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

Sofia Zaganelli,Pedro Rebelo-Guiomar,Kinsey Maundrell,Agata Rozanska,Sandra Pierredon,Christopher A Powell,Alexis A Jourdain,Nicolas Hulo,Robert N Lightowlers,Zofia M Chrzanowska-Lightowlers,Michal Minczuk,Jean-Claude Martinou

doi:10.1074/jbc.m116.771105

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.

Highlights

Both strands of the mitochondrial DNA (mtDNA) are transcribed in the form of long polycistronic precursor RNAs, which are processed to release the individual mt-tRNAs, mt-rRNAs, and mt-mRNAs [4, 5]
We added a number of other mitochondrial proteins, which not necessarily binding directly to RNA appeared from previous studies to play a role in mitochondrial gene expression
We identified 12 new mitochondrial RNA granules (MRGs)-associated proteins (Table 1), including proteins involved in RNA processing (FASTKD1, PTCD1, and PTCD2), RNA modification (TFB1M, TRUB2, RPUSD3, and RPUSD4), mitoribosome assembly (ERAL1 and NOA1/C4orf14), and structural components of mitoribosome (MRPL47, MRPS7, and MRPS9)

Summary

Introduction

Both strands of the mtDNA are transcribed in the form of long polycistronic precursor RNAs, which are processed to release the individual mt-tRNAs, mt-rRNAs, and mt-mRNAs [4, 5]. Little attention was given to these compartments until more recently when some groups, including ours, reported that a number of proteins involved in mtRNA processing, mitoribosome subunit assembly, and translation-associated factors was found to localize in these structures (for a review, see Ref. 15). The identification of such a panel of MRG-associated proteins led us to conclude that many, if not all, stages of mitochondrial gene expression are centered on these granules. We report that RPUSD4 interacts physically with the 16S mt-rRNA, mttRNAMet, and mt-tRNAPhe, and we present evidence that indicates that RPUSD4 is responsible for the formation of pseudouridine in the mt-tRNAPhe

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Results

Conclusion