Translation platforms at organelles surface – POLYGLOT

- Identifying cytosolic mRNAs associated with organelles, and mechanisms of mRNA targeting
- Characterizing the translation platforms at the mitochondrial and chloroplastic surfaces
- Exploring the functions and regulations of mRNA targeting and translation on organelles

In order to characterize the cytosolic ribosome associated with the mitochondrial surface, we have used an Arabidopsis line expressing an epitope-tagged ribosomal protein (Tag-Rib). Total and mitochondrial proteins were prepared from wild type and Tag-Rib plants, then immunoprecipitated with anti-Tag beads, and enriched proteins were identified by mass spectrometry. About 70 proteins were found enriched in IP with mitochondria from Tag-Rib plants. One of the most enriched proteins has orthologs in mammals, drosophila, yeast, protozoans, and is known for its role on mitochondria. Reverse co-IP has confirmed the interaction of this protein with cytosolic ribosomes at the surface of mitochondria.

The most outstanding result is the identification of a ubiquitous trans-factor in association with ribosomes at the mitochondrial surface. This protein could thus be involved in mRNA targeting or translation at the mitochondrial surface in all eukaryotes. Determining the exact function of this protein and its mechanism of action is now a major axis of the project.

In international journals:
Arabidopsis Voltage-Dependent Anion channels (VDACs): overlapping and specific functions in mitochondria, Hemono et al, Cells (2020) 9, 1023

Intracellular trafficking of mRNAs and localized protein synthesis are fundamental mechanisms to control protein localization. It has been proposed for years that translation was mostly cytosolic or associated with the endoplasmic reticulum (ER). However cytosolic mRNAs and ribosomes were also found associated with the mitochondrial surface in fungi, animals and plants, and with the chloroplastic surface in plants. In parallel, recent studies suggest that ribosomes are heterogeneous and specialized, and directly implicated in the spacio-temporal regulation of translation. A new vision of protein synthesis in eukaryotic cells is thus emerging, with translation platforms localized in different subcellular compartments: at the surface of ER, mitochondria, chloroplasts, or in the cytosol. Moreover, defects in mRNA targeting and localized translation have dramatic consequences in the cells. For example, Parkinson's disease associated proteins PINK1 and Parkin control translation of cytosolic mRNAs on the mitochondrial outer membrane. We have also shown that, in plant, mitochondrial biogenesis is influenced by the mitochondrial targeting of mRNAs coding for VDAC, a mitochondrial protein involved in apoptosis.
Therefore, it is necessary to re-evaluate our understanding of how mRNA translation is spatially organized and regulated in eukaryotic cells. Up to now, only tenuous clues have been obtained during the characterization of different translation systems in yeast, human or plants. In this light, the diversification of experimental models appears crucial to attain an integrated view of the core components of translation platforms in eukaryotes. We propose to use plants as a main model to characterize the translation complexes associated with mitochondria and chloroplasts. Plants indeed offer the unique opportunity to characterize translation platforms at the surface of two endosymbiotic organelles.
Our main objective aims at characterizing the translation platforms associated with mitochondria and chloroplasts. This will be organized into three tasks: (i) identifying cytosolic mRNAs associated with mitochondria and chloroplasts, and mechanisms allowing mRNA targeting, (ii) characterizing the translation platforms at the mitochondrial and chloroplastic surfaces, (iii) exploring the functions and regulations of mRNA targeting and translation at the surface of organelles.
To carry out this project, the consortium consists of two partners of world-renown, with complementary expertise and technology, at the “Institut de biologie moléculaire des plantes” (P1) in Strasbourg, and the “Laboratoire de Physiologie Cellulaire et Végétale ” (P2) in Grenoble.
Results already obtained by P1 and P2 suggest that the project success is achievable. Indeed RNA-Seq data already determined an array of mRNAs specifically accumulated at the surface of mitochondria, and proteomic results identified a set of cytosolic ribosomal proteins at the surface of mitochondria or chloroplasts. Cis-elements in 5' and 3'UTRs of mRNAs were also demonstrated to impact mRNAs localization in the vicinity of mitochondria. Moreover, a number of tools that will be required for the project have already been obtained.
Understanding the spatial regulation of translation and ribosomes specificities is essential in the light of the dramatic impact of delocalized translation on cells and entire organisms. This would also offer new strategies to correct certain cellular dysfunctions. Moreover, the identification of mRNA trafficking signals to mitochondria and chloroplasts will give new insights toward manipulating RNA and protein contents of organelles.