Investigating the assembly of ribonucleoprotein complexes (RNPs) [Elektronische Ressource] / vorgelegt von Annemarie Schultz

Christopher Merrett

Sujets

• Gesundheit

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2007
• Nombre de lectures: 23
• Langue: Deutsch
• Poids de l'ouvrage: 10 Mo

Extrait

Aus dem Institut für Molekularbiologie und Tumorforschung
Leiter: Prof. Dr. R. Müller

des Fachbereichs Medizin der Philipps-Universität Marburg

in Zusammenarbeit mit dem Max-Planck-Institut für biophysikalische Chemie in Göttingen
Geschäftsführender Direktor: Prof. Dr. Christian Griesinger

Investigating the Assembly of
Ribonucleoprotein complexes
(RNPs)


Inaugural Dissertation

zur Erlangung des Doktorgrades der Humanbiologie
dem Fachbereich Medizin der Philipps-Universität Marburg
vorgelegt von

Annemarie Schultz
aus Bremen


Marburg 2007
















Angenommen vom Fachbereich Medizin der Philipps-Universität
Marburg am: 20.07.2007

Gedruckt mit Genehmigung des Fachbereichs.

Dekan: Prof. Dr. B. Maisch
Referent: Prof. Dr. R. Lührmann
1. Korreferent: PD Dr. Heike Krebber


Für Lenie und Christopher

I. Table of Contents
I. TABLE OF CONTENTS................................................................................... I
II. TABLE OF FIGURES.....................................................................................IV
III. INDEX OF TABLES .......................................................................................VI
1. SUMMARY ...................................................................................................... 1
1.1. Summary ...................................................................................................................................1
1.2. Zusammenfassung...................................................................................................................2
2. INTRODUCTION ............................................................................................. 5
2.1. Spliceosomal snRNPs..............................................................................................................5
2.1.1. The spliceosome cycle ................................................................................................................................... 7
2.1.2. The spliceosomal U4/U6.U5 tri-snRNP ........................................................................................................ 10
2.1.3. Assembly of the tri-snRNP..................... 15
2.1.4. U4/U6.U5 tri-snRNP proteins and retinitis pigmentosa ................................................................................ 16
2.2. Small nucleolar RNPs (snoRNPs) .........................................................................................17
2.2.1. Pre-rRNA processing....................... 17
2.2.2. Small nucleolar RNP are involved in pre-rRNA processing .........................................................................19
2.2.3. Structure and function of the box C/D snoRNPs.......................................................................................... 20
2.3. Similarities between the U4/U6 snRNP and box C/D snoRNPs ..........................................23
2.4. Aim of the work presented in this dissertation....................................................................28
3. MATERIALS AND METHODS...................................................................... 30
3.1. Materials ..................................................................................................................................30
3.1.1. Fine Chemicals............................................................................................................................................. 30
3.1.2. Markers ........................................................................................................................................................ 32
3.1.3. Radio Chemicals .......................................................................................................................................... 32
3.1.4. Enzymes und Inhibitors ................................................................................................................................ 32
3.1.5. Used Organisms 33
3.1.6. Plasmids and Vectors................................................................................................................................... 33
3.1.7. Antibodies..................................................................................................................................................... 34
3.1.8. Reaction Sets (Kits)........................... 35
3.1.9. Chromatography Columns und Column Materials ....................................................................................... 35
3.1.10. Antibiotics ..................................................................................................................................................... 36
3.1.11. Miscellaneous Material........................ 36
3.1.12. Devices........................................................................................................................ 36
3.1.13. DNA Oligonucleotides .................................................................................................................................. 38
3.1.14. RNA Oligonucleotides 40
I
3.1.15. siRNAs ......................................................................................................................................................... 41
3.1.16. Peptide sequences....................................................................................................................................... 42
3.1.17. Common Buffers ................................................................................................................. 42
3.2. Methods ...................................................................................................................................43
3.2.1. Standard Molecular Biological Methods....................................................................................................... 43
3.2.2. Protein Biochemical Methods....................................................................................................................... 55
3.2.3. Immunological Methods ............................................................................................................................... 59
3.2.4. Microbiological Methods............................................................................................................................... 62
3.2.5. Cell Culture .................................................................................................................................................. 65
3.2.6. Microscopic Methods ................................................................................................................................... 68
3.2.7. Special Methods........................................................................................................................................... 72
4. RESULTS ......................................................................................................78
4.1. Protein-protein and protein-RNA contacts both contribute to the 15.5K
protein-mediated assembly of the U4/U6 snRNP and the box C/D snoRNPs....................78
4.1.1. Mutation of conserved amino acids on the surface of protein 15.5K not involved in RNA binding .............. 78
4.1.2. Conserved amino acids on the surface of protein 15.5K are required for hPrp31 association
with the U4 snRNA....................................................................................................................................... 82
4.1.3. Numerous mutations on the surface of protein 15.5K disrupt the binding of the CYPH/hPrp4/hPrp3
complex to U4/U6 snRNA ............................................................................................................................ 83
4.1.4. Box C/D snoRNP assembly requires multiple regions on the surface of protein 15.5K............................... 85
4.1.5. Alpha helix 4 of protein 15.5K is required for hU3–55K binding to the U3 snoRNA .................................... 87
4.1.6. A direct interaction between proteins hU3–55K and 15.5K.......................................................................... 88
4.2. Protein 15.5K is located in nucleoplasmic foci and is associated with the
U4/U6 di–snRNP and the U3 snoRNA in vivo .......................................................................90
4.2.1. The U4/U6 di–snRNP and box C/D snoRNP-associated protein 15.5K localises to nucleoplasmic foci ..... 90
4.2.2. EYFP–15.5K fusion protein in vivo is complexed specifically with the same RNA targets that
protein 15.5K was shown to bind directly in vitro ........................................................................................ 96
4.3. Association of the spliceosomal hPrp31 protein with the U4 snRNP:
Requirement for U4 snRNA's kink-turn stem II and the Nop domain .................................99
4.3.1. Hydroxyl-radical footprinting reveals direct contact of protein hPrp31 with various regions
of the U4 snRNA .......................................................................................................................................... 99
4.3.2. Protein hPrp31 requires stem I and stem II of the 5 ′ stem-loop of U4 for binding .....................................