Ce travail de thèse porte sur l’étude des régulations post-transcriptionnelles en réponse à la lumière chez A.thaliana. Nous avons étudié deux systèmes de réponse à la lumière, la régulation traductionnelle des antennes photosynthétiques (Lhc) et la régulation de la voie des anthocyanes par le RNA silencing permettant la photoprotection. Dans une première partie nôtre approche a permis de montrer que la lumière a un impact sur le niveau de traduction global. De plus nous avons pu mettre en évidence que certaines Lhc sont régulées de façon traductionnelles en réponse à la lumière. Cette régulation pourrait être une composante du signal rétrograde entre le chlorolaste et le noyau. En parrallèle dans une seconde partie nous avons caractérisé la voie TAS4 de RNA silencing chez les plantes. Cette voie est mise en place en réponse à la forte lumière et régule l’accumulation des anthocyanes. / This work concerned post-transcriptional regulations in response to light in Arabidopsis thaliana. We are interested in two light responsive systems, translational regulation of the photosynthetic antenna protein and the regulation of the anthocyanin pathway by RNA silencing.In a first part we have shown that light affects global translation level. Furthermore our data indicate that some Lhc proteins are regulated at translational level in response to light. It seems that transaltional regulation of Lhc is a part of retrograde signaling between chlroroplast and nuclear. In a second part we have characterized the TAS4 RNA silencing pathway in Arabidopsis. We show that TAS4 regulate the accumulation of anthocyanin pathway in respose to high light.

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We have previously shown that the insulin-like growth factor 1 receptor (IGF-1R) translocates to the cell nucleus, where it binds to enhancer-like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF-1R (nIGF-1R) physically and functionally interacts with some nuclear proteins, i.e. the lymphoid enhancer-binding factor 1 (Lef1), histone H3, and Brahma-related gene-1 proteins. In this study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF-1R-binding partner. PCNA is a pivotal component of the replication fork machinery and a main regulator of the DNA damage tolerance (DDT) pathway. We found that IGF-1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA co-incubated with the IGF-1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF-1R targets, and PCNA phosphorylation was followed by mono- and polyubiquitination. Co-immunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT-dependent E2/E3 ligases (e.g. RAD18 and SHPRH/HLTF). Absence of IGF-1R or mutation of Tyr-60, Tyr-133, or Tyr-250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF-1R, externally induced DNA damage in IGF-1R-negative cells caused G(1) cell cycle arrest and S phase fork stalling. Taken together, our results suggest a role of IGF-1R in DDT.

It is increasingly becoming clear in the post-genomic era that proteins in a cell do not work in isolation but rather work in the context of other proteins and cellular entities during their life time. This has lead to the notion that cellular components can be visualized as wiring diagrams composed of different molecules like proteins, DNA, RNA and metabolites. These systems-approaches for quantitatively and qualitatively studying the dynamic biological systems have provided us unprecedented insights at varying levels of detail into the cellular organization and the interplay between different processes. The work in this thesis attempts to use these systems or network-based approaches to understand the design principles governing different cellular processes and to elucidate the functional and evolutionary consequences of the observed principles. Chapter 1 is an introduction to the concepts of networks and graph theory summarizing the various properties which are frequently studied in biological networks along with an overview of different kinds of cellular networks that are amenable for graph-theoretical analysis, emphasizing in particular on transcriptional, post-transcriptional and functional networks. In Chapter 2, I address the questions, how and why are genes organized on a particular fashion on bacterial genomes and what are the constraints bacterial transcriptional regulatory networks impose on their genomic organization. I then extend this one step further to unravel the constraints imposed on the network of TF-TF interactions and relate it to the numerous phenotypes they can impart to growing bacterial populations. Chapter 3 presents an overview of our current understanding of eukaryotic gene regulation at different levels and then shows evidence for the existence of a higher-order organization of genes across and within chromosomes that is constrained by transcriptional regulation. The results emphasize that specific organization of genes across and within chromosomes that allowed for efficient control of transcription within the nuclear space has been selected during evolution. Chapter 4 first summarizes different computational approaches for inferring the function of uncharacterized genes and then discusses network-based approaches currently employed for predicting function. I then present an overview of a recent high-throughput study performed to provide a 'systems-wide' functional blueprint of the bacterial model, Escherichia coli K-12, with insights into the biological and evolutionary significance of previously uncharacterized proteins. In Chapter 5, I focus on post-transcriptional regulatory networks formed by RBPs. I discuss the sequence attributes and functional processes associated with RBPs, methods used for the construction of the networks formed by them and finally examine the structure and dynamics of these networks based on recent publicly available data. The results obtained here show that RBPs exhibit distinct gene expression dynamics compared to other class of proteins in a eukaryotic cell. Chapter 6 provides a summary of the important aspects of the findings presented in this thesis and their practical implications. Overall, this dissertation presents a framework which can be exploited for the investigation of interactions between different cellular entities to understand biological processes at different levels of resolution.

The oocyte-to-zygote transition represents the only physiological event in mammalian life cycle, during which a differentiated cell is reprogrammed to become pluripotent. For its most part, the reprogramming relies on the accurate post-transcriptional control of maternally deposited mRNAs. Therefore, understanding the mechanisms of post-transcriptional regulation in the oocyte will help improve our knowledge of cell reprogramming. Short non- coding microRNAs have recently emerged as an important class of post-transcriptional regulators in a wide range of cellular and developmental processes. MicroRNAs repress their mRNA targets via recruitment of deadenylation and decapping complexes, which typically accumulate in cytoplasmic Processing bodies (P-bodies). The presented work uncovers an unexpected feature of the microRNA pathway which is found to be suppressed in fully-grown mouse oocytes and through the entire process of oocyte-to-zygote transition. This finding is consistent with the observation that microRNA-related P-bodies disassemble early during oocyte growth and are absent in fully-grown oocytes. Some of the proteins normally associated with P-bodies localize to the oocyte cortex. At the final stage of oocyte growth, these proteins, together with other RNA-binding factors, form subcortical...

The oocyte-to-zygote transition represents the only physiological event in mammalian life cycle, during which a differentiated cell is reprogrammed to become pluripotent. For its most part, the reprogramming relies on the accurate post-transcriptional control of maternally deposited mRNAs. Therefore, understanding the mechanisms of post-transcriptional regulation in the oocyte will help improve our knowledge of cell reprogramming. Short non- coding microRNAs have recently emerged as an important class of post-transcriptional regulators in a wide range of cellular and developmental processes. MicroRNAs repress their mRNA targets via recruitment of deadenylation and decapping complexes, which typically accumulate in cytoplasmic Processing bodies (P-bodies). The presented work uncovers an unexpected feature of the microRNA pathway which is found to be suppressed in fully-grown mouse oocytes and through the entire process of oocyte-to-zygote transition. This finding is consistent with the observation that microRNA-related P-bodies disassemble early during oocyte growth and are absent in fully-grown oocytes. Some of the proteins normally associated with P-bodies localize to the oocyte cortex. At the final stage of oocyte growth, these proteins, together with other RNA-binding factors, form subcortical...

No description available.

The GADD45 family of proteins consists of three small nuclear proteins, GADD45A, GADD45B, and GADD45G, which are implicated in modulating the cellular response to various types of genotoxic/physiological stress. This family of proteins has been shown to interact with and modulate the function of cell-cycle control proteins, such as p21 and cdc2/cyclin B1, the DNA repair protein, PCNA, key stress response MAP kinases, including MEKK4 (an upstream regulator of JNK kinase), and p38 kinase. Despite similarities in amino acid sequence, structure and function, each gadd45 gene is induced differentially, depending on the type of stress stimuli. For example, the alkylating agent, methylmethane sulfonate (MMS), rapidly induces all three genes, whereas hydrogen peroxide and sorbitol preferentially induce gadd45a and gadd45b, respectively. Studies of the mechanisms of the stress-mediated induction of the gadd45 genes have predominantly focused on gadd45a, with knowledge of gadd45b and gadd45g regulation lacking. Thus, in order to generate a more complete understanding of the collective regulation of the gadd45 genes, a comprehensive analysis of the stress-mediated induction of gadd45b has been carried out. Towards this end, a gadd45b promoter-reporter construct was generated, consisting of 3897bp sequence upstream of the transcription start site of gadd45b, fused to a luciferase reporter. In a human colorectal carcinoma cell line (RKO), in which gadd45b mRNA levels profoundly increase by various stress stimuli, we observe similar, high levels of induction of the gadd45b-luciferase construct with MMS or UVC treatments, but surprisingly not with sorbitol or anisomycin. Linker-scanning mutagenesis of the gadd45b promoter reveals several important MMS and UVC cis-acting responsive elements contained within the proximal promoter, including a GC-rich region and the CCAAT box. Furthermore, we have identified three constitutively bound transcription factors, Sp1, MZF1, and NFY, and one inducible factor, Egr1, which bind to these regions and which contribute to MMS-responsiveness. In contrast, a post-transcriptional mechanism appears to regulate gadd45b induction upon sorbitol treatment, as this treatment increases the gadd45b mRNA half-life, compared to MMS treatment. Interestingly, with the exception of a common cis-element, the stress-mediated induction of gadd45b appears to be mechanistically distinct from gadd45a. In conclusion, this study provides novel evidence that gadd45b induction by distinct stress agents, MMS and sorbitol, is regulated differentially at the level of mRNA transcription or mRNA stability, respectively. / Molecular Biology and Genetics

Stochasticity is a ubiquitous feature of cellular processes such as gene expression that can give rise to phenotypic differences for genetically identical cells. Understanding how the underlying biochemical reactions give rise to variations in mRNA/protein levels is thus of fundamental importance to diverse cellular processes. Recent technological developments have enabled single-cell measurements of cellular macromolecules which can shed new light on processes underlying gene expression. Correspondingly, there is a need for the development of theoretical tools to quantitatively model stochastic gene expression and its consequences for cellular processes. In this dissertation, we address this need by developing general stochastic models of gene expression. By mapping the system to models analyzed in queueing theory, we derive analytical expressions for the noise in steady-state protein distributions. Furthermore, given that the underlying processes are intrinsically stochastic, cellular regulation must be designed to control the`noise' in order to adapt and respond to changing environments. Another focus of this dissertation is to develop and analyze stochastic models of post-transcription regulation. The analytical solutions of the models proposed provide insight into the effects of different mechanisms of regulation and the role of small RNAs in fine-tunning the noise in gene expression. The results derived can serve as building blocks for future studies focusing on regulation of stochastic gene expression. / Ph. D.

Gene expression regulation encompasses a wide range of mechanisms that govern cellular processes. Among these, post-transcriptional regulation, including translation control, plays a pivotal role in ensuring precise protein synthesis, timing, and quantity. Perturbations of mechanisms such as RNA modifications, and interactions between RNA-binding proteins (RBPs) and specific RNA motifs, can lead to dysregulation of essential cellular processes. These alterations contribute to the development of various disorders, including cancer, neurodegenerative diseases, and metabolic disorders. Many publicly available datasets and studies offer opportunities to investigate the link between alterations in these mechanisms and disease manifestations. However, the limited availability of datasets for certain conditions or notable inconsistencies among reported associations prevent complete understanding of the underlying processes. Therefore, extending the investigations to encompass a diverse range of genes and/or diseases will enhance our comprehension of these intricate regulatory and disease mechanisms, aiding in the identification of potential therapeutic targets and innovative interventions to mitigate pathological conditions. In particular, we focused on three separate aspects involved in gene expression regulation: RNA modifications, RBPs interactions with RNA secondary structures, and the Kozak consensus sequence as a translational modulator. Each part uncovers essential mechanisms that govern post-transcriptional control of gene expression, shedding light on their roles in cellular processes and disease contexts. At first, we performed a comprehensive exploration of 15 RBPs involved in the regulation of the N6-methyladenosine (m6A) methylation. Leveraging data from The Cancer Genome Atlas (TCGA), we conducted a pan-cancer analysis across 31 tumor types to uncover the distribution of alterations of these factors, and we developed a user-friendly web application to enable users to conduct similar analyses. Additionally, we performed a parallel analysis focused on neuroblastoma, using data from publicly available and in-house datasets. These investigations unveil the potential impact of a subset of m6A factors on cancer development and progression. While in the first case, VIRMA and YTHDF reader proteins, emerged as the most frequently altered genes with significant pan-cancer prognostic implications, in the context of neuroblastoma, the writer METTL14 and the reader ALKBH5, showed the most prominent roles. Subsequently, our focus shifted to a distinct subset of RBPs capable of interacting with RNA secondary structures, particularly with RNA G-quadruplexes (RG4s). We established a comprehensive database cataloging RBPs with potential RG4-binding capabilities. This resource represents a valuable tool for researchers aiming to explore the intricate interplays between RBPs and RG4s, and their putative implications in diverse biological processes and diseases. Finally, attention was directed to the Kozak sequence, a pivotal determinant of the regulation of translation initiation. Exploiting the power of base editors, we developed a method to optimize translation initiation by modifying the Kozak sequence. This strategy offers promise in addressing haploinsufficiency-related disorders, where enhancing the functional protein is essential. Overall, these findings present opportunities for the identification of potential therapeutic targets and precision medicine strategies to alleviate a spectrum of pathological conditions, thus fostering advancements in the field of molecular biology and disease management.