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.
Using a proximity biotinylation assay, the authors identify a pseudouridine synthase module in mitochondrial RNA granules that is essential for epigenetic modification of the mitochondrial transcriptome, ribosome biogenesis, and mitochondrial protein synthesis.
Mitochondrial RNA granules contain a pseudouridine synthase module.
Pseudouridylation of 16S rRNA by RPUSD4 is essential for mitochondrial ribosome assembly.
Pseudouridylation of specific mitochondrial mRNAs by TRUB2/RPUSD3 is necessary for their translation.
Epitranscriptomic modification of mitochondrial RNA is thus essential for cell viability.
- Received September 22, 2016.
- Revision received November 21, 2016.
- Accepted November 22, 2016.
- © 2016 The Authors
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