On the basis of the sequence of the mitochondrial genome in the flowering plant Arabidopsis thaliana, RNA editing events were systematically investigated in the respective RNA population. A total of 456 C to U, but no U to C, conversions were identified exclusively in mRNAs, 441 in ORFs, 8 in introns, and 7 in leader and trailer sequences. No RNA editing was seen in any of the rRNAs or in several tRNAs investigated for potential mismatch corrections. RNA editing affects individual coding regions with frequencies varying between 0 and 18.9% of the codons. The predominance of RNA editing events in the first two codon positions is not related to translational decoding, because it is not correlated with codon usage. As a general effect, RNA editing increases the hydrophobicity of the coded mitochondrial proteins. Concerning the selection of RNA editing sites, little significant nucleotide preference is observed in their vicinity in comparison to unedited C residues. This sequence bias is, per se, not sufficient to specify individual C nucleotides in the total RNA population in Arabidopsis mitochondria.

The ubiquitous endonuclease RNase P is responsible for the 5' maturation of tRNA precursors. Until the discovery of human mitochondrial RNase P, these enzymes had typically been found to be ribonucleoproteins, the catalytic activity of which is associated with the RNA component. Here we show that, in Arabidopsis thaliana mitochondria and plastids, a single protein called 'proteinaceous RNase P' (PRORP1) can perform the endonucleolytic maturation of tRNA precursors that defines RNase P activity. In addition, PRORP1 is able to cleave tRNA-like structures involved in the maturation of plant mitochondrial mRNAs. Finally, we show that Arabidopsis PRORP1 can replace the bacterial ribonucleoprotein RNase P in Escherichia coli cells. PRORP2 and PRORP3, two paralogs of PRORP1, are both localized in the nucleus.

Mitochondria fulfill a wide range of metabolic functions in addition to the synthesis of ATP and contain a diverse array of proteins to perform these functions. Here, we present the unexpected discovery of the presence of the enzymes of glycolysis in a mitochondrial fraction of Arabidopsis cells. Proteomic analyses of this mitochondrial fraction revealed the presence of 7 of the 10 enzymes that constitute the glycolytic pathway. Four of these enzymes (glyceraldehyde-3-P dehydrogenase, aldolase, phosphoglycerate mutase, and enolase) were also identified in an intermembrane space/outer mitochondrial membrane fraction. Enzyme activity assays confirmed that the entire glycolytic pathway was present in preparations of isolated Arabidopsis mitochondria, and the sensitivity of these activities to protease treatments indicated that the glycolytic enzymes are present on the outside of the mitochondrion. The association of glycolytic enzymes with mitochondria was confirmed in vivo by the expression of enolase-and aldolase-yellow fluorescent protein fusions in Arabidopsis protoplasts. The yellow fluorescent protein fluorescence signal showed that these two fusion proteins are present throughout the cytosol but are also concentrated in punctate regions that colocalized with the mitochondrion-specific probe Mitotracker Red. Furthermore, when supplied with appropriate cofactors, isolated, intact mitochondria were capable of the metabolism of 13 C-glucose to 13 C-labeled intermediates of the trichloroacetic acid cycle, suggesting that the complete glycolytic sequence is present and active in this subcellular fraction. On the basis of these data, we propose that the entire glycolytic pathway is associated with plant mitochondria by attachment to the cytosolic face of the outer mitochondrial membrane and that this microcompartmentation of glycolysis allows pyruvate to be provided directly to the mitochondrion, where it is used as a respiratory substrate.

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RNase P is an essential enzyme that cleaves the 59 leader sequence of tRNA precursors. RNase Ps were believed until now to occur universally as ribonucleoproteins in organisms performing RNase P activity. Here we find that protein-only RNase P enzymes called PRORP (for proteinaceous RNase P) support RNase P activity in vivo in both organelles and the nucleus in Arabidopsis. Beyond tRNA, PRORP proteins are involved in the maturation of small nucleolar RNA (snoRNA) and mRNA. Finally, ribonucleoprotein RNase MRP is not involved in tRNA maturation in plants. Altogether, our results indicate that ribonucleoprotein enzymes have been entirely replaced by proteins for RNase P activity in plants.Supplemental material is available for this article.Received February 10, 2012; revised version accepted April 5, 2012.RNase P is a virtually universal enzyme involved in the maturation of tRNAs, as it cleaves the 59 leader sequence of tRNA precursors. It is thus essential to obtain functional tRNAs and is therefore pivotal for translation (Lai et al. 2010;Reiter et al. 2010). RNase P activities from all phyla of life were assumed to be universally performed by ribonucleoprotein enzymes whose catalytic activities are held by ribozymes (Altman 2007). This concept was first challenged with the proposition that spinach chloroplast and human mitochondria RNase Ps would not contain any RNA moiety (Wang et al. 1988;Rossmanith and Karwan 1998). More recently, protein-only RNase P enzymes called PRORP (for proteinaceous RNase P) have been characterized at the molecular level in endosymbiotic organelles in both humans and Arabidopsis (Holzmann et al. 2008;Gobert et al. 2010). Still, the dogma remained that RNase P enzymes would nonetheless universally occur as ribonucleoproteins in living organisms performing RNase P activity, with protein-only RNase Ps being marginal exceptions restricted to only some organelles (e.g., Esakova and Krasilnikov 2010).The putative PRORP RNase P enzymes are characterized by the occurrence of a conserved ''NYN'' metallonuclease domain (Anantharaman and Aravind 2006). PRORP proteins also belong to the huge pentatricopeptide repeat (PPR) protein family. These proteins, typically from eukaryotes, are involved in a wide variety of posttranscriptional mechanisms (Schmitz-Linneweber and Small 2008). However, no functional information was available for PRORP proteins. We previously established that Arabidopsis PRORP1, a protein localized in organelles, can act in vitro as an RNase P enzyme that is a single protein (Gobert et al. 2010), although its function remained elusive in planta. We also used localization experiments (YFP fusions and immunodetections) to determine that PRORP2 and PRORP3, two paralogs of PRORP1, were present in Arabidopsis nuclei (Gobert et al. 2010). In addition, the fast-growing amount of genomic data has revealed that some important groups of eukaryotes, such as land plants and kinetoplastids, do not encode any recognizable genes for RNase P RNA or for proteins specific for ribonucleoprotein...

Mitochondria are the powerhouses of eukaryotic cells. They are considered as semi-autonomous because they have retained genomes inherited from their prokaryotic ancestor and host fully functional gene expression machineries. These organelles have attracted considerable attention because they combine bacterial-like traits with novel features that evolved in the host cell. Among them, mitochondria use many specific pathways to obtain complete and functional sets of tRNAs as required for translation. In some instances, tRNA genes have been partially or entirely transferred to the nucleus and mitochondria require precise import systems to attain their pool of tRNAs. Still, tRNA genes have also often been maintained in mitochondria. Their genetic arrangement is more diverse than previously envisaged. The expression and maturation of mitochondrial tRNAs often use specific enzymes that evolved during eukaryote history. For instance many mitochondria use a eukaryote-specific RNase P enzyme devoid of RNA. The structure itself of mitochondrial encoded tRNAs is also very diverse, as e.g., in Metazoan, where tRNAs often show non canonical or truncated structures. As a result, the translational machinery in mitochondria evolved adapted strategies to accommodate the peculiarities of these tRNAs, in particular simplified identity rules for their aminoacylation. Here, we review the specific features of tRNA biology in mitochondria from model species representing the major eukaryotic groups, with an emphasis on recent research on tRNA import, maturation and aminoacylation.

Mitochondrial biogenesis and function require the regulated and coordinated expression of nuclear and mitochondrial genomes throughout plant development and in response to cellular and environmental signals. To investigate the levels at which the expression of nuclear and mitochondrially encoded proteins is coordinated, we established an Arabidopsis thaliana cell culture system to modulate mitochondrial biogenesis in response to sugar starvation and refeeding. Sucrose deprivation led to structural changes in mitochondria, a decrease in mitochondrial volume, and a reduction in the rate of cellular respiration. All these changes could be reversed by the readdition of sucrose. Analysis of the relative mRNA transcript abundance of genes encoding nuclear and mitochondrially encoded proteins revealed that there was no coordination of expression of the two genomes at the transcript level. An analysis of changes in abundance and assembly of nuclear-encoded and mitochondrially encoded subunits of complexes I to V of the mitochondrial inner membrane in organello protein synthesis and competence for protein import by isolated mitochondria suggested that coordination occurs at the level of protein-complex assembly. These results further suggest that expression of the mitochondrial genome is insensitive to the stress imposed by sugar starvation and that mitochondrial biogenesis is regulated by changes in nuclear gene expression and coordinated at the posttranslational level.

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