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.

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.

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.