No description available.

According to UNAIDS there were 34 million people living with human immunodeficiency virus (HIV) infection at the end of 2010. HIV is the causative agent of acquired immunodeficiency syndrome (AIDS) and the number of people dying of AIDS-related causes at the end of 2010 was 1.8 million. Due to the high mutability of the virus, there is a constant need for new approaches to attack the virus. Splicing of HIV-1 pre-mRNA is a highly regulated process. In order to produce all mRNAs needed to be infectious HIV-1 utilizes alternative splicing ­- from one single transcript more than 35 differently spliced mRNAs can be produced. A new approach to fight HIV-1 could be to interfere with the essential splicing. In this thesis, I describe the regulation of HIV-1 pre-mRNA splicing. SR proteins are involved in the regulation of splicing, both in an early and a late stage. We find that the intracellular concentration of SR proteins is of great importance for HIV-1 to be able to produce the correct amounts of mRNAs. Variations in concentrations of SR proteins lead to big changes in the HIV-1 pre-mRNA splicing pattern. The functions of HIV-1 protein Vpr are diverse and it is essential in vivo. HIV-1 vpr mRNA 13a7 is partially spliced, containing an intron, and the regulation of it is not fully understood. We find that SRp55 and SRp75 induce the production of HIV-1 vpr mRNA 13a7 by inhibiting splice donor 3. We also conclude that this inhibition at least for SRp55 is due to an interaction with the viral RNA element GAR. In the presence of SRp55 we also see an increase in cytoplasmic amounts of intron containing vpr mRNA due to increased nuclear export. Our results show that SRp55 can have several functions in the regulation of HIV-1 splicing: by inhibiting splice donors and by facilitating the export of incompletely spliced mRNAs to the cytoplasm. In conclusion, this thesis describes SRp55 as a regulator of HIV-1 vpr mRNA, both in splicing as well as in nuclear export. These discoveries provide an insight into the regulation of HIV-1 mRNA processing.

Serine/Arginine-rich proteins (SR-proteins) have been well characterized in metazoans and in the flowering plant Arabidopsis thaliana. But so far no attempts on characterizing SR-proteins in the moss Physcomitrella patens have been done. SR-proteins are a conserved family of splicing regulators essential for constitutive- and alternative splicing. SR-proteins are mediators of alternative splicing (AS) and may be alternatively spliced themselves as a form of gene regulation. Three novel SR-proteins of the SR-subfamily were identified in P. patens. The three genes show conserved intron-exon structure and protein domain distribution, not surprising since the gene family has evidently evolved through gene duplications. The SR-proteins PpSR40 and PpSR36 show differential tissue-specific expression, whereas PpSR39 does not. Tissue-specific expression of SR-proteins has also been seen in A. thaliana. SR-proteins determine splice-site usage in a concentration dependent manner. SR-protein overexpression experiments in A. thaliana and Oryza sativa have shown alteration of splicing patterns of endogenous SR-proteins. Overexpression of PpSR40 did not alter the splicing patterns of PpSR40, PpSR36 and PpSR39. This suggests that they might not be a substrate for PpSR40. These first results of SR-protein characterization in P. patens may provide insights on the SR-protein regulation mechanisms of the common land plant ancestor.

No description available.

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.

La protéine EB2 du virus d'Epstein-Barr (EBV) est une protéine du cycle réplicatif du virus indispensable à la production de particules virales. Elle permet l’accumulation dans le cytoplasme de certains ARNm viraux issus de gènes dépourvus d’intron. Pour mettre en évidence les mécanismes qui permettent à EB2 d’exporter ses ARNm cibles dans le cytoplasme, nous avons identifié différents partenaires cellulaires d’EB2 et nous avons étudié certaines de ces interactions d’un point de vue fonctionnel. Nous avons pu montrer qu’EB2 recrute directement le facteur général d'export des ARNm, TAP/NXF1, ce qui lui permet d’être exportée du noyau vers le cytoplasme. Puis nous avons montré qu’EB2 interagit avec SRp20, une protéine impliquée notamment dans la régulation de l'épissage et l'export des ARNm cellulaires. Cette interaction entre EB2 et SRp20 est indispensable pour l’accumulation dans le cytoplasme de certains ARNm cibles d’EB2, notamment parce que SRp20 semble permettre le recrutement d'EB2 sur ces ARNm. Enfin, nous avons montré qu’EB2 forme un dimère et nous avons caractérisé le domaine de la protéine responsable de cette interaction. La dimérisation d'EB2 semble essentielle pour que la protéine interagisse avec certains de ses partenaires comme SRp20 ou encore REF. / The Epstein-Barr virus (EBV) protein EB2 is an early protein essential for the production of infectous virions. EB2 allows the cytoplasmic accumulation of a subset of viral mRNAs derived from intronless genes. To highlight the mecanisms by which EB2 exports his targets mRNA, we identified cellular partners and studied the functional role of some of these interactions. We showed that EB2 recruits directly the cellular mRNA export factor TAP/NXF1 and this interaction allows EB2’s shuttling between the nucleus and the cytoplasm. The we showed that EB2 interacts with SRp20, a cellular protein implicated in splicing regulation and mRNA export. This interaction is essential for the efficient cytoplasmic accumulation of some EB2 target mRNAs, partly because SRp20 appears to be able to recruit EB2 on these mRNAs. Then we showed that EB2 dimerises and we characterized the domain necessary for this interaction. This dimerisation appears to be essential for EB2’s interaction with several partners, including SRp20 and REF.

Die intakte Signalübertragung im animalischen Nervensystem erfordert eine an richtiger Stelle ausgebildete funktionsfähige Synapse zwischen zwei Nervenzellen bzw. zwischen Nerv und Muskel. In der vorliegenden Arbeit wurde eine Mutante von Drosophila melanogaster untersucht, bei der es zu Veränderungen der Verteilung eines wichtigen Organisationsproteins der synaptischen aktiven Zone kommt. Ein wichtiges Ergebnis der Untersuchungen ist die Beobachtung, dass es in der Mutante zu einer ektopen Ausbildung von Elementen aktiver Zonen in Axonen kommt. In den Arbeitsgruppen von E. Buchner und S. Sigrist ist bereits das Protein Bruchpilot (BRP) charakterisiert worden, das Bestandteil der präsynaptischen Ribbons, bei Drosophila als T-bars bezeichnet, ist. Bei der Suche nach Interaktionspartnern von BRP, ist eine Serin-Arginin-Protein spezifische Kinase SRPK79D entdeckt worden, die offenbar an der Regulation des Aufbaus der Tbars beteiligt ist (Nieratschker et al., 2009). Es gibt vier verschiedene Isoformen der Kinase. Werden nur zwei Isoformen der Kinase (SRPK79D-RB und -RE) exprimiert bzw. das Gen der Kinase komplett ausgeschaltet, findet man Ansammlungen von BRP als immunreaktive Aggregate in der Immunfluoreszenz- Färbung von larvalen Motoneuron-Axonen (Nieratschker, 2008). Es ist unser übergeordnetes Ziel, die Funktion und den molekularen Signalweg der Kinase SRPK79D zu entschlüsseln. Ein Ziel der vorliegenden Arbeit war es, PB-Protein in Reinform für eine Affinitätsreinigung eines PB-Antikörpers zu gewinnen, um in nachfolgenden Untersuchungen die Lokalisation dieser Kinase-Isoform zu untersuchen. Die Proteinreinigung war erfolgreich, aber es gelang nicht, eine für eine Affinitätsreinigung ausreichende Menge des Proteins zu isolieren. Ein weiterer Versuch, Lokalisationsuntersuchungen zur Expression der Kinase in Drosophila- Embryonen durchzuführen, war ebenfalls nicht erfolgreich. Obwohl die Herstellung einer für die SRPK79D mRNA spezifischen RNA Sonde für die in-Situ-Hybridisierung gelang, war die Sensitivität dieser Sonde nicht hoch genug, um die Lokalisation vornehmen zu können. Eindeutige und aufschlussreiche Ergebnisse dagegen ergab die Untersuchung der Ultrastruktur der BRP-Ansammlungen in den larvalen Motornerven. Als deren Korrelat fanden sich elektronenmikroskopisch charakteristische Ansammlungen elektronendichter intraaxonaler Strukturen, deren Form Ähnlichkeiten zu T-bars aufwies und die von Vesikeln umgeben waren. Die elektronendichten Strukturen zeigten zahlreiche Formvariationen, die wie Ansammlungen von T-bars nebeneinander bzw. „miteinander verklebte“ T-bars oder wie zerstörte T-bars aussahen. In einer nachfolgenden Studie wurde durch eine immun-elektronenmikroskopische Untersuchung gezeigt, dass diese Strukturen in der Tat BRP enthalten (Nieratschker et al., 2009). Ergebnis der Untersuchungen der vorliegenden Arbeit war der Nachweis, dass prinzipiell ähnliche Aggregate auch im Wildtyp gelegentlich gefunden werden, dass sie aber in Mutanten signifikant häufiger vorkommen und auch einen signifikant höheren Durchmesser aufweisen. Doppelimmunreaktionen mit Antikörpern, die den C- bzw. N-terminalen Bereich von BRP erkennen, belegten darüber hinaus, dass in den Aggregaten das vollständige BRP-Protein vorliegt. Angeregt durch die Ultrastrukturbefunde von mit den elektronendichten Strukturen in den Aggregaten assoziierten Vesikeln wurde in weiteren Doppelimmunreaktionen untersucht, ob ein typisches Protein synaptischer Vesikel neuromuskulärer Synapsen in Drosophila, der vesikuläre Glutamattransporter (DVGlut), in den BRP-Ansammlungen nachweisbar ist. Während Kolokalisation von BRP und DVGlut in aktiven Zonen präsynaptischer Boutons nachgewiesen werden konnte, war der Vesikelmarker in BRP-Aggregaten nicht kolokalisiert. Die Ergebnisse belegen, dass die Kinase SRPK79D für die Vermeidung einer ektopen Bildung von BRP-enthaltenden, elektronenmikroskopisch atypischen aktiven Zonen ähnelnden Strukturen in larvalen Motoneuronaxonen notwendig ist. Die in diesen Aggregaten regelmäßig zu beobachtenden Vesikel ähneln morphologisch synaptischen Vesikeln, besitzen aber keine dafür typischen Vesikelmarker. / Intact signal transmission in an animal’s nervous system requires a properly localized and functional synapse between two neurons or between neuron and muscle. This dissertation is part of the investigation of a Drosophila melanogaster mutant which displays alterations in the distribution of a synaptic active zone protein. An important result of the present study is the documentation of an ectopic formation of active zone structural elements in this mutant. Analyses carried out in the laboratories of E. Buchner and S. Sigrist contributed to the characterization of the protein Bruchpilot (BRP), a constituent of the T-bar, the characteristic presynaptic ribbon in Drosophila. Searching for interaction partners of BRP, a serine-arginine-protein specific kinase was identified that apparently regulates T-bar assembly (Nieratschker et al., 2009). There are four kinase isoforms. Knocking out two of these isoforms (SRPK79D-RB and -RE) results in accumulations of BRP-immunoreactive aggregates in the larval ventral nerves (Nieratschker, 2008). Further studies were designed to identify the function and molecular signalling pathways of the kinase SRPK79D. One objective of the present experiments was to produce purified PB-protein in order to enable affinity-purification of an antibody against this isoform of the kinase for subsequent specific immunohistochemical localization analyses. Although production of the antigen was successful, the amount of protein produced was too low to allow efficient affinity purification. An attempt to show the expression pattern of the kinase in Drosophila embryos with in-situ hybridization resulted in production of a SRPK79D specific RNAprobe, however, the probe sensitivity was not high enough to yield conclusive results for mRNA localization. Ultrastructural analyses of the BRP-ir aggregates in the larval ventral nerves, on the other hand, yielded definite and conclusive results. These aggregates corresponded to extensive intraaxonal electron-dense, ribbon-like structures surrounded by vesicles. These electron-dense structures were differently shaped and resembled accumulations of regularly shaped, clotted or dysmorphic T-bars, which in subsequent immuno-electronmicroscopic analyses carried out by another investigator were proven to contain BRP (Nieratschker et al., 2009). An important result of the present study was the observation that similar intraaxonal aggregates were occasionally also present in wild type nerves, however, the aggregates found in the mutants were significantly more frequent and of significantly larger size than those observed in wild-type larvae. Moreover, double-immunostaining using BRP-antibodies recognizing specifically the C- and the N-terminal part of the protein, respectively, provided evidence that the complete BRP protein is localized in the aggregates. Since electron microscopy had showed that numerous vesicles were associated with the electron dense aggregates, we tested whether the vesicular glutamate transporter (DVGlut), a marker protein for synaptic vesicles of motoneurons in Drosophila, could be localized in BRP-ir aggregates. While colocalization of BRP and DVGlut was observed at the presynaptic active zones, no colocalization of the synaptic vesicle marker was observed in the BRP-ir aggregates in the larval nerves. In conclusion, the results provide evidence that the kinase SRPK79D is required for the prevention of ectopic formation of BRP-containing ribbon-like structures in larval ventral nerves. These structures include vesicles resembling synaptic vesicles, which however do not display immunoreactivity for a typical synaptic vesicle marker protein.

<p>Pre-mRNA splicing and alternative pre-mRNA splicing are key regulatory steps controlling geneexpression in higher eukaryotes. The work in this thesis was focused on a characterization of thesignificance of exonic and intronic splicing enhancer elements for pre-mRNA splicing.</p><p>Previous studies have shown that removal of introns with weak and regulated splice sitesrequire a splicing enhancer for activity. Here we extended these studies by demonstrating thattwo "strong" constitutively active introns, the adenovirus 52,55K and the Drosophila Ftzintrons, are absolutely dependent on a downstream splicing enhancer for activity <i>in vitro</i>.</p><p>Two types splicing enhancers were shown to perform redundant functions as activators ofSplicing. Thus, SR protein binding to an exonic splicing enhancer element or U1 snRNP bindingto a downstream 5'splice site independently stimulated upstream intron removal. The datafurther showed that a 5'splice site was more effective and more versatile in activating splicing.Collectively the data suggest that a U1 enhancer is the prototypical enhancer element activatingsplicing of constitutively active introns.</p><p>Adenovirus IIIa pre-mRNA splicing is enhanced more than 200-fold in infected extracts. Themajor enhancer element responsible for this activation was shown to consist of the IIIa branchsite/polypyrimidne tract region. It functions as a Janus element and blocks splicing in extractsfrom uninfected cells while functioning as a splicing enhancer in the context of infected extracts.</p><p>Phosphorylated SR proteins are essential for pre-mRNA splicing. Large amount recombinantSR proteins are needed in splicing studies. A novel expression system was developed to expressphosphorylated, soluble and functionally active ASF/SF2 in <i>E. Coli</i>.</p>

Pre-mRNA splicing and alternative pre-mRNA splicing are key regulatory steps controlling geneexpression in higher eukaryotes. The work in this thesis was focused on a characterization of thesignificance of exonic and intronic splicing enhancer elements for pre-mRNA splicing. Previous studies have shown that removal of introns with weak and regulated splice sitesrequire a splicing enhancer for activity. Here we extended these studies by demonstrating thattwo "strong" constitutively active introns, the adenovirus 52,55K and the Drosophila Ftzintrons, are absolutely dependent on a downstream splicing enhancer for activity in vitro. Two types splicing enhancers were shown to perform redundant functions as activators ofSplicing. Thus, SR protein binding to an exonic splicing enhancer element or U1 snRNP bindingto a downstream 5'splice site independently stimulated upstream intron removal. The datafurther showed that a 5'splice site was more effective and more versatile in activating splicing.Collectively the data suggest that a U1 enhancer is the prototypical enhancer element activatingsplicing of constitutively active introns. Adenovirus IIIa pre-mRNA splicing is enhanced more than 200-fold in infected extracts. Themajor enhancer element responsible for this activation was shown to consist of the IIIa branchsite/polypyrimidne tract region. It functions as a Janus element and blocks splicing in extractsfrom uninfected cells while functioning as a splicing enhancer in the context of infected extracts. Phosphorylated SR proteins are essential for pre-mRNA splicing. Large amount recombinantSR proteins are needed in splicing studies. A novel expression system was developed to expressphosphorylated, soluble and functionally active ASF/SF2 in E. Coli.

Regulation of adenoviral gene expression is a complex process directed by viral proteins controlling a multitude of different activities at distinct phases of the virus life cycle. This thesis discusses adenoviral regulation of transcription and splicing by two proteins expressed at the late phase: L4-22K and L4-33K. These are closely related with a common N-terminus but unique C-terminal domains. The L4-33K protein is an alternative RNA splicing factor inducing L1-IIIa mRNA splicing, while L4-22K is stimulating transcription from the major late promoter (MLP). The L4-33K protein contains a tiny RS-repeat in its unique C-terminal end that is essential for the splicing enhancer function of the protein. Here we demonstrate that the tiny RS-repeat is required for localization of the protein to the nucleus and viral replication centers. Further, we describe an auto-regulatory loop where L4-33K enhances splicing of its own intron. The preliminary characterization of the responsive RNA-element suggests that it differs from the previously defined L4-33K-responsive element activating L1-IIIa mRNA splicing. L4-22K lacks the ability to enhance L1-IIIa splicing in vivo, and here we show that the protein is defective in L1-IIIa or other late pre-mRNA splicing reactions in vitro. Interestingly, we found a novel function for the L4-22K and L4-33K proteins as regulators of E1A alternative splicing. Both proteins selectively upregulated E1A-10S mRNA accumulation in transfection experiments, by a mechanism independent of the tiny RS-repeat. Although L4-22K is reported to be an MLP transcriptional enhancer protein, here we show that L4-22K also functions as a repressor of MLP transcription. This novel activity depends on the integrity of the major late first leader 5’ splice site. The model suggests that at low concentrations L4-22K activates MLP transcription while at high concentrations L4-22K represses transcription. So far, characterizations of the L4-22K and L4-33K proteins have been limited to human adenoviruses 2 or 5 (HAdV-2/5). We expanded our experiments to include HAdV-3, HAdV-4, HAdV-9, HAdV-11 and HAdV-41. The results demonstrated that the transcription- or splicing-enhancing properties of L4-22K and L4-33K, respectively, are evolutionarily conserved and non-overlapping. Thus, the sequence-based conservation is mirrored by the functions, as expected for functionally important proteins.