Large differences exist in the decay rates of individual mRNAs yet the molecular basis for such differences is substantially unknown. We have developed a procedure for the measurement of individual mRNAs in the yeast Saccharomyces cerevisiae which utilizes northern or dot blotting to quantitate the levels of individual mRNAs after thermal inactivation of RNA polymerase II in an rpb1-1 temperature-sensitive mutant strain (RY260). To assess the reliability of half-life measurements obtained in this manner, we have compared the results of this procedure to results obtained by three other procedures (pulse-chase analysis, approach to steady-state labeling, and inhibition of transcription with thiolutin) and also evaluated whether heat-shock alters mRNA decay rates. We find that: i) for most mRNAs, all four procedures yield comparable relative decay rates and ii) there are no significant differences in the mRNA decay rates measured in heat-shocked or non-heat-shocked cells. Of the 20 mRNAs studied, 11, including those encoded by HIS3, STE2, STE3, and MATα1, were unstable (t1/21/2> 25 min). We have begun to assess the basis and significance of such differences in the decay rates of these two classes of mRNA. The following parameters have been analyzed to determine their role in mRNA decay: i) mRNA size; ii) poly(A) tail metabolism; iii) translational status; iv) relative content of rare codons; and v) structures and sequences within the 3'-untranslated region (UTR). To identify the structural determinants responsible for the rapid decay of the unstable HIS3 and STE2 mRNAs, recombinants of their respective genes were constructed and transformed into strain RY260 on centromere-containing vectors, and the half-lives of the resulting chimeric mRNAs were measured in vivo. Chimeric genes were constructed in which the 3'-UTR of ACT1 was replaced with the corresponding region of the unstable HIS3 or STE2 mRNAs. The decay rate of the ACT1-5'-HIS3-3' mRNA was very similiar to that of the stable endogenous ACT1 mRNA, implying that the 3'-end of HIS3 is not sufficient to transfer the instability phenotype of the HIS3 mRNA. The HIS3-5'-ACT1-3' mRNA from the reciprocal construct was unstable, suggesting that HIS3 instability determinants are located within its 5'-UTR or coding sequence. A 411 nucleotide (nt) deletion within the HIS3 coding region (with either the HIS3 or ACT1 3'-UTR) was stabilized 3-fold suggesting this region is necessary for the rapid decay of HIS3 mRNA. Insertion of these 411 nts in-frame into the entire ACT1 gene had no significant effect on the stability of the hybrid mRNA implying that these HIS3 sequences are not sufficient to function on their own and that they may have to interact with HIS3 5'- sequences. The ACT1-5' -STE2-3' hybrid mRNA decayed with an intermediate half-life of 12 min. Furthermore, an 82% deletion of the STE2 coding region increased the half-life by nearly 2-fold. Both results suggest that instability determinants of STE2 mRNA are not restricted to the 3'-UTR. Our overall conclusion is that mRNA stability is not dictated by simple, transferable elements (sequences or structures), but may involve interactions between multiple determinants in the mRNA.

nÃo hà / A peÃa em anÃlise neste trabalho, Agamemnon de LÃcio Aneu SÃneca, possui, dentro de seu terceiro ato, uma longa parte narrativa (v. 421-578), realizada por um mensageiro, EurÃbates. EurÃbates narra para a rainha argiva, Clitemnestra, todos os percalÃos por que passaram os gregos no retorno da guerra de Troia. O discurso do mensageiro constitui, por seu carÃter narrativo e sua temÃtica, uma espÃcie de grande interlÃdio Ãpico dentro de uma tragÃdia, o que nos leva a alguns questionamentos: estaria SÃneca colocando em xeque as leis do gÃnero trÃgico ao construir tÃo grande narrativa Ãpica em sua peÃa? A que nÃvel levaria SÃneca a jà tÃo conhecida aproximaÃÃo entre o trÃgico e o Ãpico na Antiguidade? O discurso de EurÃbates, uma vez que possui essa natureza Ãpica, faria alusÃo a poemas Ãpicos e seriam eles importantes para a formaÃÃo de sentido no AgamÃmnon? Como um suposto âdiscurso Ãpicoâ funcionaria nessa tragÃdia? Guiados por tais questionamentos, propomos neste trabalho um estudo do relato da personagem EurÃbates, do ponto de vista do diÃlogo entre o gÃnero trÃgico e o Ãpico e do diÃlogo com outras obras, estabelecido essencialmente atravÃs de alusÃes.

Orientador: Jorg Kobarg / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-11T07:55:23Z (GMT). No. of bitstreams: 1 Quaresma_AlexandreJoseChristino_D.pdf: 9068041 bytes, checksum: 9661a43a5c28440721d55ca90f6c94ac (MD5) Previous issue date: 2008 / Resumo: Os membros da família de proteínas chamada hnRNPs (heterogenous nuclear ribonuclein proteins) apresentam importantes papeis no controle da expressão gênica e no metabolismo dos mRNAs. Os membros hnRNPD (AUF1) e hnRNPQ (NSAP1) foram alvos deste estudo. AUF1 apresenta dois domínios de ligação à RNA do tipo RRM (RNA recognition motif) e participa ativamente no processo de desestabilização de uma classe de mRNAs que apresentam um motivo rico em AU na região 3' não traduzida. Demonstramos, através do sistema de duplo híbrido em levedura, que a isoforma p37 de AUF1 interagiu com as proteínas hnRNPQ, IMP-2, NSEP1 (YB-1) e UBC9. Além disso, a proteína hnRNPQ também foi pescada num outro ensaio de duplo híbrido em levedura, que utilizou como isca a proteína humana arginina metiltransferase (PRMT1). hnRNPQ apresenta, na sua região Cterminal, um ¿motivo rico em argininas e glicinas¿ (RGG box). Demonstramos que ela é alvo de metilação pela PRMT1 in vitro e in vivo. Funcionalmente, sua metilação é importante para sua localização nuclear. NSAP1 têm uma constituição modular com um domínio ácido (AcD) no seu Nterminal, seguido por três domínios de ligação à RNA do tipo RRM e o já mencionado RGG box no seu C-terminal. Funcionalmente hnRNPQ está envolvido em vários aspectos do etabolismo de RNA, incluindo a edição do mRNA da proteína humana ApoB. Para isso, ela interage não somente com o mRNA de ApoB, mas com a enzima efetora da edição Apobec1 e com a proteína que ativadora do Apobec1 (ACF1). Mostramos que o domínio ácido, de NSAP1 é capaz de interagir com Apobec1 e que sua fosforilação in vitro pela PKC inibe esta interação. Ainda identificamos que hnRNPQ interage com proteínas da família heat shock (incluindo HSP70 e BiP), e vimos que hnRNPQ é um alvo de fosforilação principalmente pela PKCd, in vitro. A localização sub-celular de hnRNPQ é modificada pela ativação in vivo das PKCs. Em conseqüência desta ativação ou da aplicação de estresse oxidativo, térmico ou indução de estresse do reticulo endoplasmático (tratamento com tapsigargina) hnRNPQ se desloca do núcleo para o citoplasma aonde se encontra em vesículas/corpúsculos definidas. Em resumo, nossos dados sugerem que as diversas funções da hnRNPQ relacionadas ao metabolismo de mRNAs, sofrem diferentes regulações, mediadas por modificações pós-traducionais (fosforilação e metilação), que interferem tanto na sua localização celular quanto na sua afinidade por determinados proteínas parceiras / Abstract: The members of the hnRNPs family (heterogenous nuclear ribonuclein proteins) play important roles in gene expression control and mRNAs metabolism. The proteins hnRNPD (AUF1) and hnRNPQ (NSAP1) were the main targets of this study. AUF1 has two RNA recognition motifs (RRM) and participates in the process of destabilization of a class of mRNAs that contain AU-rich sequences in their 3' untranslated regions (3'-UTR). We found, using the ¿yeast two-hybrid system¿ (Y2HS), that the isoform p37 of AUF1 (AUF1p37) interacts with the proteins: hnRNPQ, IMP-2, NSEP1 (YB-1) and UBC9. Moreover, the protein hnRNPQ was also identified as a prey protein in another Y2HS screen, which used as bait the human protein Arginine methyltransferase (PRMT1). HnRNPQ presents, in its C-terminal region, an "Arginine/Glicine-rich sequence" (RGG box). We are able to show that this RGG box is a target for methylation by PRMT1 in vitro and is methylated in vivo. Functionally, this methylation is important for its nuclear localization. hnRNPQ has a modular organization with an acid domain (AcD) in its N-terminal, followed by three RNA-binding domains (RRM) and the previously mentioned RGG box in its C-terminal. Functionally, hnRNPQ is involved in diverse aspects of RNA metabolism, including editing of the mRNA encoding the human protein ApoB. It has been shown previously to interact with the mRNA of ApoB, and also with the editing enzyme Apobec1 and the Apobec1 activation protein (ACF1). Here we show that the acid domain of hnRNPQ mediates the interaction with Apobec1 and that its in vitro phosphorylation (by PKC) inhibits this interaction. Furthermore, we found that hnRNPQ interacts with members the heat shock family of proteins (including HSP70 and BiP), and demonstrated that hnRNPQ can be in vitro phosphorylated by PKCd. Finally, we discovered that the sub-cellular localization of hnRNPQ undergoes modification after activation of PKC pathways. This also occurs after application of endoplasmic reticulum stress (using tarpsigargin), oxidative or heat stress. Under all of these conditions hnRNPQ translocated from the nucleus to the cytoplasm, where it is found at defined vesicles or granules. In summary, our data suggest that the diverse functions of hnRNPQ in the context of mRNA metabolism, may suffer specific regulations, by post-translational modifications, including phosphorylation and methylation, which modify both the proteins sub-cellular localizations as well as its affinity to interacting protein partners / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

A presente dissertação tem por objetivo a realização de uma análise geral dos mensageiros divinos designados pelo termo malakh relatados na Bíblia hebraica. No texto bíblico o termo malakh é empregado por fazer referência a mensageiros humanos e ainda aos enviados sobrenaturais subordinadas a Deus. Todo o primeiro capítulo é reservado à investigação da etimologia do vocábulo em questão. Com o intuito de discernir nitidamente a relação ou até mesmo a oposição entre mensageiros humanos e divinos, o segundo capítulo trata de diversos episódios bíblicos onde homens exercem a função de mensageiros, não somente como portadores de uma mensagens divina, mas também com funções políticas e diplomáticas. Ainda tratando dessa modalidade de mensageiro, é destacada a presença dos profetas e sacerdotes que a despeito de pertencerem à esfera humana, desempenham a função de mensageiros da divindade. A seguir, o terceiro e último capítulo ocupa-se dos mensageiros divinos propriamente ditos, geralmente traduzidos ou compreendidos como anjos. Neste ponto, interpretações relacionadas à identidade, nomenclaturas e funções são destrinchadas pontualmente. Por fim, a atividade do mensageiro bíblico será analisada ainda à luz dos mensageiros correspondentes da literatura do antigo Oriente Médio. / This thesis aims at conducting a general analysis of the divine messengers described by the term malakh reported in the Hebrew Bible. In the biblical text is the term used by malakh refer to human messengers sent to the supernatural and still subordinate to God. The entire first chapter is devoted to the investigation of the etymology of the word in question. In order to clearly discern the relationship or even the opposition between human and divine messengers, the second chapter deals with various biblical episodes where men play the role of messengers, not only as bearers of a divine message, but also with political functions and diplomatic. Still dealing with this type of messenger, it is emphasized the presence of the prophets and priests who despite belonging to the human sphere, play the role of messengers of divinity. Then the third and final chapter deals with the divine messengers themselves, usually translated or understood as \"angels\". At this point, interpretations related to identity, classifications and functions are fleshed out on time. Finally, the activity of biblical Bible will be examined further in light of relevant messengers literature of the ancient Middle East.

Orientador: Célia Marisa Rizzatti Barbosa / Tese (Doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-21T22:52:21Z (GMT). No. of bitstreams: 1 Meloto_CarolinaBeraldo_D.pdf: 1035478 bytes, checksum: c619d52bbc7d2858940483c9c5fc660e (MD5) Previous issue date: 2013 / Resumo: Catecolamina-O-metiltransferase (COMT) é uma enzima com amplas funções biológicas, inclusive a modulação da dor, exercidas através da metabolização de substratos como a dopamina, adrenalina, e noradrenalina. Já é sabido que a atividade da COMT é geneticamente polimórfica em humanos, e se correlaciona com a percepção individual da dor e eficiência na sua remissão entre pacientes com disfunção temporomandibular (DTM) tratados com propranolol. Por isso, nosso primeiro objetivo foi investigar novos polimorfismos de nucleotídeo único (SNP) que pudessem marcar para este benefício. De fato, encontramos um novo SNP, rs165774 (G>A), que se mostrou associado à DTM ou fenótipos intermediários em duas coortes diferentes. Este polimorfismo, por sua vez, se localiza em proximidade a um segundo SNP, rs165895 (T>C), ambos na região 3' não traduzida (3'UTR) de um mRNA alternativo da COMT ainda não caracterizado. Assim, também foi nossos objetivos (i) verificar a expressão deste transcrito em diferentes tecidos humanos e linhas de células, rastreando-o por RT-PCR (Reverse Transcriptase - Polymerase Chain Reaction); (ii) predizer a estrutura cristalina da enzima codificada por ele, através de modelagem pelo método dinâmico molecular discreto; (iii) expressá-lo em um sistema celular e comparar sua expressão relativa e atividade enzimática às da isoforma convencional, determinando-as por RT-PCR e HPLC (High Performance Liquid Chromatography), respectivamente; e (iv) verificar os efeitos dos SNPs rs165774 e rs165895 sobre este transcrito, criando-se diferentes mutantes por mutação sítio-dirigida e observando seus efeitos sobre a expressão relativa e atividade enzimática. Nossos resultados mostraram que (i) o transcrito alternativo da COMT é expresso em diferentes tecidos humanos e sistemas celulares; (ii) a estrutura cristalina de sua enzima exibe uma região C-terminal único que é distinta da isoforma convencional; e que este transcrito é (iii) menos relativamente expresso e sua enzima menos ativa que o transcrito e a enzima convencionais, respectivamente, e (iv) sofre regulação em nível transcricional pelos SNPs rs165774 e rs165895. Com este estudo, pudemos concluir que o SNP rs165774 é um forte marcador genético para DTM; que juntamente com o SNP rs165895, localizam-se na região 3'UTR de uma forma alternativa de mRNA da COMT que, pela primeira vez, provou ser capaz de codificar para uma isoforma alternativa da enzima que exibe atividade enzimática; e que esta isoforma alternativa de mRNA da COMT sofre efeitos regulatórios, em nível transcricional, causados por ambos os SNPs / Abstract: Catecolamine-O-methyltransferase (COMT) is an enzyme with broad biological functions, including pain modulation, exerted through metabolization of substrates such as dopamine, epinephrine, and norepinephrine. It is well known that COMT activity is genetically polymorphic in humans, and correlates to individual pain perception and efficiency in its remission among temporomandibular disorder (TMD) patients treated with propranolol. Thus, our first aim was to investigate new single nucleotide polymorphism (SNP) that might mark for this benefit. Indeed, we have found a new SNP, rs165774 (G>A), that was associated with TMD or intermediate phenotypes in two different cohorts. This polymorphism, in turn, is in close proximity to a second SNP, rs165895 (T>C), both in the 3' untranslated region (3'UTR) of an alternative COMT mRNA that has not yet been characterized. Therefore, we also aimed to (i) verify the expression of this transcript in different human tissues and cell systems, tracking it through RT-PCR (Reverse Transcriptase - Polymerase Chain Reaction); (ii) predict the crystalline structure of the enzyme encoded by it, modeling it with discrete molecular dynamics; (iii) express it in a cell system and compare its relative expression and enzymatic activity to the conventional isoform, using RT-PCR and HPLC (High Performance Liquid Chromatography), respectively; and (iv) verify the effects of SNPs rs165774 and rs165895 on the transcript, creating different mutants by site-directed mutagenesis and observing their effects on the relative expression and enzymatic activity. Our findings show that (i) the alternative COMT transcript is expressed in different human tissues and cell systems; (ii) the crystalline structure of its enzyme exhibits a unique C-terminus that is distinct from the conventional isoform; and that this transcript is (iii) less relatively expressed and its enzyme is less active than the conventional transcript and enzyme, respectively, and (iv) is regulated, at transcriptional level, by the SNPs rs165774 e rs165895. With this study, we conclude that SNP SNPs rs165774 is a strong genetic marker for TMD; that along with SNPs rs165895, they are located in the 3'UTR of an alternative COMT mRNA which proved, for the first time, to be able of encoding an alternative isoform of the enzyme that exhibits enzymatic activity; and that this alternative COMT mRNA is regulated, at transcriptional level, by both SNPs / Doutorado / Protese Dental / Doutora em Clínica Odontológica

RESUME<p><p><p>Le proto-oncogène c-myc code pour un facteur de transcription qui est impliqué dans de multiples processus cellulaires tels que la prolifération, la différenciation et l’apoptose. Une dérégulation de son expression suite à des altérations génétiques (mutation, translocation, amplification) est retrouvée dans plusieurs tumeurs telles que le lymphome de Burkitt, des plasmacytomes murins ainsi que des tumeurs non-lymphoïdes.<p>c-myc est un gène dont l’expression est régulée à différents niveaux. Chez l’homme, le gène c-myc est transcrit à partir de quatre promoteurs alternatifs appelés respectivement P0, P1, P2 et P3. P1 et P2 sont les deux promoteurs les plus utilisés. Ensemble, ils permettent de former 90% des transcrits c-myc dans des cellules normales. <p>Les promoteurs P0, P1 et P2 permettent la transcription de trois ARNms qui comportent deux codons d’initiation de la traduction (un CUG et un AUG). L’utilisation alternative de ces deux codons d’initiation est à l’origine de la synthèse de deux protéines (c-Myc 1 et c-Myc 2) ayant à la fois des fonctions identiques et distinctes. <p> La grande taille des parties 5’ non-traduites ainsi que la présence dans celles-ci de phases ouvertes de lecture sont des éléments défavorables à la traduction de l’ORF codant pour les protéines Myc par un mécanisme classique d’initiation de la traduction. Notre laboratoire avait précisément montré que les protéines c-Myc sont synthétisées par un processus d’initiation interne de la traduction. Les ARNms dont l’initiation de la traduction s’effectue par entrée interne des ribosomes présentent une structure spécifique appelée IRES (Internal Ribosome Entry Site). Cette structure permet la fixation du ribosome directement à proximité du codon d’initiation. Dans le cas des ARNms c-myc, on retrouve une IRES se situant en amont des codons CUG et AUG qui permet la synthèse des protéines c-Myc1 et 2 respectivement. Un tel mécanisme permet la synthèse des protéines c-Myc dans des conditions où toute traduction dépendante de la coiffe est inhibée (mitose, apoptose).<p><p>Au cours de mon travail, tout d’abord j’ai montré qu’une séquence de 40 nt dans les transcrits P2 permet à elle seule une initiation interne efficace de la traduction. Nous avons déterminé aussi que cette séquence, appelée B4, est active dans quatre types cellulaires différents avec une efficacité variable et qu’elle active la traduction indépendamment de l’ORF placée en aval. D’autre part, il a été déterminé que la séquence B4 recrute le complexe de préinitiation 43S, qui ensuite scanne le messager jusqu’aux codons initiateurs comme c’est le cas de l’IRES du rhinovirus. <p>Une analyse plus détaillée de la séquence B4 a permis d’identifier trois plus petites séquences de plus ou moins 14 nt (Ti1, Boucle, Ti2), qui indépendamment l’une de l’autre permettent une entrée interne des ribosomes. Il a été déterminé que la présence du motif A-N6-AC dans la séquence de Ti2 était importante pour l’activité IRES de celle-ci. Cependant, ce même motif également présent dans la séquence Ti1 n’est pas essentiel à l’activité IRES de Ti1. <p>Par la suite, nous avons démontré que l’IRES de c-myc nécessite pour son activité un évènement nucléaire. Nous avons donc entrepris la recherche de facteurs cellulaires impliqués dans l’activité de l’IRES de c-myc. Dans un premier temps, nous avons exclu le rôle de certaines protéines connues pour activer d’autres IRES dont le mécanisme de recrutement du complexe de préinitiation est similaire. Ainsi, nous avons montré, par des expériences de complémentation d’un RRL, que les protéines PTB et unr connues pour activer l’IRES du rhinovirus ne contribuent pas à l’activité de l’IRES de c-myc. De plus, la complémentation de RRL avec des extraits S10 ou nucléaires de cellules HeLa n’a pas permis d’identifier des protéines impliquées dans l’activité IRES de c-myc.<p>D’autre part, des méthodes alternatives d’interaction d’ARN et de protéine comme le triple hybride ou la chromatographie d’affinité d’ARN n’a pas permis dans un premier temps de détecter une interaction entre un facteur non canonique et l’IRES de c-myc. Dès lors, l’existence de facteurs cellulaires impliqués dans l’activité de l’IRES de c-myc reste à déterminer.<p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

Cette thèse cherche à discriminer au niveau génomique entre le développement d'embryons vers un mode de reproduction sexué et le développement vers un mode asexué chez le puceron du pois, Acyrthosiphon pisum. Cette discrimination passe par la création du réseau de régulation post-transcriptionnelle des microARN et des ARNm qui possèdent des cinétiques d'expression différentes entre ces deux embryogenèses ainsi que par l'analyse des modules d'interactions de ce réseau par l'utilisation de l'analyse de concepts formels. Pour ce faire, une stratégie en plusieurs étapes a été mise en place : la création d'un réseau d'interactions entre les microARN et les ARNm du puceron du pois ; l'extraction et la réduction du réseau aux microARN et ARNm qui possèdent des cinétiques différentes entre les deux embryogenèses à partir des données d'expression tirées du séquençage haut-débit ; l'analyse du réseau d'interactions réduit aux éléments d’intérêt par l'analyse de concepts formels. L'analyse du réseau a permis l'identification de différentes fonctions potentiellement importantes comme l'ovogenèse, la régulation transcriptionnelle ou encore le système neuroendocrinien. En plus de l'analyse du réseau, l'analyse de concepts formels a été utilisée pour définir une méthode de réparation de graphe biparti basée sur une topologie en "concepts" ainsi qu'une méthode de visualisation de graphes bipartis par ses concepts. / This thesis aims to discriminate between embryos development towards either sexual or asexual reproduction types in pea aphids, Acyrthosiphon pisum, at the genomic level. This discrimination involves the creation of a post-transcriptional regulation network between microRNAs and mRNAs whose kinetic expressions change depending on the embryogenesis. It also involves a study of this network's interaction modules using formal concept analysis. To do so, a three-step strategy was set up. First the creation of an interaction network between the pea aphid's microRNAs and mRNAs. The network is then reduced by keeping only microRNAs and mRNAs which possess differential kinetics between the two embryogeneses, these are obtained using high-throughput sequencing data. Finally the remaining network is analysed using formal concept analysis. Analysing the network allowed for the identification of several functions of potential interest such as oogenesis, transcriptional regulation or even neuroendocrine system. In addition to network analysis, formal concept analysis was used to create a new method to repair a bipartite graph based on its topology and a method to visualise a bipartite graph using its formal concepts.

No description available.

The aim of this work is to simplify access to information for citizens of the city of Brno and at the same time to innovate the way of communication between the citizen and his city. The problem is solved by creating a conversational agent - chatbot Kroko. Using artificial intelligence and a Czech language analyzer, the agent is able to understand and respond to a certain set of textual, natural language queries. The agent is available on the Messenger platform and has a knowledge base that includes data provided by the city council. After conducting an extensive user testing on a total of 76 citizens of the city, it turned out that up to 97\% of respondents like the idea of a city-oriented chatbot and can imagine using it regularly. The main finding of this work is that the general public can easily adopt and effectively use a chatbot. The results of this work motivate further development of practical applications of conversational agents.

The eukaryotic gene expression requires extensive regulations to enable the homeostasis of the cell and to allow dynamic responses due to external stimuli. Although many regulatory mechanisms involve the transcription as the first step of the gene expression, intensive regulation occurs also in the post-transcriptional mRNA metabolism. Thereby, the particular composition of the mRNPs plays a central role as the components associated with the mRNA form a specific “mRNP code” which determines the fate of the mRNA. Many proteins which are involved in this regulation and the mRNA metabolism are affected in diseases and especially neurological disorders often result from an aberrant mRNP code which leads to changes in the regulation and expression of mRNPs. The focus of this work was on a trimeric protein complex which is termed TTF complex based on its subunits TDRD3, TOP3β and FMRP. Biochemical investigations revealed that the three components of the TTF complex are nucleo-cytosolic shuttle proteins which localize in the cytoplasm at the steady-state, associate with mRNPs and are presumably connected to the translation. Upon cellular stress conditions, the TTF components concentrate in stress granules. Thus, the TTF complex is part of the mRNP code, however its target RNAs and function are still completely unknown. Since the loss of functional FMRP results in the fragile X syndrome and TOP3β is associated with schizophrenia and intellectual disability, the TTF complex connects these phenotypically related neuro-psychiatric disorders with each other on a molecular level. Therefore, the aim of this work was to biochemically characterize the TTF complex and to define its function in the mRNA metabolism. In this work, evidence was provided that TDRD3 acts as the central unit of the TTF complex and directly binds to FMRP as well as to TOP3β. Thereby, the interaction of TDRD3 and TOP3β is very stable, whereas FMRP is a dynamic component. Interestingly, the TTF complex is not bound directly to mRNA, but is recruited via the exon junction complex (EJC) to mRNPs. This interaction is mediated by a specific binding motif of TDRD3, the EBM. Upon biochemical and biological investigations, it was possible to identify the interactome of the TTF complex and to define the role in the mRNA metabolism. The data revealed that the TTF complex is mainly associated with “early” mRNPs and is probably involved in the pioneer round of translation. Furthermore, TOP3β was found to bind directly to the ribosome and thus, establishes a connection between the EJC and the translation machinery. A reduction of the TTF components resulted in selective changes in the proteome in cultured cells, whereby individual protein subsets seem to be regulated rather than the global protein expression. Moreover, the enzymatic analysis of TOP3β indicated that TOP3β is a type IA topoisomerase which can catalytically attack not only DNA but also RNA. This aspect is particularly interesting with regard to the connection between early mRNPs and the translation which has been revealed in this work. The data obtained in this work suggest that the TTF complex plays a role in regulating the metabolism of an early mRNP subset possibly in the course of the pioneer round of translation. Until now, the link between an RNA topoisomerase and the mRNA metabolism is thereby unique and thus provides a completely new perspective on the steps in the post-transcriptional gene expression and its regulation. / Die eukaryotische Genexpression bedarf einer umfassenden Regulation um die Homöostase der Zelle zu gewährleisten und um dynamische Reaktionen auf externe Einflüsse zu ermöglichen. Obwohl viele der regulatorischen Mechanismen die Transkription als ersten Schritt der Genexpression betreffen, findet auch eine intensive Regulierung auf der Ebene des post-transkriptionellen mRNA-Metabolismus statt. Dabei spielt die jeweilige Zusammensetzung der mRNPs eine zentrale Rolle, da je nachdem, mit welchen Faktoren eine mRNA assoziiert ist, ein sog. „mRNP-Code“ entsteht, der das Schicksal der mRNA bestimmt. Viele der an der Regulierung und dem mRNA-Metabolismus beteiligten Proteine sind in Krankheiten betroffen und gerade neurologische Erkrankungen resultieren häufig von einem fehlerhaften mRNP-Code, der zu Veränderungen in der Regulation und Expression von mRNPs führt. Im Zentrum dieser Arbeit stand ein trimerer Proteinkomplex, der aufgrund seiner Untereinheiten TDRD3, TOP3β und FMRP als TTF-Komplex bezeichnet wird. Biochemische Daten haben gezeigt, dass die drei Komponenten des TTF-Komplexes nucleo-cytoplasmatische „Shuttle“-Proteine sind, die sich im „steady-state“ hauptsächlich im Cytoplasma befinden, mit mRNPs assoziieren und vermutlich mit der Translation in Verbindung stehen. Unter zellulären Stressbedingungen konzentrieren sich die TTF-Komponenten in Stress Granula. Der TTF-Komplex ist damit Teil des mRNP-Codes, dessen zelluläre Ziel-RNAs und Funktion bislang aber völlig unbekannt sind. Da der Verlust von funktionellem FMRP zu der Ausprägung des fragilen X Syndroms (FXS) führt und TOP3β mit Schizophrenie und geistiger Retardation in Verbindung steht, verbindet der TTF-Komplex phänotypisch verwandte neuro-psychiatrische Krankheiten auf molekularer Ebene miteinander. Das Ziel dieser Arbeit war es daher, den TTF-Komplex biochemisch zu charakterisieren und seine Funktion im mRNA-Metabolismus zu definieren. Im Zuge dieser Arbeit gelang der Nachweis, dass TDRD3 als zentrale Einheit des TTF-Komplexes agiert und sowohl FMRP als auch TOP3β direkt bindet. Die Interaktion von TDRD3 und TOP3β ist hierbei sehr stabil, FMRP ist hingegen eine dynamische Komponente. Interessanterweise wird der TTF-Komplex nicht direkt an mRNA gebunden, sondern über den Exon-Junction-Komplex (EJC) an mRNPs rekrutiert. Diese Interaktion wird durch ein spezifisches Bindungsmodul in TDRD3, dem sog. EBM vermittelt. In einer Reihe von biochemischen und systembiologischen Studien konnte das Interaktom des TTF-Komplexes bestimmt und seine Rolle im mRNA-Metabolismus definiert werden. Die Daten offenbarten, dass der TTF-Komplex primär mit „frühen“ mRNPs assoziiert ist und sehr wahrscheinlich an der „pioneer round of translation“ beteiligt ist. Weiterhin zeigte sich, dass TOP3β das Ribosom direkt bindet und somit eine Verbindung des EJC und der Translationsmaschinerie etabliert. Die Reduktion von Komponenten des TTF-Komplexes in kultivierten Zellen führte zu selektiven Änderungen im Proteom, wobei einzelne Proteinteilgruppen, jedoch nicht die globale Expression durch den TTF-Komplex reguliert zu sein scheinen. Die enzymatische Analyse von TOP3β hat darüber hinaus gezeigt, dass es sich um eine Topoisomerase vom Typ IA handelt, die nicht nur DNA sondern auch RNA angreifen kann. Dieser Aspekt ist besonders interessant im Zusammenhang der in dieser Arbeit aufgedeckten Verbindung von frühen mRNPs mit der Translation. Die im Rahmen dieser Arbeit erhaltenen Daten legen nahe, dass der TTF-Komplex eine Rolle bei der Regulation des Metabolismus „früher“ mRNP-Teilgruppen möglicherweise im Zuge der „Pionierrunde“ der Translation spielt. Dabei ist die Verbindung einer RNA-Topoisomerase mit dem mRNA-Metabolismus bisher einzigartig und eröffnet so eine ganz neue Sichtweise auf die post-transkriptionellen Schritte der Genexpression und ihre Regulation.