Caractérisation moléculaire et fonctionnelle de la protéine DYW1 dans le complexe d'édition chloroplastique d'Arabidopsis thaliana

Dans les organites des plantes, l édition de l ARN consiste majoritairement en une désamination de cytidines à des sites spécifiques de l ARNm. Trente-quatre sites d édition ont été découverts dans les transcrits chloroplastiques d Arabidopsis thaliana et plus de 500 dans les transcrits mitochondriaux. Depuis 2005, beaucoup de facteurs d édition ont été trouvés. La majorité de ces protéines appartiennent à la famille des PentatricoPeptide Repeat (PPR). Parmi ces PPR, certaines contiennent un domaine DYW possédant de faibles similarités avec les cytidines désaminases (CDA), alors que d autres en sont dénuées, générant un doute sur le fait qu il ait une activité CDA. Le gène At1g47580 (DYW1) code une protéine unique chez Arabidopsis thaliana contenant seulement un domaine DYW. Il a été proposé que DYW1 puisse interagir avec les PPR ne contenant pas de domaine DYW, pour former un hétérodimère, capable d éditer spécifiquement un site. En accord avec cette hypothèse, nous avons montré que DYW1 agissait sur le même site d édition que CRR4, une PPR sans domaine DYW, et que ces protéines interagissaient in vivo. De plus, nous avons montré que DYW1 remplaçait les parties manquantes de CRR4 pour l édition. Pour obtenir plus d informations sur la fonction du domaine DYW, des mutations ont été introduites dans DYW1. Nous avons montré que la signature CDA dans les protéines DYW était essentielle à l édition de l ARN ainsi qu à l interaction avec les ions zinc. Les données sont en accord avec l hypothèse d une activité CDA dans le domaine DYW. Cependant, aucune activité CDA n a pu être mise à jour in vitro. Il est vraisemblable qu au moins un cofacteur doive encore être identifié.In plant organelles, RNA editing mostly takes the form of conversions of cytidines to uridines at specific sites in mRNAs. Thirty-four editing sites have been found in Arabidopsis thaliana chloroplast transcripts and more than 500 sites in mitochondrial transcripts. Since 2005, lots of proteins have been found to act as RNA editing factors. Most of these proteins belong to the PentatricoPeptide Repeat (PPR) family. Amongst these PPR, some contain a DYW domain with weak similarity to cytidine deaminases (CDA), whilst others lack such a domain, creating doubts about whether this domain is required for editing. The gene At1g47580 (named DYW1) encodes a protein in Arabidopsis thaliana that contains only a DYW domain. Our initial hypothesis was that DYW1 might interact with PPR proteins that lack a DYW domain, in order to form a heterodimer, able to perform site-specific editing. In accordance with this hypothesis, we discovered that DYW1 is involved in editing the same site as CRR4, a PPR lacking a DYW domain, and that these two proteins interact together in vivo. Moreover, we showed that DYW1 replaces all the missing parts of CRR4 for editing. So, other partners need to be hypothesized for other DYW-lacking editing factors if this hypothesis is to be generalized. The highly conserved residues making up the CDA signature in DYW proteins were found to be essential for RNA editing and are also required for zinc binding, which is a known characteristic of CDAs. All the data so far are consistent with the DYW domain being (part of) a CDA activity; nevertheless, no CDA activity could be detected in vitro. It is likely that at least one required cofactor remains to be identified.EVRY-Bib. électronique (912289901) / SudocSudocFranceF

Limiting etioplast gene expression induces apical hook twisting during skotomorphogenesis of Arabidopsis seedlings

When covered by a layer of soil, seedling development follows a dark-specific program (skotomorphogenesis). In the dark, seedlings consist of small, non-green cotyledons, a long hypocotyl, and an apical hook to protect meristematic cells. We recently highlighted the role played by mitochondria in the high energy-consuming reprogramming of Arabidopsis skotomorphogenesis. Here, the role played by plastids, another energy-supplying organelle, in skotomorphogenesis is investigated. This study was conducted in dark conditions to exclude light signals so as to better focus on those produced by plastids. It was found that limitation of plastid gene expression (PGE) induced an exaggerated apical hook bending. Inhibition of PGE was obtained at the levels of transcription and translation using the antibiotics rifampicin (RIF) and spectinomycin, respectively, as well as plastid RPOTp RNA polymerase mutants. RIF-treated seedlings also showed expression induction of marker nuclear genes for mitochondrial stress, perturbation of mitochondrial metabolism, increased ROS levels, and an augmented capacity of oxygen consumption by mitochondrial alternative oxidases (AOXs). AOXs act to prevent overreduction of the mitochondrial electron transport chain. Previously, we reported that AOX1A, the main AOX isoform, is a key component in the developmental response to mitochondrial respiration deficiency. In this work, we suggest the involvement of AOX1A in the response to PGE dysfunction and propose the importance of signaling between plastids and mitochondria. Finally, it was found that seedling architecture reprogramming in response to RIF was independent of canonical organelle retrograde pathways and the ethylene signaling pathway

Identification of the RNA editing enzyme in plant organelles

Chez les plantes, l édition des ARN dans les organites conduit principalement à des conversions de cytidines en uraciles. Des protéines de la famille PPR (pentatricopeptide repeat) sont impliquées dans ce processus. Mon travail a permis de montrer que le domaine DYW présent chez certaines protéines PPR présente des homologies avec le site catalytique de cytidines désaminases et que sa distribution phylogénétique corrèle avec celle de l édition dans la lignée verte. Par ailleurs, chez A. thaliana, l analyse de mutants affectés dans l expression du gène AtDYW1, codant une protéine uniquement constituée du domaine DYW et d un peptide signal pour les organites, a révélé l existence de plantules au développement très affecté et pour lesquelles plusieurs défauts d édition dans des ARNm chloroplastiques ont pu être caractérisés. Pris ensemble, ces éléments suggèrent que le domaine DYW ou la protéine AtDYW1 pourraient être l enzyme centrale catalysant les réactions d édition.RNA editing in plants organelle transcripts is a proccess leading to specific post-transcriptional pyrimidine interconversions (mainly C-to-U). Recently a few pentratricopeptide repeat (PPR) proteins were described to be essential in this process. During my PhD, I found that the DYW domain of PPR proteins show similarities with the active site of cytidine deaminases, and that the phylogenetic distribution of this domain is strictly correlated with RNA editing in green plants. In addition, in A. thaliana, the AtDYW1 gene encodes a protein made of a targeting signal to the organelles and a DYW domain. Mutant lines in which this gene has been targeted for knock-down exhibit strongly altered development. In the affected seedlings, AtDYW1 gene expression is specifically decreased, and several editing defects in plastids transcripts were characterized.Taken together, these data support the hypothesis that the DYW domain and the AtDYW1 protein may be the central enzyme in the RNA editing.EVRY-Bib. électronique (912289901) / SudocSudocFranceF

Bioinformatic analysis of chloroplast gene expression and RNA posttranscriptional maturations using RNA Sequencing

International audienceSequencing of total RNA enables the study of the whole plant transcriptome resulting from the simultaneous expression of the three genomes of plant cells (located in the nucleus, mitochondrion and chloroplast). While commonly used for the quantification of the nuclear gene expression, this method remains complex and challenging when applied to organellar genomes and/ or when used to quantify posttranscriptional RNA maturations. Here we propose a complete bioinformatical and statistical pipeline to fully characterize the differences in the chloroplast transcriptome between two conditions. Experimental design as well as bioinformatics and statistical analyses are described in order to quantify both gene expression and RNA posttranscriptional maturations, i.e., RNA splicing, editing, and processing, and identify statistically significant differences

Characterising the RNA editing machinery and function in plants

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