This work focused on the further characterisation of Idhp and of the Krebs cycle enzymes citrate synthase 1 (Cit1p) and malate dehydrogenase 1 (Mdh1p) both of which have been identified as RNA-binding proteins without known RNA recognition motifs. Besides analysing their effects on mitochondrial translation and their organisation in protein complexes the work focused on the characterisation of the RNA-binding properties of recombinant Cit1p and Mdh1p: · Cit1p and Mdh1p play no essential role in mitochondrial protein synthesis. · Idhp is in a complex of molecular weight larger than the cytochrome c oxidase (250 kDa). · Cit1p and Mdh1p are in mitochondrial complexes smaller than 250 kDa. · 1000-fold molar excess of tRNA referring to COX2 leader RNA did not inhibit the RNA-binding of Cit1p and Mdh1p. · Cit1p and Mdh1p bind mitochondrial mRNAs (sense and antisense). The influence of cofactors and substrates on RNA-binding was analysed in order to reveal a possible link between the enzymatic function and the property of RNA-binding: · Acetyl-CoA and ATP inhibited the RNA-binding of Cit1p and Mdh1p at a concentration of 5 mM.

For the past 45 years, QKI has been studied for its role in the processes of development and central nervous system myelination using the qkv mouse. The presence of a single KH domain and the recent identification of a high-affinity binding site in mRNAs, suggests that it can bind to and regulate mRNAs through processes such as stability, splicing and transport. As a member of the STAR RNA binding family of proteins the QKI isoforms may also be involved in cell signaling pathways. / QKI's involvement in all of these processes, lead us to examine both the protein partners and the mRNA targets of the QKI complex in order to identify potentially new pathways regulated by QKI. In doing so, we identified a novel direct protein-protein interaction with PABP and for the first time described the relocalization of QKI to cytoplasmic granules following oxidative stress. In addition, in vivo mRNA interaction studies were performed and allowed the identification of approximately 100 new mRNA targets in human glioblastoma cells. One of the targets identified was VEGF mRNA. / Another QKI target mRNA is MBP, a major protein component of the myelin sheath and the candidate auto-antigen in multiple sclerosis (MS). In vivo MBP is symmetrically dimethylated on a single arginine residue. To further establish the role of the methylation of MBP in myelination, a methyl-specific antibody and an adenovirus expressing a recombinant protein arginine methyltransferase 5 (PRMT5) was generated. We show that methylated MBP is found in areas of mature myelin and that overexpression of the PRTM5 blocked the differentiation of oligodendrocytes. / Taken together these datas implicate QKI for the first time in the process of human cancer angiogenesis and could explain the vascularization defects observed in some of the qkI mutant mice. In addition, arginine methylation of MBP may prove to have an important role in the process of myelination and in the pathogenesis of demyelination and the autoimmune reaction in diseases such as MS.

The innate immune pathways that stimulate the expression of cytokines and proapoptotic factors in response to infection are triggered by the activation of the cytosolic receptors retinoic acid-inducible gene I (RIG-I) and melanoma differentiationassociated gene 5 (MDA5). Activation of both receptors occurs as a result of binding to RNA. MDA5 only recognizes double stranded forms of RNA, whereas RIG-I is capable of recognizing both single and double stranded RNA. In vivo, MDA5 is known to be stimulated by long (>1 kb) strands of RNA, forming filaments along the phosphate backbone. However, the manner in which MDA5 can recognize the terminal end of its RNA ligand is uncertain. I have examined the mechanism of binding of the MDA5 protein by comparing MDA5 binding to short (<18 bp) blunt RNA, 5’ triphosphate RNA, and RNA with a 3’ or 5’ overhang. It is shown that while the MDA5 protein regulatory domain (RD) is essential for RNA recognition, the MDA5 RD only weakly recognizes short double stranded RNA ligands with overhangs or a 5’ triphosphate group. The Cys951 residue was shown to disrupt stability of the MDA5 RD-RNA complex. Binding analyses were performed using a combination of SDS-PAGE, gel filtration analysis, and nondenaturing gel electrophoresis. In addition, structural data was gathered by crystallization of the MDA5 RD-RNA complex using X-ray crystallography. These results help to establish the manner in which MDA5 is regulated predominantly to the binding of long RNA ligands. Also included in this document is structural data on the dimer form of the PepQ protein from E. coli. PepQ is a highly conserved proline peptidase that has a secondary activity of hydrolyzing organophosphorus triesters, toxic compounds found in many pesticides. The PepQ protein was crystallized and analyzed by X-ray diffraction. The dimer interface was clearly defined within the structure and provides insight into how the active dimer forms from the PepQ monomer.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 122-140.

A soja constitui uma das culturas mais importantes mundialmente, tanto social quanto economicamente. Consequentemente, informações moleculares sobre processos de desenvolvimento, bem como conhecimento detalhado das interações entre condições estressoras e a resposta da planta a fatores ambientais são necessários. A identificação e caracterização de genes que respondem a condições ambientais específicas constituem um passo inicial no entendimento dos processos adaptativos. Proteínas ricas em glicina (GRPs) são polipeptídeos contendo um grande número do aminoácido glicina em sua estrutura primária. Os genes codificantes de GRPs são regulados ao longo do desenvolvimento e regulados por auxina, ABA, frio, ferimentos, luz, ritmo circadiano, salinidade, seca, patógenos e encharcamento. Entretanto, há pouca informação sobre GRPs de plantas e seus papéis no desenvolvimento e resposta a estresses. As GRPs podem ser divididas em quatro classes (I, II, III, IV) de acordo com sua estrutura primária e presença de domínios característicos. A classe IV é composta por proteínas ligantes de RNA. Domínios adicionais permitem dividir a classe IV de GRPs em quatro subclasses (IVa, IVb, IVc, IVd). A subclasse IVc é representada por proteínas contendo um cold-schock domain (CSD) e dedos de zinco CCHC tipo retrovirais. O objetivo do presente estudo foi: (i) identificar e caracterizar os genes codificantes de classe IV de GRPs, (ii) verificar a padrão de expressão dos genes codificantes da subclasse IVc de GRPs e (iii) produzir plantas de soja transgênicas expressando o gene AtGRP2, o qual foi mostrado estar envolvido na floração e desenvolvimento da semente em Arabidopsis, e também poderia desempenhar um papel na aclimatação ao frio. Um total de 47 genes codificantes da classe IV de GRPs foi identificado no genoma da soja: 19 da subclasse IVa, sete da IVb, seis da IVc e 15 da IVd. Análises in silico indicaram uma expressão preferencial de todos os genes codificantes da subclasse IVc em tecidos em desenvolvimento. Análises de RT-qPCR revelaram que plantas jovens e maduras exibem uma expressão mais alta em folhas do que em outros órgãos, com exceção dos genes GRP2L_4/5 que tiveram expressão mais alta em sementes. GRP2L_4/5 e GRP2L_2 foram induzidos em resposta a baixas temperaturas. Sob estresse com ABA a expressão de todos os genes foi reprimida em folhas e/ou raízes, com exceção do gene GRP2L_2 que foi induzido em raízes. Em resposta a infecção com Phakopsora pachyrhizi, a expressão de GRP2L_2 e GRP2L_3 foi mais alta e precoce no genótipo suscetível quando comparada com o resistente, enquanto que a resposta de GRP2L_4/5 e GRP2L_6 foi mais tardia no genótipo resistente. Ainda, embriões somáticos secundários das cultivares Bragg, IAS-5 e BRSMG 68 Vencedora de soja foram usados para introduzir o gene AtGRP2 no genoma da soja por bombardeamento e sistema bombardeamento/Agrobacterium. Seis eventos de transformação independentes foram confirmados por PCR. No presente momento as plantas estão em desenvolvimento em frascos de vidro. No presente estudo a classe IV de GRPs em soja foi identificada e caracterizada. Este é o primeiro passo para elucidar o papel destas proteínas em plantas. / Molecular information on plant developmental process, as well as detailed knowledge of the interaction between stress conditions and plant response to environmental factors are essential for understanding the adaptive response. Glycine-Rich Proteins (GRP) have the amino acid glycine well represented in their primary structure. The genes encoding GRPs are developmentally regulated and induced by auxin, ABA, cold, wound, light, circadian rhythm, salinity, drought, pathogens, and flooding. However, there is scarce information about plant GRPs and its role on development and stress response. The GRPs can be divided into four classes (I, II, II and IV) according to their primary structure and the presence of characteristic domains. Class IV is composed by RNA-binding proteins. Additional domains permit to split class IV GRPs into four subclasses (IVa, IVb, IVc and IVd). Subclass IVc is represented by proteins containing a Cold-Shock Domain (CSD) and retroviral-like CCHC zinc fingers. The goal of the present study was: (i) to identify and characterize the genes encoding class IV GRPs, (ii) to verify the relative expression of genes encoding subclass IVc GRPs and (iii) to produce transgenic soybean plants expressing the AtGRP2 gene, which was shown to be involved in Arabidopsis flower and seed development, and can also play a role in cold acclimation. A total of 47 genes encoding class IV GRPs were found in the soybean genome: 19 from IVa, seven from IVb, six from IVc and 15 from IVd subclasses. In silico analyses indicated a preferential expression of all genes encoding subclass IVc GRPs in tissues under development. RT-qPCR analyses revealed that both young and mature plants exhibit relative higher expression of subclass IVc GRPs in leaves than in other organs, with exception of GRP2L_4/5 genes that have higher expression in seeds. The GRP2L_4/5 and GRP2L_2 were up-regulated in response to low temperatures. Under ABA stress the expression of all genes was down-regulated in leaves and roots, with exception of GRP2L_2 gene that was up-regulated in roots. In response to Phakopsora pachyrhizi infection, GRP2L_2 and GRP2L_3 expression was higher and earlier in the susceptible genotype when compared with that of the resistant one, while GRP2L_4/5 and GRP2_6 respond later in the resistant genotype. Furthermore, secondary somatic embryos of Bragg, IAS-5 and BRSMG 68 Vencedora soybean cultivars were used to introduce the AtGRP2 gene into the soybean genome by particle bombardment and bombardment/Agrobacterium system. Six independent Bragg transformation events were confirmed by PCR. In the present moment the plants are under development in glass flasks. In the present study the soybean class IV GRPs were identified and characterized. This is the first step to elucidate the role of these proteins in plants.

A soja constitui uma das culturas mais importantes mundialmente, tanto social quanto economicamente. Consequentemente, informações moleculares sobre processos de desenvolvimento, bem como conhecimento detalhado das interações entre condições estressoras e a resposta da planta a fatores ambientais são necessários. A identificação e caracterização de genes que respondem a condições ambientais específicas constituem um passo inicial no entendimento dos processos adaptativos. Proteínas ricas em glicina (GRPs) são polipeptídeos contendo um grande número do aminoácido glicina em sua estrutura primária. Os genes codificantes de GRPs são regulados ao longo do desenvolvimento e regulados por auxina, ABA, frio, ferimentos, luz, ritmo circadiano, salinidade, seca, patógenos e encharcamento. Entretanto, há pouca informação sobre GRPs de plantas e seus papéis no desenvolvimento e resposta a estresses. As GRPs podem ser divididas em quatro classes (I, II, III, IV) de acordo com sua estrutura primária e presença de domínios característicos. A classe IV é composta por proteínas ligantes de RNA. Domínios adicionais permitem dividir a classe IV de GRPs em quatro subclasses (IVa, IVb, IVc, IVd). A subclasse IVc é representada por proteínas contendo um cold-schock domain (CSD) e dedos de zinco CCHC tipo retrovirais. O objetivo do presente estudo foi: (i) identificar e caracterizar os genes codificantes de classe IV de GRPs, (ii) verificar a padrão de expressão dos genes codificantes da subclasse IVc de GRPs e (iii) produzir plantas de soja transgênicas expressando o gene AtGRP2, o qual foi mostrado estar envolvido na floração e desenvolvimento da semente em Arabidopsis, e também poderia desempenhar um papel na aclimatação ao frio. Um total de 47 genes codificantes da classe IV de GRPs foi identificado no genoma da soja: 19 da subclasse IVa, sete da IVb, seis da IVc e 15 da IVd. Análises in silico indicaram uma expressão preferencial de todos os genes codificantes da subclasse IVc em tecidos em desenvolvimento. Análises de RT-qPCR revelaram que plantas jovens e maduras exibem uma expressão mais alta em folhas do que em outros órgãos, com exceção dos genes GRP2L_4/5 que tiveram expressão mais alta em sementes. GRP2L_4/5 e GRP2L_2 foram induzidos em resposta a baixas temperaturas. Sob estresse com ABA a expressão de todos os genes foi reprimida em folhas e/ou raízes, com exceção do gene GRP2L_2 que foi induzido em raízes. Em resposta a infecção com Phakopsora pachyrhizi, a expressão de GRP2L_2 e GRP2L_3 foi mais alta e precoce no genótipo suscetível quando comparada com o resistente, enquanto que a resposta de GRP2L_4/5 e GRP2L_6 foi mais tardia no genótipo resistente. Ainda, embriões somáticos secundários das cultivares Bragg, IAS-5 e BRSMG 68 Vencedora de soja foram usados para introduzir o gene AtGRP2 no genoma da soja por bombardeamento e sistema bombardeamento/Agrobacterium. Seis eventos de transformação independentes foram confirmados por PCR. No presente momento as plantas estão em desenvolvimento em frascos de vidro. No presente estudo a classe IV de GRPs em soja foi identificada e caracterizada. Este é o primeiro passo para elucidar o papel destas proteínas em plantas. / Molecular information on plant developmental process, as well as detailed knowledge of the interaction between stress conditions and plant response to environmental factors are essential for understanding the adaptive response. Glycine-Rich Proteins (GRP) have the amino acid glycine well represented in their primary structure. The genes encoding GRPs are developmentally regulated and induced by auxin, ABA, cold, wound, light, circadian rhythm, salinity, drought, pathogens, and flooding. However, there is scarce information about plant GRPs and its role on development and stress response. The GRPs can be divided into four classes (I, II, II and IV) according to their primary structure and the presence of characteristic domains. Class IV is composed by RNA-binding proteins. Additional domains permit to split class IV GRPs into four subclasses (IVa, IVb, IVc and IVd). Subclass IVc is represented by proteins containing a Cold-Shock Domain (CSD) and retroviral-like CCHC zinc fingers. The goal of the present study was: (i) to identify and characterize the genes encoding class IV GRPs, (ii) to verify the relative expression of genes encoding subclass IVc GRPs and (iii) to produce transgenic soybean plants expressing the AtGRP2 gene, which was shown to be involved in Arabidopsis flower and seed development, and can also play a role in cold acclimation. A total of 47 genes encoding class IV GRPs were found in the soybean genome: 19 from IVa, seven from IVb, six from IVc and 15 from IVd subclasses. In silico analyses indicated a preferential expression of all genes encoding subclass IVc GRPs in tissues under development. RT-qPCR analyses revealed that both young and mature plants exhibit relative higher expression of subclass IVc GRPs in leaves than in other organs, with exception of GRP2L_4/5 genes that have higher expression in seeds. The GRP2L_4/5 and GRP2L_2 were up-regulated in response to low temperatures. Under ABA stress the expression of all genes was down-regulated in leaves and roots, with exception of GRP2L_2 gene that was up-regulated in roots. In response to Phakopsora pachyrhizi infection, GRP2L_2 and GRP2L_3 expression was higher and earlier in the susceptible genotype when compared with that of the resistant one, while GRP2L_4/5 and GRP2_6 respond later in the resistant genotype. Furthermore, secondary somatic embryos of Bragg, IAS-5 and BRSMG 68 Vencedora soybean cultivars were used to introduce the AtGRP2 gene into the soybean genome by particle bombardment and bombardment/Agrobacterium system. Six independent Bragg transformation events were confirmed by PCR. In the present moment the plants are under development in glass flasks. In the present study the soybean class IV GRPs were identified and characterized. This is the first step to elucidate the role of these proteins in plants.

The development of an organism and its adult homeostasis rely on regulatory mechanisms that control the underlying gene expression programs. In certain biological contexts, such as germ cell development, gene expression regulation is largely executed at the post-­‐transcriptional level. This relies on RNA-­‐binding proteins (RBPs), whose activity and expression are also heavily controlled. While the RNA-­‐binding potential of RBPs is currently of intense scrutiny, surprisingly little is known to date about the molecular mechanisms that control RNA-­‐binding proteins abundance in the context of germ cell development. This work identifies the molecular mechanisms that shape expression patterns of two evolutionarily conserved RNA-­‐binding proteins, CPB-­‐3 and GLD-­‐ 1, which belong to CPEB and STAR protein family, respectively. By focusing on their regulation in the C. elegans germ line, this work reveals an involvement of the proteasome in reducing levels of CPB-­‐3/CPEB and GLD-­‐1/STAR at the pachytene-­‐to-­‐diplotene transition during meiotic prophase I. Furthermore, it documents that CPB-­‐3 and GLD-­‐1 are targeted to proteasomal degradation by a conserved SCF ubiquitin ligase complex that utilises SEL-­‐10/Fbxw7 as a substrate recognition subunit. Importantly, destabilisation of both RBPs is likely triggered by their phosphorylation, which is regulated by the mitogen-­‐activated protein kinase, MPK-­‐1, and restricted to the meiotic timepoint of pachytene exit. Lastly, this work investigates the potential consequences of target mRNA regulation upon delayed RBP degradation. Altogether, the collected data characterise a molecular pathway of CPEB and STAR protein turnover, and suggest that MPK-­‐1 signaling may couple RBP-­‐mediated regulation of gene expression to progression through meiosis during oogenesis.

Le syndrome de l’X fragile représente la première cause de déficience intellectuelle héréditaire. Ce syndrome résulte de l’absence de la protéine FMRP. FMRP est proposée réguler, sous contrôles des mGluR-I et d’autres récepteurs, l’expression de protéines importantes pour la plasticité synaptique en se fixant spécifiquement sur leur ARNm et en modulant leur traduction. Des milliers d’ARNm cibles ont déjà été proposées dans la littérature, mais très peu ont pu être validées. Par approche de pontage covalent aux UV et immunoprecipitation (CLIP) couplé à une analyse microarray, nous avons identifié un ARNm comme cible unique de FMRP dans les neurones corticaux. Cet ARNm code pour une kinase contrôlant le niveau de deux seconds messagers lipidiques importants pour le remodelage des épines dendritiques. De plus, nous avons montré que l’activation mGluR-I dépendante de la kinase est absente dans les neurones Fmr1 KO, avec pour conséquence une altération de plusieurs espèces lipidiques du neurone. Ces défauts peuvent expliquer les altérations morphologiques et fonctionnelles des épines dendritiques, cause principale proposée du syndrome de l’X fragile. / Fragile X syndrome is the leading cause of inherited intellectual disability and is due to the absence of the RNA binding protein FMRP (Fragile X Mental Retardation Protein). FMRP is proposed to bind and regulate synaptic expression of mRNA targets upon mGluR-I activation. Thousands of mRNA targets have already been proposed in the literature, but only a few have been validated leaving unsolved the question of the genes mostly affected by the absence of FMRP in the brain of fragile Xpatients. The main project of the thesis was to identify the mRNAs associated with FMRP in cortical neurons by performing cross-linking immunoprecipitation approach (CLIP). We found that FMRP principally targets one unique mRNA which encodes an important synaptic kinase. This enzyme controls the level of two second lipid messengers important for remodeling of dendritic spines. Consequently, the mGluR-I-dependant activation of the enzyme is lost in absence of FMRP, leading to several lipid species alterations in the neuron. These defects may explain the morphological and functional alterations of dendritic spines, the hallmark of fragile X syndrome.

Il a été observé que l’altération des fonctions des granules de stress, entités cytoplasmiques non-membranaires composées d’ARN et de protéines liant l’ARN (RBPs), peut conduire au développement de maladies telles que la sclérose latérale amyotrophique, la démence fronto-temporale, la myopathie à inclusions et la maladie de Paget des os. Ces pathologies sont caractérisées par un dépôt cytoplasmique d’inclusions solides enrichies en RBPs et comprenant des fibrilles. Une connexion génétique a été suggérée entre la persistance des granules de stress et l’accumulation de ces inclusions pathologiques dans le cytoplasme des patients. Dans mon manuscrit de thèse, il est mis en évidence le fait que la protéine hnRNPA1, dont les mutations entrainent les maladies mentionnées plus haut, subit une séparation de phases entre deux liquides connue également sous l’appellation « Séparation de Phases Liquide-Liquide » (LLPS) dans des gouttelettes enrichies en protéines. Bien que le domaine composé d’une séquence à faible complexité (Low Complexity sequence Domains ou LCD) soit suffisant pour obtenir cette séparation de phases, les domaines de liaison à l’ARN y contribuent également en présence d’ARN. Cela a permis d’envisager l’existence de plusieurs mécanismes intervenant dans la régulation de l’assemblage de ces granules. Un autre résultat a mis en exergue le fait que la formation de fibrilles n’est pas une obligation pour permettre la séparation de phases mais que les gouttelettes, enrichies en protéines, entrainent, par ailleurs, une augmentation de la formation de ces fibrilles. La séparation de phases liquide-liquide induite par le domaine composé d’une séquence à faible complexité semble contribuer à l’assemblage des granules de stress et à leurs propriétés liquides. Finalement, cette étude propose d’établir une réelle corrélation entre la formation des granules de stress qui deviennent persistants et l’accumulation d’inclusions pathologiques dans le cytoplasme des patients. / Stress granules are membrane-less organelles composed of RNA-binding proteins (RBPs) and RNA. Functional impairment of stress granules has been implicated in amyotrophic lateral sclerosis, inclusion body myopathy, Paget’s disease of bone and frontotemporal dementia; these diseases are characterized by solid, fibrillar, cytoplasmic inclusions that are rich in RNA binding proteins (RBPs). Genetic evidence suggests a link between persistent stress granules and the accumulation of pathological inclusions. In this thesis manuscript, I demonstrate that the disease-related RBP hnRNPA1 undergoes liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by a low complexity sequence domain (LCD). While the LCD of hnRNPA1 is sufficient to mediate LLPS, the folded RNA recognition motifs contribute to LLPS in the presence of RNA, potentially giving rise to several mechanisms for regulating assembly of stress granules. Importantly, while not required for LLPS, fibrillization is enhanced in protein-rich droplets. I suggest that LCD-mediated LLPS contributes to the assembly of stress granules and their liquid properties, and provides a mechanistic link between persistent stress granules and fibrillar protein pathology in disease.

Dans les cellules eucaryotes, l'expression des gènes est régulée à de multiples étapes. Les régulations post-transcriptionnelles regroupent l'ensemble des contrôles qui s'exercent sur un ARN, en particulier sa maturation nucléaire, sa stabilité et son contrôle traductionnel. Les protéines de liaison aux ARN sont des acteurs majeurs de ces régulations post-transcriptionnelles. Les protéines CELF1 et ELAVL1, abondantes et quasiment ubiquitaires, participent à ces différents niveaux de régulation et sont susceptibles de modifier le devenir des transcrits. Au cours de ces travaux, nous avons étudié l'impact de CELF1 sur l'abondance et la maturation des ARN par des approches transcriptomiques. Nous avons classé les transcrits en fonction de leurs réponses aux différentes inactivations. L'ensemble de ces données montre que i) CELF1 et ELAVL1 ont des rôles bivalents, leur déplétion étant associée à une répression ou une activation de certains gènes ; ii) dans des cellules HeLa, les effets des différentes déplétions sont majoritairement des effets indirects ; iii) CELF1 et ELAVL1 ont très majoritairement le même effet sur l'abondance des ARNm qu'elles contrôlent directement ; iv) CELF1 et ELAVL1 ont le plus souvent des effets coopératifs ou redondants sur l'abondance des transcrits liés. L'effet combinatoire de CELF1 et ELAVL1 dépend de l'ARNm considéré, révélant une complexité des régulations post-transcriptionnelles critiques pour le devenir d'une cellule. / In eukaryotic cells, after transcription gene expression is controlled at multiple steps. These qualitative and quantitative post-transcriptional regulations are specified by RNA binding proteins (RBP). By combining transcriptomic analysis and binding site information for CELF1, we showed that CELF1 regulates both nuclear and cytoplasmic steps of gene expression. CELF1 directly controls the stability of cyclin D1 mRNA and the splicing of several RNA including KLC1 (light chain kinesin 1). Because mRNAs are in complexes consisting of multiple RBP we studied whether CELF1 and ELAVL1 would interact and control the abundance of their bound mRNAs. This analysis unravel a surprising redundancy or cooperativity of CELF1 and ELAVL1. The combinatorial effects of CELF1 and ELAVL1 were highly dependent on the considered RNA. Interestingly, we showed that both proteins cooperate and interact physically.