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NMR investigation of protein-RNA interactions involved in ribosomal gene silencing.

NMR Untersuchung von Protein-RNA Wechselwirkungen in der Inaktivierung von ribosomalen Genen.

München, Technische Universität, Fakultät für Chemie, Diss., 2012, 189 S.
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Genes encoding ribosomal RNA (rRNA) exist in two distinct states of chromatin, an ‘open’ one that is permissive to transcription and a ‘closed’ one that is transcriptionally refractive. Even in metabolically active mammalian cells a significant part of the tandemly repeated rRNA genes is epigenetically silenced. Silencing of rDNA is mediated by NoRC, a SNF2h-containing chromatin remodeling complex with TIP5 as its largest subunit, which recruits chromatin-modifying activities to the rDNA promoter, thereby triggering heterochromatin formation. The evolutionary conserved TAM domain of TIP5 shows sequence homology to MBD domains of methyl-CpG-binding proteins. However, in contrast to MBD domains, TIP5 TAM domain specifically binds to a phylogenetically conserved RNA hairpin. This small non-coding pRNA (promoter RNA) is produced from Pol I promoters in the intergenic spacer regions (IGS) that separate rDNA repeats, and matches the rDNA promoter sequence. Interaction of TIP5 with pRNA was shown to be required for nucleolar localization of NoRC and heterochromatin formation in the rDNA regions. Thus, the TAM/pRNA interaction plays a major role in the regulation of eukaryotic gene expression. However, the molecular details of this interaction have not been described yet. The goal of the thesis was to perform structural and biochemical studies of the TAM/pRNA interaction, by starting from an analysis of the individual binding partners. In Chapter 1 of this thesis the biological background of rDNA silencing and the role of the NoRC complex are presented. Chapter 2 reviews the basic principles of NMR spectroscopy and its application for structure calculation and investigation of biomolecules in solution. Chapters 3-5 present a summary of materials and methodology used for biochemical experiments and structural analysis. In Chapter 6 the boundaries of the functional TAM domain of human TIP5 are defined based on sequence analysis of homologous MBD and TAM domains and NMR and biochemical studies of the derived constructs. Chapter 7 presents the three-dimensional solution structure of the TIP5 TAM domain. The structure was solved using state-of-the-art NMR methods and examined using information from paramagnetic relaxation enhancement and hydrogen-deuterium exchange. The conformational dynamics of the TAM domain was analyzed using 15N NMR relaxation experiments. In Chapter 8 the minimal pRNA sequence sufficient for TIP5 interaction and suitable for NMR studies is defined and investigated by NMR and biochemically. In Chapter 9 the TAM/pRNA interaction is characterized using NMR titrations, biochemical experiments and mutational analysis. The pRNA binding interface was mapped onto the structure of the TAM domain. Residues that are crucial for the TAM/pRNA interaction were identified by mutations and tested experimentally in electrophoretic mobility shift and filter binding assays. Biochemical and NMR studies of a previously reported TIP5 mutant, deficient in pRNA binding are shown as well. In Chapter 10 the results of the thesis are discussed. The results of this thesis show, that the TAM domain adopts a novel variant of the MBD-domain fold, which is extended by additional structural elements at the N- and C-termini. The 35 nucleotide stem of pRNA is sufficient to promote TIP5 binding in vitro. NMR analysis suggests that the free RNA is dynamic and may adopt multiple conformations. Therefore, further optimization of the RNA construct will be required for structural analysis by NMR. The most distinct feature of the TAM domain structure is an extended antiparallel β-sheet, which is not conserved within the MBD domains, but mediates the pRNA binding to the TAM domain. The TAM/pRNA interaction is dependent on the residues W546 and R545, as point mutation of these amino acids to alanine abolishes the pRNA binding activity of TAM or considerably decreases its specify to pRNA respectively. In contrast, the double mutation W551G/Y552A, which has been previously shown to abolish the silencing activity of TIP5, strongly affects the tertiary fold of the TAM domain as indicated by NMR and biochemical analysis, thus demonstrating the importance of the native TAM domain fold for its functional activity. The structural and biochemical data presented in this thesis provide novel insight into the molecular function of the TIP5 TAM domain and indicate a novel mode of RNA binding by the TAM domain fold, distinct to the DNA binding by MDB domains. Given that the TAM/pRNA interaction is important for the functional activity of TIP5 and the NoRC for heterochromatin formation and transcriptional silencing of eukaryotic genes, the results provide new insight into molecular details of the processes involved in genome stability and cancer development.
Ein wichtiger Prozess in der Genregulation eukaryontischer Zellen ist die epigenetische Stillegung eines grossen Teils der ribosomalen RNA Gene. Die damit einhergehende Bildung von Heterochromatin wird durch den nukleolären Remodellierungskomplex NoRC eingeleitet. Die Bindung der TIP5 Untereinheit des NoRC an die kleine, nichtkodierende pRNA (Promoter RNA) spielt eine zentrale Rolle für die NoRC Funktion. In der vorliegenden Arbeit wurden die strukturellen Grundlagen TIP5/pRNA-Bindung mittels NMR und biochemischer Methoden untersucht. Die Struktur der TIP5 TAM Domäne wurde mittels NMR-Spektroskopie in Lösung bestimmt. Die TAM/pRNA Interaktion wurde durch NMR Titrationen und biochemische Experimente untersucht und durch Mutationsanalyse validiert. Die TAM Domäne ist ein neues RNA Bindungsmotiv das durch Bindung an die pRNA eine wichtige Rolle für die ribosomale Genregulation durch den NoRC Komplex spielt.
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Publikationstyp Sonstiges: Hochschulschrift
Typ der Hochschulschrift Dissertationsschrift
Schlagwörter NMR; Structural Biology; Epigenetics; Gene Silencing; Protein-RNA interaction; rRNA; rDNA; NoRC; TAM; pRNA
Quellenangaben Band: , Heft: , Seiten: 189 S. Artikelnummer: , Supplement: ,
Verlag Universitätsbibliothek der TU München
Verlagsort München
Hochschule Technische Universität
Hochschulort München
Fakultät Fakultät für Chemie