Refine
Has Fulltext
- yes (2)
Is part of the Bibliography
- yes (2)
Year of publication
- 2009 (2) (remove)
Document Type
- Doctoral Thesis (2)
Keywords
- 7SK RNA (1)
- Biogenese (1)
- Biopolymere (1)
- Catalyst (1)
- Chaperone (1)
- Elongation (1)
- Enzym (1)
- Enzyme (1)
- Katalysator (1)
- LARP7 (1)
Institute
- Lehrstuhl für Biochemie (2) (remove)
Macromolecular complexes, also termed molecular machines, facilitate a large spectrum of biological reactions and tasks crucial to the survival of cells. These complexes are composed of either protein only, or proteins bound to nucleic acids (DNA or RNA). Prominent examples for each class are the proteosome, the nucleosome and the ribosome. How such units are assembled within the context of a living cell is a central question in molecular biology. Earlier studies had indicated that even very large complexes such as ribosomes could be reconstituted from purified constituents in vitro. The structural information required for the formation of macromolecular complexes, hence, lies within the subunits itself and, thus, allow for self- assembly. However, increasing evidence suggests that in vivo many macromolecular complexes do not form spontaneously but require assisting factors (“assembly chaperones”) for their maturation. In this thesis the assembly of RNA-protein (RNP) complexes has been studied by a combination of biochemical and structural approaches. A resourceful model system to study this process is the biogenesis pathway of the uridine-rich small nuclear ribonucleoproteins (U snRNPs) of the spliceosome. This molecular machine catalyzes pre-mRNA splicing, i.e. the removal of non-coding introns and the joining of coding exons to functional mRNA. The composition and architecture of U snRNPs is well defined, also, the nucleo-cytoplasmic transport events enabling the formation of these particles in vivo have been analyzed in some detail. Furthermore, recent studies suggest that the formation of U snRNPs in vivo is mediated by an elaborate assembly machinery consisting of protein arginine methyltransferase (PRMT5)- and survival motor neuron (SMN)-complexes. The elucidation of the reaction mechanism of cellular U snRNP assembly would serve as a paradigm for our understanding of how RNA-protein complexes are formed in the cellular environment. The following key findings were obtained as part of this study: 1) Efforts were made to establish a full inventory of the subunits of the SMN-complex. This was achieved by the biochemical definition and characterization of an atypical component of this complex, the unrip protein. This protein is associated with the SMN-complex exclusively in the cytoplasm and influences its subcellular localization. 2) With a full inventory of the components in hand, the architecture of the SMN-complex was defined on the basis of an interaction map of all subunits. This study elucidated that the proteins SMN, Gemin7 and Gemin8 form a backbone, onto which the remaining subunits adhere in a modular manner. 3) The two studies mentioned above formed the basis to elucidate the reaction mechanism of cellular U snRNP assembly. Initially, an early phase in the SMN-assisted formation of U snRNPs was analyzed. Two subunits of the U7 snRNP (LSm10 and 11) were found to interact with the PRMT5-complex, without being methylated. This report suggests that the stimulatory role of the PRMT5-complex is independent of its methylation activity. 4) Key reaction intermediates in U snRNP assembly were found and characterized by a combination of biochemistry and structural studies. Initially, a precursor to U snRNPs with a sedimentation coefficient of 6S is formed by the pICln subunit of the PRMT5-complex and Sm proteins. This intermediate was shown to constitute a kinetic trap in the U snRNP assembly reaction. Progression towards the assembled U snRNP depends on the activity of the SMN-complex, which acts as a catalyst. The formation of U snRNPs is shown to be structurally similar to the way clamps are deposited onto DNA to tether poorly processive polymerases. 5) The human SMN-complex is composed of several subunits. However, it is unknown whether all subunits of this entity are essential for U snRNP assembly. A combination of bioinformatics and biochemistry was applied to tackle this question. By mining databases containing whole-genome assemblies, the SMN-Gemin2 heterodimer is recognized as the most ancestral form of the SMN-complex. Biochemical purification of the Drosophila melanogaster SMN-complex reveals that this complex is composed of the same two subunits. Furthermore, evidence is provided that the SMN-Gemin2 heterodimer is necessary and sufficient to promote faithful U snRNP assembly. Future studies will adress further details in the reaction mechanism of cellular U snRNP assembly. The results obtained in this thesis suggest that the SMN and Gemin2 subunits are sufficient to promote U snRNP formation. What then is the function of the remaining subunits of the SMN-complex? The reconstitution schemes established in this thesis will be instrumental to address this question. Furthermore, additional mechanistic insights into the U snRNP assembly reaction will require the elucidation of structures of the assembly machinery trapped at various states. The prerequisite for these structural studies, the capability to generate homogenous complexes in sufficient amounts, has been accomplished in this thesis.
LARP7 – ein La ähnliches Protein reguliert die Elongation der PolII Transkription durch das 7SK RNP
(2009)
Genexpression in Eukaryoten beschreibt einen mehrstufigen Prozess, welcher auf Ebene der Transkription durch den positiven Transkriptionselongationsfaktor P-TEFb entscheidend reguliert wird. PTEFb bildet einen heterodimeren Komplex aus der Cyclin abhängigen Kinase 9 und deren Kofaktor Cyclin T1/2. Dieser Komplex aktiviert die Elongation der Transkription durch Phosphorylierung der negativen Elongationsfaktoren DSIF und NELF. Darüber hinaus phosphoryliert PTEFb Serin2 Reste in der C-terminalen Domäne von RNA PolII und stimuliert so die kotranskriptionelle Prozessierung der synthetisierten prä-mRNA. In Anpassung an unterschiedliche Wachstumsbedingungen wird die Aktivität dieses Faktors durch reversible Interaktion mit 7SK RNA und HEXIM Proteinen innerhalb eines katalytisch inaktiven Ribonukleoproteinpartikels (7SK RNP) streng kontrolliert. Dieses sensible Gleichgewicht zwischen P-TEFb auf der einen und dem 7SK RNP auf der anderen Seite bildet die Grundlage der Regulation der Transkriptionselongation. Trotz der hohen Abundanz von 7SK RNA in der Zelle, assoziiert in vivo jedoch nur ein relativ kleiner Teil hiervon mit P-TEFb, sodass die effektiv zur Verfügung stehende RNA-Menge für die Bildung des 7SK RNP vermutlich limitierend wirkt. Ziel der vorliegenden Arbeit war es daher neue 7SK RNA interagierende Faktoren zu identifizieren, welche die Interaktion von PTEFb mit dem 7SK RNP steuern und so die PolII abhängige Transkription regulieren. Anhand verschiedener chromatographischer Reinigungen konnte zunächst ein bislang uncharakterisiertes La ähnliches Protein (LARP7) mit einer spezifischen Affinität für Pyrimidinreiche RNAs isoliert werden. LARP7 bindet, wie durch immunbiochemische Analysen und RNA- Bindungsstudien gezeigt werden konnte, quantitativ an das hoch konservierte uridylreiche 3´- Ende von 7SK RNA. Diese Assoziation erfordert dessen La- und RRMDomänen und erhöht wesentlich die Stabilität der RNA. Darüber hinaus kofraktioniert LARP7 mit weiteren Faktoren des 7SK RNP, bindet direkt an HEXIM1 und P-TEFb und stellt somit ebenfalls eine integrale Komponente des 7SK RNP dar. Die gewonnenen Daten weisen außerdem erstmals darauf hin, dass P-TEFb durch einen vorgeformten trimeren Komplexes, bestehend aus HEXIM1, 7SK RNA und LARP7 inhibiert wird. Reportergenanalysen in TZMbl-Zellen, welche Luziferase unter der Kontrolle des streng P-TEFb abhängigen HIV-1-LTRPromotors exprimieren zeigten, dass diese Inhibition im Wesentlichen durch LARP7 vermittelt wird. So ließ sich nach Reduktion der LARP7 Expression mittels RNAi eine signifikante Steigerung der Transkription vom HIV-1-LTR-Promotor beobachten. Eine ähnliche Stimulation der Transkription von PolII konnte in LARP7 defizienten HeLa-Zellen durch quantitative Real-Time-PCR auch für eine Reihe zellulärer Gene nachgewiesen werden. Die Beobachtung, dass LARP7 die generelle PolII Transkription reprimiert, korreliert zudem mit einer bereits beschriebenen Tumorsupressorfunktion des LARP7 homologen mxc Proteins aus D. melanogaster. Somit beeinflusst LARP7 das zelluläre Gleichgewicht zwischen freiem und 7SK RNP-gebundenem P-TEFb und fungiert somit als negativer Regulator der PolII Transkription in vivo.