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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.

Le Glypican-3 (GPC3) est surexprimé dans 72% des carcinomes hépatocellulaire (CHC). C’est un co-récepteur membranaire du récepteur WNT, qui appartient à la famille des protéoglycanes à sulfates d'héparane. L'objectif général de ma thèse vise à étudier les mécanismes de régulation post-transcriptionnelle de l’expression du GPC3 dans le CHC. Pour cela, j’ai développé un test fonctionnel qui m’a permis de cribler une bibliothèque de 876 microARNs humains. Ceci a conduit à l’identification de 5 microARNs régulateurs de l’expression de l’ARNm codant pour le GPC3 via sa région 3’ non traduite (NT). Mon travail de thèse porte plus particulièrement sur le miR-1271 et le miR-1291 car ils sont dérégulés dans le CHC et sont respectivement inhibiteur et inducteur de l’expression du GPC3. Dans un premier projet, j’ai démontré que le miR-1271 cible directement la région 3’NT du GPC3 et diminue la stabilité de son ARNm. Ce microARN est sous-exprimé dans le CHC et son expression corrèle négativement avec celle de l'ARNm du GPC3 dans les CHC associés à une infection par le virus de l’hépatite B. Dans un deuxième projet, j’ai démontré que le miR-1291 régule positivement l’expression du GPC3 en inhibant un facteur intermédiaire. Une analyse in silico a permis d’identifier IRE1α comme candidat. IRE1α est une protéine transmembranaire du réticulum endoplasmique (RE) qui participe à « l’Unfolded Protein Response », une réponse adaptative activée lors de l’accumulation de protéines mal conformées dans le RE. J’ai démontré qu’IRE1α clive l’ARNm codant pour le GPC3 grâce à son activité endoribonucléase. D’autre part, le miR-1291 cible directement l’ARNm codant pour IRE1α dans sa région 5’NT ce qui inhibe son expression et induit une surexpression du GPC3. Le miR-1291 est surexprimé dans le CHC et son expression corrèle positivement avec celle de l’ARNm du GPC3. En conclusion, mon travail de thèse m’a permis de mettre en évidence et de caractériser deux nouveaux microARNs (miR-1271 et miR-1291) contrôlant l’expression du GPC3 par des mécanismes directs ou indirects. La pertinence physiopathologique de ces régulations dans le CHC est en accord avec les niveaux d’expression respectifs de ces microARNs, qui pourraient contribuer à la surexpression du GPC3 dans ces tumeurs. / Glypican-3 (GPC3) is overexpressed in 72% of hepatocellular carcinoma (HCC). It is a co-receptor for WNT receptor and belongs to the heparan sulfate proteoglycans family. The general objective of my PhD thesis was to study the mechanisms by which GPC3 is post-transcriptionnally regulated in HCC. To this end, I developed a functional test that allowed me to screen a library of 876 human microRNAs. This led me to identify 5 microRNAs that regulate the expression of GPC3 mRNA through its 3’Untranslated Region (UTR). The work presented in this thesis particulary focuses on miR-1271 and miR-1291 as both microRNAs present a deregulated expression in HCC and are respectively inhibitor and activator of GPC3 mRNA expression. In a first project, I demonstrated that miR-1271 directly binds to GPC3 mRNA 3’UTR and affects its stability. This microRNA is underexpressed in HCC and its expression negatively correlates with that of GPC3 mRNA in a subgroup of HCC corresponding to those associated with hepatitis B virus infection. In a second project, I demonstrated that miR-1291 postively regulates the expression of GPC3 mRNA by targeting an intermediate factor. An in silico analysis led to the identification of the Inositol Requiring Enzyme 1 alpha (IRE1α) as a potential candidate. IRE1α is an endoplasmic reticulum (ER) resident type I transmembrane protein and contributes to the signaling of the Unfolded Protein Response (UPR). The UPR is an adaptive response activated upon accumulation of improperly folded proteins in the ER. I showed that IRE1α cleaves GPC3 mRNA through its endoribonuclease activity. Moreover I demonstrated that miR-1291 directly targets IRE1α mRNA through its 5’UTR, thereby decreasing its expression and contributing to GPC3 mRNA overexpression. MiR-1291 is overexpressed in HCC and its expression positively correlates with that of GPC3 mRNA. In summary, the work carried out during my PhD allowed the identification and the characterization of two new microRNAs (miR-1271 and miR-1291) that control the expression of GPC3 mRNA through direct or indirect mechanisms. The pathophysiological relevance of these regulatory mechanisms is in agreement with the respective expression levels of these microRNAs in HCC, which could therefore contribute to the overexpression of GPC3 in those tumors.

Diabetes é uma doença metabólica caracterizada por hiperglicemia associada a prejuízos na captação e utilização de glicose, em que reduções na expressão da proteína GLUT4 (codificada pelo gene SLC2A4), bem como das enzimas Hexokinase-2 e Glycogen synthase (codificadas pelos genes HK2 e GYS1), desempenham papel importante. Recentemente, um novo elemento vem sendo relacionado à etiopatogenia e à fisiopatologia do diabetes, os microRNAs (miRNAs), que são pequenos RNAs envolvidos na regulação da expressão gênica, geralmente afetando a degradação de mRNAs. Entretanto, a participação de miRNAs envolvidos na redução da expressão de mRNAs relacionados a proteínas envolvidas na captação e utilização de glicose, sobretudo em músculo esquelético, permanece desconhecida. O objetivo desse estudo foi investigar a expressão de miRNAs potencialmente reguladores da expressão de Slc2a4/GLUT4, Hk2/HK2 e Gys1/GYS1 em músculo esquelético de ratos diabéticos. Utilizamos ratos Wistar machos que foram tornados diabéticos pela administração de estreptozotocina. Após 13 dias, 3 grupos foram formados: não-diabético (ND) e diabético tratado com placebo (DP) ou insulina (DI). O tratamento foi conduzido por 7 dias, totalizando 21 dias de diabetes. Variáveis metabólicas foram avaliadas e os músculos sóleos foram removidos para avaliar a expressão de mRNAs, miRNAs e proteínas. Uma abrangente análise in silico foi conduzida para determinar miRNAs candidatos a regularem a expressão de Slc2a4, Hk2 e Gys1. Os animais diabéticos apresentaram perda de peso, poliúria, glicosúria, hiperglicemia e aumento de frutosamina plasmática; a insulinoterapia melhorou estas variáveis. O diabetes reduziu a expressão dos mRNAs Slc2a4 (~55%), Hk2 (~47%) e Gys1 (~45%), e das proteínas GLUT4 (~77%), HK2(~52%) e GYS1 (~49%); a insulinoterapia restaurou essas variáveis. A expressão de 20 miRNAs foi avaliada neste estudo; 8 foram modulados pelo diabetes, sendo três supra-regulados, miR-1 (~28%), miR-29b (~118%) e miR-29c (~51%); e cinco infra-regulados, miR-93 (~39%), miR-199a (~30%), miR-345-3p (~23%), miR-532-3p (~26%) e miR-150 (~32%). Exceto pelo miR-1 e miR-150, a insulinoterapia reverteu as demais alterações. Além disso, miR-29b e miR-29c correlacionaram-se negativamente com GLUT4 e HK2, e positivamente com glicemia, glicosúria e frutosamina, sugerindo uma possível relação causal; enquanto que miR-199a e miR-532-3p correlacionaram-se positivamente com GLUT4 e HK2, e também com as variáveis metabólicas, sugerindo uma regulação indireta sobre os mRNAs dessas proteínas. No último caso, demonstrou-se que o miR-199a tem como alvo o NFKB1, um repressor do gene Slc2a4, o qual diminuiu no diabetes, explicando, pelo menos parcialmente, o efeito indireto sobre o GLUT4. Em suma, o diabetes aumenta a expressão de miR-29b e miR-29c, e reduz a expressão de miR-199a e miR-532-3p; o primeiro efeito, potencialmente age diretamente na tradução do mRNA Slc2a4 e Hk2, e o segundo, potencialmente age indiretamente, via NFKB, na transcrição dos genes. Como consequência, as proteínas GLUT4 e HK2 diminuem, o que reduziria a utilização de glicose pelo músculo, contribuindo para a hiperglicemia do diabetes. / Diabetes is a metabolic disease characterized by hyperglycemia associated with impaired glucose metabolism and uptake, in which reductions of GLUT4, hexokinase 2 (HK2) and glycogen synthase 1 (GYS1) proteins, encoded respectively by SLC2A4, HK2 and GYS1 genes, play an important role. Recently, a new element have been related to etiopathogeny and pathophysiology of diabetes, the microRNAs (miRNAs), which are small RNAs involved in the regulation of gene expression, usually by affecting the degradation of mRNAs. However, the participation of miRNAs diabetes-induced reduction of expression of genes related to glucose uptake and metabolism in skeletal muscle remains unknown. Thus, the objective of this study was to investigate the expression of miRNAs potentially regulators of the Slc2a4/GLUT4, Hk2/HK2 and Gys1/GYS1 in skeletal muscle of diabetic rats. Male Wistar rats were rendered diabetic by receiving streptozotocin. After 13 days, 3 groups were formed: non-diabetic (ND), and diabetic treated with placebo (DP) or insulin (DI) (NPH insulin, 6U/day). Treatment was conducted for 7 days, totalizing 21 days of diabetes. At the end of the experimental period, metabolic variables were evaluated and the soleus muscle was removed for evaluation of mRNA, miRNA and protein expression. A broad in silico analysis was performed to determine candidate miRNAs as potential regulators of Slc2a4, Hk2 and Gys1. Diabetic rats shown weight loss, polyuria, glycosuria, hyperglycemia and increased plasma fructosamine; insulin treatment improved these variables. Diabetes reduced Slc2a4 (~55%), Hk2 (~47%) and Gys1 (~45%) mRNAs, as well as GLUT4 (77%), HK2 (52%) and GYS1 (49%) proteins; insulin treatment restored these variables. Twenty miRNAs were assessed in this study. Eight miRNAs were modulated by diabetes in skeletal muscle; three were upregulated: miR-1 (28%), miR-29b (118%) and miR-29c (51%), whereas five were downregulated: miR-93 (39%), miR-150 (32%), miR-199a (30%), miR-345-3p (23%) and miR-532-3p (26%). Except for miR-1 and miR-150, all regulations were reverted by insulin treatment. Besides, miR-29b and miR-29c were negatively correlated with GLUT4 and HK2 proteins, and positively with glucose, glycosuria and plasma fructosamine suggesting a direct causal relationship; while miR-199a and miR-532-3p were positively correlated with GLUT4 and HK2 proteins, and also with the metabolic variables, suggesting an indirect causal relationship. In the last case, it was demonstrated that miR-199a has the Slc2a4 repressor Nfkb1 as target, which was reduced in muscle from diabetic rats, explaining, at least partially, the indirect effect upon GLUT4. In conclusion, diabetes increase the expression of miR-29b and miR-29c, and reduce the expression of miR-199a e miR-532-3p; the first effect, potentially acts directly in the translation of Slc2a4 and Hk2 mRNAs, and the second one, potentially acts indirectly, via NFKB, in the transcription of these genes. As a result, the expression of GLUT4 and HK2 decreases, which would reduce the muscle glucose uptake and metabolization, contributing to the hyperglycemia of the diabetes.

abstract: Multicellular organisms use precise gene regulation, executed throughout development, to build and sustain various cell and tissue types. Post-transcriptional gene regulation is essential for metazoan development and acts on mRNA to determine its localization, stability, and translation. MicroRNAs (miRNAs) and RNA binding proteins (RBPs) are the principal effectors of post-transcriptional gene regulation and act by targeting the 3'untranslated regions (3'UTRs) of mRNA. MiRNAs are small non-coding RNAs that have the potential to regulate hundreds to thousands of genes and are dysregulated in many prevalent human diseases such as diabetes, Alzheimer's disease, Duchenne muscular dystrophy, and cancer. However, the precise contribution of miRNAs to the pathology of these diseases is not known. MiRNA-based gene regulation occurs in a tissue-specific manner and is implemented by an interplay of poorly understood and complex mechanisms, which control both the presence of the miRNAs and their targets. As a consequence, the precise contributions of miRNAs to gene regulation are not well known. The research presented in this thesis systematically explores the targets and effects of miRNA-based gene regulation in cell lines and tissues. I hypothesize that miRNAs have distinct tissue-specific roles that contribute to the gene expression differences seen across tissues. To address this hypothesis and expand our understanding of miRNA-based gene regulation, 1) I developed the human 3'UTRome v1, a resource for studying post-transcriptional gene regulation. Using this resource, I explored the targets of two cancer-associated miRNAs miR-221 and let-7c. I identified novel targets of both these miRNAs, which present potential mechanisms by which they contribute to cancer. 2) Identified in vivo, tissue-specific targets in the intestine and body muscle of the model organism Caenorhabditis elegans. The results from this study revealed that miRNAs regulate tissue homeostasis, and that alternative polyadenylation and miRNA expression patterns modulate miRNA targeting at the tissue-specific level. 3) Explored the functional relevance of miRNA targeting to tissue-specific gene expression, where I found that miRNAs contribute to the biogenesis of mRNAs, through alternative splicing, by regulating tissue-specific expression of splicing factors. These results expand our understanding of the mechanisms that guide miRNA targeting and its effects on tissue-specific gene expression. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2019

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