Tesi sul tema "Messenger RNA-binding proteins"

Although the reason for insufficient release of insulin in diabetes mellitus may vary depending on the type and stage of the disease, it is of vital importance that an amplified insulin biosynthesis can meet the increased need during periods of hyperglycemia. The insulin mRNA is highly abundant in beta cells and changes in insulin mRNA levels are, at least in part, controlled by altered rates of mRNA degradation. Since the mechanisms behind the control of insulin messenger stability and translation are still largely obscure, the work presented in this thesis therefore aimed to further investigate the role of insulin mRNA binding proteins in the control of insulin mRNA break-down and utilization for insulin biosynthesis. To clarify how glucose regulates insulin mRNA stability and translation we studied the correlation between polypyrimidine tract binding protein (PTB) gene expression and insulin mRNA levels. It was found that an increase in PTB mRNA and protein levels is paralleled by an increase in insulin mRNA levels. It was also found that PTB binds to the 5’-untranslated region of the insulin mRNA and that insulin mRNA can be translated through a cap-independent mechanism in human islets of Langerhans, possibly due to the interaction with PTB. Further it was discovered that the suppressed insulin biosynthesis in human islets during glucotoxicity is partly due to an induction of the microRNA miR-133a. This induction leads to decreased levels of PTB and insulin biosynthesis rates in human islets. Finally, we were able to identify two proteins, hnRNP U and TIAR, that bind specifically to the insulin mRNA in vitro, and show that the stability and translation of insulin mRNA is oppositely affected by these proteins. In conclusion, insulin producing cells seem to be able to regulate insulin messenger stability and translation by a control mechanism in which the binding of specific proteins to the insulin messenger dictates the outcome. A better understanding of the events leading to insulin production may in the future aid in optimal diagnosis and treatment of type 2 diabetes.

Maternally supplied mRNAs encode for necessary developmental regulators that pattern early embryos in many species until zygotic transcription is activated. In Caenorhabditis elegans, post-transcriptional regulatory mechanisms guide early development during embryogenesis. Maternal transcripts remain in a translationally silenced state until fertilization. A suite of RNA-binding proteins (RBP’s) regulate these maternally supplied mRNAs during oogenesis, the oocyte-to-embryo transition, and early embryogenesis. Identifying the target specificity of these RNA-binding proteins will reveal their contribution to patterning of the embryo. We are studying post-transcriptional regulation of maternal mRNAs during oocyte maturation, which is an essential part of meiosis that prepares oocytes for fertilization. Although the physiological events taking place during oocyte maturation have been well studied, the molecular mechanisms that regulate oocyte maturation are not well understood. OMA-1 and OMA-2 are essential CCCH-type tandem zinc finger (TZF) RBP’s that function redundantly during oocyte maturation. This dissertation shows that I defined the RNA-binding specificity of OMA-1, and demonstrated that OMA-1/2 are required to repress the expression of 3ʹUTR reporters in developing oocytes. The recovered sequences from in vitro selection demonstrated that OMA-1 binds UAA and UAU repeats in a cooperative fashion. Interestingly, OMA-1 binds with high affinity to a conserved region of the glp-1 3ʹUTR that is rich in UAA and UAU repeats. Multiple RNA-binding proteins regulate translation of GLP-1 protein, a homolog of Notch receptor. In addition to previously identified RBP’s, we showed that OMA-1 and OMA-2 repress glp-1 reporter expression in C. elegans oocytes. Mapping the OMA-1 dependent regulatory sites in the glp-1 mRNA and characterizing the interplay between OMA-1 and other factors will help reveal how multiple regulatory signals coordinate the transition from oocyte to embryo but the abundance of OMA-1 binding motifs within the glp-1 3ʹUTR makes it infeasible to identify sites with a functional consequence. I therefore first developed a strategy that allowed us to generate transgenic strains efficiently using a library adaptation of MosSCI transgenesis in combination with rapid RNAi screening to identify RBP-mRNA interactions with a functional consequence. This allowed me to identify five novel mRNA targets of OMA-1 with an in vivo regulatory connection. In conclusion, the findings in this dissertation provide new insights into OMA-1 mediated mRNA regulation and provide new tools for C. elegans transgenesis. Development of library MosSCI will advance functional mapping of OMA-1 dependent regulatory sites in the target mRNAs. Extending this strategy to map functional interactions between mRNA targets and RNAbinding proteins in will help reveal how multiple regulatory binding events coordinate complex cellular events such as oocyte to embryo transition and cell-fate specification.

Maternally supplied mRNAs encode for necessary developmental regulators that pattern early embryos in many species until zygotic transcription is activated. In Caenorhabditis elegans, post-transcriptional regulatory mechanisms guide early development during embryogenesis. Maternal transcripts remain in a translationally silenced state until fertilization. A suite of RNA-binding proteins (RBP’s) regulate these maternally supplied mRNAs during oogenesis, the oocyte-to-embryo transition, and early embryogenesis. Identifying the target specificity of these RNA-binding proteins will reveal their contribution to patterning of the embryo. We are studying post-transcriptional regulation of maternal mRNAs during oocyte maturation, which is an essential part of meiosis that prepares oocytes for fertilization. Although the physiological events taking place during oocyte maturation have been well studied, the molecular mechanisms that regulate oocyte maturation are not well understood. OMA-1 and OMA-2 are essential CCCH-type tandem zinc finger (TZF) RBP’s that function redundantly during oocyte maturation. This dissertation shows that I defined the RNA-binding specificity of OMA-1, and demonstrated that OMA-1/2 are required to repress the expression of 3ʹUTR reporters in developing oocytes. The recovered sequences from in vitro selection demonstrated that OMA-1 binds UAA and UAU repeats in a cooperative fashion. Interestingly, OMA-1 binds with high affinity to a conserved region of the glp-1 3ʹUTR that is rich in UAA and UAU repeats. Multiple RNA-binding proteins regulate translation of GLP-1 protein, a homolog of Notch receptor. In addition to previously identified RBP’s, we showed that OMA-1 and OMA-2 repress glp-1 reporter expression in C. elegans oocytes. Mapping the OMA-1 dependent regulatory sites in the glp-1 mRNA and characterizing the interplay between OMA-1 and other factors will help reveal how multiple regulatory signals coordinate the transition from oocyte to embryo but the abundance of OMA-1 binding motifs within the glp-1 3ʹUTR makes it infeasible to identify sites with a functional consequence. I therefore first developed a strategy that allowed us to generate transgenic strains efficiently using a library adaptation of MosSCI transgenesis in combination with rapid RNAi screening to identify RBP-mRNA interactions with a functional consequence. This allowed me to identify five novel mRNA targets of OMA-1 with an in vivo regulatory connection. In conclusion, the findings in this dissertation provide new insights into OMA-1 mediated mRNA regulation and provide new tools for C. elegans transgenesis. Development of library MosSCI will advance functional mapping of OMA-1 dependent regulatory sites in the target mRNAs. Extending this strategy to map functional interactions between mRNA targets and RNAbinding proteins in will help reveal how multiple regulatory binding events coordinate complex cellular events such as oocyte to embryo transition and cell-fate specification.

RNA-binding proteins (RBPs) are important for a wide variety of biological processes involved in gene regulation. However, the structural and dynamic contributions to their biological activity are poorly understood. The tristetraprolin (TTP) family of RBPs, including TTP, TIS11b and TIS11d, regulate the stability of mRNA transcripts encoding for key cancer-related proteins, such as tumor necrosis factor- and vascular endothelial growth factor. Biophysical studies have shown that the RNA binding domain, consisting of two CCCH zinc fingers (ZFs), is folded in the absence of RNA in TIS11d and TIS11b. In TTP, however, only ZF1 adopts a stable fold, while RNA is required to completely fold the tandem zinc finger (TZF). The focus of this research was to understand the origin and biological significance of the structural differences observed for the TZF domains of TTP and TIS11d. Three residues were shown to control the affinity for the structural Zn2+ and determine the folding of ZF2 in the absence of RNA. The partially-folded TZF domain of TTP has greater selectivity for RNA sequences than the fully folded TZF domain of TIS11d. The mRNA destabilizing activity of TTP was increased when the partially disordered RBD of TTP was replaced with the fully structured TZF domain of TIS11d. Disruption of the structure and/or dynamics of the TZF domain observed in the disease-associated mutations of TIS11d, P190L and D219E, results in aberrant cytoplasmic localization. This work demonstrates that the extent of RBD folding in the TTP family is important for differential RNA recognition, mRNA turnover, and protein localization in vivo.

Retroviruses have evolved sophisticated mechanisms to ensure timely export of incompletely spliced viral messenger ribonucleic acids (mRNAs) for gene expression and for viral packaging. For example, the Human Immunodeficiency Virus type 1 (HIV-1) encodes the Rev regulatory protein, a sequence-specific RNA-binding protein that is responsible for the cytoplasmic accumulation of intron-containing viral mRNAs. The HIV-1 Rev protein contains an amino terminal (N-terminal) Arginine-Rich Motif (ARM) RNA-binding domain (RBD) and a carboxy terminal (C-terminal) leucine-rich activation domain which functions as a Nuclear Export Signal (NES). The Rev ARM interacts in a sequence-specific manner with a cis-acting viral RNA stem-loop structure, the Rev Responsive Element (RRE), located in all incompletely spliced viral mRNAs. This initial interaction is followed by the recruitment of additional Rev molecules to form a RiboNucleoProtein (RNP) complex involving the RRE and Rev molecules. The cytoplasmic accumulation of the Rev:RRE RNP complex is dependent on the interaction of Rev with key cellular cofactors. Rev activation domain mutants exhibit a trans-dominant negative phenotype, suggesting that this domain of Rev interacts with cellular proteins required for Rev function. Biochemical and genetic studies have identified several cellular proteins that bind to the activation domain of Rev and are therefore candidate cofactors for Rev function. Amongst these is the human Rev Interacting Protein [hRIP, 79], which is also known as the Rev/Rex activation domain-binding protein [Rab, 18]. hRIP was identified in a yeast two-hybrid assay with the HIV-1 Rev and its functionally equivalent Human T-cell Leukemia Virus type-1 (HTLV-1) Rex protein as baits. The interaction between hRIP and HIV-1 Rev is dependent on a functional Rev NES, as predicted for a bona fide Rev cellular cofactor, and the Nucleoporin-like (Nup-like) repeats in the C-terminus of hRIP (18, 79]. Additional genetic studies indicated that the interaction between hRIP and Rev is indirect and is most likely mediated by the cellular export receptor CRM1 (Chromosomal Region Maintenance 1) [1, 153]. A role for hRIP in Rev function or HIV-1 replication has remained elusive. The goal of this dissertation was to determine whether hRIP is required for Rev function and HIV-1 replication. We used two approaches, a dominant-negative mutant and RNA interference (RNAi), to ablate hRIP activity and analyzed Rev function and HIV-1 replication using standard assays. The results of this dissertation demonstrate that hRIP is required for Rev function and HIV-1 replication. We show that Rev function is inhibited upon ablation of hRIP activity by either a trans-dominant negative mutant or RNAL Furthermore, we find that depletion of endogenous hRIP by RNAi results in the loss of viral replication in human cell lines and primary human macrophages. Unexpectedly, in the absence of functional hRIP, RRE-containing viral RNAs accumulate in the nuclear periphery where hRIP is localized. Comparable ablation of hRIP activity did not affect the intracellular localization or trafficking of a variety of proteins or cellular poly (A+ mRNA, suggesting that the inhibition of Rev-directed RNA export is specific. In conclusion, the results of this dissertation demonstrate that hRIP is involved in the movement of Rev-directed RNAs from the nuclear periphery to the cytoplasm. Therefore, hRIP is required for Rev function and HIV-1 replication. The hRIP protein is not essential for the maintenance of cell viability and thus might represent a novel target for the development of antiviral agents for HIV-1.

Les protéines de liaison à l'ARN (RBP) jouent un rôle majeur dans la régulation de la traduction de l'ARN messager et l'adaptation du métabolisme cellulaire à divers signaux environnementaux. La présence dans de nombreuses RBP d'une combinaison unique de domaines fonctionnels structurés et de régions intrinsèquement désordonnées, leur permet de générer des condensats riches en ARN par le processus de séparation de phase liquide-liquide. G3BP1 est une RBP centrale dans le processus de protection de l'ARNm lorsque les cellules sont exposées à des stress environnementaux via la formation des granules de stress - des condensats de ribonucléoprotéines qui s'assemblent en réponse au stress. Les granules de stress (SG) pourraient servir de point de contrôle dans la destinée des ARNm : stockage de l'ARNm pour éviter l'expression de protéines inutiles à la survie cellulaire, transfert des transcrits d'ARNm vers les p-bodies pour dégradation ou transfert dans des polysomes pour traduction de protéines essentielles à la survie cellulaire. G3BP1 est un élément central dans l'assemblage des SGs car elle possède des domaines de liaison à l'ARN et une activité hélicase, et interagit spécifiquement avec plusieurs protéines présentes dans les SG comme Caprin1.Le but de cette étude est d'analyser la coopération entre G3BP1 et Caprin1 dans la liaison à l'ARN et la formation de condensats. Des études antérieures mettent en évidence le caractère central de G3BP1 dans l'assemblage de SG, mais G3BP1 ne possède pas de domaine de type prion contrairement aux nombreuses protéines impliquées dans la nucléation des SG et son affinité pour l'ARNm est assez faible. On propose que Caprin1, en tant que partenaire de G3BP1 à travers le domaine NTF2L et également une protéine impliquée dans la formation de stress granules, favorise la liaison de G3BP1 à l'ARNm et son recrutement dans les granules de stress. De plus, on a décortiqué la fonction des différents domaines de G3BP1 dans cette interaction.Pour démontrer l'interaction G3BP1-Caprin1-ARNm, nous avons utilisé plusieurs méthodes de biologie structurale et cellulaire. Nous proposons que Caprin1, qui est à la fois un partenaire connu de G3BP1 via le domaine NTF2-L de G3BP1 et une protéine de liaison à l'ARNm via son domaine de faible complexité riche en RGG, puisse améliorer l'interaction entre ARNm et G3BP1 et favoriser le recrutement de l'ARNm dans les SGs. L'analyse de l'influence des différents domaines de G3BP1 dans cette interaction démontre que l'amélioration du recrutement de l'ARNm via l'interaction avec Caprin1 ne fonctionne que si G3BP1 conserve ses domaines de liaison à l'ARNm. La coopération entre Caprin1 et G3BP1 permet de mieux comprendre la centralité de G3BP1 dans l'assemblage des SGs et ouvre des perspectives sur le recrutement d'ARNm spécifiques lors d'épisodes de stress
RNA-binding proteins play major role in regulation of messenger RNA translation and the adaptation of cellular metabolism to various environmental signals. This is accomplished due to RBPs possessing unique combination of structured functional domains and non-structured intrinsically disordered regions, which allows them to undergo liquid-liquid phase separation and form separate condensates with mRNA. G3BP1 is a central protein in a network of RBPs that participate in protection of mRNA from environmental stress by forming stress granules - ribonucleoprotein condensates that assemble in response to stress. Stress granules (SGs) might function as checkpoint for mRNA fate: storage of translationally silent mRNA, transfer of mRNA transcripts to P-bodies for degradation or transfer back into polysomes for translation. G3BP1 possesses RNA-binding domains, helicase activity and recruits several proteins into SGs, with some of them considered central nucleators in SG assembly, Caprin1 among them. The aim of this study is to investigate the cooperation between G3BP1 and Caprin1 in RNA-binding and condensate formation. Previous studies evidence the centrality of G3BP1 in SG assembly but, unlike other SG-nucleating proteins, G3BP1 lacks a prion-like domain and its direct mRNA binding is not clear. We propose that Caprin1, which is a known G3BP1 partner through the NTF2L domain of G3BP1 and a SG protein, may promote the G3BP1 mRNA binding and improve the mRNA recruitment in SG. In addition, we analyzed the function of the different G3BP1 domains in this interaction To demonstrate G3BP1-Caprin1-mRNA interplay, we used several methods of structural and cellular biology. We confirmed that G3BP1 and Caprin1 can co-localize and recruit mRNA in vivo, moreover, NTF2L-domain of G3BP1 is needed for this interaction. The mRNA recruiting capabilities of G3BP1 are improved in presence of Caprin1, and the RNA-binding domains of G3BP1 are of fundamental importance. Similarly, the enhanced mRNA recruitment of G3BP1-Caprin1 complex to SGs is at play only when full length G3BP1 is present. The consequence of G3BP1-Caprin1 interaction explain the centrality of G3BP1 in SG assembly and complement the model in which the shift RNA concentration triggers the conformational switch of G3BP1 at the heart of SG assembly by liquid-liquid phase separation

Local protein synthesis is required for long-term memory formation in the brain. One protein family, Cytoplasmic Polyadenylation Element binding Protein (CPEB) that regulates protein synthesis is found to be important for long-term memory formation possibly through regulating local protein synthesis in neurons. The well-studied member of this family, CPEB1, mediates both translational repression and activation of its target mRNAs by regulating mRNA polyadenylation. Mouse with CPEB1 KO shows defect in memory extinction but not long-term memory formation. Three more CPEB1 homologs (CPEB2-4) are identified in mammalian system. To test if CPEB2-4 may have redundant role in replacing CPEB1 in mediating local protein synthesis, the RNA binding specificity of these homologs are studied by SELEX. The result shows CPEB2-4 bind to RNAs with consensus sequence that is distinct from CPE, the binding site of CPEB1. This distinction RNA binding specificity between CPEB1 and CPEB2-4 suggests CPEB2-4 cannot replace CPEB1 in mediating local protein synthesis. For CPEB2-4 have distinct RNA binding specificity compared to CPEB1, they are referred as CPEB-like proteins. One of CPEB-like protein, CPEB3, binds GluR2 mRNA and represses its translation. The subcellular localization of CPEB family proteins during glutamate over stimulation is also studied. The CPEB family proteins are identified as nucleus/cytoplasm shuttling proteins that depend on CRM1 for nuclear export. CPEB-like proteins share similar nuclear export ciselement that is not present in CPEB1. Over-stimulation of neuron by glutamate induces the nuclear accumulation of CPEB family proteins possibly through disrupted nuclear export. This nuclear accumulation of CPEB family protein is induced by imbalance of calcium metabolism in the neurons. Biochemical and cytological results suggest CPEB4 protein is associated with ER membrane peripherally in RNA independent manner. This research provides general description of biochemical, cytological properties of CPEB family proteins.

Mestrado em Biologia Molecular e Celular
Alternative polyadenylation (APA) is an important mechanism of gene regulation that occurs in 70% of eukaryotic organisms. This process comprises the formation of alternative 3’ ends of an mRNA by cleavage of the pre-mRNA and polyadenylation at different sites according to the polyadenylation signals (pAs). The choice of pAs in APA is a co-transcriptional mechanism that depends on auxiliary cis- and trans-acting factors. The usage of the proximal or the distal pAs has been related to global physiologic events. It is consensually assumed that in proliferative conditions there is preferential usage of proximal pAs, while during development and in differentiated cellular states occurs lengthening of the 3’UTRs by selection of the distal pAs. This pattern is also confirmed in brain tissues, where most of the cells are differentiated, and where it was observed a lengthening of the 3’ UTRs. However, there is not a complete switch for the distal pA, since the shortest mRNA is still expressed. Rho GTPases are key molecular switchers essential for several cellular processes, including differentiation, however nothing is known about transcriptional regulation in these genes. Therefore, we started to explore if Rho GTPases genes undergo APA. We found by 3’RACE analyses, that classical Rho GTPAses express two alternative mRNA isoforms. However during oligodendrocytes differentiation, they preferentially express the shortest mRNA isoform, and we did not observe a switch towards the distal pA usage, in contrast with the published genome-wide data obtained from brain tissues. Since Rho GTPases are tightly regulated at the protein level by GEFs and GAPs, they may not require this mode of co-transcriptional regulation. The atypical RhoBTB2, which is constitutively active, present a global induction of distal pA sites, distinct from the classical Rho GTPases. Interestingly, this pattern suggests that APA is a gene specific mechanism. As longer 3'UTRs contain more binding sites for miRNAs and RNA binding proteins (RBPs) this suggests that atypical Rho GTPases require a fine-tune regulation at the co-transcriptional level, by APA. Additionally, we showed that APA is also cell-specific, by analyzing the expression of the different mRNA isoforms of Rho GTPases in other glial cells (microglia, astrocytes) and different types of neurons (cortical, striatal and hippocampal). We observed the same APA profile for the selected Rho GTPases in all glial cells types. However, in cortical and striatal neurons we observed a lengthening in the 3’UTR Rac1 mRNA during axonal growth, which results in the increase of the total protein levels. Taken together, our results indicate for the first time that APA is a gene- and cell- specific mechanism. In addition, we have found a differential expression of both Cdc42 isoforms during OL and sciatic nerve differentiation. During in vitro OL and in vivo sciatic nerve differentiation we observed an increase in the expression ratio between Cdc42 Iso1/Cdc42 Iso2. Further, constitutive expression of Cdc42 Iso2 in OLs induces a delay in differentiation, whereas constitutive expression of Cdc42 Iso1 induces an increase in OL branching, suggesting an exacerbation of the differentiated phenotype. Thus, these observations suggest a distinct role for the different Cdc42 isoforms during OL differentiation. Overall, this thesis opens new avenues to explore in the future that can impact our understanding on the regulation of the myelination/remyelination processes.
A poliadenilação alternativa (APA) é um mecanismo importante de regulação genética que ocorre em 70% dos organismos eucariotas. Este mecanismo compreende a formação de extremidades 3’ alternativas por poliadenilação em diferentes locais do mRNA, de acordo com os sinais de poliadenilação (pAs). Na APA, a escolha dos pAs é um mecanismo co-transcripcional que depende de factores auxiliares cis e trans necessários para os processos de clivagem e poliadenilação de todos os pré-mRNAs. Além disso, o uso dos pAs proximais ou distais está relacionado com eventos fisiológicos gerais. Consensualmente assume-se que em estados de proliferação ocorre o encurtamento, enquanto em estados de desenvolvimento e diferenciação ocorre o alongamento das extremidades 3’ não traduzidas (3’UTRs). Este padrão de APA é confirmado em tecidos cerebrais, onde a maior parte das células são diferenciadas, no entanto não existe uma alteração completa para a isoforma de mRNA longa uma vez que a isoforma curta continua a ser expressa. As Rho GTPases são ‘interruptores’ moleculares essenciais a vários processos celulares, incluindo a diferenciação, no entanto nada é conhecido sobre a sua regulação transcripcional. Assim, começamos a explorar se estes genes são regulados por APA. Descobrimos por análise de 3´RACE que, as Rho GTPases clássicas, expressam duas formas alternativas de mRNA. Contudo durante a diferenciação dos oligodendrócitos (OLs), eles expressam preferencialmente a isoforma mRNA mais curta, e não se observou uma alteração para a escolha da isoforma mais longa, em contraste com os dados de estudos globais do genoma em tecido cerebral. Uma vez que estas proteínas são altamente reguladas por GEFs e por GAPs, provavelmente não necessitam de regulação a nível transcripcional. As Rho GTPases atípicas, que estão constitutivamente activas, apresentam um indução global dos pAs distais, distintas das Rho GTPases clássicas. Curiosamente, este padrão sugere que APA é um mecanismo específico do gene. Como 3’UTRs mais longas providenciam mais locais de ligação para microRNA ou proteínas de ligação ao RNA (RBPs), isto sugere que as Rho GTPases atípicas requerem uma regulação mais fina ao nível co-transcriptional, por APA. Adicionalmente, mostramos que a APA é também específica de cada tipo celular, pela análise da expressão do mRNA em outras células da glia (microglia, astrócitos), e em diferentes tipos de neurónios (corticais, estriatais e hipocampais). Nós observamos o mesmo padrão de APA para as Rho GTPases selecionadas em todas as células da glia. No entanto, em neurónios corticais e do estriado, observámos a existência do alongamento do 3’UTR no mRNA da Rac1 durante o crescimento axonal, o que resulta num aumento da quantidade total de proteína. Em resumo, estes resultados indicam, pela primeira vez, que a APA é um mecanismo específico de cada gene e de cada tipo celular. Para além disso, descobrimos uma expressão diferencial de ambas as isoformas da Cdc42 durante a diferenciação dos OLs e do nervo ciático. Durante a diferenciação in vitro de OLs e in vivo do nervo ciático, observámos um aumento do rácio da expressão entre Cdc42 Iso1/Cdc42 Iso2. Mais ainda, a expressão constitutiva de Cdc42 Iso2 em OLs induz um atraso na diferenciação, enquanto a expressão constitutiva da Cdc42 Iso1 induz um aumento das ramificações, sugerindo uma exacerbação do fenótipo de diferenciação. Assim, estas observações sugerem um papel distinto para as diferentes isoformas de Cdc42 durante a diferenciação de OLs. Globalmente, esta tese abre novos caminhos para explorar no futuro, que podem ter um impacto no nosso conhecimento, na regulação do processo de mielinização/remielinização.

RNA-binding proteins interact specifically with RNA strands to regulate important cellular processes. Knowing the binding partners of a protein is a crucial issue in biology and it is essential to understand the protein function and its involvement in diseases. The identification of the interactions is currently resolvable only through in vivo and in vitro experiments which may not detect all binding partners. Computational methods which capture the protein-dependent nature of the binding phenomena could help to predict, in silico, the binding and could be resistant against experimental biases. This thesis addresses the creation of models based on support vector machines and trained on experimental data. The goal is the identification of RNAs which bind specifically to a regulatory protein. Starting from a case study, done with protein CELF1, we extend our approach and propose three methods to predict whether an RNA strand can be bound by a particular RNA-binding protein. The methods use support vector machines and different features based on the sequence (method Oli), the motif score (method OliMo) and the secondary structure (method OliMoSS). We apply them to different experimentally-derived datasets and compare the predictions with two methods: RNAcontext and RPISeq. Oli outperforms OliMoSS and RPISeq affirming our protein specific prediction and suggesting that oligo frequencies are good discriminative features. Oli and RNAcontext are the most competitive methods in terms of AUC. A Precision-Recall analysis reveals a better performance for Oli. On a second experimental dataset, where negative binding information is available, Oli outperforms RNAcontext with a precision of 0.73 vs. 0.59. Our experiments show that features based on primary sequence information are highly discriminative to predict the binding between protein and RNA. Sequence motifs can improve the prediction only for some RNA-binding proteins. Finally, we can conclude that experimental data on RNA-binding can be effectively used to train protein-specific models for in silico predictions.

RNA-binding proteins interact specifically with RNA strands to regulate important cellular processes. Knowing the binding partners of a protein is a crucial issue in biology and it is essential to understand the protein function and its involvement in diseases. The identification of the interactions is currently resolvable only through in vivo and in vitro experiments which may not detect all binding partners. Computational methods which capture the protein-dependent nature of the binding phenomena could help to predict, in silico, the binding and could be resistant against experimental biases. This thesis addresses the creation of models based on support vector machines and trained on experimental data. The goal is the identification of RNAs which bind specifically to a regulatory protein. Starting from a case study, done with protein CELF1, we extend our approach and propose three methods to predict whether an RNA strand can be bound by a particular RNA-binding protein. The methods use support vector machines and different features based on the sequence (method Oli), the motif score (method OliMo) and the secondary structure (method OliMoSS). We apply them to different experimentally-derived datasets and compare the predictions with two methods: RNAcontext and RPISeq. Oli outperforms OliMoSS and RPISeq affirming our protein specific prediction and suggesting that oligo frequencies are good discriminative features. Oli and RNAcontext are the most competitive methods in terms of AUC. A Precision-Recall analysis reveals a better performance for Oli. On a second experimental dataset, where negative binding information is available, Oli outperforms RNAcontext with a precision of 0.73 vs. 0.59. Our experiments show that features based on primary sequence information are highly discriminative to predict the binding between protein and RNA. Sequence motifs can improve the prediction only for some RNA-binding proteins. Finally, we can conclude that experimental data on RNA-binding can be effectively used to train protein-specific models for in silico predictions.

Thesis (Ph. D.)--Michigan State University. Dept. of Microbiology and Molecular Genetics, 2006.
Title from PDF t.p. (viewed on Nov. 20, 2008) Includes bibliographical references. Also issued in print.

Clostridioides difficile est une cause majeure de diarrhée nosocomiale. La physiopathologie de C. difficile est régie par des réseaux de régulation complexes, incluant des mécanismes basés sur l'ARN tels que les riboswitches. Les riboswitches, situés dans la région 5' non traduite des ARNm, se lient à des ligands spécifiques, induisant des changements de conformation qui modulent l'expression du gène en aval. Chez C. difficile, 16 riboswitches répondent à la molécule de signalisation c-di-GMP. Le c-di-GMP est un régulateur contrôlant la transition d'un mode de vie planctonique libre à un mode de vie sessile associé à la régulation des facteurs de virulence. Plusieurs riboswitches à c-di-GMP régulent des gènes impliqués dans la formation des flagelles, l'assemblage des pili de type IV, le développement du biofilm, l'adhésion et la production de facteurs de virulence tels que les toxines chez C. difficile. De plus, le c-di-GMP inhibe la sporulation chez C. difficile, mais le mécanisme sous-jacent n'est pas connu.Nous avons caractérisé ici des riboswitches à c-di-GMP qui n'ont pas encore été étudiés. Nos analyses bioinformatiques ont révélé que 5 d'entre eux sont situés directement en amont de gènes codant des petites protéines (PPs) de 58 acides aminés (AA). De manière intéressante, un alignement de ces 5 protéines a montré qu'elles sont presque identiques en séquence. De plus, une recherche d'homologie a permis de découvrir 2 protéines supplémentaires de 60 AA, très similaires aux cinq premières, bien que leurs gènes ne soient pas précédés d'un riboswitch. Cette nouvelle famille de protéines est conservée dans toutes les souches de C. difficile, mais n'a pas d'homologues en dehors de l'espèce. Nous avons construit une version étiquetée d'une PP et l'avons détectée par immunoblotting de fractions cellulaires, confirmant sa nature protéique et révélant qu'elle est associée à la membrane.Des données de séquençage de l'ARN (RNA-seq) ont démontré que le c-di-GMP régule négativement l'expression des 5 gènes de PP en aval des riboswitches ainsi que des 2 gènes supplémentaires. Nous avons également observé que le c-di-AMP, un autre dinucléotide cyclique impliqué dans l'osmorégulation, réprime l'expression des 7 gènes. Des expériences de fusions transcriptionnelles entre la région promotrice d'une PP et un gène rapporteur ont confirmé que le c-di-GMP nécessite le riboswitch pour moduler l'expression des gènes en aval. En revanche, le c-di-AMP régule leur expression indépendamment du riboswitch en modulant l'activité du promoteur. Ainsi, le c-di-GMP et le c-di-AMP influencent l'expression des PP par des mécanismes distincts.Pour étudier le rôle des PP chez C. difficile, nous avons surexprimé l'une d'elles et comparé son transcriptome à celui de la souche sauvage. Cela a révélé l'induction de l'expression de plus de 100 gènes impliqués dans la sporulation dans la souche surexprimant la PP. Conformément à ces données, la surexpression de cette PP a conduit à un phénotype d'hypersporulation. En outre, la délétion des 7 gènes de PP (mutant Δ7) a entraîné une réduction de la sporulation, avec des phénotypes intermédiaires pour les souches où seuls certains gènes des PP ont été délétés. Le défaut de sporulation de Δ7 est similaire à celui d'une souche produisant des niveaux élevés de c-di-GMP, suggérant que l'impact du c-di-GMP sur la sporulation pourrait être médié par la régulation des PP. Pour tester cette hypothèse, nous avons créé un mutant Δ7 produisant de fortes concentrations de c-di-GMP. Le défaut de sporulation de cette souche était équivalent à celui du mutant Δ7 non affecté dans sa production de c-di-GMP, indiquant que les effets des délétions des PP et de la surproduction de c-di-GMP ne sont pas cumulatifs.Dans l'ensemble, nos résultats démontrent que cette nouvelle famille de petites protéines est régulée à la fois par le c-di-GMP et le c-di-AMP et joue un rôle clé dans le contrôle de la sporulation chez C. difficile
Clostridioides difficile is the leading cause of nosocomial diarrhea in adults in industrialized countries. The pathophysiology of C. difficile is governed by complex regulatory networks, including RNA-based mechanisms like riboswitches. Riboswitches, located in the 5' untranslated region of mRNAs, bind specific ligands, inducing conformational changes that either promote or inhibit the expression of the downstream gene. In C. difficile, 16 riboswitches respond to the signaling molecule cyclic di-GMP (c-di-GMP). C-di-GMP acts as a second messenger and is recognized as a central regulator controlling the transition from a free planktonic to a sessile lifestyle associated with biofilm formation and virulence factor regulation. Several of the c-di-GMP-responding riboswitches have been well-studied in C. difficile and shown to regulate genes involved in flagella formation, type IV pili assembly, biofilm development, adhesion, and the production of virulence factors such as toxins. Moreover, c-di-GMP inhibits sporulation in C. difficile, but the underlying mechanism remains unclear.In this PhD work, we sought to characterize c-di-GMP-responding riboswitches that have not yet been studied. Our bioinformatics analyses revealed that 5 of them are located directly upstream of predicted genes encoding small proteins (SPs) of 58 amino acids. Interestingly, an alignment of these 5 proteins showed that they are almost identical in sequence. Moreover, a homology search uncovered two additional proteins of 60 amino acids, highly similar to the first five, though their genes are not preceded by a c-di-GMP riboswitch. This novel family of proteins is conserved across C. difficile strains but lacks homologs outside the species. We built a tagged version of one SP and detected it by immunoblotting of cell fractions, confirming its protein nature and revealing that it is primarily localized to the cell membrane.RNA sequencing (RNA-seq) data demonstrated that c-di-GMP negatively regulates not only the expression of the 5 SP genes downstream of the riboswitches but also the 2 additional genes. Unexpectedly, we also observed that c-di-AMP, another cyclic dinucleotide primarily involved in osmoregulation, repressed the expression of all seven genes. We performed reporter assays in different strain backgrounds to explore how these small proteins are regulated by both c-di-GMP and c-di-AMP. These experiments indicated that c-di-GMP required the riboswitch for modulation of downstream gene expression. In contrast, c-di-AMP regulated their expression independently of the riboswitch by modulating the promoter activity. Thus, c-di-GMP and c-di-AMP influence SP expression through distinct mechanisms.To investigate the role of these small proteins in C. difficile physiology, we overexpressed one SP and compared its transcriptome to that of the wild-type strain using RNA-seq. This revealed the upregulation of more than 100 genes involved in sporulation in the overexpressing strain. Consistent with these data, overexpression of this SP led to a hypersporulation phenotype. Furthermore, deletion of all 7 SP genes (Δ7 mutant) resulted in a significant reduction in sporulation, with intermediate phenotypes in strains where only some of the SP genes were deleted. Interestingly, the sporulation defect in the Δ7 mutant was mirrored in a strain producing elevated levels of c-di-GMP, suggesting that the impact of c-di-GMP on sporulation could be mediated by SP regulation. To test this hypothesis, we created a Δ7 mutant producing high concentrations of c-di-GMP. The sporulation defect in this strain was equivalent to that of the Δ7 mutant unaffected in its c-di-GMP production, indicating that the effects of SP gene deletions and c-di-GMP overproduction were not cumulative.Overall, our findings demonstrate that this novel family of small proteins is regulated by both c-di-GMP and c-di-AMP and plays a key role in controlling sporulation in C. difficile

Chez la levure fissipare Schizosaccharomyces pombe, un sous-ensemble de gènes méiotiques est transcrit de manière constitutive au cours de la mitose. Afin d’éviter l'expression prématurée du programme méiotique et l'initiation de la différenciation sexuelle, les cellules ont développé un système de dégradation de l'ARN qui élimine sélectivement les transcrits méiotiques correspondants. Ce processus nécessite la protéine de liaison à l'ARN Mmi1 (à domaine YTH), qui reconnaît en cis les molécules d'ARN (motifs UNAAAC) et les cible pour la dégradation par l'exosome nucléaire. Au début de la méiose, Mmi1 est séquestrée au sein d’une particule ribonucléoprotéique composée de la protéine de liaison à l'ARN Mei2 et du long ARN non-codant (lncRNA) meiRNA, permettant ainsi l'expression des gènes méiotiques et le déroulement de la méiose. Mon travail de thèse a consisté à étudier les mécanismes par lesquels Mmi1 assure la dégradation des transcrits méiotiques et qui régulent son activité au cours des cycles mitotiques et méiotiques. Pendant la croissance végétative, Mmi1 s'associe étroitement à la protéine conservée Erh1 pour former le complexe hétérotétramérique Erh1-Mmi1 (EMC) qui est essentiel pour la dégradation des transcrits méiotiques. Par des approches de biologie structurale et de biochimie, nous avons montré qu'Erh1 s'assemble en homodimère in vitro et in vivo, en accord avec des analyses récentes. Des mutations qui empêchent l'homodimérisation d'Erh1 mais préservent son interaction avec Mmi1 entraînent l'accumulation de transcrits méiotiques en raison d'un défaut de liaison de Mmi1 à ses cibles ARN. L'homodimérisation d’Erh1 est également nécessaire pour séquestrer Mmi1 dans le complexe Mei2-meiRNA et assurer la progression de la méiose. Ainsi, l'assemblage d’EMC est essentiel pour la reconnaissance et la dégradation des transcrits méiotiques par Mmi1 dans les cellules mitotiques et contribue à l'inactivation de cette dernière au début de la méiose. Des travaux antérieurs ont montré que, pendant la croissance végétative, Mmi1 recrute le complexe Ccr4-Not pour ubiquitinyler et limiter l’accumulation de son propre inhibiteur Mei2, maintenant ainsi son activité dans la dégradation des ARNs méiotiques. Nous avons identifié un lncRNA, différent de meiRNA et appelé mamRNA (Mmi1- and Mei2-associated RNA), qui sert de plateforme à Mmi1 pour cibler Mei2 vers le complexe Ccr4-Not. Réciproquement, lorsque cette régulation négative de Mei2 est défectueuse, mamRNA est nécessaire pour l'inactivation de Mmi1 par les niveaux élevés de Mei2. Des expériences d’hybridation in situ par fluorescence en molécules uniques (smFISH) ont également montré que mamRNA est localisé dans un corps nucléaire contenant Mmi1, suggérant que le contrôle mutuel de Mmi1 et Mei2 est confiné dans l’espace. mamRNA peut également relayer meiRNA pour inhiber Mmi1 et favoriser la progression de la méiose. mamRNA apparait donc comme un régulateur critique des activités de Mmi1 et Mei2 pour ajuster la dégradation des ARNs méiotiques et modeler la transition de la mitose vers la méiose
In the fission yeast S. pombe, a subset of meiosis-specific genes is constitutively transcribed during the mitotic cell cycle. To prevent untimely expression of the meiotic program and premature initiation of sexual differentiation, cells have evolved an RNA degradation system that selectively eliminates the corresponding meiotic transcripts. This process requires the YTH-family RNA-binding protein Mmi1, which recognizes cis-elements within RNA molecules (UNAAAC motifs) and targets them for degradation by the nuclear exosome. At the onset of meiosis, Mmi1 is sequestered in a ribonucleoparticle composed of the RNA-binding protein Mei2 and the long non-coding RNA (lncRNA) meiRNA, thereby allowing expression of meiotic genes and meiosis progression. My PhD work consisted in studying the mechanisms by which Mmi1 promotes the degradation of meiotic transcripts and how its activity is regulated during both the mitotic and meiotic cell cycles. During vegetative growth, Mmi1 tightly associates with the evolutionarily conserved Erh1 protein to form the heterotetrameric Erh1-Mmi1 complex (EMC) that is essential for the degradation of meiotic transcripts. Using biochemical and structural approaches, we have shown that Erh1 assembles as a homodimer in vitro and in vivo, consistent with recent analyses. Mutations that disrupt Erh1 homodimerization but preserve interaction with Mmi1 result in the accumulation of meiotic transcripts due to inefficient binding of Mmi1 to its RNA targets. Erh1 homodimerization is also required for Mmi1 luring by the Mei2-meiRNA complex and meiosis progression. Thus, EMC assembly is essential for the recognition and degradation of meiotic transcripts by Mmi1 in mitotic cells and contributes to Mmi1 inactivation at meiosis onset. Previous work showed that, during vegetative growth, Mmi1 recruits the conserved Ccr4-Not complex to ubiquitinylate and downregulate a pool of its own inhibitor Mei2, thereby maintaining its activity in meiotic RNA degradation. We have identified a lncRNA, different from meiRNA and termed mamRNA (Mmi1- and Mei2-associated RNA), to which Mmi1 associates to target Mei2 to the Ccr4-Not complex. Conversely, when Mei2 downregulation is impaired, mamRNA is necessary for Mmi1 inactivation by increased Mei2 levels. Single molecule RNA FISH experiments also indicated that mamRNA localizes to a nuclear body enriched in Mmi1, suggesting that the mutual control of Mmi1 and Mei2 is spatially confined. mamRNA can also take over meiRNA to inhibit Mmi1 and promote meiosis progression. Therefore, mamRNA emerges as a critical regulator of Mmi1 and Mei2 activities to fine tune meiotic RNA degradation and shape the mitosis to meiosis transition

L'etude des facteurs intervenant dans la regulation post-transcriptionnelle de l'expression genique a permis de caracteriser les prosomes (classe de particules ribonucleoproteiques (rnp)) qui s'associent aux complexes mrnp reprimes et d'etudier le role de la proteine poly(a) associee aux rnam traduits dans les polyribosomes. Le prosome est un complexe rnp, de coefficient sedimentation 19s, constitues de petits rna (prna) et de proteines presentant une structure hautement organisee. La caracterisation biochimique et immunologique des prosomes de plusieurs cellules montrent que certaines proteines des prosomes sont phylogenetiquement conservees au cours de l'evolution, alors que d'autres peuvent varier selon l'espece. Les prosomes sont presents a la fois dans le noyau et le cytoplasme, en partie en association avec le cytosquelette. Aux stades initiaux du developpement, les prosomes sont d'origine maternelle. La proteine poly a semble jouer un role dans la traduction des rna messagers cytoplasmiques

Chan Kam Leung.
Thesis (M.Phil.)--Chinese University of Hong Kong, 1999.
Includes bibliographical references (leaves 175-182).
Abstract also in Chinese.
Acknowledgements --- p.i
Abstract --- p.ii
Abbreviations --- p.v
Table of Contents --- p.vii
Chapter Chapter 1 --- General Introduction
Chapter 1.1 --- Drosophila as a model for studying development --- p.1
Chapter 1.2 --- The formation of the body axis in Drosophila --- p.2
Chapter 1.3 --- The maternal genes are essential for development --- p.9
Chapter 1.4 --- Maternal gene bicoid is essential for formation of the anterior structures in the embryo --- p.11
Chapter 1.5 --- The formation of the biocid protein gradient from anterior pole to posterior pole of the embryo --- p.13
Chapter 1.6 --- The bed protein gradient controls the downstream zygotic target genes in a concentration-dependent manner --- p.15
Chapter 1.7 --- The formation of the bed protein gradient in embryo --- p.17
Chapter 1.8 --- Components required for bcd mRNA localization at anterior pole of oocyte --- p.21
Chapter 1.8.1 --- Cis-acting elements --- p.21
Chapter 1.8.2 --- Trans-acting elements --- p.21
Chapter 1.9 --- The properties of exuperantia protein --- p.25
Chapter 1.9.1 --- The function of exu protein --- p.25
Chapter 1.9.2 --- Exuperantia is a phosphoprotein --- p.26
Chapter 1.9.3 --- Phosphorylation pattern of exuperantia protein is stage-specific --- p.28
Chapter 1.9.4 --- Reversible phosphorylation is one of the major mechanisms to control protein activity in all eukaryotic cells --- p.29
Chapter 1.9.5 --- The relationship between the exu protein phosphorylation and the bcd mRNA localization --- p.30
Chapter 1.10 --- Aim of project --- p.31
Chapter CHAPTER 2 --- Preparation of the exuperantia genomic DNA and complement DNA (cDNA) mutant Constructs
Chapter 2.1 --- Introduction --- p.33
Chapter 2.2 --- Materials and methods --- p.35
Chapter 2.2.1 --- DNA preparation methods --- p.35
Chapter 2.2.1.1 --- Preparation of double-stranded DNA by polyethylene glycol6000 --- p.35
Chapter 2.2.1.2 --- Preparation of M13mp8 single-stranded DNA --- p.37
Chapter 2.2.1.3 --- "Preparation of double-stranded DNA by Biol prep (Modified from Maniatis et al.,1989)" --- p.38
Chapter 2.2.2 --- "Preparation of DH5α，JM109, TG1 competent cells" --- p.39
Chapter 2.2.3 --- Bacteria transformation --- p.40
Chapter 2.2.4 --- Restriction enzyme digestion --- p.40
Chapter 2.2.5 --- Phenol/chloroform extraction --- p.41
Chapter 2.2.6 --- Purification of DNA fragment by electro-elution --- p.42
Chapter 2.2.7 --- DNA ligation --- p.43
Chapter 2.2.8 --- DNA dephosphorylation --- p.43
Chapter 2.2.9 --- In vitro site-directed mutagenesis --- p.44
Chapter 2.2.9.1 --- The Sculptor´ёØ in vitro mutagenesis --- p.44
Chapter 2.2.9.2 --- The GeneEditor´ёØ in vitro site-directed mutagenesis --- p.47
Chapter 2.2.10 --- The double-stranded or single-stranded DNA sequencing by T7 DNA polymerase sequencing system --- p.50
Chapter 2.2.11 --- Denatured polyacrylamide gel electorphoresis --- p.51
Chapter 2.2.11 --- Nucleotide sequence of the sequencing primers and the mutageneic oligonucleotides --- p.54
Chapter 2.3 --- Results --- p.55
Chapter 2.3.1 --- Design exuperantia mutant constructs --- p.55
Chapter 2.3.1.1 --- Comparison of exu protein amino acids sequence with different Drosophila species --- p.56
Chapter 2.3.2 --- The exu genomic mutant constructs --- p.63
Chapter 2.3.3 --- The exu cDNA mutant constructs --- p.63
Chapter 2.4 --- Discussion --- p.76
Chapter CHAPTER 3 --- Epitope tagging of exuperantia protein with c-myc eptiope
Chapter 3.1 --- Introduction --- p.79
Chapter 3.2 --- Materials and methods --- p.84
Chapter 3.2.1 --- Preparation of the c-myc eptiope DNA fragment --- p.84
Chapter 3.2.2 --- End-filling of 5'overhang DNA fragment by Klenow fragment --- p.86
Chapter 3.2.3 --- In vitro translation of protein by TNT® Quick coupled transcription and translation system --- p.86
Chapter 3.2.4 --- Immunoprecipitation of recombinant exu protein --- p.87
Chapter 3.2.5 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.88
Chapter 3.2.5.1 --- SDS-PAGE preparation --- p.88
Chapter 3.2.5.2 --- SDS-PAGE electrophoresis --- p.90
Chapter 3.2.6 --- Western blot analysis --- p.90
Chapter 3.2.6.1 --- Transfer the protein to a nitro-cellulose membrane by semi-dried blotting --- p.90
Chapter 3.2.6.2 --- Western blot blocking and antibody recognition --- p.91
Chapter 3.3 --- Results --- p.92
Chapter 3.3.1 --- Construction of the plasmid containing exu cDNA tagging with a c-myc epitope --- p.92
Chapter 3.3.2 --- In vitro translation of c-myc epitope tagged exu protein --- p.102
Chapter 3.3.3 --- Immunoprecipitation of c-myc labeled exu protein by a polyclonal rabbit anti-exu antibody and monoclonal mouse anti-myc antibody --- p.104
Chapter 3.4 --- Discussion --- p.109
Chapter CHAPTER 4 --- In vitro phosphorylation of exuperantia Protein
Chapter 4.1 --- Introduction --- p.111
Chapter 4.2 --- Materials and methods --- p.113
Chapter 4.2.1 --- Exogenous kinase phsophorylation reactions --- p.113
Chapter 4.2.2 --- Separation of the phosphorylated exu protein variants by SDS- PAGE --- p.114
Chapter 4.3 --- Results --- p.115
Chapter 4.3.1 --- Western blot analysis of in vitro translated exu protein variants --- p.115
Chapter 4.3.2 --- Phosphorylation of in vitro translated exu protein variants by exogenous cAMP-dependent protein kinase --- p.118
Chapter 4.3.3 --- Phosphorylation of in vitro translated exu protein variants by exogenous cGMP-dependent protein kinase --- p.123
Chapter 4.3.4 --- Phosphorylation of in vitro translated exu protein variants by exogenous protein kinase C --- p.128
Chapter 4.4 --- Discussion --- p.133
Chapter CHAPTER 5 --- Introduction of the exuperantia genomic constrcuts into the germline of Drosophila by P element-mediated transformation
Chapter 5.1 --- Introduction --- p.136
Chapter 5.2 --- Materials and methods --- p.138
Chapter 5.2.1 --- Construction of a genomic construct for production of transgenic flies --- p.138
Chapter 5.2.2 --- Preparation of double-stranded DNA by ultra-centrifugation --- p.142
Chapter 5.2.3 --- P-element mediated transformation --- p.143
Chapter 5.2.3.1 --- Eggs collection --- p.143
Chapter 5.2.3.2 --- Dechorionating the eggs --- p.143
Chapter 5.2.3.3 --- Orientating the eggs --- p.144
Chapter 5.2.3.4 --- Microinjection --- p.145
Chapter 5.2.4 --- Collecting virgin female Drosophila --- p.146
Chapter 5.2.5 --- Setup a crossing experiment --- p.146
Chapter 5.2.6 --- Preparation of total ovaries and testes extracts exu protein from Female and male Drosophila --- p.147
Chapter 5.2.7 --- Immunohistochemical distribution of exuperantia protein --- p.147
Chapter 5.3 --- Results --- p.150
Chapter 5.3.1 --- Insertion of the mutated exu fragments into the Drosophila Transformation vector (pCaSpeR) --- p.150
Chapter 5.3.2 --- Introduction of the mutated exu gene into the genome of Drosophila by P-element mediated transformation --- p.153
Chapter 5.3.3 --- Western blot analysis of the exu protein in the exu (ES2.1) transgenic fly --- p.160
Chapter 5.3.4 --- Immunohistochemical distribution of exu protein in exuES21 mutants --- p.162
Chapter 5.3.5 --- Rescue test of exuES2.1 trangenic flies --- p.165
Chapter 5.4 --- Discussion --- p.168
Chapter CHAPTER 6 --- General Discussion --- p.171
References --- p.173
Chapter Appendix I: --- List of reagents --- p.183
Chapter Appendix II: --- Publication --- p.187

Indiana University-Purdue University Indianapolis (IUPUI)
My thesis comprises of two individual projects which revolve around the importance of post-transcriptional regulation in liver. My first project is studying the integrated miRNA – mRNA network in NAFLD. For fulfillment of the study we conducted a genome-wide study to identify microRNAs (miRs) as well as the miR-mRNA regulatory network associated with hepatic fat and NAFLD. Hepatic fat content (HFC), miR and mRNA expression were assessed in 73 human liver samples. Liver histology of 49 samples was further characterized into normal (n=33) and NAFLD (n=16). Liver miRNome and transcriptome were significantly associated with HFC and utilized to (a) build miR-mRNA association networks in NAFLD and normal livers separately based on the potential miR-mRNA targeting and (b) conduct pathway enrichment analyses. We identified 62 miRs significantly correlated with HFC (p < 0.05 with q < 0.15), with miR-518b and miR-19b being most positively and negatively correlated with HFC, respectively (p < 0.008 for both). Integrated network analysis showed that six miRs (miRs-30b*, 612, 17*, 129-5p, 204 and 20a) controlled ~ 70% of 151 HFC-associated mRNAs (p < 0.001 with q < 0.005). Pathway analyses of these HFC-associated mRNA revealed their key effect (p<0.05) in inflammation pathways and lipid metabolism. Further, significant (p<2.47e-4, Wilcoxon test) reduction in degree of negative associations for HFC-associated miRs with HFC-associated mRNAs was observed in NAFLD as compared to normal livers, strongly suggesting highly dysfunctional miR-mRNA post-transcriptional regulatory network in NAFLD. Our study makes several novel observations which provide clues to better understand the pathogenesis and potential treatment targets of NAFLD. My second project is based on uncovering important players of post-transcriptional regulation (RBPs) and how they are associated with age and gender during healthy liver development. For this study, we performed an association analysis focusing on the expression changes of 1344 RNA Binding proteins (RBPs) as a function of age and gender in human liver. We identify 88 and 45 RBPs to be significantly associated with age and gender respectively. Experimental verification of several of the predicted associations in the mouse model confirmed our findings. Our results suggest that a small fraction of the gender-associated RBPs (~40%) are likely to be up-regulated in males. Altogether, these observations show that several of these RBPs are important developmentally conserved regulators. Further analysis of the protein interaction network of RBPs associated with age and gender based on the centrality measures like degree, betweenness and closeness revealed that several of these RBPs might be prominent players in liver development and impart gender specific alterations in gene expression via the formation of protein complexes. Indeed, both age and gender-associated RBPs in liver were found to show significantly higher clustering coefficients and network centrality measures compared to non-associated RBPs. The compendium of RBPs and this study will help us gain insight into the role of post-transcriptional regulatory molecules in aging and gender specific expression of genes.

PTB (HnRNP I) is a multifunctional RNA binding protein which participates in a variety of RNA metabolic processes put together called as post transcriptional gene regulation. It interacts with shuttling hnRNPs L, K and E2 of the spliceosomal machinery and also with other RNA binding proteins like PSF, Raver1 and Raver2, which assists PTB in splicing. Based on the complexity of these processes and multifunctional nature of PTB, we hypothesized that; it might interact with various additional proteins not identified till date. Keeping this objective in mind, we set out to screen the custom made 18 day old mouse testes cDNA library in pGAD10 vector available in the laboratory, to hunt for novel interacting partners of PTB using the Clontech’s Matchmaker Gal4 yeast two hybrid system III. PTB1, the prototype of PTB was chosen and the above mentioned cDNA library was screened for novel PTB interacting partners. Twenty five large scale library transformations (spanning 8*106 independent clones) were performed and 99 putatives were obtained. By re-transformation of these library plasmids with bait construct to check for the interaction phenotype and eliminating bait independent activation of reporter genes and elimination of known false positives, only 5 clones were consistent with the interaction phenotype. All these library plasmids were sequenced with vector specific primers, ORF was identified and BLAST analysis for the identification of insert was done. Two of these clones encoded the partial CDS of mouse Protein Inhibitor of Activated STAT3-PIAS3. One of these encoded the partial CDS of mouse TOLL Interacting Protein-TOLLIP. The other two encoded the partial CDS of mouse importin-α and mouse hnRNP K, both of which were already known interacting partners of PTB. GST pull down assay and mammalian matchmaker co-immunoprecipitation was used for confirming the in vitro one to one physical interaction between PTB and these newly identified protein partners. Indirect Immunofloresence was used for demonstrating the co-localization of PTB and PIAS3 in Gc1Spg mouse spermatogonial cell line. The fact that PIAS3 an E3 SUMO ligase was picked up as an interacting partner of PTB was interesting and we hypothesized that PTB might be a sumoylation substrate. Towards this, we first resorted to the prediction of sumoylation consensus motif by using SUMOPLOT. PTB indeed was found to have sumoylation consensus sites. Subsequently, in vivo sumoylation of PTB was demonstrated, where in over expression of donor protein [SUMO-1] and acceptor protein [PTB] in RAG-1 mouse kidney cell line had resulted in the identification of an approximately 67 kDa slow moving SUMO modified myc tagged PTB band apart from the bulk of unmodified 57 kDa myc-PTB. This confirmed the fact that PTB is SUMO modified only at a single consensus target site in vivo and attempts are made to map this site of modification. SUMOylation regulates diverse biological processes in vivo ranging from nucleo- cytoplasmic shuttling, alteration of protein-protein interaction, DNA protein interaction etc. PTB shuttles rapidly between the nucleus and cytoplasm in a transcription sensitive manner and the translocation of PTB to the cytoplasm, happens under the conditions of cell stress, viral infections, apoptosis and exposure of cells to genotoxic agents like doxorubicin. Phosphorylation of PTB at Ser-16 residue has been shown to modulate the nucleo-cytoplasmic shuttling of PTB, albeit shuttling can also occur irrespective of this modification. Interaction of PTB with an E3 SUMO ligase-PIAS3 and the fact that it is SUMOylated in vivo, we hypothesize that K-47 residue present in the NLS/NES might be the most probable site of this SUMO modification and SUMOylation of PTB by PIAS3 might regulate the nucleo-cytoplasmic shuttling of PTB.

PTB (HnRNP I) is a multifunctional RNA binding protein which participates in a variety of RNA metabolic processes put together called as post transcriptional gene regulation. It interacts with shuttling hnRNPs L, K and E2 of the spliceosomal machinery and also with other RNA binding proteins like PSF, Raver1 and Raver2, which assists PTB in splicing. Based on the complexity of these processes and multifunctional nature of PTB, we hypothesized that; it might interact with various additional proteins not identified till date. Keeping this objective in mind, we set out to screen the custom made 18 day old mouse testes cDNA library in pGAD10 vector available in the laboratory, to hunt for novel interacting partners of PTB using the Clontech’s Matchmaker Gal4 yeast two hybrid system III. PTB1, the prototype of PTB was chosen and the above mentioned cDNA library was screened for novel PTB interacting partners. Twenty five large scale library transformations (spanning 8*106 independent clones) were performed and 99 putatives were obtained. By re-transformation of these library plasmids with bait construct to check for the interaction phenotype and eliminating bait independent activation of reporter genes and elimination of known false positives, only 5 clones were consistent with the interaction phenotype. All these library plasmids were sequenced with vector specific primers, ORF was identified and BLAST analysis for the identification of insert was done. Two of these clones encoded the partial CDS of mouse Protein Inhibitor of Activated STAT3-PIAS3. One of these encoded the partial CDS of mouse TOLL Interacting Protein-TOLLIP. The other two encoded the partial CDS of mouse importin-α and mouse hnRNP K, both of which were already known interacting partners of PTB. GST pull down assay and mammalian matchmaker co-immunoprecipitation was used for confirming the in vitro one to one physical interaction between PTB and these newly identified protein partners. Indirect Immunofloresence was used for demonstrating the co-localization of PTB and PIAS3 in Gc1Spg mouse spermatogonial cell line. The fact that PIAS3 an E3 SUMO ligase was picked up as an interacting partner of PTB was interesting and we hypothesized that PTB might be a sumoylation substrate. Towards this, we first resorted to the prediction of sumoylation consensus motif by using SUMOPLOT. PTB indeed was found to have sumoylation consensus sites. Subsequently, in vivo sumoylation of PTB was demonstrated, where in over expression of donor protein [SUMO-1] and acceptor protein [PTB] in RAG-1 mouse kidney cell line had resulted in the identification of an approximately 67 kDa slow moving SUMO modified myc tagged PTB band apart from the bulk of unmodified 57 kDa myc-PTB. This confirmed the fact that PTB is SUMO modified only at a single consensus target site in vivo and attempts are made to map this site of modification. SUMOylation regulates diverse biological processes in vivo ranging from nucleo- cytoplasmic shuttling, alteration of protein-protein interaction, DNA protein interaction etc. PTB shuttles rapidly between the nucleus and cytoplasm in a transcription sensitive manner and the translocation of PTB to the cytoplasm, happens under the conditions of cell stress, viral infections, apoptosis and exposure of cells to genotoxic agents like doxorubicin. Phosphorylation of PTB at Ser-16 residue has been shown to modulate the nucleo-cytoplasmic shuttling of PTB, albeit shuttling can also occur irrespective of this modification. Interaction of PTB with an E3 SUMO ligase-PIAS3 and the fact that it is SUMOylated in vivo, we hypothesize that K-47 residue present in the NLS/NES might be the most probable site of this SUMO modification and SUMOylation of PTB by PIAS3 might regulate the nucleo-cytoplasmic shuttling of PTB.

The hTREX complex mediates cellular bulk mRNA nuclear export by recruiting the nuclear export factor, TAP, via a direct interaction with the export adaptor, Aly. Intriguingly however, depletion of Aly only leads to a modest reduction in cellular mRNA nuclear export, suggesting the existence of additional mRNA nuclear export adaptor proteins. In order to efficiently export Kaposi's sarcoma-associated herpesvirus (KSHV) intronless mRNAs from the nucleus, the KSHV ORF57 protein recruits hTREX onto viral intronless mRNAs allowing access to the TAP-mediated export pathway. Similarly however, depletion of Aly only leads to a modest reduction in the nuclear export of KSHV intronless mRNAs. Herein, we identify a novel interaction between ORF57 and the cellular protein, UIF. We provide the first evidence that the ORF57-UIF interaction enables the recruitment of hTREX and TAP to KSHV intronless mRNAs in Aly-depleted cells. Strikingly, depletion of both Aly and UIF inhibits the formation of an ORF57-mediated nuclear export competent ribonucleoprotein particle and consequently prevents ORF57-mediated mRNA nuclear export and KSHV protein production. Importantly, these findings highlight that redundancy exists in the eukaryotic system for certain hTREX components involved in the mRNA nuclear export of intronless KSHV mRNAs.

A cellular pre-mRNA undergoes various post-transcriptional processing events, including capping, splicing and polyadenylation prior to nuclear export. Splicing is particularly important for mRNA nuclear export as two distinct multi-protein complexes, known as human TREX (hTREX) and the exon-junction complex (EJC), are recruited to the mRNA in a splicing-dependent manner. In contrast, a number of Kaposi's sarcoma-associated herpesvirus (KSHV) lytic mRNAs lack introns and are exported by the virus-encoded ORF57 protein. Herein we show that ORF57 binds to intronless viral mRNAs and functions to recruit the complete hTREX complex, but not the EJC, in order assemble an export component viral ribonucleoprotein particle (vRNP). The formation of this vRNP is mediated by a direct interaction between ORF57 and the hTREX export adapter protein, Aly. Aly in turn interacts directly with the DEAD-box protein UAP56, which functions as a bridge to recruit the remaining hTREX proteins to the complex. Moreover, we show that a point mutation in ORF57 which disrupts the ORF57-Aly interaction leads to a failure in the ORF57-mediated recruitment of the entire hTREX complex to the intronless viral mRNA and inhibits the mRNAs subsequent nuclear export and virus replication. Furthermore, we have utilised a trans-dominant Aly mutant to prevent the assembly of the complete ORF57-hTREX complex; this results in a vRNP consisting of viral mRNA bound to ORF57, Aly and the nuclear export factor, TAP. Strikingly, although both the export adapter Aly and the export factor TAP were present on the viral mRNP, a dramatic decrease in intronless viral mRNA export and virus replication was observed in the absence of the remaining hTREX components (UAP56 and hTHO-complex). Together, these data provide the first direct evidence that the complete hTREX complex is essential for the export of KSHV intronless mRNAs and infectious virus production.

Cellular response to various stress conditions involves regulation of gene expression by different mechanisms. Translation is the final step in the flow of genetic information and regulation at this level allows an early response to changes in physiological conditions. Initiation of translation is the rate-limiting step of protein synthesis and hence is tightly regulated. Translation initiation in mammalian cells is mainly by “cap dependent pathway” wherein the 5’methyl guanosine “cap” structure is recognized by certain canonical initiation factors along with 40S ribosomal subunit and the complex scans the 5’UTR till it recognizes initiator AUG. This leads to the joining of the 60S ribosomal subunit and the initiation of translation. In an alternate mode of translation initiation called as the Internal ribosome entry site mediated translation (IRES), the ribosomes are recruited closer to the initiator AUG in a 5’ cap independent manner. Efficient translation by IRES mode requires some canonical initiation factors like eIF2 and eIF3 and other non-canonical IRES-trans-acting factors (ITAFs), which include human La antigen, polypyrimidine-tract binding protein (PTB),Upstream of N-Ras (Unr), Poly (rC) binding protein (PCBP2) etc. Various types of stress conditions, such as starvation of growth factors, heat shock, hypoxia, viral infection lead to down regulation of protein synthesis. However, translation of a subset of mRNAs continues or is up-regulated. Many of these mRNA may be translated by an IRES mode. It is believed that cellular IRESs become active during such conditions that abrogate the cap-dependent mode of translation so that the pool of vital proteins is maintained in the cell. In this thesis, presence of ‘IRES’ element has been investigated in the 5’UTR of Interferon regulatory factor -2 (IRF2) mRNA and the possible physiological significance has been studied. Further, it has been shown that polypyrimidine tract binding protein or PTB is important for the IRES activity. The probable mechanism of action of PTB has also been investigated which suggests that PTB interaction alters the IRF2 IRES conformation thus facilitating translation initiation. In the first part of the thesis, mRNAs that continue to be translated under heat-shocked condition, which is known to abrogate cap-dependent translation initiation, has been investigated by cDNA micro-array hybridization analysis of the ribosome bound RNA. The global protein synthesis was severely impaired under heat shock; however a number of mRNAs continued translation under this condition. Some of these mRNAs encode proteins that are likely to be involved in the heat shock response. Few of these genes are also reported to contain IRES element. Since the micro-array was performed from the RNA extracted from ribosome bound mRNA fraction in a condition when cap-dependent translation is impaired, it was hypothesized that some of the genes, which are up regulated under such condition, might operate via cap-independent mode of translation initiation. Based on this study, one candidate gene, the ‘interferon regulatory factor 2 (IRF2)’ was selected from the pool of up regulated genes and presence of an IRES element was investigated. Interferon regulatory factors are DNA-binding proteins that control interferon (IFN) gene expression. IRF2 has been shown to function as repressor of IFN and IFN-inducible genes. Real–Time and semi-quantitative RT-PCR assays were performed which validated the micro-array data. In the second part of the thesis, the presence of IRES element in the 5’UTR of IRF2 was investigated. Bicistronic assay showed comparable IRES activity with a known representative IRES, BiP, thus suggesting the presence of an IRES element in the IRF2 5’UTR. Stringent assays were then performed to rule out cryptic promoter activity, re-initiation/scanning or alternative splicing in the 5’UTR of the IRF2. RNA transfections using in vitro synthesized bicistronic RNAs further validated the presence of the IRES element. To understand the physiological significance of an IRES element in IRF2 mRNA, the cells were subjected to various stress conditions and IRES activity was studied. It seems IRF2 IRES function might not be sensitive to eIF4G cleavage, since its activity was only marginally affected in presence of Coxsackievirus 2A protease, which is known to cleave eIF 4G and thus inhibit the cap-dependent translation. Incidentally, Hepatitis A virus IRES was affected under such condition. Additionally, it was observed that compared to HCV or Bip IRES, the effect of Interferon α treatment was not so pronounced on the IRF2 IRES. This was further evidenced by its unchanged protein level post-treatment with interferon α. Furthermore, in cells treated with tunicamycin (a known agent causing ER stress), the IRF2 IRES activity and the protein levels were unaffected, although the cap dependent translation was severely impaired. The observations so far suggested that the IRF2 protein level is practically unchanged under conditions of ER stress and interferon treatment. Metabolic labeling followed by immunoprecipitation of IRF2 in cells treated with either tunicamycin or interferon suggested that de novo synthesis of the protein is continued under the above conditions thus validating our earlier data. In the third part of the thesis, the role of an IRES trans acting factor, PTB, in modulating the IRF2 IRES activity has been investigated. Analysis of the cellular protein binding with the IRF2 IRES suggested that certain cellular factors might influence its function under stress conditions. The IRF2 IRES was found to interact with a known trans-acting factor or PTB. To study the possible role of this trans acting factor, the PTB gene was partially silenced by PTB specific siRNA. This led to a decrease in the IRF2 IRES activity, suggesting that PTB is probably essential for the IRES activity. Interestingly, when Hela cells (with partially silenced PTB) were treated with tunicamycin (inducer of ER stress) the level of IRF2 protein was also found to be less thus pointing to an important role of PTB in IRF2 protein synthesis under such conditions. Western blot analysis and immunofluoroscence assay suggested that there was no significant nuclear-cytoplasmic relocalization of PTB under the condition studied. Primer extension inhibition assay or Toe-printing analysis was performed to detect the contact points of PTB on the IRF2 5’UTR. Many toe-prints were found on the 3’ end of the 5’UTR RNA. A 3’ deletion mutant was generated that showed reduced PTB binding. Incidentally the IRES activity of the mutant was also found to be less than the wt IRF2 RNA. Subsequently, structural analysis of the RNA was performed using enzymatic (CV1, RNase T1) and chemical modification (DMS) agents. Footprinting assay in presence of PTB suggested that there is change in the structure when PTB interacts with the RNA. To investigate this further, CD spectrum analysis of the IRF2 RNA in the presence of PTB was performed which indicated that there was a conformational change under such condition thus validating our earlier observation. The thesis reveals a novel cellular IRES element in the 5’UTR of IRF2 mRNA. The characterization of the IRES and possible role played by PTB protein in modulating its activity suggests that the regulated expression of IRF2 protein by its IRES element under various stress conditions would have major implications on the cellular response. Incidentally, this study constitutes the first report on translational control of interferon regulatory factors by internal initiation. The results might have far reaching implications on the possible role of IRF2 in controlling the intricate balance of cellular gene expression under stress conditions in general.

Cellular response to various stress conditions involves regulation of gene expression by different mechanisms. Translation is the final step in the flow of genetic information and regulation at this level allows an early response to changes in physiological conditions. Initiation of translation is the rate-limiting step of protein synthesis and hence is tightly regulated. Translation initiation in mammalian cells is mainly by “cap dependent pathway” wherein the 5’methyl guanosine “cap” structure is recognized by certain canonical initiation factors along with 40S ribosomal subunit and the complex scans the 5’UTR till it recognizes initiator AUG. This leads to the joining of the 60S ribosomal subunit and the initiation of translation. In an alternate mode of translation initiation called as the Internal ribosome entry site mediated translation (IRES), the ribosomes are recruited closer to the initiator AUG in a 5’ cap independent manner. Efficient translation by IRES mode requires some canonical initiation factors like eIF2 and eIF3 and other non-canonical IRES-trans-acting factors (ITAFs), which include human La antigen, polypyrimidine-tract binding protein (PTB),Upstream of N-Ras (Unr), Poly (rC) binding protein (PCBP2) etc. Various types of stress conditions, such as starvation of growth factors, heat shock, hypoxia, viral infection lead to down regulation of protein synthesis. However, translation of a subset of mRNAs continues or is up-regulated. Many of these mRNA may be translated by an IRES mode. It is believed that cellular IRESs become active during such conditions that abrogate the cap-dependent mode of translation so that the pool of vital proteins is maintained in the cell. In this thesis, presence of ‘IRES’ element has been investigated in the 5’UTR of Interferon regulatory factor -2 (IRF2) mRNA and the possible physiological significance has been studied. Further, it has been shown that polypyrimidine tract binding protein or PTB is important for the IRES activity. The probable mechanism of action of PTB has also been investigated which suggests that PTB interaction alters the IRF2 IRES conformation thus facilitating translation initiation. In the first part of the thesis, mRNAs that continue to be translated under heat-shocked condition, which is known to abrogate cap-dependent translation initiation, has been investigated by cDNA micro-array hybridization analysis of the ribosome bound RNA. The global protein synthesis was severely impaired under heat shock; however a number of mRNAs continued translation under this condition. Some of these mRNAs encode proteins that are likely to be involved in the heat shock response. Few of these genes are also reported to contain IRES element. Since the micro-array was performed from the RNA extracted from ribosome bound mRNA fraction in a condition when cap-dependent translation is impaired, it was hypothesized that some of the genes, which are up regulated under such condition, might operate via cap-independent mode of translation initiation. Based on this study, one candidate gene, the ‘interferon regulatory factor 2 (IRF2)’ was selected from the pool of up regulated genes and presence of an IRES element was investigated. Interferon regulatory factors are DNA-binding proteins that control interferon (IFN) gene expression. IRF2 has been shown to function as repressor of IFN and IFN-inducible genes. Real–Time and semi-quantitative RT-PCR assays were performed which validated the micro-array data. In the second part of the thesis, the presence of IRES element in the 5’UTR of IRF2 was investigated. Bicistronic assay showed comparable IRES activity with a known representative IRES, BiP, thus suggesting the presence of an IRES element in the IRF2 5’UTR. Stringent assays were then performed to rule out cryptic promoter activity, re-initiation/scanning or alternative splicing in the 5’UTR of the IRF2. RNA transfections using in vitro synthesized bicistronic RNAs further validated the presence of the IRES element. To understand the physiological significance of an IRES element in IRF2 mRNA, the cells were subjected to various stress conditions and IRES activity was studied. It seems IRF2 IRES function might not be sensitive to eIF4G cleavage, since its activity was only marginally affected in presence of Coxsackievirus 2A protease, which is known to cleave eIF 4G and thus inhibit the cap-dependent translation. Incidentally, Hepatitis A virus IRES was affected under such condition. Additionally, it was observed that compared to HCV or Bip IRES, the effect of Interferon α treatment was not so pronounced on the IRF2 IRES. This was further evidenced by its unchanged protein level post-treatment with interferon α. Furthermore, in cells treated with tunicamycin (a known agent causing ER stress), the IRF2 IRES activity and the protein levels were unaffected, although the cap dependent translation was severely impaired. The observations so far suggested that the IRF2 protein level is practically unchanged under conditions of ER stress and interferon treatment. Metabolic labeling followed by immunoprecipitation of IRF2 in cells treated with either tunicamycin or interferon suggested that de novo synthesis of the protein is continued under the above conditions thus validating our earlier data. In the third part of the thesis, the role of an IRES trans acting factor, PTB, in modulating the IRF2 IRES activity has been investigated. Analysis of the cellular protein binding with the IRF2 IRES suggested that certain cellular factors might influence its function under stress conditions. The IRF2 IRES was found to interact with a known trans-acting factor or PTB. To study the possible role of this trans acting factor, the PTB gene was partially silenced by PTB specific siRNA. This led to a decrease in the IRF2 IRES activity, suggesting that PTB is probably essential for the IRES activity. Interestingly, when Hela cells (with partially silenced PTB) were treated with tunicamycin (inducer of ER stress) the level of IRF2 protein was also found to be less thus pointing to an important role of PTB in IRF2 protein synthesis under such conditions. Western blot analysis and immunofluoroscence assay suggested that there was no significant nuclear-cytoplasmic relocalization of PTB under the condition studied. Primer extension inhibition assay or Toe-printing analysis was performed to detect the contact points of PTB on the IRF2 5’UTR. Many toe-prints were found on the 3’ end of the 5’UTR RNA. A 3’ deletion mutant was generated that showed reduced PTB binding. Incidentally the IRES activity of the mutant was also found to be less than the wt IRF2 RNA. Subsequently, structural analysis of the RNA was performed using enzymatic (CV1, RNase T1) and chemical modification (DMS) agents. Footprinting assay in presence of PTB suggested that there is change in the structure when PTB interacts with the RNA. To investigate this further, CD spectrum analysis of the IRF2 RNA in the presence of PTB was performed which indicated that there was a conformational change under such condition thus validating our earlier observation. The thesis reveals a novel cellular IRES element in the 5’UTR of IRF2 mRNA. The characterization of the IRES and possible role played by PTB protein in modulating its activity suggests that the regulated expression of IRF2 protein by its IRES element under various stress conditions would have major implications on the cellular response. Incidentally, this study constitutes the first report on translational control of interferon regulatory factors by internal initiation. The results might have far reaching implications on the possible role of IRF2 in controlling the intricate balance of cellular gene expression under stress conditions in general.

La localisation subcellulaire de l’ARN permet un déploiement prompt et spatialement restreint autant des activités protéiques que des ARN noncodant. Le trafic d’ARN est dirigé par des éléments de séquences (sous-séquences primaires, structures secondaires), aussi appelés motifs de régulation, présents en cis à même la molécule d’ARN. Ces motifs sont reconnus par des protéines de liaisons aux ARN qui médient l’acheminement des transcrits vers des sites précis dans la cellule. Des études récentes, chez l’embryon de Drosophile, indiquent que la majorité des ARN ont une localisation subcellulaire asymétrique, suggérant l’existence d’un « code de localisation » complexe. Cependant, ceci peut représenter un exemple exceptionnel et la question demeurait, jusqu’ici, si une prévalence comparable de localisation d’ARN est observable chez des cellules standards développées en culture. De plus, des informations facilement disponibles à propos des caractéristiques de distribution topologique d’instances de motifs à travers des transcriptomes complets étaient jusqu’à présent manquantes. Afin d’avoir un aperçu de l’étendue et des propriétés impliquées dans la localisation des ARN, nous avons soumis des cellules de Drosophile (D17) et de l’humain (HepG2) à un fractionnement biochimique afin d’isoler les fractions nucléaire, cytosolique, membranaire et insoluble. Nous avons ensuite séquencé en profondeur l’ARN extrait et analysé par spectrométrie de masse les protéines extraites de ces fractions. Nous avons nommé cette méthode CeFra-Seq. Par des analyses bio-informatiques, j’ai ensuite cartographié l’enrichissement de divers biotypes d’ARN (p. ex. ARN messager, ARN long non codant, ARN circulaire) et protéines au sein des fractions subcellulaires. Ceci a révélé que la distribution d’un large éventail d’espèces d’ARN codants et non codants est asymétrique. Une analyse des gènes orthologues entre mouche et humain a aussi démontré de fortes similitudes, suggérant que le processus de localisation est évolutivement conservé. De plus, j’ai observé des attributs (p. ex. la taille des transcrits) distincts parmi les populations d’ARN messagers spécifiques à une fraction. Finalement, j’ai observé des corrélations et anti-corrélations spécifiques entre certains groupes d’ARN messagers et leurs protéines. Pour permettre l’étude de la topologie de motifs et de leurs conservations, j’ai créé oRNAment, une base de données d’instances présumée de sites de liaison de protéines chez des ARN codants et non codants. À partir de données de motifs de liaison protéique par RNAcompete et par RNA Bind-n-Seq, j’ai développé un algorithme permettant l’identification rapide d’instances potentielles de ces motifs dans un transcriptome complet. J’ai pu ainsi cataloguer les instances de 453 motifs provenant de 223 protéines liant l’ARN pour 525 718 transcrits chez cinq espèces. Les résultats obtenus ont été validés en les comparant à des données publiques de eCLIP. J’ai, par la suite, utilisé oRNAment pour analyser en détail les aspects topologiques des instances présumées de ces motifs et leurs conservations évolutives relatives. Ceci a permis de démontrer que la plupart des motifs sont distribués de façon similaire entre espèces. De plus, j’ai discerné des points communs entre les sous-groupes de protéines liant des biotypes distincts ou des régions d’ARN spécifiques. La présence de tels patrons, similaires ou non, entre espèces est susceptible de refléter l’importance de leurs fonctions. D’ailleurs, l’analyse plus détaillée du positionnement d’un motif entre régions transcriptomiques comparables chez les vertébrés suggère une conservation synténique de ceux-ci, à divers degrés, pour tous les biotypes d’ARN. La topologie régionale de certaines instances de motifs répétées apparaît aussi comme évolutivement conservée et peut être importante afin de permettre une liaison adéquate de la protéine. Finalement, les résultats compilés avec oRNAment ont permis de postuler sur un nouveau rôle potentiel pour l’ARN long non codant HELLPAR comme éponge de protéines liant l’ARN. La caractérisation systématique d’ARN localisés et de motifs de régulation en cis présentée dans cette thèse démontre comment l’intégration d’information à l’échelle transcriptomique permet d’évaluer la prévalence de l’asymétrie, les caractéristiques distinctes et la conservation évolutive de collections d’ARN.
The subcellular localization of RNA allows a rapid and spatially restricted deployment of protein and noncoding RNA activities. The trafficking of RNA is directed by sequence elements (primary subsequences, secondary structures), also called regulatory motifs, present in cis within the RNA molecule. These motifs are recognized by RNA-binding proteins that mediate the transport of transcripts to specific sites in the cell. Recent studies in the Drosophila embryo indicate that the majority of RNAs display an asymmetric subcellular localization, suggesting the existence of a complex "localization code". However, this may represent an exceptional example and the question remained, until now, whether a comparable prevalence of RNA localization is observable in standard cells grown in culture. In addition, readily available information about the topological distribution of pattern instances across full transcriptomes has been hitherto lacking. In order to have a broad overview of the extent and properties involved in RNA localization, we subjected Drosophila (D17) and human (HepG2) cells to biochemical fractionation to isolate the nuclear, cytosolic, membrane and insoluble fractions. We then performed deep sequencing on the extracted RNA and analyzed through mass spectrometry the proteins extracted from these fractions. We named this method CeFra-Seq. Through bioinformatics analyses, I then profiled the enrichment of various RNA biotypes (e.g. messenger RNA, long noncoding RNA, circular RNA) and proteins within the subcellular fractions. This revealed the high prevalence of asymmetric distribution of both coding and noncoding RNA species. An analysis of orthologous genes between fly and human has also shown strong similarities, suggesting that the localization process is evolutionarily conserved. In addition, I have observed distinct attributes (e.g. transcript size) among fraction-specific messenger RNA populations. Finally, I observed specific correlations and anti-correlations between defined groups of messenger RNAs and the proteins they encode. To study motifs topology and their conservation, I created oRNAment, a database of putative RNA-binding protein binding sites instances in coding and noncoding RNAs. Using data from protein binding motifs assessed by RNAcompete and by RNA Bind-n-Seq experiments, I have developed an algorithm allowing their rapid identification in a complete transcriptome. I was able to catalog the instances of 453 motifs from 223 RNA-binding proteins for 525,718 transcripts in five species. The results obtained were validated by comparing them with public data from eCLIP. I then used oRNAment to further analyze the topological aspects of these motifs’ instances and their relative evolutionary conservation. This showed that most motifs are distributed in a similar fashion between species. In addition, I have detected commonalities between the subgroups of proteins linking preferentially distinct biotypes or specific RNA regions. The presence or absence of such pattern between species is likely a reflection of the importance of their functions. Moreover, a more precise analysis of the position of a motif among comparable transcriptomic regions in vertebrates suggests a syntenic conservation, to varying degrees, in all RNA biotypes. The regional topology of certain motifs as repeated instances also appears to be evolutionarily conserved and may be important in order to allow adequate binding of the protein. Finally, the results compiled with oRNAment allowed to postulate on a potential new role for the long noncoding RNA HELLPAR as an RNA-binding protein sponge. The systematic characterization of RNA localization and cis regulatory motifs presented in this thesis demonstrates how the integration of information at a transcriptomic scale enables the assessment of the prevalence of asymmetry, the distinct characteristics and the evolutionary conservation of RNA clusters.

De nombreuses voies de signalisation cellulaire complexes permettent de répondre à la présence de dommages à l’ADN. Cette réponse cellulaire est indispensable afin d’éviter l’accumulation de mutations pouvant éventuellement conduire à la transformation tumorale. Ces différentes voies de réponse aux dommages à l’ADN sont hautement coordonnées et sont regroupées au sein d’un mécanisme global appelé DNA damage response (DDR). Les facteurs du DDR sont régulés à plusieurs niveaux de la cascade de l’expression des gènes. De façon notable, plusieurs protéines de liaison à l’ARN (RBP) participent à la régulation de l’expression des gènes du DDR via la régulation post- transcriptionnelle de leur ARN messager. La RBP STAU2 est connue pour lier plusieurs ARNm codant pour des protéines impliquées dans le contrôle du cycle cellulaire ainsi que dans les voies du DDR. La protéine STAU2 est elle-même régulée au niveau transcriptionnel par le facteur de transcription E2F1. De récentes observations laissent penser que la kinase centrale du DDR, CHK1, pourrait être impliquée dans la régulation de la stabilité de STAU2. Par ailleurs, les conséquences cellulaires de la diminution du niveau d’expression de STAU2 sont à ce jour très peu connues. Ce mémoire a d’abord été entrepris dans le but de mieux comprendre l’implication de la voie de la kinase CHK1 dans la régulation de la protéine de liaison à l’ARN STAU2. CHK1 est une protéine centrale des voies du DDR ainsi que du contrôle de la progression du cycle cellulaire en l’absence de dommages à l’ADN. Nos résultats montrent que la diminution de CHK1 induit une dégradation rapide de STAU2 par les caspases d’une façon indépendante de l’apoptose. Nous avons également renforcé ce lien entre STAU2 et les mécanismes de réparation des dommages à l’ADN en identifiant plusieurs protéines des voies de réparation dans l’environnement immédiat de STAU2. D’autre part nos travaux visent à mettre en évidence les conséquences de la déplétion de STAU2 dans plusieurs types cellulaires. STAU2 étant une RBP, sa dérégulation impacte inévitablement le devenir de plusieurs ARNm. Afin de caractériser ces différentes conséquences, nous avons dans un premier temps réalisé la déplétion totale de STAU2 dans des cellules hTert-RPE par la technique de CRISPR/Cas9. Nos résultats montrent que ces cellules accumulent anormalement des dommages à l’ADN et prolifèrent plus rapidement que des cellules normales. En outre plusieurs gènes impliqués dans la réparation des dommages à l’ADN se retrouvent diminués dans ces cellules. Dans un second temps, afin de définir si cet effet est dépendant du type cellulaire, nous avons induit la diminution de l’expression de STAU2 dans des cellules IMR90. Nous avons montré que dans ce cas, la diminution de STAU2 induit un arrêt du cycle cellulaire et une entrée des cellules en sénescence. Ainsi, les données présentées dans ce mémoire contribuent à mieux comprendre l’implication de STAU2 dans les processus cellulaires majeurs que sont la régulation du DDR et le contrôle du cycle cellulaire.
Many complex cellular pathways are induced in response to DNA damages. This cellular response is indispensable to prevent the accumulation of mutations and to avoid malignant transformation. These different pathways are highly coordinated and are organized in a global mechanism called DNA damage response (DDR). Proteins involved in the DDR are regulated at different levels of the gene expression process. Notably, several RNA binding proteins are involved in the regulation of DDR gene expression through the post-transcriptional control of their mRNA. The RBP STAU2 is known to bind various mRNAs coding for proteins involved in the DDR or cell cycle control. STAU2 is regulated at the transcriptional levels by the major transcription factor E2F1. Recent observations suggest that CHK1 could be implicated in the control of the steady-state level of STAU2. Otherwise, the cellular consequences of STAU2 downregulation remain elusive. The purpose of this research was first to elucidate the implication of CHK1 pathway in STAU2 regulation. CHK1 is a major protein involved in the DDR regulation as well as in the control of cell cycle progression in the absence of DNA damage. Our data show that the downregulation of CHK1 rapidly leads to a caspase-dependent degradation of STAU2 independently of apoptosis. The link between STAU2 and mechanisms of DNA repair was reinforced by our BioID2 experiment that identified several proteins of the DDR in close proximity with STAU2. On the other hand, the aim of this study was to determine the consequences of STAU2 downregulation in different cell lines. Given that STAU2 is an RBP, its dysregulation will inevitably change the fate of several mRNA. In order to increase our understanding of theses consequences, we generated an hTert-RPE1 STAU2-KO cell line using the CRISPR/Cas9 technique. Our data show that these cells accumulate DNA damage and have an increased proliferation rate. Moreover, several genes involved in the DNA repair pathway are downregulated. We also downregulated STAU2 in IMR90 to determine if the previous observations are cell-type specifics. In the latter case, STAU2 diminution triggers cell cycle arrest and cellular senescence. Altogether, these results contribute to improve our knowledge of STAU2 function, especially in DNA damage response pathway and in cell cycle regulation.