1728–1736 Nucleic Acids Research, 2012, Vol. 40, No. 4 doi:10.1093/nar/gkr941 Published online 27 October 2011 Yeast mitochondrial RNase P, RNase Z and the RNA degradosome are part of a stable supercomplex Rachid Daoud, Lise Forget and B. Franz Lang* Robert-Cedergren Centre for Bioinformatics and Genomics, Department of Biochemistry, Universite´ de Montre´ al, 2900 Edouard-Montpetit, Montreal, Quebec, H3T 1J4 Canada Received August 19, 2011; Revised September 30, 2011; Accepted October 12, 2011 ABSTRACT Initial steps in the synthesis of functional tRNAs require 50- and 30-processing of precursor tRNAs (pre-tRNAs), which in yeast mitochondria are achieved by two endonucleases, RNase P and RNase Z. In this study, using a combination of detergent-free Blue Native Gel Electrophoresis, proteomics and in vitro testing of pre-tRNA matur- ation, we reveal the physical association of these plus other mitochondrial activities in a large, stable complex of 136 proteins. It contains a total of seven proteins involved in RNA processing including RNase P and RNase Z, five out of six subunits of the mitochondrial RNA degradosome, components of the fatty acid synthesis pathway, translation, me- tabolism and protein folding. At the RNA level, there are the small and large rRNA subunits and RNase P RNA. Surprisingly, this complex is absent in an oar1" deletion mutant of the type II fatty acid syn- thesis pathway, supporting a recently published functional link between pre-tRNA processing and the FAS II pathway—apparently by integration into a large complex as we demonstrate here. Finally, the question of mt-RNase P localization within mitochondria was investigated, by GFP-tracing of a known protein subunit (Rpm2p). We find that about equal fractions of RNase P are soluble versus membrane-attached. INTRODUCTION To synthesize functional tRNAs, precursor tRNAs (pre-tRNA) need to be processed at their 50- and 30-termini. An almost ubiquitous RNase P is responsible for endonucleolytic 50-processing (1). Until the discovery that human and Arabidopsis mitochondrial RNase P are protein-only enzymes (2,3), these activities were thought to be always ribonucleo-proteins, composed of a catalytic RNA plus one or several proteins, throughout Archaea, Bacteria and eukaryotes (4–9). Processing of tRNA 30-termini is achieved by exonucleolytic and/or endonucleolytic activities, depending on the organism and the cellular compartment (10–14). In Escherichia coli, processing of 30-termini requires a multi-step process initiated by an endonucleotic cleavage, followed by exonucleolytic trimming (11). In other bacteria and most eukaryotes (including nuclear, mitochondria, and plastid) and in all Archaea, 30-tRNA processing is an endonucleolytic cleavage catalyzed by tRNase Z that exists in two forms, a long form of 750–930 amino acids only present in Eukarya (tRNase ZL), and a short form of 280–360 amino acids (tRNaseZ S) (15). Most information on mitochondrial (mt) pre-tRNA processing comes from studies of Saccharomyces cerevisiae. A highly puriﬁed form of mitochondrial RNase P (mt-RNase P) has been obtained by lysis of mitochondria in the presence of detergent and high salt, and a series of chromatographic steps. It contains the nucleus-encoded Rpm2p protein (Rpm2p; 119 kDa), and only an incomplete set of RNA fragments covering the mtDNA-encoded RPM1 RNA subunit (mt-P RNA) of 427 nt (16–18). Yet, given that yeast mt-P RNA has a highly reduced RNA structure compared to its cytosolic counterpart (19), we strongly expect that native mt-RNase P contains more than just one protein, to compensate for the lack of RNA structure [for comparison, cytosolic RNase P contains nine proteins; (20)]. Previous studies revealed that Rpm2p is involved not only in mt-RNase P activity, but also effects mitochondrial import (21), fer- mentative growth (22), and transcriptional activation of several nucleus-encoded mitochondrial components (23). In addition (and most curiously), tRNA processing inter- sects with the type II fatty acid synthesis pathway (24). Disruption of any enzyme in the FAS II pathway leads to a defective mt-RNase P (24). One possibility to explain *To whom correspondence should be addressed. Tel: 514 343 5842; Fax: 514 343 2210; Email: Franz.Lang@Umontreal.ca The Author(s) 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.