Détail du poste
Établissement : Université Paris-Saclay GS Biosphera - Biologie, Société, Ecologie & Environnement, Ressources, Agriculture & Alimentation École doctorale : Sciences du Végétal : du gène à l'écosystème Laboratoire de recherche : IJPB - Institut Jean-Pierre Bourgin-Sciences du Végétal Direction de la thèse : Hakim MIREAU ORCID 0000000222995139 Début de la thèse : 2026-10-01 Date limite de candidature : 2026-05-06T23:59:59 Ce projet multidisciplinaire vise à analyser de manière mécanistique des processus spécifiques liés à la traduction mitochondriale chez les plantes, en particulier ceux impliqués dans l'étape d'initiation. En raison de leur origine bactérienne, les mitochondries possèdent un génome réduit codant une trentaine de protéines essentielles, traduites au sein de l'organite selon des mécanismes qui diffèrent à plusieurs égards de la traduction bactérienne. L'objectif de ce projet est de comprendre comment les sites d'initiation de la traduction sont reconnus sur les ARN messagers mitochondriaux. Autrement dit, il s'agit d'élucider comment les mitoribosomes sont recrutés et guidés vers les codons d'initiation en l'absence de séquences de recrutement ribosomique analogues à celles présentes dans les régions 5 des ARN messagers bactériens. Le projet se concentrera sur un ensemble de protéines à répétitions pentatricopeptides (PPR), chacune s'étant révélée essentielle à la traduction d'un ARNm mitochondrial spécifique. À l'aide d'approches génétiques complémentaires, nous chercherons à démontrer que ces protéines doivent se lier à des régions précises situées immédiatement en amont des codons AUG des ARNm qu'elles régulent afin d'activer l'initiation traductionnelle. Nous testerons également l'hypothèse selon laquelle leur mode d'action repose sur une modification de la structure locale de l'ARNm à proximité des codons initiateurs. Les résultats issus de ce projet contribueront à une meilleure compréhension de la diversité et de l'évolution des mécanismes de traduction chez les eucaryotes. Mitochondria (mt) are essential energy-producing organelles responsible for generating most of the cellular ATP. Since their acquisition from a bacterial ancestor (Martijn et al., 2018), mitochondria have undergone continuous evolutionary diversification. In most eukaryotes, mitochondria retain a small, bacteria-like genome encoding approximately 30 proteins, the majority of which correspond to highly hydrophobic subunits of the respiratory chain complexes. As a result, mitochondrial function relies on the import of a vast cohort of nuclear-encoded factors from the cytosol, numbering over 1,000 proteins.
The coordinated action of components derived from distinct genetic compartments has been a major driver of mitochondrial diversification. This diversity is particularly striking in processes related to mitochondrial gene expression, and especially in those governing mRNA translation (Ott et al., 2015; Greber and Ban, 2016). At the extreme of this variation lie the remarkably diverse structures and compositions of mitochondrial ribosomes (mitoribosomes) observed across eukaryotes (Greber et al., 2015; Amunts et al., 2015; Desai et al., 2017; Waltz et al., 2020). Consequently, the study of mitochondrial translation represents a compelling research area for exploring the divergence and specialization of translational mechanisms (Al-Faresi et al., 2019).
An astonishing divergence from bacterial translation concerns the initiation step, as mitochondrial mRNAs lack Shine-Dalgarno (S/D) sequences in their 5 leaders to facilitate or regulate translation initiation (Ott et al., 2015; Hammani and Giegé, 2014; Saito et al., 2020). Consequently, how correct translational start sites are recognized and how the small ribosomal subunit is guided to the appropriate AUG codon remain largely unknown at the molecular level in plant mitochondria. Our analysis of the Arabidopsis mitochondrial translatome, however, revealed substantial differences in ribosome loads across mitochondrial mRNAs, indicating that their translation is finely regulated (Planchard et al., 2018). While the precise molecular mechanisms remain unclear, it is likely that combinatorial interactions between cis-acting elements in 5 UTRs and mRNA-specific trans-factors drive the differential recruitment of ribosomes to mitochondrial transcripts.
To dissect these regulatory mechanisms and understand how start codons are identified, we applied the Ribo-Seq technology (Ingolia et al., 2009) to a large collection of Arabidopsis T-DNA insertion mutants affecting mitochondria-targeted P-type pentatricopeptide repeat (PPR) proteins (Lurin et al., 2004). We focused on PPR proteins because their diverse roles in plant mitochondrial mRNA expression suggested they might also play key roles in translational initiation (Barkan and Small, 2014). This strategy proved highly effective: we identified 5 PPR proteins that are required for the translation of specific mitochondrial mRNAs. Most of these translational PPRs (tPPRs) act mono-specifically, facilitating translation of a single target mRNA. Surprisingly, efficient translation of one mitochondrial mRNA was found to require the coordinated action of two distinct tPPRs. Recently, we mapped the in vivo RNA-binding sites of all these tPPRs and found that they bind to short RNA sequences located 20-50 nucleotides upstream of the AUG start codons of their target mRNAs. The identified tPPRs are therefore essential for orchestrating the translation initiation of their target mRNAs, yet the precise mechanism by which they perform this function remains completely unclear. The present PhD project is part of a broader effort to investigate mitochondrial translation in plants, with a particular focus on understanding how translation initiation complexes are recruited to plant mitochondrial 5 UTRs and how they accurately identify translation start sites. We have identified key factors, termed translational PPR proteins (tPPRs), that facilitate translation initiation on specific mitochondrial mRNAs, and the current project aims to elucidate the molecular mechanisms underlying their function. The model organism for this project will be Arabidopsis thaliana. Work Package 1: Modifying tPPR binding sites in vivo to unravel their impact on translation initiation.
The requirement for tPPRs to bind their short RNA targets in vivo will be genetically validated by modifying these binding sites in mitochondrial DNA using MitoTALED technology. This approach employs modular, programmable DNA-binding proteins called TALEs (transcription activator-like effector nucleases), which, when targeted to mitochondria and fused to specific deaminases, can edit selected bases in plant mitochondrial DNA. Possible editing events include C-to-T (Kang et al., 2021; Mok et al., 2020) or A-to-G (Mok et al., 2022) conversions. The student will use this technology by expressing TALED proteins in planta, programmed to bind and modify the sequences corresponding to the identified tPPR binding sites. Constructs will be generated using GoldenBraid cloning, allowing efficient assembly of TALE modules and enabling testing of multiple MitoTALED variants per binding site if necessary. Base changes will be prioritized within the first 4-5 nucleotides of the PPR binding sites, which are critical for specific PPR/RNA interactions (Miranda et al., 2018). Sequence modifications in the mitochondrial DNA will be confirmed by PCR amplification and sequencing of the relevant regions. Plants exhibiting altered phenotypes will undergo further analysis, including assessment of the translation efficiency of the corresponding mitochondrial gene via Ribo-Seq. Finally, the effect of the mutations on tPPR binding will be validated through molecular analyses like gel shift assays.
In a preliminary analysis, mitochondrial mutant plants have already been generated for one of the tPPRs, demonstrating the feasibility of the approach.
Work Package 2: Introduction of compensatory changes in tPPR to restore normal translation.
The inability of tPPRs to efficiently promote translation of mitochondrial mRNAs harboring mutations in their binding sites (WP1) will most likely be attributable to reduced or abolished binding affinity for the altered sequences. To rigorously validate the requirement for tPPR binding to these rRNA regions in vivo, compensatory mutations will be introduced into the tPPR sequences to restore specific recognition and binding of the mutated sites. It has been clearly established that specific combinations of amino acids at positions 5 and 35 within each PPR repeat determine the recognition of the four RNA bases by individual repeats in PPR proteins (Barkan et al., 2012). Since its discovery this PPR recognition code has been widely validated, including in our laboratory. In this phase of the project, codons specifying amino acids 5 and 35 in selected PPR repeats of the tPPRs will be altered according to the PPR code, generating compensatory changes that will restore binding to the mutated sites identified in WP1. For each tPPR, this strategy will target only one mutated binding site from WP1, chosen for producing plants with altered growth phenotypes but that are still suitable for transformation. Recovery of wild-type growth will validate the functionality of the compensatory tPPRs, which will be further examined by molecular analyses, including Ribo-Seq, to confirm proper translation of their target mRNA. The re-establishment of normal binding between the tPPRs and their mutated binding sites will be confirmed using molecular assays, such as gel shift experiments.
Work Package 3: Investigating the RNA chaperone activity of tPPRs on the 5 UTRs of their target mRNA.
It has been proposed that tPPRs facilitate translation initiation either by directly recruiting the small ribosomal subunit through physical interactions or by modifying the local RNA structure near the translation start codon to create a structural environment favorable for ribosome anchoring and translation initiation. We have recently ruled out the first hypothesis by extensively characterizing the in vivo protein interactome of tPPRs, which revealed no association with any small mitoribosomal subunit. This finding provides strong evidence that the second model represents the most likely mechanism underlying tPPR-mediated translation initiation in plant mitochondria. To investigate this mechanism, we will analyze mitochondrial mRNA structures in vivo using mitoDMS-MaPseq (Moran et al., 2024). This approach relies on the ability of dimethyl sulfate (DMS) to methylate unpaired adenine and cytidine residues, which subsequently induce misincorporations during reverse transcription by the TGIRT-III reverse transcriptase, enabling nucleotide-resolution RNA structure probing. We have recently implemented this technology successfully in our laboratory and generated a first draft of the mitochondrial RNA structurome of Arabidopsis thaliana, revealing a high degree of RNA structuration in plant mitochondria, in contrast to animal mitochondria (Moran et al., 2024). In this work package, mitoDMS-MaPseq will be applied to individual tPPR mutants to characterize the in vivo structure of the 5 UTRs of their target mRNAs and to compare these profiles with those of wild-type plants. In addition, double mutants corresponding to pairs of tPPRs acting on the same mitochondrial mRNA will be included to assess potential collaborative or synergistic effects on 5 UTR structuration. This approach will identify the regions surrounding AUG start codons that must be locally de-structured by tPPR binding to permit translation initiation and will, for the first time, provide a mechanistic model for the orchestration of mitochondrial translation initiation in plants.
Beyond its mechanistic insights, the resulting mitochondrial RNA structurome data will constitute an unprecedented resource for the field by itself and will lead to a high-quality publication in a leading international molecular biology journal, thereby securing a strong publication outcome for the student.
Work Package 4: Writing of scientific articles and thesis manuscript.
A first article will be written based on the data produced in the frame of WP1 and WP2. A second paper relating the results obtained with mitoDMS-MaPseq technology (WP3) will follow at the end of the thesis. The last months of the thesis will focus on the writing of articles, the thesis manuscript and the preparation of the thesis defense.
Le profil recherché
Publiée le 14/04/2026 - Réf : b144313b9a57308a607d5c7319aff673
