Protein kinases comprise a fundamental class of cell function regulators that modify proteins by transferring phosphate groups from a nucleoside triphosphate such as ATP to specific amino acid residues on target proteins, altering protein conformation, function, and activity. As such, protein kinases are major regulators of many biological processes, including gene expression, which consists of the transfer of hereditary information in two major processing steps, transcription of DNA into a complementary precursor RNA transcript (pre-mRNA) and its subsequent translated into protein by the ribosome, where it can then go on to perform various processes in the cell. One particular family of protein kinases, otherwise known as serine/arginine protein-specific protein kinases (SRPKs), is conserved throughout eukaryotes and has been shown to be important in regulating gene expression, yet their roles in the gene expression pathway have yet to be elucidated. SRPK are known to phosphorylate serine/arginine (SR) splicing factor proteins, which are involved in mRNA splice site recognition and recruitment of splicing machinery. Members of the LAMMER kinase subfamily of SRPKs have also been shown to be required for efficient pre-mRNA splicing and important for mediating cellular progression through the cell cycle. To determine what other roles SRPKs play in mRNA processing, it is of use to study the homologous SRPK and LAMMER kinases in fission yeast, S. pombe, Dsk1 and Kic1, respectively. S. pombe provides a genetically valuable model for studying kinase function in RNA processing as both RNA processing machinery and SRPKs are conserved through higher eukaryotes. Using a novel green fluorescent protein tagging system based on properties of the MS2 bacteriophage genome, we are able to label specific mRNA transcripts of interest and visualize their locations in the cell using fluorescence microscopy. By visualizing the mRNA trafficking patterns of intron-containing and intronless mRNA transcripts, we show for the first time that deletions of the Dsk1 and Kic1 genes result in the nuclear retention of mRNA, such that Dsk1 and Kic1 are distinctly involved in mRNA export out of the nucleus.

A velocidade de renovação das proteínas musculares define o crescimento muscular, e afetam a qualidade da carne, destacando-se a renovação de colágeno e taxa proteolítica que tem implicação no fenótipo de maciez. No entanto, existe pouca informação na literatura sobre mudanças no processo de remodelação de proteínas musculares em resposta à taxa de recuperação do ganho de peso, após um período de subnutrição em animais fisiologicamente maduros. Dessa forma, o uso de sequenciamento de RNA pode indicar mudanças no tecido muscular através de um perfil de expressão diferencial que explicam mudanças nos fenótipos associados ao crescimento muscular. Portanto, o objetivo deste estudo foi avaliar as mudanças no transcriptoma associado a fenótipos de carne de vacas adultas Nelore submetidas a diferentes taxas de recuperação de peso. Neste experimento foram utilizadas 28 vacas adultas de 5 a 16 anos, sendo que 23 animais, por apresentarem baixo escore corporal no inicio do experimento como resultado de perda de peso durante uma estação seca, foram aleatoriamente distribuídas em 3 grupos baseados em ganho diário de peso vivo: GB - Base (abatidos com baixo escore corporal, n=4); GL - Lento (0,6 kg/dia, n=9); GR - Rápido (1,2 kg/dia, n=10). Um quarto grupo (GM - Manutenção, n=5), foi formado de animais com alto escore corporal do início ao final do experimento. Os animais foram abatidos em quatro pontos definidos por dias (d) de confinamento (A1(0d) = GB; A2(51d) = GL e GR; A3(74d) = GR; A4(104d) = GL e GM), e as amostras do musculo Longissimus thoracis foram coletadas para obtenção do RNA total. Após a obtenção do mRNA e a preparação das bibliotecas de cDNA, o sequenciamento das amostras foi feito em um equipamento HiScanSQ. Vinte e quatro horas após o abate, foram determinados o pH e a cor instrumental (L*, a*, b*) medidos no músculo Longissimus thoracis. Dois bifes foram amostrados para a análise de força de cisalhamento às 24h e 21 dias. Na comparação entre fenótipos e genes, os contrastes utilizaram o grupo GB como referência. As análises estatísticas foram realizadas no programa R, usando ANOVA e teste de Tukey. Menores força de cisalhamento aos 21 dias (P<=0,05) foram encontradas para o grupo GL (A4) e GM (A4) comparadas ao GB. Valores de L*, a*, b* no músculo do GR (A3) também foram maiores em relação ao GB. Somente o valor de b* na gordura foi diferente (P<=0,05) em GR (A2 e A3) em relação ao GB. Foram identificados o total de 578 genes diferencialmente expressos (DE; FDR 5%) nos cinco contrastes avaliados. A análise funcional de genes DE entre contrastes identificou uma via KEEG para o grupo GL (\"Ribosome\") e cinco para o grupo GR (\"ECM receptor interaction\", \"Focal Adhesion\", \"Protein digestion and absorption\", \"PI3K-Akt signaling pathway\"). Para o enriquecimento GO, foram obtidas quatro categorias, sendo \"Semaphorin receptor activity\" para o grupo GL, enquanto \"Cellular response to amino acid stimulus\", \"Collagen trimer\" e \"Extracellular matrix structural constituent\" foram identificadas para GR. As vias em geral estão associadas a alterações no tecido conjuntivo do músculo. As diferenças encontradas entre fenótipos e genes indicam que as taxas de crescimento afetam de forma diferente os fenótipos associados à qualidade da carne. A realimentação em animais adultos, ao alterar o transcriptoma com possíveis impactos no tecido conjuntivo, parece mostrar um cenário de fibrose muscular no ganho rápido de peso, diferentemente do ganho lento, que mostrou ser uma alternativa para melhorar a qualidade da carne. Todavia, os impactos na estrutura muscular e proteica que possam explicar incremento em aspectos de textura da carne precisam ser determinados. / The speed of renewal of muscle proteins defines muscle growth, and affect meat quality, especially the renewal the of collagen turnover and proteolytic rate that has implication in the tenderness phenotype. There is little information in the literature about changes in the muscle protein remodeling process in response to the rate of recovery of weight gain after malnutrition in physiologically mature animals. In this way, the use of RNA sequencing may indicate changes in muscle tissue through a differential expression profile that explains changes in phenotypes associated with muscle growth. Therefore, the objective of this study was to evaluate changes in the transcriptome associated with meat phenotypes of Nellore adult cows submitted to different rates of weight recovery. In this experiment, 28 adult cows from 5 to 16 years old were used, being that twenty-three animals were distributed randomly in 3 groups based on daily gain of live weight, since they presented low body score at the beginning of the experiment as a result of weight loss during a dry season: GB - Base (slaughtered with low body score, n=4); GL - Slow (0.6kg/day, n=9); GR - High (1.2 kg/day, n=10). A fourth group (GM - Maintenance, n = 5) was formed from animals with high body score from the beginning to the end of the experiment. The animals were slaughtered at four points defined by confinement days(d) (A1(0d) = GB; A2(51d) = GL and GR; A3(74d) = GR; A4(104d) = GL and GM), and Longissimus thoracis muscle samples were collected to obtain the total RNA. After obtaining the mRNA and preparation of the libraries, the sequencing of the samples was done in a HiScanSQ equipment. Twenty-four hours after slaughter, the pH and instrumental color (L*, a*, b*) were determined in the Longissimus thoracis muscle. Two steaks were collected for shear force analysis at 24h and 21 days. In the comparison of the phenotypes and genes, contrasts were used the GB group as reference. Statistical analysis were performed in R software, using ANOVA and Tukey test. Lower shear forces at 21 days (P<=0.05) were found for the GL (A4) and GM (A4) groups compared to GB. Values of L*, a*, b* in the GR (A3) muscle were also higher in relation to GB. Only the value of b* in fat was different (P<=0.05) in GR (A2 and A3) in relation to GB. A total of 578 differentially expressed genes (DE; false discovery rate 5%) in the five contrasts evaluated. Functional analysis of DE genes between contrasts identified a KEEG pathway for the GL group (\"Ribosome\") and five for the GR group (\"ECM receptor interaction\", \"Focal Adhesion\", \"Protein digestion and absorption\", \"PI3K-Akt signaling pathway\"). For GO enrichment, four categories were obtained, being \"Semaphorin receptor activity\" for GL, while \"Cellular response to amino acid stimulus\", \"Collagen trimer\" and \"Extracellular matrix structural constituent\" were identified for GR. Pathways in general are associated with changes in connective tissue of muscle. Re-feeding in adult animals by altering the transcriptome with possible impacts on connective tissue, seems to show a scenario of muscle fibrosis in the fast gain of weight, unlike the slow gain, which proved to be an alternative to improve meat quality. However, the impacts on muscle and protein structure that may explain the increase in meat texture aspects need to be determined.

In recent years it has become apparent that neuronal development and function relies not just on the regulation of transcription but also on post-transcriptional events. Two prevalent mRNA-based regulatory mechanisms in neurons are asymmetric mRNA localisation and the generation of different 3’UTR isoforms by alternative polyadenylation (APA). While experiments in mammalian systems indicate that subcellular mRNA localisation plays an important role in regulating local expression of proteins in neuronal processes, little is known about how mRNAs reach their destinations. It has been proposed that APA allows the production of mRNA isoforms with different roles. However, the importance of 3’UTR extensions has not been addressed in detail, particularly at the organismal level. In my PhD, I investigated the mechanisms of mRNA localisation and functional consequences of APA using the Drosophila embryonic nervous system as a genetically tractable model. I screened for mRNAs that localise in embryonic axons using an available transgenic library of 3’UTR sequences, as well as publically available in situ hybridisation data. I found that Ankyrin2 (Ank2) mRNA localises in Drosophila embryonic sensory neurons, and showed that this is dependent on the kinesin-1 motor and microtubules. These data reveal an active mRNA transport system in embryonic neurons. I also showed that the Ank2 mRNA has an extended 3’UTR that is found in axons, suggesting that APA could be relevant to axonal functions of Ank2. I demonstrated that while mRNA molecules could still localise to axons upon CRISPR-Cas9-mediated deletion of the Ank2 3’UTR extension, a fraction of the mutant embryos had a disrupted nervous system. Interestingly, embryos that lack the ability to make Ank2 protein have an overtly normal embryonic nervous system. This observation reveals that the extension does not simply promote Ank2 protein function. Further experiments revealed that the extended 3’UTR is required for efficient locomotion of adult flies. While the exact function of the Ank2 3’UTR extension requires future investigation, I show that it is unlikely to be associated with the trafficking of associated proteins into axons. RNA affinity purifications from embryonic extracts provide evidence that the 3’UTR extension selectively binds conserved RNA-binding proteins. I speculate that the extension plays a role in regulating axonal morphogenesis by regulating the relative expression level of different Ank2 protein isoforms.

Made available in DSpace on 2015-03-26T13:51:51Z (GMT). No. of bitstreams: 1 texto completo.pdf: 528396 bytes, checksum: 2b516aa2ea3ea48bfb173971c8b65f4c (MD5) Previous issue date: 2010-08-18 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The low germination percentages of P. microcarpus basidiospores represents a major drawback for obtaining monokaryotic strains for quantitative genetic studies on the mycorrhizal associations, the use of spores in mutagenesis experiments aiming at the identification of genes important to the symbiosis, and the use of these propagules as inoculants in forest nurseries. The characterization of the mRNA present within the fungal basidiospores may provide information on the level of preparedness of the spores to germinate and sustain initial hyphal growth. The aim of this work was to characterize the mRNA present in the basidiospores of the ectomycorrhizal fungus P. microcarpus after basidiosporogenesis. Total RNA was extracted from the mature basidiospores and mycelium and mRNA was used for cDNA synthesis. An analysis of the presence of 14 gene transcripts involved in lipid metabolism, glycogen mobilization, trehalose mobilization, nitrogen assimilation, glycolysis, pentose phosphate pathway and glucan degradation using cDNA from the fungal basidiospores and dikaryotic mycelium was done. qPCR analysis was performed to compare the amount of transcripts of the genes d15fa, ntrh, and ag13 which code respectively, for &#916;15 fatty acid desaturase, neutral &#945;-trehalase, and &#945;-1,3-glucosidase in the dikaryotic mycelium and basidiospores. The 14 transcripts studied were present both in the dikaryotic mycelium and in the basidiospores, indicating a preparedness of these propagules to initiate and sustain germination. qPCR analysis indicated a higher amount of transcripts of genes the d15fa, ntrh, and ag13 inside the basidiospores when compared to the dikaryotic mycelium. These results suggest a higher need for enzymes involved in the synthesis of unsaturated fatty acids, trehalose mobilization, and glucan degradation during basidiospore germination. A cDNA library was constructed from basidiospore mRNAs and 288 clones were sequenced. One hundred and nineteen sequences were obtained, resulting in a total of 12 ESTs (expressed sequence tags). The amino acid sequence deduced from the EST corresponding to clone 277 showed significant similarity to a protein involved in respiratory metabolism and may be important during the germination process. Other ESTs showed significant identity to unknown ESTs deposited in the NCBI database. These ESTs may code for proteins that are specific of the basidiospores of P. microcarpus. However, to confirm this hypothesis, other clones must be sequenced to better characterize the cDNA library. / As baixas percentagens de germinação dos basidiósporos do fungo ectomicorrízico Pisolithus microcarpus dificultam a obtenção de estirpes monocarióticas, material para estudos de genética quantitativa da associação micorrízica, inviabilizam a utilização de esporos em experimentos de mutagênese visando à identificação de genes importantes para a simbiose, além de dificultar a utilização desses propágulos como inoculantes em viveiros florestais. A caracterização do mRNA presente no interior desses basidiósporos poderá fornecer informações sobre o nível de preparação que têm para iniciar o processo de germinação e sustentar o crescimento inicial das hifas. O objetivo deste trabalho foi o de caracterizar os mRNA presentes nos basidiósporos do fungo ectomicorrízico P. microcarpus após a basidiosporogênese. O RNA total foi extraído de basidiósporos maduros e micélio, procedendo posteriormente a obtenção mRNA para a síntese de cDNA. Determinou-se a presença de transcritos de 14 genes envolvidos no metabolismo de lipídeos, na mobilização de glicogênio, na mobilização de trealose, na assimilação de nitrogênio, na via glicolítica, na via das pentoses fosfato e na degradação de glicanas. A análise por qPCR foi realizada visando comparar a quantidade de transcritos dos genes d15fa, ntrh e ag13 que codificam respectivamente as enzimas ácido graxo-&#916;15 desaturase, &#945;- trealase neutra e 1,3-&#945;-glicosidase, nos basidiósporos e no micélio dicariótico. Os 14 x transcritos avaliados foram encontrados tanto no micélio dicariótico quanto nos basidiósporos, sugerindo a preparação desses propágulos para iniciar e sustentar o processo de germinação. A análise por qPCR indicou maior quantidade de transcritos dos genes d15fa, ntrh e ag13 no interior dos basidiósporos quando comparado ao micélio dicariótico. Esse resultado sugere a participação de enzimas responsáveis pela síntese de ácidos graxos insaturados, mobilização de trealose e degradação de glicanas durante o processo de germinação. Uma biblioteca de cDNA foi construída a partir dos fragmentos de cDNA dos basidiósporos, sendo caracterizada por meio do sequenciamento de 288 clones. Foram obtidas 119 sequências que, ao serem agrupadas, resultaram em 12 ESTs (expressed sequence tags). A sequência de aminoácidos deduzida da EST referente ao clone 277 apresentou similaridade significativa com proteína envolvida no metabolismo respiratório, podendo ser importante durante o processo de germinação. Outras ESTs apresentaram identidade significativa com ESTs depositadas no banco de dados do NCBI ainda não identificadas. Alternativamente, sugere-se que essas ESTs possam ser codificadoras de proteínas específicas dos basidiósporos de P. microcarpus, importantes para a etapa de germinação desses propágulos.

Chez les Eucaryotes, l’étape d’initiation est de loin la plus complexe et la plus lente du processus de traduction. Elle nécessite l’intervention de 12 facteurs protéiques, d’une coiffe m7GpppN située à l’extrémité 5’ des ARNm et d’une queue poly(A) en 3’. Les ARNm des histones « réplication-dépendantes » sont particuliers car dépourvus d’extrémité 3’ polyadénylée et dotés d’une extrémité 5’ non traduite extrêmement courte, de 9 nt seulement chez l’ARNm H4 de la souris. Pour traduire ces ARNm, un processus d’initiation non conventionnel a été décrit au laboratoire. L’objectif de ma thèse a été d’établir les bases structurales de ce mécanisme en combinant différentes approches expérimentales. Deux protocoles originaux de repliement ont été mis au point afin d’isoler l’ARNm H4 dans deux conformations distinctes et stables. Une caractérisation fonctionnelle et structurale de ces deux formes de l’ARNm a ensuite été réalisée. La stabilité et la structure de ces deux formes ont été étudiées par DLS, par SAXS et par équilibre de sédimentation. Puis, nous avons étudié la capacité de ces deux formes d’ARNm H4 à fixer le facteur d’initiation eIF4E et les ribosomes assemblés sur le codon d’initiation ainsi que leur aptitude à être traduits in vitro. Un modèle de repliement de la structure secondaire de l’ARNm H4 a été construit après sondage enzymatique et chimique des deux formes de l’ARNm. Ce modèle a servi de base pour le travail d’ingénierie de l’ARNm H4 qui a conduit à son découpage en sous-domaines. Des essais de cristallisation ont porté sur 18 de ces fragments ainsi que sur les deux formes de l’ARNm H4 complet. / In eukaryotes, the initiation step is far more complex and the slowest inside the translation process. It requires the intervention of 12 protein factors, an m7GpppN cap located to the 5 'end of the mRNA and a poly (A) tail at the 3'. Histone mRNAs "replication-dependent" are specific because they lack of polyadenylated tail at the 3’ end and have a 5' end untranslated extremely short (9 nt only in the mouse). To translate these mRNAs, a process of unconventional initiation has been described in the laboratory. The aim of my thesis was to establish the structural basis of the mechanism by combining different experimental approaches. Two original refolding protocols have been developed to isolate mRNA H4 in two distinct and stable conformations. A functional and structural characterization of these two shapes of H4 mRNA was then performed. The stability and the structure of these two shapes have been studied by DLS, SAXS and sedimentation equilibrium. Then, we studied the ability of these two conformations of H4 mRNA to bind the eIF4E initiation factor and ribosomes assembled on the start codon as well as their ability to be translated in vitro. Models of the secondary structure has been constructed after enzymatic and chemical probing of the two shapes of the mRNA. This model was the basis for the engineering of the H4 mRNA that led to its division into sub-domains. Crystallization trials focused on 18 of these fragments as well as on both H4 mRNA shapes.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-04-04T13:30:03Z No. of bitstreams: 1 sabinewohlresviana.pdf: 1957296 bytes, checksum: 29394666e22c25c01e78f33c22873af2 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-04-04T15:37:11Z (GMT) No. of bitstreams: 1 sabinewohlresviana.pdf: 1957296 bytes, checksum: 29394666e22c25c01e78f33c22873af2 (MD5) / Made available in DSpace on 2017-04-04T15:37:11Z (GMT). No. of bitstreams: 1 sabinewohlresviana.pdf: 1957296 bytes, checksum: 29394666e22c25c01e78f33c22873af2 (MD5) Previous issue date: 2009-03-09 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Embriões produzidos in vivo e in vitro apresentam diferenças morfológicas e de desenvolvimento as quais podem ser influenciadas pelo de cultivo. Além disso, existem poucos estudos mostrando o efeito do ambiente in vitro sobre os embriões provenientes de diferentes raças bovinas como, por exemplo, Gir (Bos indicus) e Holandesa (Bos taurus). Alguns genes importantes para a formação, sobrevivência e posterior desenvolvimento de embriões bovinos podem ter seu padrão de expressão influenciado por diversos fatores relacionados ao sistema de produção e a raça. O objetivo deste estudo foi avaliar a abundância relativa de transcritos relacionados ao transporte de água e solutos (Aquaporinas), formação da blastocele (Na/KATPases), apoptose (BAX) e resposta a estresse celular (Peroxiredoxina 1) em blastocistos bovinos das raças Gir e Holandesa produzidos in vivo e in vitro para identificar efeitos de sistema de produção e/ou raça. Para cada grupo (Gir-in vivo; Gir-in vitro; Holandês-in vivo; Holandês-in vitro), blastocistos (n=15) divididos em três conjuntos foram utilizados para a extração do RNA, amplificação, e transcrição reversa. Os cDNAs obtidos foram submetidos à PCR em Tempo-Real, utilizando o gene H2a como controle endógeno, e analisados pelo software REST©. Na avaliação de efeito do sistema de produção para a raça Gir, a expressão relativa do gene PRDX1 apresentou-se sub-regulada (p<0,01) assim como nos genes Na/KATPase-β2 e BAX (p<0,05), enquanto os genes AQP3, AQP11 e Na/K-ATPase-α1 não apresentaram diferenças significativas (p>0,05) nas amostras de embriões produzidos in vitro comparados com embriões in vivo. Na raça Holandesa, a expressão relativa do gene BAX apresentou-se sobre-regulada (p<0,01), enquanto o gene Na/K-ATPase-β2 apresentou uma tendência a sub-regulação (p=0,06) e os genes AQP3, AQP11, Na/K-ATPase-α1, PRDX1 não apresentaram diferenças significativas (p>0,05) nas amostras de embriões produzidos in vitro comparados com embriões in vivo. Quando a produção in vivo foi avaliada entre as raças, os embriões da raça Holandesa apresentaram os genes AQP3, Na/K-ATPase-α1, PRDX1, e BAX sub-regulados (p<0,05), enquanto os genes Na/K-ATPase-β2 e AQP11 não demonstraram diferenças significativas na expressão (p>0,05) quando comparados com embriões da raça Gir. Para a produção in vitro, apenas o gene BAX apresentou-se sobre-regulado (p<0,05) nos embriões da raça Holandesa quando comparados com embriões da raça Gir. Com base nestes resultados podese concluir que, para os genes avaliados, existem diferenças na expressão gênica inerentes às raças observadas nos embriões produzidos in vivo e que o sistema de produção in vitro pode influenciar a expressão gênica, atenuando estas variações. A avaliação de outros transcritos é necessária para uma melhor compreensão dos efeitos relacionados aos processos de produção de embriões bovinos em diferentes raças. / Embryos produced in vivo and in vitro show morphological and developmental differences, which can be influenced by culture environment. Nevertheless, there are few studies showing the effect of in vitro environment on embryos from different bovine breeds, such as Gyr (Bos indicus) and Holstein (Bos taurus). Some important genes associated to formation, survival and subsequent development of bovine embryos can be affected by various factors related to the production system and breed. The aim of this study was to evaluate the relative abundance of transcripts associated to water transport (Aquaporins), blastocoel formation (Na/K-ATPases), apoptosis (BAX) and response to cellular stress (Peroxiredoxin 1) in bovine blastocysts from Gyr and Holstein breed produced in vivo and in vitro to identify breed and/or production system effects. For each group (Gyr-in vivo; Gyr-in vitro; Holstein-in vivo; Holstein-in vitro), blastocysts (n=15) distributed in 3 pools were used for RNA extraction, followed by RNA amplification and reverse transcription. The cDNAs obtained were submitted to Real-Time PCR, using the H2a gene as endogenous control, and analyzed with REST software©. For the production system in the Gyr embryos, the relative expression of PRDX1 gene was down-regulated (p<0.01) as well as Na/K-ATPase-β2 and BAX genes (p<0.05), and the AQP3, AQP11 and Na/K-ATPase-α1 genes were not significantly different (p>0.05) when in vitro embryos were compared with in vivo produced ones. In Holstein embryos, BAX was up-regulated in in vitro embryos (p<0.01), while AQP3, AQP11, Na/K-ATPaseα1, Na/K-ATPase-β2 and PRDX1 genes were not significantly different (p>0.05) from in vivo produced embryos. When the breed effect was evaluated, Holstein in vivo produced blastocysts presented AQP3, Na/K-ATPase-α1, PRDX1, and BAX genes down-regulated (p<0.05) while Na/K-ATPase-β2 and AQP11 genes were not significantly different (p>0.05) from Gyr embryos. For the in vitro produced embryos, only Bax was up-regulated (p<0.05) in Holstein embryos compared with Gyr embryos. Based on those results, for the evaluated genes, it is possible to conclude that are inherent breed differences in gene expression observed in in vivo produced embryos and that the in vitro production system influences in the relative expression in both breeds, attenuating the inherent breed differences. Other genes should be evaluated for a better understanding of these breeds differences.

Les P-bodies sont des granules cytoplasmiques de fonction inconnue. Ils sont néanmoins conservés de la levure à l’homme, suggérant un rôle important chez les eucaryotes. L’analyse de l’influence de l’expression de certaines protéines pouvant se localiser dans ces granules a permis de mettre en évidence le rôle crucial de DDX6 dans l’assemblage des P-bodies. En effet, l’inhibition de l’expression de DDX6 dans les cellules humaines empêche l’assemblage des P-bodies. L’étude de la protéine DDX6 semblait donc être un bon point de départ pour comprendre d’avantage le rôle des P-bodies. Cette hélicase à motif DEAD de 54 kDa interagit avec des protéines du complexe répression de la traduction CPEB chez le Xénope, mais également avec des protéines des complexes de dégradation 5’-3’ et 3’-5 ‘ des ARNm chez les mammifères, les levures et les drosophiles, ou encore avec les protéines Argonautes du complexe miRISC chez les mammifères. Nos travaux se sont donc intéressés à déterminer les principales fonctions de DDX6 dans les cellules humaines. Les complexes protéiques contenant DDX6 ont été purifiés à partir de lysats cytoplasmiques de cellules HEK293 transfectées avec un plasmide codant pour la protéine FLAG-DDX6-HA. Plus de 300 partenaires protéiques ont été identifiés en spectrométrie de masse. Trois complexes majeurs contenant DDX6 ont été mis en évidence : un complexe de répression « CPEB-like », le complexe de décoiffage des ARNm, et un complexe contenant les ataxin-2 et ataxin-2-like. Les protéines du cœur du complexe de jonction d’exons sont également en interaction avec DDX6, suggérant que DDX6 interagit avec des ARNm tout juste sortis du noyau. Enfin, le grand nombre et les hauts scores avec lesquels ont été identifiés les protéines ribosomales nous ont conduit à analyser la présence de DDX6 au niveau des polysomes. L’analyse de lysats cytoplasmiques sur gradient de sucrose nous a permis de mettre en évidence l’association d’une fraction de DDX6 aux polysomes. Toutes ces observations mettent en évidence le rôle multiple de DDX6 entre répression et dégradation des ARNm, dans les cellules humaines. L’hélicase pourrait permettre le recrutement du complexe de répression par des ARNm activement traduits. La fixation multiple de DDX6 à l’ARNm pourrait être un moyen de recruter simultanément les complexes de répression et de dégradation des ARNm sur un même ARNm. / P-bodies are cytoplasmic granules. Their function is unknown, but they are conserved from the yeast to humans, suggesting an important role through eukaryotes. The expression inhibition of several proteins localized in P-bodies leads to their disassembly. In most cases, a cellular stress induced by arsenite treatment causes P-body assembly, except in cells depleted for DDX6. Since this observation showed that DDX6 is necessary for P-body assembly, to study this protein is a good starting point to further understand the role of P-bodies. This 54 kDa DEAD-box helicase interacts with proteins of the CPEB repression complex in xenopus oocytes, but also with protein of the déadénylation and dacapping complex in yeasts, drosophila and mammals, and with proteins of the miRISC complex. Our project was to determine the DDX6 main protein partners in human cells. To do so, DDX6 containing complexes were purified using HEK293 cells transfected with a plasmid encoding for the FLAG-DDX6-HA. Over 300 partners were identified by mass spectrometry. Three main DDX6 containing complexes were highlighted in human cells: A “CPEB-like” repression complex, the decapping complex, and a complex containing ATXN2 and ATXN2L proteins. Exon junction complex core proteins were also identified as DDX6 partners, raising the possibility that DDX6 interacts with mRNA not yet translated. A large number of ribosomal proteins were also identified with high scores, suggesting that DDX6 interacts with actively translated mRNA. Analyze of cytoplasmic lysates on sucrose gradients showed that DDX6 is partially associated with polysomes. To conclude, these observations showed the multiple roles of DDX6 in human cell, between mRNA repression and degradation. The helicase could allow the recruitment of the repression complex on actively translated mRNA. In a nutshell, the multiple binding of DDX6 on one mRNA could be a way to enable the fixation of repression and degradation complexes at the same time, on the same mRNA.

Das Neuroblastom ist der häufigste extrakraniell gelegene solide Tumor der pädiatrischen Onkologie. Der Verlauf der Erkrankung geht von spontaner Regression oder Differenzierung bis hin zu tödlich verlaufenden Erkrankungen. Die Mortalität von Patienten mit Tumoren in fortgeschrittenen Stadien ist immer noch sehr hoch. Die aggressivsten Tumoren sind die, die eine Amplifikation des Protoonkogens MYCN aufweisen. Eine Untergruppe dieser MYCN amplifizierten Tumoren weist eine Coamplifikation von DDX1 auf. Die Prognose dieser Patienten ist besser als die mit allein MYCN amplifizierten Tumoren, wenn auch immer noch schlechter als die von Patienten ohne MYCN Amplifikation. Das DDX1-Protein ist eine putative RNA-Helikase. Über seine genaue Funktion ist noch nicht viel bekannt. Ziel dieser Arbeit war es, potentielle Ziel-mRNAs von DDX1 zu identifizieren, um einen besseren Einblick in die Funktionen von DDX1 und mögliche Wege der Beeinflussung von Tumorverhalten und Prognose zu erhalten. Hierzu wurden eine DDX1 amplifizierte und eine nicht amplifizierte Zelllinie in Kultur genommen und eine Immunopräzipitation mit Zelllysaten der beiden Zelllinien durchgeführt – jeweils mit einem spezifischen Antikörper gegen DDX1 und einem unspezifischen Kontrollantikörper. Die Identifizierung der an DDX1 gebundenen mRNAs erfolgte mittels Microarray. Validiert wurden einige der im Microarray identifizierten RNAs mittels RT-PCR. CDK1, ATM und p18 ließen sich als spezifische Ziel-mRNAs von DDX1 identifizieren.

U5 small ribonucleoprotein particle (U5 snRNP) is a crucial component of the spliceosome, the complex responsible for pre-mRNA splicing. Despite the importance of U5 snRNP, not much is known about its biogenesis. When we depleted one of the core U5 components, protein PRPF8, the other U5-specific proteins do not associate with U5 snRNA and the incomplete U5 was accumulated in nuclear structures known as Cajal bodies. To further clarify the role of PRPF8 in U5 snRNP assembly, we studied PRPF8 mutations that cause an autosomal dominant retinal disorder, retinitis pigmentosa (RP). We prepared eight different PRPF8 variants carrying RP-associated mutations and expressed them stably in human cell culture. We showed that most mutations interfere with the assembly of snRNPs which consequently leads to reduced efficiency of splicing. The mutant PRPF8 together with EFTUD2 are stalled in the cytoplasm in a form of U5 snRNP assembly intermediate. Strikingly, we identified several chaperons including the HSP90/R2TP complex and ZNHIT2 as new PRPF8's interactors and potential U5 snRNP assembly factors. Our results further imply that these chaperons preferentially bind the unassembled U5 complexes and that HSP90 is required for stability of...

The spliceosome is one of the largest and most dynamic molecular machines in the cell. The central part of the complex is formed by five small nuclear ribonucleoproteins (snRNPs) which are generated in a multi-step biogenesis pathway. Moreover, the snRNPs undergo extensive rearrangements during the splicing and require reassembly after every intron removal. Both de novo assembly and post-splicing recycling of snRNPs are guided and facilitated by specific chaperones. Here, I reveal molecular details of function of two snRNP chaperones, SART3 and TSSC4. While TSSC4 is a previously uncharacterized protein, SART3 has been described before as a U6 snRNP-specific factor which assists in association of U6 and U4 particles into di-snRNP, and is important for the U4/U6 snRNP recycling. However, the mechanism of its function has been unclear. Here, I provide an evidence that SART3 interacts with a post-splicing complex and propose that SART3 could promote its disassembly. Our data further suggest that SART3 binds U6 snRNP already within the post-splicing complex and thus participates in the whole recycling phase of U6 snRNP. Then, I show that TSSC4 is a novel U5 snRNP-specific chaperone which promotes an assembly of U5 and U4/U6 snRNPs into a splicing-competent tri-snRNP particle. We identified...