRNA-specific child (tRNA pseudouridine(55) synthase activity) is annotated separately; no mRNA-specific pseudouridine synthase activity term exists in GO, so this general term is as precise as the mRNA-directed evidence allows and is kept. TRUB2 retains the TruB fold and the predicted catalytic aspartate (by similarity). supported_by: - *id001 - *id008 - *id009 - term: id: GO:0009982 label: pseudouridine synthase activity evidence_type: IDA original_reference_id: PMID:31477916 qualifier: enables review: summary: Recombinant TRUB2 pseudouridylated a single site in an in vitro-transcribed pool of human mRNA fragments. action: ACCEPT reason: Recombinant human TRUB2 converts U55 to pseudouridine in tRNA transcripts in vitro and also modified one mRNA site in an in vitro mRNA pool, so pseudouridine synthase activity is directly established. The tRNA-specific child (tRNA pseudouridine(55) synthase activity) is annotated separately; no mRNA-specific pseudouridine synthase activity term exists in GO, so this general term is as precise as the mRNA-directed evidence allows and is kept. Note that the single TRUB2 mRNA site was in a cytosolic mRNA and the authors doubt it is physiological. supported_by: - &id013 reference_id: PMID:31477916 supporting_text: TRUB1 modified the largest number of sites (61), followed by PUS7 (22), PUS1 (20), RPUSD2 (17), and TRUB2 (1). - &id014 reference_id: PMID:31477916 supporting_text: The modification of a cytosolic mRNA by TRUB2 was unexpected given the predominantly mitochondrial localization of this enzyme - term: id: GO:0009982 label: pseudouridine synthase activity evidence_type: IDA original_reference_id: PMID:33023933 qualifier: enables review: summary: Recombinant TRUB2 synthesizes Psi55 in tRNA transcripts in vitro. action: ACCEPT reason: Recombinant human TRUB2 converts U55 to pseudouridine in tRNA transcripts in vitro and also modified one mRNA site in an in vitro mRNA pool, so pseudouridine synthase activity is directly established. The tRNA-specific child (tRNA pseudouridine(55) synthase activity) is annotated separately; no mRNA-specific pseudouridine synthase activity term exists in GO, so this general term is as precise as the mRNA-directed evidence allows and is kept. supported_by: - *id001 - term: id: GO:0009982 label: pseudouridine synthase activity evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: enables supporting_entities: - InterPro:IPR020103 - InterPro:IPR039048 - RHEA:56644 review: summary: Electronic assignment from InterPro (IPR020103, IPR039048) and Rhea:56644. action: ACCEPT reason: Recombinant human TRUB2 converts U55 to pseudouridine in tRNA transcripts in vitro and also modified one mRNA site in an in vitro mRNA pool, so pseudouridine synthase activity is directly established. The tRNA-specific child (tRNA pseudouridine(55) synthase activity) is annotated separately; no mRNA-specific pseudouridine synthase activity term exists in GO, so this general term is as precise as the mRNA-directed evidence allows and is kept. supported_by: - *id001 - reference_id: file:human/TRUB2/TRUB2-uniprot.txt record_section: ["DR:InterPro"] supporting_text: IPR039048; Trub2 - term: id: GO:0009982 label: pseudouridine synthase activity evidence_type: TAS original_reference_id: Reactome:R-HSA-9937369 qualifier: enables review: summary: 'Reactome: RPUSD3 or TRUB2 pseudouridylates MT-CO1 and MT-CO3 mRNA.' action: ACCEPT reason: Recombinant human TRUB2 converts U55 to pseudouridine in tRNA transcripts in vitro and also modified one mRNA site in an in vitro mRNA pool, so pseudouridine synthase activity is directly established. The tRNA-specific child (tRNA pseudouridine(55) synthase activity) is annotated separately; no mRNA-specific pseudouridine synthase activity term exists in GO, so this general term is as precise as the mRNA-directed evidence allows and is kept. The Reactome reaction relies on Antonicka et al., who consider RPUSD3 the major enzyme for these mt-mRNA sites. supported_by: - *id010 - *id001 - term: id: GO:0016556 label: mRNA modification evidence_type: TAS original_reference_id: Reactome:R-HSA-9937008 qualifier: involved_in review: summary: Reactome includes TRUB2 in mitochondrial mRNA modification via pseudouridylation of MT-CO1/MT-CO3 mRNA. action: MODIFY reason: Correct in direction but general; the specific modification is pseudouridylation, captured by mRNA pseudouridine synthesis (already annotated and treated as non-core because TRUB2's contribution to mt-mRNA pseudouridylation appears secondary to RPUSD3). proposed_replacement_terms: - *id007 supported_by: - *id010 - *id011 - term: id: GO:0035770 label: ribonucleoprotein granule evidence_type: IDA original_reference_id: PMID:28082677 qualifier: located_in review: summary: Immunofluorescence shows TRUB2 enrichment in mitochondrial RNA granules (MRGs). action: ACCEPT reason: Mitochondrial RNA granules are matrix ribonucleoprotein foci; GO has no mitochondrion-specific RNA granule term, so ribonucleoprotein granule is the most precise available term. Independently supported by BioID with granule proteins. supported_by: - *id012 - *id004 - term: id: GO:0070131 label: positive regulation of mitochondrial translation evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: involved_in supporting_entities: - PANTHER:PTN000324318 - UniProtKB:O95900 review: summary: Phylogenetic inference of positive regulation of mitochondrial translation. The IBA derives from a PAINT IBD at Bilateria node PTN000324318, seeded solely by human TRUB2 (O95900); TRUB2's appearance in its own WITH/FROM reflects its experimental grounding, not circularity. action: KEEP_AS_NON_CORE reason: 'TRUB2 depletion reduces mitochondrial protein synthesis (to 76% of control), most strongly for ATP8/ATP6, and disrupts mitoribosome large-subunit assembly; CRISPR disruption of module members abolishes 16S rRNA abundance and mitochondrial translation. These are necessity data: TRUB2 is a modifying enzyme required for efficient mitochondrial translation, not a demonstrated regulator of it. The term is retained as non-core because the PAINT curator deliberately propagated it and the gene product does act on mitochondrial translation substrates, but it should not be read as a core regulatory activity.' supported_by: - reference_id: PMID:27974379 supporting_text: was decreased to 76, 20, and 53% of control in cells in which TRUB2, RPUSD4, and RPUSD3 were depleted - reference_id: PMID:27974379 supporting_text: Remarkably, in TRUB2 knockdown cells, the synthesis of ATP6 and ATP8 proteins (encoded on a bicistronic transcript) was severely reduced - term: id: GO:0070131 label: positive regulation of mitochondrial translation evidence_type: IMP original_reference_id: PMID:27667664 qualifier: involved_in review: summary: CRISPR disruption of TRUB2 and other module members reduces 16S rRNA and mitochondrial translation. action: KEEP_AS_NON_CORE reason: 'TRUB2 depletion reduces mitochondrial protein synthesis (to 76% of control), most strongly for ATP8/ATP6, and disrupts mitoribosome large-subunit assembly; CRISPR disruption of module members abolishes 16S rRNA abundance and mitochondrial translation. These are necessity data: TRUB2 is a modifying enzyme required for efficient mitochondrial translation, not a demonstrated regulator of it. The term is retained as non-core because the PAINT curator deliberately propagated it and the gene product does act on mitochondrial translation substrates, but it should not be read as a core regulatory activity.' supported_by: - reference_id: PMID:27667664 supporting_text: Finally, we used CRISPR to disrupt all members of the module and found that they were all required for 16S rRNA abundance and mitochondrial translation - reference_id: PMID:27667664 supporting_text: Using protein immunoprecipitation and mass spectrometry, we identified and confirmed the interaction of NGRN with WBSCR16, RPUSD3, RPUSD4, TRUB2, and FASTKD2. - term: id: GO:0070902 label: mitochondrial tRNA pseudouridine synthesis evidence_type: IMP original_reference_id: PMID:33023933 qualifier: involved_in review: summary: TRUB2 knockdown reduces tRNA Psi55 synthase activity of mitochondrial extracts. action: ACCEPT reason: 'TRUB2 is the mitochondrial tRNA Psi55 synthase: recombinant enzyme makes Psi55 and its knockdown removes Psi55 activity from mitochondrial extracts. Caveat: the assay substrates were tRNA transcripts (tRNATrp, tRNAAla) rather than mitochondrial tRNAs, and the four mt-tRNAs bearing Psi55 are proposed, not shown, as in vivo TRUB2 targets. PAINT did not propagate this term to node PTN000324318, so it is human-experimental only.' supported_by: - *id005 - &id016 reference_id: PMID:33023933 supporting_text: TRUB2 is present in the mitochondria and is suggested to produce Ψ55 in all four human mitochondrial tRNAs (out of 22 total) with this modification - *id001 - term: id: GO:0160148 label: tRNA pseudouridine(55) synthase activity evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: enables supporting_entities: - RHEA:42532 - EC:5.4.99.25 review: summary: Electronic assignment via Rhea:42532 / EC 5.4.99.25. action: ACCEPT reason: Recombinant human TRUB2 has tRNA Psi55 synthase activity in vitro, requiring the U54-A58 reverse Hoogsteen pair and enhanced by m1A58, and knockdown removes Psi55 activity from mitochondrial extracts. This is the best biochemically established activity of TRUB2 and the ancestral activity of the TruB family. supported_by: - *id001 - *id008 - term: id: GO:0160148 label: tRNA pseudouridine(55) synthase activity evidence_type: IMP original_reference_id: PMID:33023933 qualifier: enables review: summary: Knockdown reduces Psi55 synthesis by mitochondrial extracts. action: ACCEPT reason: Recombinant human TRUB2 has tRNA Psi55 synthase activity in vitro, requiring the U54-A58 reverse Hoogsteen pair and enhanced by m1A58, and knockdown removes Psi55 activity from mitochondrial extracts. This is the best biochemically established activity of TRUB2 and the ancestral activity of the TruB family. PAINT did not propagate this term to the Bilateria TRUB2 node, so transfer to orthologs should treat it as an experimental human finding. supported_by: - *id005 - *id001 - term: id: GO:1990481 label: mRNA pseudouridine synthesis evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: involved_in supporting_entities: - PANTHER:PTN000324318 - UniProtKB:O95900 review: summary: Phylogenetic inference of mRNA pseudouridine synthesis. The IBA derives from a PAINT IBD at Bilateria node PTN000324318, seeded solely by human TRUB2 (O95900); TRUB2's appearance in its own WITH/FROM reflects its experimental grounding, not circularity. action: KEEP_AS_NON_CORE reason: TRUB2 depletion lowers pseudouridylation at COX1 and COX3 mt-mRNA sites, and recombinant TRUB2 modified one site in vitro. However, the authors of the in vivo study argue RPUSD3 is the major enzyme and the TRUB2 effect secondary (TRUB2 knockdown also halves RPUSD3 mRNA), and the in vitro site was a cytosolic mRNA. The process is therefore plausible but only weakly established as a direct TRUB2 activity; kept as non-core. supported_by: - *id011 - &id015 reference_id: PMID:27974379 supporting_text: three lines of evidence suggest that RPUSD3 is the major modifying enzyme for COXI and COXIII mRNA and that the effect of TRUB2 is secondary - term: id: GO:1990481 label: mRNA pseudouridine synthesis evidence_type: IDA original_reference_id: PMID:31477916 qualifier: involved_in review: summary: Recombinant TRUB2 modified a single site in an in vitro mRNA pool. action: KEEP_AS_NON_CORE reason: TRUB2 depletion lowers pseudouridylation at COX1 and COX3 mt-mRNA sites, and recombinant TRUB2 modified one site in vitro. However, the authors of the in vivo study argue RPUSD3 is the major enzyme and the TRUB2 effect secondary (TRUB2 knockdown also halves RPUSD3 mRNA), and the in vitro site was a cytosolic mRNA. The process is therefore plausible but only weakly established as a direct TRUB2 activity; kept as non-core. supported_by: - *id013 - *id014 - term: id: GO:1990481 label: mRNA pseudouridine synthesis evidence_type: IMP original_reference_id: PMID:27974379 qualifier: involved_in review: summary: TRUB2 knockdown reduces pseudouridylation at COX1 and COX3 mt-mRNA sites (Psi-seq). action: KEEP_AS_NON_CORE reason: TRUB2 depletion lowers pseudouridylation at COX1 and COX3 mt-mRNA sites, and recombinant TRUB2 modified one site in vitro. However, the authors of the in vivo study argue RPUSD3 is the major enzyme and the TRUB2 effect secondary (TRUB2 knockdown also halves RPUSD3 mRNA), and the in vitro site was a cytosolic mRNA. The process is therefore plausible but only weakly established as a direct TRUB2 activity; kept as non-core. supported_by: - *id011 - *id015 references: - id: GO_REF:0000002 title: Gene Ontology annotation through association of InterPro records with GO terms findings: [] - id: GO_REF:0000033 title: Annotation inferences using phylogenetic trees findings: [] - id: GO_REF:0000044 title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt findings: [] - id: GO_REF:0000120 title: Combined Automated Annotation using Multiple IEA Methods findings: [] - id: PMID:22658674 title: Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. findings: [] reference_review: relevance: LOW correctness: VERIFIED review_notes: HeLa mRNA interactome capture; generic RNA binding only. - id: PMID:22681889 title: The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts. findings: [] reference_review: relevance: LOW correctness: VERIFIED review_notes: HEK293 mRNA-bound proteome; generic RNA binding only. - id: PMID:27667664 title: A Genome-wide CRISPR Death Screen Identifies Genes Essential for Oxidative Phosphorylation. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Full text read. CRISPR death screen; TRUB2 in NGRN-centred module required for 16S rRNA abundance and mitochondrial translation. - id: PMID:27974379 title: A pseudouridine synthase module is essential for mitochondrial protein synthesis and cell viability. findings: [] reference_review: relevance: HIGH correctness: VERIFIED review_notes: Full text read. Primary study of TRUB2 localization (BioID, IF), module membership, knockdown translation phenotype and Psi-seq; authors judge TRUB2's effect on COX1/COX3 mRNA pseudouridylation secondary to RPUSD3. - id: PMID:28082677 title: The Pseudouridine Synthase RPUSD4 Is an Essential Component of Mitochondrial RNA Granules. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Full text read. TRUB2 enriched in mitochondrial RNA granules by immunofluorescence. - id: PMID:31477916 title: mRNA structure determines modification by pseudouridine synthase 1. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Full text read. TRUB2 modified one site, in a cytosolic mRNA, in an in vitro mRNA-pool assay; authors doubt physiological relevance. - id: PMID:33023933 title: Mammalian nuclear TRUB1, mitochondrial TRUB2, and cytoplasmic PUS10 produce conserved pseudouridine 55 in different sets of tRNA. findings: [] reference_review: relevance: HIGH correctness: VERIFIED review_notes: Full text read. Recombinant TRUB2 tRNA Psi55 synthase activity and knockdown loss of mitochondrial-extract Psi55 activity; in vivo mt-tRNA targets only suggested. - id: PMID:34800366 title: Quantitative high-confidence human mitochondrial proteome and its dynamics in cellular context. findings: [] reference_review: relevance: LOW correctness: VERIFIED review_notes: High-throughput mitochondrial proteome; supports mitochondrial localization only. - id: Reactome:R-HSA-9936243 title: RPUSD4 in the mitochondrial pseudouridylation module transforms uridine-1397 to pseudouridine-1397 in mitochondrial 16S rRNA findings: [] - id: Reactome:R-HSA-9937008 title: Mitochondrial mRNA modification findings: [] - id: Reactome:R-HSA-9937369 title: RPUSD3,TRUB2 pseudouridylates MT-CO1,MT-CO3 mRNA findings: [] - id: file:human/TRUB2/TRUB2-uniprot.txt title: TRUB2 UniProt entry (O95900) findings: [] core_functions: - description: 'tRNA pseudouridine(55) synthase in the mitochondrial matrix: isomerizes uridine-55 in the T-loop of tRNA to pseudouridine; accounts for the Psi55 synthase activity of mitochondria (experimental in human; not propagated by PAINT).' molecular_function: id: GO:0160148 label: tRNA pseudouridine(55) synthase activity directly_involved_in: - *id006 locations: - *id002 supported_by: - *id001 - *id005 - *id016 - description: Pseudouridine synthase acting within mitochondrial RNA granules, contributing (apparently secondarily to RPUSD3) to pseudouridylation of COX1 and COX3 mitochondrial mRNAs. molecular_function: id: GO:0009982 label: pseudouridine synthase activity directly_involved_in: - *id007 locations: - *id002 - id: GO:0035770 label: ribonucleoprotein granule supported_by: - *id011 - *id015 - *id013 suggested_questions: - question: Is TRUB2 the enzyme that installs Psi55 in the four human mitochondrial tRNAs that carry it (mt-tRNA-Glu, -Gln, -Ser(UCN), -Tyr) in vivo, and if so should PAINT propagate tRNA pseudouridine(55) synthase activity to the Bilateria TRUB2 node? - question: Does TRUB2 pseudouridylate COX1/COX3 mt-mRNA directly, or is its effect indirect through reduced RPUSD3 expression? - question: Should GO provide a mitochondrial RNA granule cellular component term, given the number of mtRNA-processing and modification enzymes localized there? - question: Is 'positive regulation of mitochondrial translation' the right process term for modifying enzymes whose loss reduces mitochondrial translation, or does it overstate a regulatory role? suggested_experiments: - description: Map Psi55 in purified mitochondrial tRNAs (e.g. by CMC/RT stop, BID-seq or LC-MS of isolated mt-tRNAs) in TRUB2 knockout cells rescued with wild-type or catalytic Asp98 mutant TRUB2. - description: Test recombinant TRUB2 on in vitro transcribed COX1 and COX3 mt-mRNA fragments, and measure COX1/COX3 Psi levels in TRUB2 knockout cells with RPUSD3 expression restored. - description: Determine whether the ATP8/ATP6 translation defect of TRUB2 depletion requires catalytic activity by complementing with a catalytically dead TRUB2.