Rapid Shifts in Mitochondrial tRNA Import in a Plant Lineage with Extensive Mitochondrial tRNA Gene Loss.

In most eukaryotes, transfer RNAs (tRNAs) are one of the very few classes of genes remaining in the mitochondrial genome, but some mitochondria have lost these vestiges of their prokaryotic ancestry. Sequencing of mitogenomes from the flowering plant genus Silene previously revealed a large range in tRNA gene content, suggesting rapid and ongoing gene loss/replacement. Here, we use this system to test longstanding hypotheses about how mitochondrial tRNA genes are replaced by importing nuclear-encoded tRNAs. We traced the evolutionary history of these gene loss events by sequencing mitochondrial genomes from key outgroups (Agrostemma githago and Silene [=Lychnis] chalcedonica). We then performed the first global sequencing of purified plant mitochondrial tRNA populations to characterize the expression of mitochondrial-encoded tRNAs and the identity of imported nuclear-encoded tRNAs. We also confirmed the utility of high-throughput sequencing methods for the detection of tRNA import by sequencing mitochondrial tRNA populations in a species (Solanum tuberosum) with known tRNA trafficking patterns. Mitochondrial tRNA sequencing in Silene revealed substantial shifts in the abundance of some nuclear-encoded tRNAs in conjunction with their recent history of mt-tRNA gene loss and surprising cases where tRNAs with anticodons still encoded in the mitochondrial genome also appeared to be imported. These data suggest that nuclear-encoded counterparts are likely replacing mitochondrial tRNAs even in systems with recent mitochondrial tRNA gene loss, and the redundant import of a nuclear-encoded tRNA may provide a mechanism for functional replacement between translation systems separated by billions of years of evolutionary divergence.

Complete Sequence, Multichromosomal Architecture and Transcriptome Analysis of the Solanum tuberosum Mitochondrial Genome.

Mitochondrial genomes (mitogenomes) in higher plants can induce cytoplasmic male sterility and be somehow involved in nuclear-cytoplasmic interactions affecting plant growth and agronomic performance. They are larger and more complex than in other eukaryotes, due to their recombinogenic nature. For most plants, the mitochondrial DNA (mtDNA) can be represented as a single circular chromosome, the so-called master molecule, which includes repeated sequences that recombine frequently, generating sub-genomic molecules in various proportions. Based on the relevance of the potato crop worldwide, herewith we report the complete mtDNA sequence of two S. tuberosum cultivars, namely Cicero and Désirée, and a comprehensive study of its expression, based on high-coverage RNA sequencing data. We found that the potato mitogenome has a multi-partite architecture, divided in at least three independent molecules that according to our data should behave as autonomous chromosomes. Inter-cultivar variability was null, while comparative analyses with other species of the Solanaceae family allowed the investigation of the evolutionary history of their mitogenomes. The RNA-seq data revealed peculiarities in transcriptional and post-transcriptional processing of mRNAs. These included co-transcription of genes with open reading frames that are probably expressed, methylation of an rRNA at a position that should impact translation efficiency and extensive RNA editing, with a high proportion of partial editing implying frequent mis-targeting by the editing machinery.

A genome-scale analysis of mRNAs targeting to plant mitochondria: upstream AUGs in 5' untranslated regions reduce mitochondrial association.

Intracellular sorting of mRNAs is an essential process for regulating gene expression and protein localization. Most mitochondrial proteins are nuclear-encoded and imported into the mitochondria through post-translational or co-translational processes. In the latter case, mRNAs are found to be enriched in the vicinity of mitochondria. A genome-scale analysis of mRNAs associated with mitochondria has been performed to determine plant cytosolic mRNAs targeted to the mitochondrial surface. Many messengers encoding mitochondrial proteins were found associated with mitochondria. These mRNAs correspond to particular functions and complexes, such as respiration or mitoribosomes, which indicates a coordinated control of mRNA localization within metabolic pathways. In addition, upstream AUGs in 5' untranslated regions (UTRs), which modulate the translation efficiency of downstream sequences, were found to negatively affect the association of mRNAs with mitochondria. A mutational approach coupled with in vivo mRNA visualization confirmed this observation. Moreover, this technique allowed the identification of 3'-UTRs as another essential element for mRNA localization at the mitochondrial surface. Therefore, this work offers new insights into the mechanism, function and regulation of the association of cytosolic mRNAs with plant mitochondria.

In vitro RNA uptake studies in plant mitochondria.

During evolution, most of the ancestral genes from the endosymbiotic α-proteobacteria at the origin of mitochondria have been either lost or transferred to the nuclear genome. To allow the comeback of proteins and RNAs [in particular transfer RNA (tRNAs)] into the organelle, macromolecule import systems were universally established. While protein import processes have been studied into details, much less is known about tRNA mitochondrial import. In plants, part of the knowledge on the tRNA import process into mitochondria has been acquired thanks to in vitro import assays. Furthermore, the development of in vitro RNA import strategies allowed the study of plant mitochondrial gene expression. The purpose of this chapter is to provide detailed protocols to perform in vitro RNA uptake into potato (Solanum tuberosum) or Arabidopsis (Arabidopsis thaliana) mitochondria as well as approaches to analyze them.

Molecular basis for the differential interaction of plant mitochondrial VDAC proteins with tRNAs.

In plants, the voltage-dependent anion-selective channel (VDAC) is a major component of a pathway involved in transfer RNA (tRNA) translocation through the mitochondrial outer membrane. However, the way in which VDAC proteins interact with tRNAs is still unknown. Potato mitochondria contain two major mitochondrial VDAC proteins, VDAC34 and VDAC36. These two proteins, composed of a N-terminal α-helix and of 19 ß-strands forming a ß-barrel structure, share 75% sequence identity. Here, using both northwestern and gel shift experiments, we report that these two proteins interact differentially with nucleic acids. VDAC34 binds more efficiently with tRNAs or other nucleic acids than VDAC36. To further identify specific features and critical amino acids required for tRNA binding, 21 VDAC34 mutants were constructed and analyzed by northwestern. This allowed us to show that the ß-barrel structure of VDAC34 and the first 50 amino acids that contain the α-helix are essential for RNA binding. Altogether the work shows that during evolution, plant mitochondrial VDAC proteins have diverged so as to interact differentially with nucleic acids, and this may reflect their involvement in various specialized biological functions.

Targeting of cytosolic mRNA to mitochondria: naked RNA can bind to the mitochondrial surface.

Mitochondria contain hundreds of proteins but only a few are encoded by the mitochondrial genome. The other proteins are nuclear-encoded and imported into mitochondria. These proteins can be translated on free cytosolic polysomes, then targeted and imported into mitochondria. Nonetheless, numerous cytosolic mRNAs encoding mitochondrial proteins are detected at the surface of mitochondria in yeast, plants and animals. The localization of mRNAs to the vicinity of mitochondria would be a way for mitochondrial protein sorting. The mechanisms responsible for mRNA targeting to mitochondria are not clearly identified. Sequences within the mRNA molecules (cis-elements), as well as a few trans-acting factors, have been shown to be essential for targeting of some mRNAs. In order to identify receptors involved in mRNA docking to the mitochondrial surface, we have developed an in vitro mRNA binding assay with isolated plant mitochondria. We show that naked mRNAs are able to bind to isolated mitochondria, and our results strongly suggest that mRNA docking to the plant mitochondrial outer membrane requires at least one component of TOM complex.

A protein shuttle system to target RNA into mitochondria.

Mitochondria play a key role in essential cellular functions. A deeper understanding of mitochondrial molecular processes is hampered by the difficulty of incorporating foreign nucleic acids into organelles. Mitochondria of most eukaryotic species import cytosolic tRNAs. Based on this natural process, we describe here a powerful shuttle system to internalize several types of RNAs into isolated mitochondria. We demonstrate that this tool is useful to investigate tRNA processing or mRNA editing in plant mitochondria. Furthermore, we show that the same strategy can be used to address both tRNA and mRNA to isolated mammalian mitochondria. We anticipate our novel approach to be the starting point for various studies on mitochondrial processes. Finally, our study provides new insights into the mechanism of RNA import into mitochondria.

Plant mitochondria use two pathways for the biogenesis of tRNAHis.

All tRNA(His) possess an essential extra G(-1) guanosine residue at their 5' end. In eukaryotes after standard processing by RNase P, G(-1) is added by a tRNA(His) guanylyl transferase. In prokaryotes, G(-1) is genome-encoded and retained during maturation. In plant mitochondria, although trnH genes possess a G(-1) we find here that both maturation pathways can be used. Indeed, tRNA(His) with or without a G(-1) are found in a plant mitochondrial tRNA fraction. Furthermore, a recombinant Arabidopsis mitochondrial RNase P can cleave tRNA(His) precursors at both positions G(+1) and G(-1). The G(-1) is essential for recognition by plant mitochondrial histidyl-tRNA synthetase. Whether, as shown in prokaryotes and eukaryotes, the presence of uncharged tRNA(His) without G(-1) has a function or not in plant mitochondrial gene regulation is an open question. We find that when a mutated version of a plant mitochondrial trnH gene containing no encoded extra G is introduced and expressed into isolated potato mitochondria, mature tRNA(His) with a G(-1) are recovered. This shows that a previously unreported tRNA(His) guanylyltransferase activity is present in plant mitochondria.

RNA trafficking in plant cells: targeting of cytosolic mRNAs to the mitochondrial surface.

Subcellular localization of mRNA is a widespread and efficient way for targeting proteins to specific regions of a cell. Messenger RNA sorting appears as a key mechanism for posttranscriptional gene regulation, and its involvement in organelle biogenesis has been described in different organisms. Here we demonstrate that mRNA targeting to the surface of mitochondria occurs in higher plants. Cytosolic mRNAs corresponding to mitochondrial proteins, but also to some particular cytosolic proteins, were found associated to mitochondria, offering new perspectives for mitochondria biogenesis in plant cells.

Dual-targeted tRNA-dependent amidotransferase ensures both mitochondrial and chloroplastic Gln-tRNAGln synthesis in plants.

Aminoacyl-tRNAs are generally formed by direct attachment of an amino acid to tRNAs by aminoacyl-tRNA synthetases, but Gln-tRNA is an exception to this rule. Gln-tRNA(Gln) is formed by this direct pathway in the eukaryotic cytosol and in protists or fungi mitochondria but is formed by an indirect transamidation pathway in most of bacteria, archaea, and chloroplasts. We show here that the formation of Gln-tRNA(Gln) is also achieved by the indirect pathway in plant mitochondria. The mitochondrial-encoded tRNA(Gln), which is the only tRNA(Gln) present in mitochondria, is first charged with glutamate by a nondiscriminating GluRS, then is converted into Gln-tRNA(Gln) by a tRNA-dependent amidotransferase (AdT). The three subunits GatA, GatB, and GatC are imported into mitochondria and assemble into a functional GatCAB AdT. Moreover, the mitochondrial pathway of Gln-tRNA(Gln) formation is shared with chloroplasts as both the GluRS, and the three AdT subunits are dual-imported into mitochondria and chloroplasts.

Four paralogues of RPL12 are differentially associated to ribosome in plant mitochondria.

Ribosomal protein L12 is the only component present in four copies in the ribosome. In prokaryotes as well as in yeast and human mitochondria, all copies correspond to the same RPL12. By contrast, we present here evidence that plant mitochondria contain four different RPL12 proteins. Compared to E. coli RPL12, the four mature RPL12 variants show a conserved C-terminal region that contains all the functional domains of prokaryotic RPL12 but three of them present an additional N-terminal extension containing either an acidic or a basic domain and a high level of proline residues. All proteins have a potential mitochondrial N-terminal targeting sequence and were imported in vitro into isolated mitochondria. Using RPL12 antibodies, the four variants were shown to be present in a potato mitochondrial ribosome fraction. Moreover, the four proteins reacted differently to the destabilization of ribosomes. This suggests either a heterogeneous RPL12 composition among each ribosome and/or a heterogeneous population of plant mitochondrial ribosomes.

The voltage-dependent anion channel, a major component of the tRNA import machinery in plant mitochondria.

In plants, as in most eukaryotic cells, import of nuclear-encoded cytosolic tRNAs is an essential process for mitochondrial biogenesis. Despite its broad occurrence, the mechanisms governing RNA transport into mitochondria are far less understood than protein import. This article demonstrates by Northwestern and gel-shift experiments that the plant mitochondrial voltage-dependent anion channel (VDAC) protein interacts with tRNA in vitro. It shows also that this porin, known to play a key role in metabolite transport, is a major component of the channel involved in the tRNA translocation step through the plant mitochondrial outer membrane, as supported by inhibition of tRNA import into isolated mitochondria by VDAC antibodies and Ruthenium red. However VDAC is not a tRNA receptor on the outer membrane. Rather, two major components from the TOM (translocase of the outer mitochondrial membrane) complex, namely TOM20 and TOM40, are important for tRNA binding at the surface of mitochondria, suggesting that they are also involved in tRNA import. Finally, we show that proteins and tRNAs are translocated into plant mitochondria by different pathways. Together, these findings identify unexpected components of the tRNA import machinery and suggest that the plant tRNA import pathway has evolved by recruiting multifunctional proteins.

Fate of a larch unedited tRNA precursor expressed in potato mitochondria.

In higher plant mitochondria, post-transcriptional C to U conversion known as editing mostly affects mRNAs. However, three tRNAs were also shown to be edited. Among them, three editing sites were identified in larch mitochondrial tRNA(His). We have previously shown that only the edited version can undergo maturation in vitro. In this paper, we introduced via direct DNA uptake the edited or unedited version of larch mitochondrial trnH into isolated potato mitochondria and expressed them under the control of potato mitochondrial 18 S rRNA promoter. As expected, the edited form of larch mitochondrial tRNA(His) precursor was processed in the isolated organelles. By contrast, no mature tRNA(His) was detected when using the unedited version of trnH. However, precursor molecules could be characterized by reverse transcription-PCR. These data demonstrate that the potato mitochondrial editing machinery is not able to recognize these "foreign" editing sites and confirm that these unedited tRNA precursor molecules are not correctly processed in organello. As a consequence, the fate of these RNA precursor molecules is likely to be degradation. Indeed, we detected by PCR two 3'-end truncated precursor RNAs. Interestingly, both RNA species exhibit poly(A) tails, a hallmark of degradation in plant mitochondria. Taken together, these data suggest that, in plant mitochondria, a defective unedited RNA precursor that cannot be processed to give a mature stable tRNA, is degraded through a polyadenylation-dependent pathway.

In vitro import of a nuclearly encoded tRNA into mitochondria of Solanum tuberosum.

Some of the mitochondrial tRNAs of higher plants are nuclearly encoded and imported into mitochondria. The import of tRNAs encoded in the nucleus has been shown to be essential for proper protein translation within mitochondria of a variety of organisms. Here, we report the development of an in vitro assay for import of nuclearly encoded tRNAs into plant mitochondria. This in vitro system utilizes isolated mitochondria from Solanum tuberosum and synthetic tRNAs transcribed from cloned nuclear tRNA genes. Although incubation of radioactively labeled in vitro-transcribed tRNA(Ala), tRNA(Phe), and tRNA(Met-e) with isolated potato mitochondria resulted in importation, as measured by nuclease protection, the amount of tRNA transcripts protected at saturation was at least five times higher for tRNA(Ala) than for the two other tRNAs. This difference in in vitro saturation levels of import is consistent with the in vivo localization of these tRNAs, since cytosolic tRNA(Ala) is naturally imported into potato mitochondria whereas tRNA(Phe) and tRNA(Met-e) are not. Characterization of in vitro tRNA import requirements indicates that mitochondrial tRNA import proceeds in the absence of any added cytosolic protein fraction, involves at least one protein component on the surface of mitochondria, and requires ATP-dependent step(s) and a membrane potential.

A family of RRM-type RNA-binding proteins specific to plant mitochondria.

Expression of higher plant mitochondrial (mt) genes is regulated at the transcriptional, posttranscriptional, and translational levels, but the vast majority of the mtDNA and RNA-binding proteins involved remain to be identified. Plant mt single-stranded nucleic acid-binding proteins were purified by affinity chromatography, and corresponding genes have been identified. A majority of these proteins belong to a family of RNA-binding proteins characterized by the presence of an N-terminal RNA-recognition motif (RRM) sequence. They diverge in their C-terminal sequences, suggesting that they can be involved in different plant mt regulation processes. Mitochondrial localization of the proteins was confirmed both in vitro and in vivo and by immunolocalization. Binding experiments showed that several proteins have a preference for poly(U)-rich sequences. This mt protein family contains the ubiquitous RRM motif and has no known mt counterpart in non-plant species. Phylogenetic and functional analysis suggest a common ancestor with RNA-binding glycine-rich proteins (GRP), a family of developmentally regulated proteins of unknown function. As with several plant, cyanobacteria, and animal proteins that have similar structures, the expression of one of the Arabidopsis thaliana mt RNA-binding protein genes is induced by low temperatures.