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Organelles ensure fundamental functions in energy production and other metabolic pathways. Owing to their bacterial origin, mitochondria and chloroplasts still contain DNA but with a limited set of genes. Nevertheless, their expression is essential and dysfunction is usually deleterious for the cell. Otherwise, most organellar proteins and half of the mitochondrial tRNA species are expressed from nuclear genes and the biogenesis of organelles relies on efficient macromolecule trafficking systems. The existence of such traffics between the nucleus, the cytosol and plant organelles is well established, but many questions remain unanswered. In particular, while protein import has been studied in great details in yeast much less is known about protein and RNA trafficking to plant mitochondria and chloroplasts.

Our team has a long history in studying two key components of the cytosolic, plastidial and mitochondrial translation machineries: aminoacyl-tRNA synthetases and tRNAs. This allowed us to demonstrate key steps of organellar biogenesis, among them we can cite tRNA editing, cytosolic tRNA mitochondrial import, dual targeting of aminoacyl-tRNA synthetases to both organelles. Thanks to our present knowledge and expertise and to various collaborations, we continue to better unravel macromolecule trafficking at the protein, mRNA and tRNA levels to plant organelles.

tRNA import process into plant mitochondria

In plants, as in most eukaryotic cells, import of nuclear-encoded cytosolic tRNAs is an essential process for mitochondrial biogenesis. For example land plant mitochondrial genomes lack at least one-third of the tRNA genes and in the green alga, Chlamydomonas reinardtii, the mitochondrial genome only encodes three tRNA genes and all other mitochondrial tRNAs are encoded by nuclear genes and imported into mitochondria.

Despite its broad occurrence, the mechanisms governing RNA transport into mitochondria are far less understood than protein import. Four major questions need to be answered: how is the selectivity of the process achieved, how the targeting step from the nucleus to the surface of mitochondria, how the translocation step through the double mitochondrial membranes are done, and finally is the import process regulated?

You will find below a summary of the major recent achievements of the team in this field.

To study tRNA translocation through the plant mitochondrial outer membrane, we set up two in vitro systems. First, we demonstrated that direct tRNA import into isolated potato mitochondria can be obtained without added protein factor. Under these in vitro conditions, VDAC (Voltage-Dependent Anion Channel) is implicated. Second, we showed that the use of a protein carrier greatly improves import. In both cases, components of the protein import machinery (The TOM complex) were shown to be involved. Whether both pathways exist in vivo is an open question we wish to answer in the near future.

 

model import tRNA
Identification of two plant RNA mitochondrial import pathways in vitro


The use of an artificial protein carrier, the pre-dHFR, allowed us to develop a new tool to address foreign RNAs  (pre-tRNAs, mRNAs) into isolated mitochondria of various organisms (plant, yeast, animal, human cells). Our data thus enlarge the set of tools for studying mitochondrial genetics.

In Chlamydomonas, we found a precise correlation between the mitochondrial codon usage and the nature and amount of imported tRNAs. By modifying the mitochondrial genome of the model green alga, in collaboration with the team of Prof. C. Remacle  (University of Liège, Belgium), we recently shed light on the fine regulation between the mitochondrial import process of tRNAs and the needs of the organelle. This work allows a better understanding of the co-evolutive aspect of this trafficking process.

mRNA trafficking at the surface of plant mitochondria

Mitochondria biogenesis involves import of more than 98% of their proteins. To be targeted and imported into mitochondria, most of these proteins possess a N-terminal targeting sequence. Some proteins also contain internal signals or have, until now, no identified signals. This last decade, it has been shown in yeast and mammals that cytosolic mRNAs can be targeted to the surface of mitochondria. These mRNAs are then translated by cytosolic polysomes found in the vicinity of mitochondria and neosynthesized proteins are directly imported into the organelle. This demonstrates that other signals can be involved in protein targeting to mitochondria. Study of this process in plants is of great interest because of the presence of plastids, the second endosymbiotic organelles found in a plant cell. Mitochondrial and plastidial targeting sequences present similar features, and the mechanisms involved in the specificity of protein targeting are not well understood. Thus, mRNA targeting to mitochondria and/or plastids could contribute to targeting specificity in plant cells. Indeed, we recently showed that cytosolic mRNAs are differentially associated to potato mitochondria, suggesting that also in plants, mRNA targeting to mitochondria represents a key process in mitochondria biogenesis.

confocal imaging mRNA trafficking

The green RNA strategy to study mRNA trafficking to mitochondria

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December 15, 2017
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