Growth-dependent regulation of ribosome biogenesis and the role of Rrn3p in RNA polymerase I transcription [Elektronische Ressource] / vorgelegt von Robert Steinbauer

Robert Steinbauer

Sujets

• Biologie

Informations

• Publié par: universitat_regensburg
• Publié le: 01 janvier 2010
• Nombre de lectures: 68
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

Growth-dependent regulation of ribosome biogenesis
and
the role of Rrn3p in RNA polymerase I transcription






DISSERTATION

ZUR ERLANGUNG DES DOKTORGRADES
DER NATURWISSENSCHAFTEN (DR. RER. NAT.)
DER FAKULTÄT FÜR BIOLOGIE UND VORKLINISCHE MEDIZIN
DER UNIVERSITÄT REGENSBURG




vorgelegt von
Robert Steinbauer

aus
Schmidmühlen

im Dezember 2010





















































Das Promotionsgesuch wurde eingereicht am: 15. Dezember 2010

Die Arbeit wurde angeleitet von: Prof. Dr. Herbert Tschochner


Prüfungsausschuss:

Vorsitzender: Prof. Dr. Reinhard Wirth
1. Prüfer: Prof. Dr. Herbert Tschochner
2. Prüfer: Prof. Dr. Michael Thomm
3. Prüfer: Prof. Dr. Wolfgang Seufert



Tag der mündlichen Prüfung: 07. Februar 2011




































Die vorliegende Arbeit wurde in der Zeit von Januar 2007 bis Dezember 2010 am Lehrstuhl
Biochemie III des Institutes für Biochemie, Genetik und Mikrobiologie der Fakultät für
Biologie und Vorklinische Medizin der Universität zu Regensburg unter Anleitung von Prof.
Dr. Herbert Tschochner angefertigt.

















Ich erkläre hiermit, dass ich diese Arbeit selbst verfasst und keine anderen als die
angegebenen Quellen und Hilfsmittel verwendet habe.
Diese Arbeit war bisher noch nicht Bestandteil eines Prüfungsverfahrens.
Andere Promotionsversuche wurden nicht unternommen.



Regensburg, den 15. Dezember 2010




Robert Steinbauer




































Table of Contents

Table of Contents

1 SUMMARY – ZUSAMMENFASSUNG ......................................................... 1
Summary .......................................................................................... 1
Zusammenfassung .......................................................................................................... 3
2 INTRODUCTION ......................... 5
2.1 Ribosome biogenesis and cell growth ................................................................ 5
2.2 RNA polymerase I transcription in eukaryotes .................................................. 6
2.2.1 Structure of ribosomal RNA genes ................................................... 6
2.2.2 Subunit composition of RNA polymerase I .................................................................... 7
2.2.3 RNA polymerase I transcription factors and their function ...... 9
2.2.4 Pre-rRNA processing and rRNA maturation ................................................................12
2.2.5 Pol5p and Mybbp1a – potential regulators of ribosomal RNA synthesis ..........15
2.3 TOR – a central component of the eukaryotic growth regulatory network .. 16
2.3.1 General description of the target of rapamycin (TOR) .............................................16
2.3.2 Upstream and downstream of the TOR signaling network ...18
2.3.3 TOR signaling in the context of growth-dependent regulation of ribosome
biogenesis ...............................................................................................................................20
2.4 Objectives............ 24
3 RESULTS ................................................................................................... 27
3.1 Effects of TOR inactivation on RNA polymerase I transcription, rRNA
production, and yeast cell growth .... 27
3.1.1 Proteasome-dependent reduction of Rrn3p-levels in growth-arrested yeast
cells............................................................................................................................................27
3.1.2 Level of Rrn3p influences Pol I-Rrn3p complex formation, Pol I recruitment to
the rDNA, and yeast cell growth but not the rDNA copy number .......................30
3.1.3 RNA polymerase I transcription is not affected at early stages of TOR
inactivation in yeast cells ...................................................................................................36
I Table of Contents

3.1.4 Inhibition of translation is sufficient to mimic severe pre-rRNA processing
defects observed at early stages of TOR inactivation in yeast cells.....................42
3.1.5 Short-term TOR inactivation predominantly affects expression of ribosomal
proteins whose abundance is important for yeast cell growth ............................46
3.1.6 Nucleolar entrapment of ribosome biogenesis factors in yeast cells is
mediated by both rapamycin and cycloheximide treatment as well as by
conditional shut-down of ribosomal protein expression .......................................50
3.2 Effects of overexpression of Rrn3p on RNA polymerase I transcription ........ 53
3.2.1 GAL1-dependent overexpression of Rrn3p results in defects of yeast cell
growth ......................................................................................................................................53
3.2.2 Overexpression of Rrn3p leads to increased amounts of Pol I-Rrn3p
complexes in yeast cells .....................................................................................................55
3.2.3 ChIP experiments reveal no increase in the association of Pol I with the rDNA
locus, but an enhanced level of Rrn3p crosslinking to the rDNA locus when
Rrn3p is overexpressed in yeast cells ............................................................................57
3.2.4 Overexpression of Rrn3p does not lead to severe pre-rRNA processing defects
or changes in mature rRNA production in yeast cells ..............................................60
3.3 Pol5p, which plays an important role in rRNA synthesis, is a putative
interaction partner of Rrn3p.............................................................................. 63
3.3.1 Co-purification of Pol5p in the course of phosphorylation analyses of Rrn3p
indicates interaction between the two proteins .......................63
3.3.2 ChIP experiments reveal no association of Pol5p with the rDNA locus .............66
4 DISCUSSION ............................................................................................. 69
4.1 The role of the proteasome in the down-regulation of Rrn3p-levels upon TOR
inactivation ......................................... 69
4.2. The role of Rrn3p-levels in the formation of Pol I-Rrn3p complexes upon TOR
inactivation ......................................................................... 70
4.3. The role of phosphorylation in the formation of Pol I-Rrn3p complexes ...... 71
4.4 Uncoupling RNA polymerase I transcription and mature rRNA production
after short-term TOR inactivation ..................................................................... 72
4.5 A model for the drastic down-regulation of ribosome production upon TOR
inactivation ......................................... 75
II Table of Contents

4.6 Overexpression of Rrn3p and its impact on ribosome biogenesis and yeast
cell growth .......................................................................................................... 76
4.7 The role of Pol5p in ribosome biogenesis and yeast cell growth ................... 78
4.8 Outlook ................................................................................................................ 79
5 MATERIAL AND METHODS ..... 81
5.1 Material ............................................................................................................... 81
5.1.1 Saccharomyces cerevisiae strains .....................81
5.1.2 Escherichia coli strains .........................................................................................................86
5.1.3 Plasmids ...................................86
5.1.4 Oligonucleotides ..................................................................................................................87
5.1.5 Probes.......................................91
5.1.6 Antibodies...............................................................................................91
5.1.7 Enzymes ...................................................................91
5.1.8 Kits .............................................................................92
5.1.9 Media ........................................................................92
5.1.10 Buffers ....................................................