La régulation de l’homéostasie du fer est cruciale chez les bactéries. Chez Salmonella, l’expression des gènes d’acquisition et du métabolisme du fer au moment approprié est importante pour sa survie et sa virulence. Cette régulation est effectuée par la protéine Fur et les petits ARN non codants RfrA et RfrB. Le rôle de ces régulateurs est d’assurer que le niveau de fer soit assez élevé pour la survie et le métabolisme de Salmonella, et assez faible pour éviter l’effet toxique du fer en présence d’oxygène. Les connaissances concernant le rôle de ces régulateurs ont été principalement obtenues par des études chez S. Typhimurium, un sérovar généraliste causant une gastro-entérite chez les humains. Très peu d’informations sont connues sur le rôle de ces régulateurs chez S. Typhi, un sérovar humain-spécifique responsable de la fièvre typhoïde. Le but de cette étude était de déterminer les rôles de Fur, RfrA et RfrB dans l’homéostasie du fer et la virulence de Salmonella, et de démontrer qu’ils ont une implication distincte chez les sérovars Typhi et Typhimurium. Premièrement, Fur, RfrA et RfrB régulent l’homéostasie du fer de Salmonella. Les résultats de cette étude ont démontré que Fur est requis pour la résistance au stress oxydatif et pour une croissance optimale dans différentes conditions in vitro. La sensibilité du mutant fur est due à l’expression des petits ARN RfrA et RfrB, et cette sensibilité est beaucoup plus importante chez S. Typhi que chez S. Typhimurium. Également, Fur inhibe la transcription des gènes codant pour les sidérophores en conditions riches en fer, tandis que les petits ARN RfrA et RfrB semblent être importants pour la production d’entérobactine et de salmochélines chez S. Typhi lors de conditions pauvres en fer. Ensuite, ces régulateurs affectent la virulence de Salmonella. Fur est important pour la motilité de Salmonella, particulièrement chez S. Typhi. Fur est nécessaire pour l’invasion des deux sérovars dans les cellules épithéliales, et pour l’entrée et la survie de S. Typhi dans les macrophages. Chez S. Typhimurium, Fur ne semble pas impliqué dans l’interaction avec les macrophages. De plus, les petits ARN RfrA et RfrB sont importants pour la multiplication intracellulaire de Salmonella dans les macrophages pour les deux sérovars. Finalement, la protéine Fur et les petits ARN RfrA et RfrB régulent l’expression de l’opéron fimbriaire tcf, absent du génome de S. Typhimurium. Un site de liaison putatif de la protéine Fur a été identifié dans la région promotrice de tcfA chez S. Typhi, mais une régulation directe n’a pas été confirmée. L’expression de tcf est induite par le fer et par Fur, et est inhibée par les petits ARN RfrA et RfrB. Ainsi, ces régulateurs affectent des gènes de virulence qui sont retrouvés spécifiquement chez S. Typhi. En somme, ce projet a permis de démontrer que les régulateurs de l’homéostasie du fer de Salmonella peuvent affecter la résistance de cette bactérie pathogène à différents stress, notamment le stress oxydatif, la croissance en conditions de carence en fer ainsi que la virulence. Ces régulateurs jouent un rôle distinct chez les sérovars Typhi et Typhimurium. / Regulation of iron homeostasis is crucial for bacteria. For Salmonella, proper timing of the expression of iron acquisition and metabolism genes is important for survival and virulence. This regulation is mediated by the protein Fur and the small non-coding RNAs (sRNAs) RfrA and RfrB. The role of these regulators is to assure that the iron level is high enough for survival and metabolism of Salmonella, and low enough to avoid the toxic effect of iron in the presence of oxygen. Thus far, information on the role of these regulators was principally obtained by studying S. Typhimurium, a generalist serovar causing gastro-enteritis in humans. Very little is known about the role of these regulators in S. Typhi, a human-specific serovar which causes typhoid fever. The goal of this study was to determine the roles of Fur, RfrA and RfrB in iron homeostasis and virulence of Salmonella, and to determine if they have a distinct implication in the serovars Typhimurium and Typhi. First, Fur, RfrA and RfrB regulate iron homeostasis in Salmonella. The results of this study have shown that Fur is required for resistance to oxidative stress and for optimal growth in different in vitro conditions. The sensitivity of the fur mutant is due to the expression of the sRNAs RfrA and RfrB, and this sensitivity is worse in S. Typhi than in S. Typhimurium. Also, Fur represses the transcription of the genes encoding siderophores in high-iron conditions, and the sRNAs RfrA and RfrB are required for enterobactin and salmochelins production in S. Typhi in low-iron conditions. Secondly, these regulators affect the virulence of Salmonella. Fur is important for the motility of Salmonella, especially in S. Typhi. Fur is required for the invasion of both serovars in epithelial cells, and for the uptake and survival of S. Typhi in macrophages. In S. Typhimurium, Fur is not required for the interaction with macrophages. Moreover, the sRNAs RfrA and RfrB are important for the intracellular multiplication of Salmonella within macrophages for both serovars. Finally, the Fur protein and the sRNAs RfrA and RfrB regulate the expression of the tcf fimbrial operon, absent from the genome of S. Typhimurium. A putative Fur binding site was identified in the tcfA promoter region of S. Typhi, but direct regulation has not been confirmed. tcf expression is activated by iron and Fur, and is inhibited by the sRNAs RfrA and RfrB. Therefore, these regulators affect virulence genes that are found specifically in S. Typhi. To conclude, this project demonstrates that the iron homeostasis regulators of Salmonella can affect the bacterial resistance to different stresses, espacially oxidative stress, the growth in iron-limiting conditions and virulence. These regulators have a distinct role in the serovars Typhi and Typhimurium.

OCL1 (Outer Cell Layer 1) est le membre fondateur, chez le maïs, de la famille multigénique regroupant les facteurs de transcription HD-ZIP IV. La plupart de ces gènes s’exprime préférentiellement dans l’épiderme, et chez Arabidopsis l’étude de mutants a montré que certains HD-ZIP IV étaient essentiels pour la différenciation de cette couche cellulaire. Lors de ma thèse je me suis intéressée à la régulation du gène OCL1 par un petit ARN non codant. En effet, la conservation au sein des 3’UTR de plusieurs gènes HD-ZIP IV d’un motif de 21 nucléotides (nt) suggérait l’existence d’un tel mécanisme. J’ai mis en évidence que ce motif de 21 nt était conservé des Bryophytes aux Angiospermes et qu’il était toujours couplé à un second motif conservé d’une taille de 19 nt avec lequel il peut s’apparier pour former une structure secondaire de type tige-boucle. J’ai démontré l’existence d’un petit ARN ayant une séquence (quasi) complémentaire au site de 21 nt. La biogenèse de ce petit ARN de 24 nt que nous avons nommé small1, dépend de RDR2/ MOP1, DCL3 et Pol IV/ RMR6, composants normalement requis pour le mécanisme de RdDM. A l’aide d’un système GFP sensor, j’ai cependant mis en évidence que small1 régulait l’expression de son gène cible par inhibition de la traduction et non par RdDM. Ces expériences ont par ailleurs démontré qu’OCL1 n’est pas régulé uniquement par small1, mais également via un second mécanisme dans lequel pourrait intervenir la structure secondaire de type tige-boucle. Enfin, j’ai montré que small1 possède une extrémité 5’modifiée, expliquant ainsi son absence des banques de données et définissant aussi une nouvelle classe de petits ARN chez les plantes. / Small non-coding RNAs are versatile riboregulators that control gene expression at the transcriptional or post-transcriptional level, governing many facets of plant development and stress responses. We previously suggested the possible regulation of OCL1 (Outer Cell Layer1) by a small RNA based on the intriguing presence of two conserved motives of 19 and 21nt in its 3’UTR. ZmOCL1 is a founding member of the HD-ZIP IV gene family encoding plant specific transcription factors mainly involved in epidermis differentiation and specialization. Here we present evidence for the existence of a 24 nt small RNA complementary to ZmOCL1 3’UTR which accumulates preferentially in maize reproductive organs but also in Arabidopsis flowers and inflorescences. The biogenesis of this 24 nt small RNA (that we named small1) depends on MOP1/RDR2 and RMR6/POLIV and DCL3, components normally required for RNA-dependent DNA-methylation. Unexpectedly GFP-sensor experiments showed that small1 may regulate its target at the post-transcriptional level, mainly through translational inhibition. These experiments further highlighted the importance of additional 3’UTR sequences required for efficient target repression, possibly implicating a secondary stem-loop structure. Finally, we showed that small1 is modified at its 5’ end, which not only explains its absence from the current databases but also defines a novel class of plant small RNAs.

Argonaute proteins are small DNA/RNA-guided endonucleases found in all domains of life. In animals, small RNAs of length 21–35 nucleotides direct the PIWI-clade of Argonautes to silence complementary target RNAs; these are called PIWI-interacting RNAs (piRNAs). During spermatogenesis in mice, piRNA-guided PIWI proteins, MIWI2, MILI, and MIWI, silence transposons, regulate expression of protein-coding genes and are necessary for fertility. A working endonuclease activity of MIWI and MILI is essential to complete spermatogenesis. Yet, both MIWI and MILI produce weak and slow target cleavage in vitro, thwarting biochemical examination of the silencing step. Here, we find that PIWI proteins require an auxiliary protein to efficiently cleave their targets, unlike any other known Argonaute. Gametocyte Specific Factor 1 (GTSF1) is a conserved zinc-finger protein essential for fertility and piRNA-directed silencing. We show GTSF1 accelerates the pre-steady-state rate of target cleavage by MIWI and MILI; this role of GTSF1 is also preserved in insects. A critical step in GTSF1 mechanism entails binding RNA. GTSF1 allowed detailed kinetic analyses of catalytic PIWIs: they require extensive 3′ complementarity between the guide and target to efficiently cleave them, but this base-pairing also limits turnover. Interestingly, within a species, different PIWI proteins have unique kinetic properties. In sum, our findings provide molecular mechanisms of GTSF1 function and target silencing by PIWIs as well as a useful method for future studies.

RNA interference (RNAi) is an important mechanism regulating gene expression. In plants, RNAi is triggered by double-stranded RNA (dsRNA) which is processed into small RNAs (sRNAs), usually 21-24 nt long. The sRNAs are loaded into Argonaut (AGO) protein and recognize the target based on sequence complementarity. When the target is mRNA, they can slice it or block translation leading to posttranscriptional gene silencing (PTGS). When the target is DNA, they can induce DNA methylation and chromatin changes, which when present in the promoter can lead to transcriptional gene silencing (TGS). The individual components of RNAi are well described, but less is known about the impact of different types of dsRNA precursors on the dynamics of RNAi. To study these aspects of RNAi, we used tobacco BY-2 cell line expressing GFP reporter and inducible silencers. The silencers used different ways of triggering the dsRNA formation by transcripts from antisense (AS), unterminated sense (UT) and inverted repeat (IR) GFP sequence to initiate PTGS. Additionally, one IR silencer based on the CaMV 35S promoter initiated TGS. This allowed us to study RNAi from the beginning throughout the steady state level and till the recovery phase, all in the highly homogeneous system. Using this system, we described several features...

Surprisingly little is known about the organization and distribution of tRNAs and tRNA-related sequences on a genome-wide scale. While tRNA complements are usually reported in passing as part of genome annotation efforts, and peculiar features such as the tandem arrangements of tRNAs in Entamoeba histolytica have been described in some detail, comparative studies are rare. We therefore set out to systematically survey the genomic arrangement of tRNAs in a wide range of eukaryotes to identify common patterns and taxon-specific peculiarities. We found that tRNA complements evolve rapidly and that tRNA locations are subject to rapid turnover. At the phylum level, distributions of tRNA numbers are very broad, with standard deviations on the order of the mean. Even within fairly closely related species, we observe dramatic changes in local organization. Consistent with this variability, syntenic conservation of tRNAs is also poor in general, with turn-over rates comparable to those of unconstrained sequence elements. We conclude that the genomic organization of tRNAs shows complex, lineage-specific patterns characterized by extensive variability, and that this variability is in striking contrast to the extreme levels of sequence-conservation of the tRNA genes themselves. Our comprehensive analysis of eukaroyotic tRNA distributions provides a basis for further studies into the interplay between tRNA gene arrangements and genome organization in general. Secondly, we focused on the investigation of small non-coding RNAs (ncRNAs) from whole transcriptome data. Since ncRNAs constitute a significant part of the transcriptome, we explore this data to detect and classify patterns derived from transcriptome-associated loci. We selected three distinct ncRNA classes: microRNAs, snoRNAs and tRNAs, all of which undergo maturation processes that lead to the production of shorter RNAs. After mapping the sequences to the reference genome, specific patterns of short reads were observed. These read patterns appeared to reflect RNA processing and, if so, should specify the RNA transcripts from which they are derived. In order to investigate whether the short read patterns carry information on the particular ncRNA class from which they orginate, we performed a random forest classification on the three distinct ncRNA classes listed above. Then, after exploring the potential classification of general groups of ncRNAs, we focused on the identification of small RNA fragments derived from tRNAs. After mapping transcriptome sequence data to reference genomes, we searched for specific short read patterns reflecting tRNA processing. In this context, we devised a common tRNA coordinate system based on conservation and secondary structure information that allows vector representation of processing products and thus comparison of different tRNAs by anticodon and amino acid. We report patterns of tRNA processing that seem to be conserved across species. Though the mechanisms and functional implications underlying these patterns remain to be clarified, our analysis suggests that each type of tRNA exhibits a specific pattern and thus appears to undergo a characteristic maturation process.

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In Chloroplasten durchlaufen primäre Transkripte eine großen Anzahl von bzw. Reifungsprozesse. Diese Ereignisse spielen eine wichtige Rolle bei der Regulation der Genexpression und sind im Wesentlichen durch Proteinfaktoren, insbesondere RNA-Bindeproteine, reguliert. Der plastidäre Spleißfaktor MatK zählt zu den prokaryotischen Gruppe-II-Intron. MatK aus Nicotiana tabacum interagiert mit seinem Heimatintron trnK und sechs weiteren Gruppe IIA Introns. In dieser Untersuchung, MatK-Bindestellen konnten unterschiedlichen Regionen der Gruppe-II-Introns zugewiesen werden mit RIP-seq in Nicotiana tabacum. Die vorliegenden Ergebnisse zeigen, dass MatK im Vergleich zu seinen bakteriellen Vorfahren an Vielseitigkeit in der RNA-Erkennung gewonnen hat. MatK zeigt somit beispielhaft, wie eine Maturase die Fähigkeit erworben haben könnte, in trans auf mehrere Introns zu wirken. Quantitative Untersuchung und mathematische Modellierung der Expression von MatK und dessen Zielen offenbart ein komplexes Muster möglicher regulatorischer Feedback-Mechanismen. In dieser Studie konnte ein möglicher Feedback- Mechanismus durch Analyse von polysomal gebundenen Transkripten ausgeschlossen werden. Stabile Bindung von Proteinen an spezifische RNA-Bindestellen und anschließender Abbau der ungeschützten RNA kann zu Akkumulation von kleinen RNAs (sRNAs) führen. Solche Footprints von RNA-Bindeproteinen wurden durch die Untersuchung von Datensätzen kleiner RNAs in Chlamydomonas reinhardtii identifiziert. Zwei der sRNAs entsprechen den 5'' Enden der reifen psbB und psbH mRNAs. Beide sRNAs sind abhängig von Mbb1, einem TPR (Tetratrico-peptide repeat) Protein. Die beiden sRNAs besitzen eine hohe Ähnlichkeit in ihrer Primärsequenz und fehlen in der mbb1 Mutante. Dies legt nahe, dass auch andere der hier identifizierten sRNAs an 5'' Enden plastidärer mRNAs Protein-Bindestellen repräsentieren, die für die korrekte RNA-Prozessierung und RNA-Stabilisierung in Chlamydomonas Chloroplasten erforderlich sind. / In chloroplasts, primary transcripts are subjected to a number of processing events. These events play important roles in the regulation of gene expression and are extensively controlled by protein factors, especially by RNA-binding proteins. Chloroplast splicing factor MatK is related to prokaryotic group II intron maturases. Nicotiana tabacum MatK interacts with its home intron trnK and six additional group IIA introns. In this study, binding sites of MatK were narrowed down to varying regions of its group II targets by RIP-seq in Nicotiana tabacum. The results obtained demonstrate that MatK has gained versatility in RNA recognition relative to its bacterial ancestors. MatK thus exemplifies how a maturase could have gained the ability to act in trans on multiple introns during the dispersion of the group II introns through the eukaryotic genome early in the eukaryote evolution. Quantitative investigation and mathematical modeling of the expression of MatK and its targets revealed a complex pattern of possible feedback regulatory interactions. In this study, one possible feedback regulation mechanism was ruled out by the analysis of polysome associated transcripts. Stable binding of proteins to specific RNA sites and subsequent degradation of the unprotected RNA regions can result in small RNA, footprint of the RNA binding protein. Such footprints were identified by examining small RNA datasets of Chlamydomonas reinhardtii. Two of the sRNAs correspond to the 5’ ends of mature psbB and psbH mRNAs. Both sRNAs are dependent on Mbb1, a nuclear-encoded TPR (Tetratrico-peptide repeat) protein. The two sRNAs have high similarity in primary sequence, and both are absent in the mbb1 mutant. This suggests that sRNAs at the 5’ ends of chloroplast mRNAs identified here generally represent the binding sites of proteins, which function in RNA processing and RNA stabilization in Chlamydomonas chloroplast.

Cis-regulatory changes in e.g. promoters or enhancers that affect the expression of a linked focal gene have long been thought to be important for adaptation. In this thesis, I investigate the selective importance and genomic correlates of cis-regulatory variation and divergence in the genus Capsella, using massively parallel sequencing data. This genus provides an opportunity to investigate cis-regulatory changes in response to polyploidization and mating system shifts, as it harbors three diploid species, the outcrosser Capsella grandiflora and the selfers Capsella orientalis and Capsella rubella, as well as the tetraploid Capsella bursa-pastoris. We first identify cis-regulatory changes associated with adaptive floral evolution in connection with the recent switch to self-fertilization in C. rubella and show that cis-regulatory changes between C. rubella and its outcrossing close relative C. grandiflora are associated with differences in transposable element content. Second, we show that variation in positive and purifying selection is important for the distribution of cis-regulatory variation across the genome of C. grandiflora. Interestingly, the presence of polymorphic transposable elements is strongly associated with cis-regulatory variation in C. grandiflora. Third, we show that the tetraploid C. bursa-pastoris is of hybrid origin and investigate the contribution of both parental species to gene expression. We show that gene expression in the tetraploid is partly explained by cis-regulatory divergence between the parental species. Nonetheless, within C. bursa-pastoris there is a great deal of variation in homeolog expression. In summary, this thesis explores the role of cis-regulatory changes for adaptive morphological changes in connection to a shift in mating system, the role of cis-regulatory divergence between progenitor species for an allopolyploid as well as the impact of positive and purifying selection on cis-regulatory variation within a species.