title: A pseudouridine synthase module is essential for mitochondrial protein synthesis and cell viability.
abstract: Pseudouridylation is a common post-transcriptional modification in RNA, but its functional consequences at the cellular level remain largely unknown. Using a proximity-biotinylation assay, we identified a protein module in mitochondrial RNA granules, platforms for post-transcriptional RNA modification and ribosome assembly, containing several proteins of unknown function including three uncharacterized pseudouridine synthases, TRUB2, RPUSD3, and RPUSD4. TRUB2 and RPUSD4 were previously identified as core essential genes in CRISPR/Cas9 screens. Depletion of the individual enzymes produced specific mitochondrial protein synthesis and oxidative phosphorylation assembly defects without affecting mitochondrial mRNA levels. Investigation of the molecular targets in mitochondrial RNA by pseudouridine-Seq showed that RPUSD4 plays a role in the pseudouridylation of a single residue in the 16S rRNA, a modification that is essential for its stability and assembly into the mitochondrial ribosome, while TRUB2/RPUSD3 were similarly involved in pseudouridylating specific residues in mitochondrial mRNAs. These results establish essential roles for epitranscriptomic modification of mitochondrial RNA in mitochondrial protein synthesis, oxidative phosphorylation, and cell survival.
authors.affiliation: Department of Human Genetics, Montreal Neurological Institute, McGill University, Montreal, QC, Canada.
authors.forename: Hana
authors.initials: H
authors.lastname: Antonicka
authors.affiliation: Department of Human Genetics, Montreal Neurological Institute, McGill University, Montreal, QC, Canada.
authors.forename: Karine
authors.initials: K
authors.lastname: Choquet
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada.
authors.forename: Zhen-Yuan
authors.initials: ZY
authors.lastname: Lin
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada.|Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada.
authors.forename: Anne-Claude
authors.initials: AC
authors.lastname: Gingras
authors.affiliation: Department of Human Genetics, Segal Cancer Centre and Lady Davis Institute, Jewish General Hospital, McGill University, Montréal, QC, Canada.
authors.forename: Claudia L
authors.initials: CL
authors.lastname: Kleinman
authors.affiliation: Department of Human Genetics, Montreal Neurological Institute, McGill University, Montreal, QC, Canada eric@ericpc.mni.mcgill.ca.
authors.forename: Eric A
authors.identifier: 0000-0003-1498-9473
authors.initials: EA
authors.lastname: Shoubridge
mesh_terms: D002352:Carrier Proteins; D002460:Cell Line; D002470:Cell Survival; D006801:Humans; D019751:Intramolecular Transferases / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000096:biosynthesis*; D011485:Protein Binding; D021381:Protein Transport; D012313:RNA / Q000235:genetics / Q000378:metabolism; D034443:RNA Transport; D000077278:RNA, Mitochondrial; D012335:RNA, Ribosomal / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D024101:Mitochondrial Proteins; D000077278:RNA, Mitochondrial; D012335:RNA, Ribosomal; D012313:RNA; D019751:Intramolecular Transferases; C096995:pseudouridine synthases
keywords: epitranscriptomic modification; mitochondrial protein synthesis; oxidative phosphorylation; pseudouridine synthase; ribosome assembly