The Pseudouridine Synthase RPUSD4 Is an Essential Component of Mitochondrial RNA Granules

Abstract

Mitochondrial gene expression is a fundamental process that is largely dependent on nuclear-encoded proteins. Several steps of mitochondrial RNA processing and maturation, including RNA post-transcriptional modification, appear to be spatially organized into distinct foci, which we have previously termed mitochondrial RNA granules (MRGs). Although an increasing number of proteins have been localized to MRGs, a comprehensive analysis of the proteome of these structures is still lacking. Here, we have applied a microscopy-based approach that has allowed us to identify novel components of the MRG proteome. Among these, we have focused our attention on RPUSD4, an uncharacterized mitochondrial putative pseudouridine synthase. We show that RPUSD4 depletion leads to a severe reduction of the steady-state level of the 16S mitochondrial (mt) rRNA with defects in the biogenesis of the mitoribosome large subunit and consequently in mitochondrial translation. We report that RPUSD4 binds 16S mt-rRNA, mt-tRNAMet, and mt-tRNAPhe, and we demonstrate that it is responsible for pseudouridylation of the latter. These data provide new insights into the relevance of RNA pseudouridylation in mitochondrial gene expression.

Mettl17, a regulator of mitochondrial ribosomal RNA modifications, is required for the translation of mitochondrial coding genes

“…For 16S rRNA, a group of 2′‐O‐ribose methyltransferases [multiple reaction monitoring (MRM) 1, MRM2/FtsJ2, and MRM3/RNMTL1] have been shown to be involved in the modification of Gm1160, Um1387, and Gm1389, respectively, 3 nt positions of the peptidyl transferase center of 16S mt‐rRNA (10, 11). In addition, pseudouridine synthase RPUSD4 has been identified as an enzyme that provide the Psi1416 modification of 16S mt‐rRNA (12).…”

Mitochondrial RNA granules are fluid condensates, positioned by membrane dynamics

“…6a-d). To test whether dynamic exchange is generalizable beyond FASTKD2, we created two additional stable cell lines expressing MRG markers, ERAL1 and DDX28, fused to eGFP 29,30 . Both recovered rapidly, at a timescale (4.4 seconds, ERAL1 and 6.3 seconds, DDX28) similar to FASTKD2 ( Fig.…”

NSUN2 introduces 5-methylcytosines in mammalian mitochondrial tRNAs

“…Our experimental work has revealed several NSUN2-dependent targets in mtDNA-encoded mammalian tRNAs localised in the V-loop region (positions 48,49,50). The m 5 C modification is most often found at positions 48 and 49 of the V-loop, located between the anticodon arm and the T-arm.…”

“…Furthermore, there is increasing evidence that some of the most common heteroplasmic pathogenic mtDNA mutations perturb the introduction of mt-tRNA post-transcriptional modifications (14,47,48). Lastly, a number of mtRNA modifying enzymes have been identified and characterised using basic molecular and systems biology approaches (23,(49)(50)(51)(52)(53). Despite this recent progress several mitochondrial RNA modifications, including m 5 C, await the responsible enzyme to be identified.…”