L’ARNm mitochondrial nad4L n’est pas traduit correctement chez rfl23 . 163

Comme pour le mutant rfl22, puisque qu’aucun rôle pour la protéine RFL23 pouvant expliquer le phénotype du mutant n’a pu être révélé en étudiant l’abondance et la maturation des transcrits mitochondriaux, nous avons fait l’hypothèse que cette protéine pouvait être impliquée dans la traduction de son ARNm cible. Pour valider ou infirmer cette hypothèse, nous avons utilisé la technique du profilage de ribosomes afin d’estimer le niveau de traduction des ARNm mitochondriaux chez rfl23 en comparaison au sauvage (Figure 51). De la même façon que pour le mutant rfl22, cette étude nous a permis d’observer que les niveaux de traduction des différents ARNm mitochondriaux n’étaient pas strictement équivalents entre le mutant rfl23 et le sauvage.

Figure 51 – Traductomes mitochondriaux de Col-0 et du mutant rfl23

Traductomes mitochondriaux mesurés par profilage de ribosomes. Cette figure permet de comparer les niveaux de traduction entre les transcrits d’un même génotype mais ne permet pas de faire de comparaisons entre génotypes.

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Pour affiner ces résultats, une comparaison statistique des niveaux d’accumulation de traduction des ARNm mitochondriaux entre le mutant et le sauvage a été réalisée. Il ressort de cette analyse que les ARNm nad4L et nad2 sont significativement moins traduits chez

rfl23 (Figure 52). Au sujet de nad2, nous observons que les exons concernés par cette baisse de niveau de traduction sont les exons 2, 3 et 4, soit ceux qui suivent intron 1, qui n’est que partiellement épissé chez ce mutant.

Figure 52 – Analyse différentielle des changements transcriptionnels et traductionnels des ARNm mitochondriaux chez le mutant rfl23.

Chaque exon mitochondrial a été quantifié par RT-PCR quantitative. Les niveaux de traduction de chaque exon ont été mesurés par profilage de ribosomes.

Les croix représentent les gènes ne comprenant qu’un exon, les ronds les gènes comprenant plusieurs

exons. Si le gène est en couleur et que le nom de l’exon est indiqué, alors le log₂ (niveau de traduction

chez rfl23/niveau de traduction chez Col-0) est supérieur à 1 ou inférieur à -1 (variation d’un facteur 2 au minimum). Si un exon (rond) est en couleur sans que le nom du gène ne soit indiqué, alors un des autres exons du même gène vérifie la condition précédente.

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La répartition et la densité des empreintes de ribosomes le long de l’ARNm nad4L

montre que la traduction est décalée en 3’ chez le mutant rfl23 en comparaison au sauvage et ne commence, qu’au début du deuxième tiers du transcrit (Figure 53). Il ressort donc que l’ARNm nad4L est pris en charge par les polysomes mitochondriaux, l’initiation traductionnelle chez rfl23 n’a néanmoins pas lieu sur le codon AUG situé au début de la phase codante (comme chez le sauvage), mais au début du 2ème tiers du transcrit.

Enfin, plusieurs transcrits apparaissent significativement plus traduits chez le mutant par rapport au sauvage. Il s’agit notamment de matR, rpl2,rpl5, rpl16, rps3 et rps12 (Figure 52), soit entre autres cinq transcrits codant des sous-unités du mitoribosome.

Figure 53 - Couverture ribosomale de l'ARNm nad4L chez Col-0 et le mutant rfl23

La couverture est exprimée en nombre d’empreintes de ribosomes par nucléotide, normalisée par la

taille des banques de séquençage. La couverture de nad4L chez Col-0 est représentée en gris, celle

chez rfl23 l’est en noir. La phase de lecture du gène nad4L est matérialisée par le rectangle noir en bas de la figure.

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Résultats

Partie 3 : Analyse du

traductome mitochondrial chez

Arabidopsis thaliana

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4.3. Projet d’article : Analyse du traductome mitochondrial chez

Arabidopsis thaliana

Analysis of the translational landscape of

Arabidopsis mitochondria

Noelya Planchard1,2,‡, Pierre Bertin3,‡, Martine Quadrado1, Céline Dargel-Graffin1, Isabelle Hatin3, Olivier Namy3,*, Hakim Mireau1,*

1Institut Jean-Pierre Bourgin, INRA, AgroParisTech, CNRS, Université Paris-Saclay, RD10, 78026 Versailles Cedex, France

2Paris-Sud University, Université Paris-Saclay, 91405 Orsay Cedex, France

3Institute for Integrative Biology of the Cell (I2BC), UMR 9198 CEA, CNRS, Univ. Paris Sud, Bâtiment 400, 91405, Orsay, France.

‡These authors contributed equally to the paper as first authors.

*To whom correspondence should be addressed. Tel: +33 130 833 070; Fax: +33 130 833 319; Email: hakim.mireau@inra.fr

Correspondence may also be addressed to Olivier Namy. Tel: +33 169 155 051; Fax: +33 169 157 296; Email: olivier.namy@i2bc.paris-saclay.fr

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Abstract

Unlike other mRNA expression processes at play in plant mitochondria, translation has remained largely unexplored up to now. Growing evidence indicate though that mitochondrial translation in most organisms differs from its bacterial counterpart in many key aspects. In this analysis, we used the ribosome profiling technology to generate a genome-wide snapshot of mitochondrial translation in Arabidopsis. We reveal that respiratory subunit-encoding mRNAs show much higher ribosome association than other mitochondrial mRNAs, implying that they are translated to higher levels. Homogenous ribosome densities were generally detected within each respiratory complex except for complex V where higher mRNA ribosome coverage corroborate with higher needs in specific subunits. In complex I respiratory mutants, a slight reorganization of mitochondrial mRNAs ribosome association was detected mostly involving an increase in ribosome densities on certain ribosomal protein encoding transcripts and a reduction in the translation of a few complex V mRNAs. Altogether, our observations reveal that plant mitochondrial translation is a dynamic process and that translational control is an important component of gene expression in plant mitochondria. This study paves the way for future advances in the understanding of translation in higher plant mitochondria.

4.3.1. Introduction

The functioning of eukaryotic cells relies on the integrated activity of several highly specialized subcellular organelles. Among them, mitochondria represent essential energy conversion powerhouses that produce most of the energy of cells through aerobic respiration. Mitochondria have an exogenous origin and the prevailing view is that they arose from the endosymbiotic adoption of an ancient α-proteobacteria (Gray, 1999, 2012). Throughout evolution, the adoption of mitochondria has been associated with a profound reduction of their genetic content. Modern mitochondrial genomes encode only a handful of mRNAs (33 in Arabidopsis thaliana) that are translated within the organelle. This requires the existence of a complete gene expression machinery whose most involved factors are encoded by nuclear genes but that necessarily coevolved with mitochondrial genomes.

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Thereby, gene expression in mitochondria involves a cooperation between highly degenerated bacterial-like features inherited from the original endosymbiont and eukaryotic-derived nuclear-encoded trans-factors that appeared throughout the evolution. This dual origin has resulted in complex mRNA expression processes that involve a high number of post-transcriptional modifications (Lightowlers et al., 2014; Hammani & Giegé, 2014). In the last years, significant progresses have been made on certain aspects of gene expression in plant mitochondria (Hammani & Giegé, 2014). However, mitochondrial translation, the last level of mRNA expression that is also the least prone to easy molecular analyses, has remained largely unexplored in plants. Yet, we know that despite of its ancient prokaryotic origin, the mitochondrial translation machinery differs profoundly from its bacterial counterpart in many essential aspects and that major translation-associated components are substantially different from the ones found in bacteria (Lightowlers et al., 2014; Janska & Kwasniak, 2014; Ott et al., 2016). In particular, the rRNA and protein compositions of mitoribosomes differ extensively from the one of E. coli ribosomes (Bonen, 2004; Greber & Ban, 2016).

Additionally, the constraints of translating few mRNAs mostly encoding hydrophobic membrane subunits have conducted to highly specialized translation mechanisms guided by membrane-associated mitochondrial ribosomes (mitoribosomes) to facilitate cotranslational insertion of mitochondria-encoded proteins into the inner mitochondrial membrane (Pfeffer et al., 2015). The divergence with bacteria is also particularly obvious for translation initiation step since plant mitochondrial mRNAs lack typical ribosome anchoring motif in their 5’ leaders used in prokaryotes to help position the start codon at the P-site of the ribosome (Bonen, 2004). Thus, we currently don’t know how mitochondrial ribosomes are recruited on 5’ untranslated regions and especially how the correct translation initiation codon is recognized by the small ribosomal subunit. The high degree of sequence divergence among 5’ leaders of plant mitochondrial mRNA has suggested a ribosome recruitment mechanism involving gene-specific cis-sequences and trans-factors (Hazle & Bonen, 2007; Choi et al., 2012).

Up to now only two proteins belonging to the large family of pentatricopeptide repeat (PPR) proteins have been shown to promote mitochondrial translation initiation in higher plants (Manavski et al., 2012; Haili et al., 2016). However, the mechanisms by which these PPR proteins help recruiting the mitoribosome on the mRNAs they target have not been determined yet.

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The recent development of the ribosome profiling technology has greatly facilitated the detailed measurement of translation at the single-nucleotide level and on a genome-wide scale (Ingolia et al., 2009). Its general principal consists in the purification and subsequent sequencing of short mRNA segments covered by the translating ribosomes. These mRNA segments are called ribosomes footprints and measure about 30-nucleotide long in most species (Brar & Weissman, 2015). The statistical analysis of their distributions reveals the identity and the boundaries of translated RNA regions and provides a measurement of the relative translational levels of mRNAs. This technology has recently revealed interesting functional aspects on ribosome behaviour in plastid and human mitochondria but had been never used to study plant mitochondrial translation (Zoschke et al., 2013; Rooijers et al., 2013; Chotewutmontri & Barkan, 2016).

Therefore, to get a comprehensive view of the Arabidopsis mitochondrial translatome and the functioning of the plant mitochondrial machinery, we prepared and sequenced total ribosome footprints from Arabidopsis inflorescences, and mapped them to the Arabidopsis mitochondrial genome. In particular, this approach allowed us to estimate and compare the translational activity of mitochondrial mRNAs, to see how mitochondrial translation is reorganized in respiratory mutants.