Functional analysis of RNA transport mediated by Pur-alpha in Drosophila melanogaster [Elektronische Ressource] / Verena Nicole Aumiller

Verena Nicole Aumiller

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2011
• Nombre de lectures: 38
• Langue: Deutsch
• Poids de l'ouvrage: 18 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München








Functional analysis of RNA transport mediated
by Pur-alpha in Drosophila melanogaster














Verena Nicole Aumiller

aus

Dachau



2011

Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29. Januar
1998 (in der Fassung der vierten Änderungssatzung vom 26. November 2004) von Herrn Prof. Dr.
Klaus Förstemann betreut.







Ehrenwörtliche Versicherung

Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.


München, 15.04.2011







Verena Nicole Aumiller










Dissertation eingereicht am 15.04.2011


1. Gutacher: Prof. Dr. Klaus Förstemann

2. Gutachter: Prof. Dr. Dietmar Martin


Mündliche Prüfung am 26.05.2011 1. SUMMARY .................................................................................................................................. 4
2. INTRODUCTION .......................................................................................................................... 5
2.1 mRNA transport ................................................................................................................. 5
2.2 Drosophila oogenesis ......................................................................................................... 6
2.2.1 Process of oogenesis .................................................................................................. 7
2.2.2 Signaling by Drosophila follicle cells during oogenesis .............................................. 8
2.3 Messenger ribonucleoprotein complexes ........................................................................ 1 0
2.4 Pur-alpha10
2.4.1 Structure of Drosophila Pur-alpha ........................................................................... 11
2.4.2 DNA bound functions of Pur-alpha .......................................................................... 13
2.4.3 Pur-alpha in cell cycle control and tumor suppression ............................................ 13
2.4.4 RNA bound functions of Pur-alpha1 4
2.4.5 Pur-alpha knockout mouse ...................................................................................... 15
2.5 Triplet-repeat associated diseases ................................................................................... 15
2.6 Fragile-X associated tremor and ataxia syndrome (FXTAS) ............................................. 15
2.7 Aims of this thesis ............................................................................................................ 17
3. MATERIALS AND METHODS ..................................................................................................... 18
3.1 Materials .......................................................................................................................... 18
3.1.1 Laboratory equipment ............................................................................................. 18
3.1.2 Laboratory chemicals ............................................................................................... 19
3.1.3 Enzymes .................................................................................................................... 20
3.1.4 Other materials ........................................................................................................ 21
3.1.5 Bacterial cells ............................................................................................................ 22
3.1.6 Drosophila melanogaster cells ................................................................................. 23
3.1.7 Fly stocks .................................................................................................................. 23
3.1.8 Flyfood ...................................................................................................................... 24
3.1.9 Plasmids .................................................................................................................... 24
3.1.10 Oligonucleotides ...................................................................................................... 25
3.1.10.1 RNA-Interference ............................................................................................. 25
3.1.10.2 Molecular Cloning ............................................................................................ 26
3.1.10.3 Fly stock mapping26
3.1.10.4 Quantitative PCR .............................................................................................. 27
3.1.10.5 MicroRNA profiling ........................................................................................... 30
3.1.10.6 Northern Blotting ............................................................................................. 32
3.1.10.7 Electrophoretic mobility shift assays (EMSA) ................................................... 32
1 3.1.11 Antibodies ............................................................................................................... 32
3.1.11.1 Primary antibodies ........................................................................................... 32
3.1.11.2 Secondary antibodies ....................................................................................... 33
3.1.12 Commonly used buffers and stock solutions .......................................................... 34
3.2 Methods ........................................................................................................................... 37
3.2.1 Methods for molecular cloning ................................................................................ 37
3.2.2 Methods with Drosophila Schneider 2 cells .............................................................. 38
3.2.2.1 RNA-Interference (RNAi) ..................................................................................... 38
3.2.2.2 Transfection of plasmid DNA .............................................................................. 39
3.2.2.3 Analysis of Schneider cells by fluorescence microscopy .................................... 40
3.2.3 Methods with flies ................................................................................................... 40
3.2.3.1 Maintenance and handling............................................................................... 40
3.2.3.2 Egg laying experiments4 1
3.2.3.3 Rescue experiments .......................................................................................... 4 1
3.2.3.4 Analysis of ovaries by fluorescence microscopy ............................................... 4 1
3.2.4 Protein analysis ....................................................................................................... 42
3.2.4.1 Protein extraction ............................................................................................. 4 2
3.2.4.2 Co-immunoprecipitation .................................................................................. 42
3.2.4.3 Western-Blotting .............................................................................................. 42
3.2.4.4 Mass spectrometry ........................................................................................... 43
3.2.4.5 Electrophoretic mobility shift assay (EMSA) .................................................... 43
3.2.5 RNA analysis ............................................................................................................ 44
3.2.5.1 RNA isolation .................................................................................................... 44
3.2.5.2 Northern Blotting ............................................................................................. 44
3.2.5.3 Quantitative RT-PCR ......................................................................................... 44
3.2.5.4 microArray ........................................................................................................ 45
4. RESULTS .................................................................................................................................... 46
4.1 Pur-alpha has no impact on small RNA biogenesis pathways .......................................... 46
4.2 Localization of Pur-alpha ................................................................................................. 49
4.2.1 Drosophila Schneider 2 cells .................................................................................... 49
4.2.1.1 Pur-alpha bodies are no P-Bodies ....................................................................... 49
4.2.1.2 Pur-alpha is associated with FMRP, Ago1 and Ago2 in Schneider cells .............. 52
4.2.2 Localisation of Pur-alpha during Drosophila oogenesis .......................................... 56
4.2.2.1 Pur-alpha is enriched in the polar cells of the follicular epithelium ...