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[EN] Viroids are small, circular, highly structured pathogens that infect a broad range of plants, causing economic losses. Since their discovery in the 1970s, they have been considered as non-coding pathogens. In the last few years, the discovery of other RNA entities, similar in terms of size and structure, that were shown to be translated (e.g., cirRNAs, precursors of miRNA, RNA satellites) as well as studies showing that some viroids are located in ribosomes, have reignited the idea that viroids may be translated. In this study, we used advanced bioinformatic analysis, in vitro experiments and LC-MS/MS to search for small viroid peptides of the PSTVd. Our results suggest that in our experimental conditions, even though the circular form of PSTVd is found in ribosomes, no produced peptides were identified. This indicates that the presence of PSTVd in ribosomes is most probably not related to peptide production but rather to another unknown function that requires further study.

Viroids are small, circular, highly structured pathogens that infect a broad range of plants, causing economic losses. Since their discovery in the 1970s, they have been considered as non-coding pathogens. In the last few years, the discovery of other RNA entities, similar in terms of size and structure, that were shown to be translated (e.g., cirRNAs, precursors of miRNA, RNA satellites) as well as studies showing that some viroids are located in ribosomes, have reignited the idea that viroids may be translated. In this study, we used advanced bioinformatic analysis, in vitro experiments and LC-MS/MS to search for small viroid peptides of the PSTVd. Our results suggest that in our experimental conditions, even though the circular form of PSTVd is found in ribosomes, no produced peptides were identified. This indicates that the presence of PSTVd in ribosomes is most probably not related to peptide production but rather to another unknown function that requires further study.

Pendant longtemps, seul un rôle d’intermédiaire était connu à l’ARN. Cependant, il a été trouvé que grâce à sa forme simple brin, cette molécule peut se replier sur elle-même et ainsi constituer des structures qui lui confèrent diverses fonctions. Parmi ces formations, le G- quadruplexe (G4) est composé d’appariements de type Hoogsteen au lieu des appariements de type Watson-Crick. Les G4 peuvent se replier dans l’ARN, mais aussi dans l’ADN et y avoir de multiples rôles. Leur étude, initialement timide, a vécu un engouement dans les dernières années après avoir été découvertes dans les télomères et ayant un impact sur les cancers. Les G4 sont aussi fortement enrichis dans des régions régulatrices, comme les promoteurs dans les génomes ou les régions non traduites dans les transcrits. Quelques recherches laissent penser qu’il en est de même pour tous les eucaryotes, mais pas pour les bactéries. À ce jour, l’évolution des G4 reste peu étudiée, principalement à cause de la mauvaise conservation des séquences. Dans la présente thèse, la distribution des G4 dans les différents royaumes du vivant a été explorée, mais aussi différentes approches pour mieux appréhender leur évolution. Pour ce faire, une première étape était de prédire les G4 dans le transcriptome humain, et de valider ces prédictions. Ensuite, cette stratégie a été appliquée à diverses espèces au travers des trois royaumes du vivant. Ces données ont été analysées puis mises à disposition pour la communauté de chercheurs dans une base de données nommées GAIA. Finalement, une approche a été développée pour tenter de mieux comprendre l’évolution des G4., For a long time, RNA only known function was its intermediate role. Yet, it has been discovered that, thanks to its single-strand form, it can fold onto itself to generate structures and thus acquire diverse functions. Among these structures stands G-quadruplexes (G4s), which are made of Hoogsteen base-pairing instead of Watson & Crick ones. This structure can fold in RNA, but also in DNA where they can have multiple functions. The G4 study remained shy until the past few years when G4s interest suddenly increased with the discovery of their presence in telomeres and the impact they have on cancer. G4s are also highly enriched in other regulatory regions like promoters in genomes or in untranslated regions of transcripts. Some studies are implying that this distribution might be similar among all eukaryotes but not bacteria. The evolution of G4s is still relatively unknown and few studies are made on it. This is mainly due to one phenomenon: G4s sequences are badly conserved, which harden the task of its study. In the present thesis, it is proposed to study more in-depth G4s distribution among living kingdoms, but also to try new approaches to apprehend their evolution. To do so, the first step was to predict G4s inside the human transcriptome and to validate these predictions. Then, this method has been applied to diverse species among the three living kingdoms. These data have been be analysed and made public thanks to the GAIA database. Finally, an approach has been be developed to look at the evolution of G4s.

Les mécanismes clefs des cellules sont finement régulés tant durant leur développement que leur cycle cellulaire. La régulation post-transcriptionnelle est importante pour moduler le devenir des ARN messagers (ARNm), de leur expression dans une région précise à leur dégradation. Ces processus reposent sur des motifs et structures sur l’ARNm. Parmi ces dernières, des séquences riches en guanines peuvent se replier en structures secondaires stables appelées G-quadruplexes (G4). Elles sont constituées de quatre séries de guanines, séparées par des boucles, et s’organisant en tétrades coplanaires. Ces structures sont largement distribuées dans le transcriptome des mammifères, et constituent des éléments de régulation clefs dans de nombreux mécanismes biologiques, dont la maturation des pré-ARNm. Il a été montré qu’un G4 dans la région en 3’ non traduite (3’UTR) a un rôle dans le choix du site de coupure et polyadénylation, dans le cas de l’ARNm codant pour la protéine FXR1. Ce phénomène aboutit à la formation de transcrits ayant des régions 3’UTR de différentes longueurs. Cette différence de taille peut induire la formation de quantités variables de protéines. Elle peut aussi moduler les interactions avec les micro ARN par la présence ou l’absence de leurs sites de liaisons. Dans cette étude, nous souhaitions définir les partenaires protéiques capable de lier le G4 en 3’UTR de l’ARNm codant pour FXR1 étudié précédemment. Initialement, il a été montré que les séquences d’ARN étudiées formaient un G4 pour la séquence sauvage et non pour la séquence mutée. La liaison de ces séquences aux billes de streptavidine-sépharose par l’aptamère S1m a été vérifiée. Les séquences liées aux billes ont été utilisées dans les trois essais de type pull down. Ces derniers ont été suivis par une analyse par spectrométrie de masse. En comparant tous les essais avec l’outil d’analyse SAINT, trois protéines ont été identifiées. DHX36, ZNFX1, et IGHMBP2 sont des hélicases, dont le rôle princip, Key mechanisms in cells are finely regulated both during their development and cell cycle. Post - transcriptional regulation is essential to modulate the fate of messenger RNAs (mRNAs), from their expression in a precise localization to their degradation. These processes are based on motifs and structures on mRNAs. Among them, guanines-rich sequences can fold in stable secondary structures called G-quadruplexes (G4). They consist of four sets of guanines, separated by loops, and organizing as coplanar tetrads. These structures are widely distributed in mammalian transcriptome, and are key regulatory elements in many biological mechanisms, including premRNA processing. It has been shown that a G4 in the 3’ untranslated region (3’UTR) plays a role in the choice of the cleavage and polyadenylation site, in the case of the mRNA coding for FXR1 protein. This phenomenon leads to the production of transcripts having 3’UTR of different lengths. This difference may lead to the formation of variable amounts of protein. It could also modulate interactions with micro-RNAs by the presence or absence of their binding sites. In this study, we wanted to define protein partners able to bind the G4 in the 3’UTR of FXR1 mRNA previously studied. Initially, it was shown that the studied RNA folded into a G4 for the wild-type sequence and not for the mutated one. The binding of these sequences to streptavidin-sepharose beads by the aptamer S1m has been verified. Sequences bound to the beads were used for three pull down assays. These were followed by mass spectrometric analysis. By comparing the assays with the SAINT algorithm, three proteins were identified. DHX36, ZNFX1, and IGHMBP2 are helicases, whose primary role is to denature secondary structures in DNA and/or RNA. DHX36 is known to bind and unfold G4s. ZNFX1 and IGHMBP2 contain a zinc finger domain, often associated with the G4s binding capacity. These three proteins were detected in the nucleus of our model cell line

Viroids are non-coding RNA plant pathogens that are characterized by their possession of a high mutation level. Although the sequence heterogeneity in viroid infected plants is well understood, shifts in viroid population dynamics due to mutations over the course of infection remain poorly understood. In this study, the ten most abundant sequence variants of potato spindle tuber viroid RG1 (PSTVd) expressed at different time intervals in PSTVd infected tomato plants were identified by high-throughput sequencing. The sequence variants, forming a quasi-species, were subjected to both the identification of the regions favoring mutations and the effect of the mutations on viroid secondary structure and viroid derived small RNAs (vd-sRNA). At week 1 of PSTVd infection, 25% of the sequence variants were similar to the "master" sequence (i.e., the sequence used for inoculation). The frequency of the master sequence within the population increased to 70% at week 2 after PSTVd infection, and then stabilized for the rest of the disease cycle (i.e., weeks 3 and 4). While some sequence variants were abundant at week 1 after PSTVd infection, they tended to decrease in frequency over time. For example, the variants with insertions at positions 253 or 254, positions that could affect the Loop E as well as the metastable hairpin I structure that has been shown important during replication and viroid infectivity, resulted in decreased frequency. Data obtained byin silicoanalysis of the viroid derived small RNAs (vd-sRNA) was also analyzed. A few mutants had the potential of positively affecting the viroid's accumulation by inducing the RNA silencing of the host's defense related genes. Variants with mutations that could negatively affect viroid abundance were also identified because their derived vd-sRNA were no longer capable of targeting any host mRNA or of changing its target sequence from a host defense gene to some other non-important host gene. Together, these findings open av

Au sein du système nerveux, les mécanismes de régulation post-transcriptionnelle contribuent grandement aux différents phénotypes cellulaires. En effet, de faibles dérégulations peuvent mener à des maladies. Les G-quadruplexes (G4) d’ARN émergent comme étant de nouveaux acteurs dans la régulation post-transcriptionnelle. Les G4 sont des structures secondaires très stables retrouvées dans l’ADN et l’ARN. Ils sont formés par l’empilement d’un minimum de deux tétrades de guanines et stabilisés par des cations monovalents, habituellement le potassium. Au niveau de l’ARN, ils sont impliqués dans différents mécanismes comme l’épissage alternatif, la polyadénylation alternative, l’activation et l’inhibition de la traduction et la maturation des microARN. Selon plusieurs, les G4 sont considérés comme étant des évènements plutôt que des entités. Des éléments trans, soit des protéines, sont alors en mesure d’assurer leur repliement et dépliement. Une analyse bio-informatique a confirmé un enrichissement de G4 d’ARN dans les transcrits du système nerveux. Cette analyse a aussi permis d’identifier la maladie de Parkinson comme l’une des maladies ayant la plus grande proportion de potentiel G4 (pG4). En effet, sur 16 gènes reliés à la maladie, 15 d’entre eux contiennent au moins un pG4. Ensuite, différentes techniques biochimiques comme des essais de fluorescence, le dichroïsme circulaire et la cartographie in-line ont été utilisées pour confirmer la formation de certains G4 situés dans des régions 5’ non traduites (5’UTR). Ces techniques ont permis d’identifier 4 nouveaux G4 d’ARN n’ayant jamais été caractérisés auparavant. Par la suite, des essais luciférase ont conclus que deux de ces G4, chez les ARNm de VPS35 et PRKN, ont un effet répresseur sur la traduction. Les protéines pouvant lier ces deux G4 ont ensuite été analysées par spectrométrie de masse. La protéine Guanine Nucleotide-Binding Protein-Like 1 (GNL1) a alors été identifiée comme étant une protéine pouvant lier le, Within the nervous system, the post-transcriptional regulatory mechanisms contribute to the different cellular phenotypes. Indeed, weak deregulations can lead to diseases. RNA G-quadruplexes (G4) are emerging as new players in post-transcriptional regulation. G4s are very stable secondary structures found in DNA and RNA. They are formed by a minimum of two tetrads of guanines and stabilized by monovalent cations usually potassium. At the RNA level, they are involved in different mechanisms, such as alternative polyadenylation, alternative splicing, activation and inhibition of translation, and maturation of microRNAs. According to many, G4s are seen as events rather than entities. Trans elements, like proteins, are able to ensure their folding and unfolding. A bioinformatic analysis confirmed that there is indeed an enrichment of RNA G4s in the transcripts of the nervous system. This analysis also identified Parkinson's disease as one of the diseases with the highest proportion of potential G4 (pG4). Out of 16 disease-related genes, 15 of them contain a least one pG4. Then, different biochemical techniques such as fluorescence assay, circular dichroism, and in-line probing were used to confirm the formation of G4s located in the different 5’ untranslated regions (5’UTR). These techniques identified 4 new RNA G4s that have never been characterized before. Subsequently, luciferase assays concluded that two of these G4s, in the mRNA of VPS35 and PRKN, have a repressive effect on translation. The proteins capable of binding these two G4s were then identified by mass spectrometry. The protein Guanine Nucleotide-Binding Protein-Like 1 (GNL1) was identified as being a protein capable of binding the two G4s. Finally, the interaction between GNL1 and the G4 from VPS35 and PRKN was confirmed by electrophoretic mobility shift assays. In brief, this study identified a new protein, GNL1, which can bind two new G-quadruplexes located in deregulated genes associated with Parkinson