Yeast mitochondrial RNase P, RNase Z and the RNA degradosome are part of a stable supercomplex

Abstract

Initial steps in the synthesis of functional tRNAs require 5′- and 3′-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 maturation, 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, metabolism 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 oar1Δ deletion mutant of the type II fatty acid synthesis 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.

Massive programmed translational jumping in mitochondria

“…Isolated mitochondria were solubilized with digitonin and homogenized, and the remaining membranes were removed by centrifugation. The supernatant was electrophoretically separated on a nondenaturating poly-acrylamide gel (37), individual bands were cut out and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) (38,39), and peptides were annotated by Mascot (40). LacZ Constructs of Byps and Expression in E. coli.…”

Transfer RNA Post-Transcriptional Processing, Turnover, and Subcellular Dynamics in the YeastSaccharomyces cerevisiae

“…rRNA, and snRNAs (van Hoof et al 2000;Copela et al 2008;Ozanick et al 2009). RNase Z, Trz1, is the endonuclease that participates in 39 end processing for both mitochondrial and nuclear encoded tRNAs (Chen et al 2005;Daoud et al 2011;Maraia and Lamichhane 2011). The exo-and endonucleases have been proposed to have differential access to particular pre-tRNAs dependent upon tRNA binding by the La protein (Lhp1), such that La binding to tRNA 39 ends inhibits access to Rex1 and 39 maturation occurs via Trz1-mediated endonucleolytic cleavage (Yoo and Wolin 1997).…”

tRNA 3′ processing in yeast involves tRNase Z, Rex1, and Rrp6

“…The main 3 ′ -5 ′ exonucleolytic activity in yeast mitochondria, responsible for mtRNA processing and turnover, is provided by the degradosome (mtEXO), composed of RNase Dss1 and helicase Suv3 (Dziembowski et al 2003;Lipinski et al 2010). Importantly, three mitochondrial RNA processing activities, RNase P, RNase Z, and mtEXO, were recently reported to form a stable RMT (RNA processing, Metabolism, Translation) supercomplex (Daoud et al 2012). However, yeast suv3Δ or dss1Δ strains exhibit a particularly high level of mitochondrial genome instability due to inhibition of translation , and it was not practical to combine these deletions with a dysfunction of Trz1, which also results in a similar mt phenotype.…”

“…However, yeast suv3Δ or dss1Δ strains exhibit a particularly high level of mitochondrial genome instability due to inhibition of translation , and it was not practical to combine these deletions with a dysfunction of Trz1, which also results in a similar mt phenotype. In addition, mtEXO was reported not to be involved in tRNA processing (Dziembowski et al 2003), and its presence in the RMT supercomplex probably reflects its participation in maturation of rRNA and/or mRNA molecules, as rRNA subunits, some ribosomal proteins, and rRNA or mRNA processing factors are also part of this complex (Daoud et al 2012).…”