Ctk1 function is crucial for efficient translation initiation and interacts with the mRNP processing factor Npl3 [Elektronische Ressource] / Britta Coordes. Betreuer: Roland Beckmann

Britta Coordes

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2011
• Nombre de lectures: 32
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

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






Ctk1 function is crucial for efficient translation initiation
and interacts with the mRNP processing factor Npl3








Britta Coordes
aus
Bremen



2011



Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom
29. Januar 1998 (in der Fassung der sechsten Änderungssatzung vom 16. August 2010)
von Herrn Prof. Dr. Roland Beckmann betreut.





Ehrenwörtliche Versicherung

Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.

München, den 30.04.11



Britta Coordes



Dissertation eingereicht am 03.05.11

1. Gutachter: Prof. Dr. Roland Beckmann
2. Gutachter: Prof. Dr. Klaus Förstemann





Mündliche Prüfung am 27.06.11






Parts of the present thesis are submitted for publication:

Coordes B., Brünger K.M., Burger K., Soufi B., Horenk J., Eick D., Olsen J.V., Sträßer
K.: “Ctk1 function is crucial for efficient translation initiation”, under revision.



As a collaboration with the laboratory of Prof. Karl-Peter Hopfner, the following
manuscript will be submitted for publication:

Schiller C., Lammens K., Guerini I., Coordes B., Schlauderer F., Möckel C., Schele A.,
Sträßer K., Jackson S. P., Hopfner K.-P.:
“Insights into DNA double-strand break repair and ataxia-telangiectasia like disease from
the structure of an Mre11-Nbs1 complex“, manuscript in preparation.



TABLE OF CONTENTS
TABLE OF CONTENTS

SUMMARY ............................................................................................................................... 1

1. INTRODUCTION .............................................................................................................. 2
1.1. The mechanism of cap dependent translation initiation 2
1.2. Translational control under stress conditions .............................................................. 4
1.3. Translation initiation by Internal Ribosome Entry Sites ............................................. 5
1.4. The transcription cycle ................................ 7
1.5. The CTDK-I complex and the related complex Bur1-Bur2 ........ 9
1.6. Ribosome biogenesis ................................................................. 11
1.7. The SR protein Npl3 .................................. 13
1.8. Aim of this work ........ 15

2. RESULTS ......................................................................................................................... 17
2.1 Ctk1 function is crucial for translation initiation ..................... 17
2.1.1. Ctk1 is needed for efficient translation initiation ................... 17
2.1.2. Loss of Ctk1 causes a decrease of 80S initiation complex formation and
inefficient subunit joining at the start codon .......................................................... 19
2.1.3 The translation defect in Ctk1 depleted cells is not caused by stress ...................... 23
2.1.4. The kinase activity of Ctk1 is important for its function in translation initiation .. 25
2.1.5. The translation initiation defect in Ctk1 depleted extracts is not due to the
missing phosphorylation of Rps2 ........................................................................... 27
2.2. Ctk1 phosphorylates distinct translation initiation factors in vitro, but in vivo no
substrate of Ctk1 could be identified ......... 27
2.2.1. Ctk1 phosphorylates distinct translation initiation factors in vitro ........................ 27
2.2.2. In vivo, no phosphorylation by Ctk1 of the identified in vitro substrates of Ctk1
can be detected ........................................................................................................ 29
2.3. Ctk1 depletion causes reduced phosphorylation of proteins involved in ribosome
biogenesis and translation, but Ctk1 is not involved in rRNA processing ................ 31
2.3.1. Ctk1 depletion causes reduced phosphorylation of proteins involved in ribosome
biogenesis and translation ...................................................................................... 31
2.3.2. In contrast to the depletion of Bur2, Ctk1 depletion does not cause rRNA
processing defects .................................................................................................. 37
2.4. Ctk1 depleted extracts have an increased sensitivity to RNAse compared to mock
depleted extracts ........ 38


TABLE OF CONTENTS
2.5. Depletion of the Bur1-Bur2 complex leads to a similar translation initiation defect
as CTDK-I depletion ................................................................................................. 39
2.6. Ctk1 and Npl3 most likely have antagonistic functions in the nucleus and Npl3
is an in vitro substrate of Ctk1 ................................................................................... 40
2.6.1. CTK1 and NPL3 interact genetically ...... 40
2.6.2. A C-terminal TAP tag leads to the mislocalization of Npl3 to the cytoplasm ........ 41
2.6.3. Ctk1 phosphorylates Npl3 on S349, S356 and S411 .............................................. 42
2.6.4. In vivo, S411 is the main phosphorylation site of Npl3, and there is no major
reduction of Npl3 phosphorylation upon Ctk1 depletion ....... 45
2.6.5. The Npl3-S349A-S356A-TAP, but not the Npl3-S349A-S356A-S411A mutant
……..has a reduced rate of translation ............................................................................. 46
2.6.6. Loss of phosphorylation of Npl3 by Ctk1 does not lead to a translation initiation
defect ....................................................................................................................... 47
2.6.7. The lethality of Npl3 overexpression in ctk1 cells is not due to the missing
phosphorylation by Ctk1 ........................ 47

3. DISCUSSION .................................................................................................................. 49
3.1. Ctk1 function is crucial for subunit joining during translation initiation ................... 49
3.2. No substrate involved in translation initiation that is phosphorylated by Ctk1
in vitroand in vivo could be identified ....................................................................... 51
3.3. The phosphorylation of proteins involved in ribosome biogenesis and translation is
decreased upon Ctk1 depletion, but no direct substrate of Ctk1 could be identified 51
3.4. The translation defect observed upon Ctk1 depletion is not due to stress ................. 53
3.5. The translation initiation defect in Ctk1 depleted cells is small ................................ 54
3.6. The translation initiation defect upon Ctk1 depletion might be direct or indirect .... 55
3.7. Is the ribosome integrity affected upon Ctk1 depletion? ........................................... 56
3.8. Ctk1 – a shuttling protein coupling multiple steps of gene expression? ................... 56
3.9. Is Ctk1 involved in translational control of global mRNA or a specific subset of
mRNAs? ..................................................................................................................... 58
3.10. The function of Bur1 and Ctk1 is similar regarding translation initiation, but
different regarding rRNA processing ........................................................................ 59
3.11. Ctk1 and Npl3 interact with each other and most likely have antagonistic functions
in the nucleus .............................................. 59
3.12. Might the function of Ctk1 in translation initiation be conserved? ........................... 61

4. MATERIALS & METHODS ................................................................ 62
4.1. MATERIALS ............................................. 62
4.1.1. Consumables and chemicals ................... 62

TABLE OF CONTENTS
4.1.2. Commercially available kits ................................................................................... 62
4.1.3 Equipment ............................................... 63
4.1.4. Radioactivity........... 64
4.1.5. Enzymes ................................................................................. 64
4.1.6. Antibodies............................................... 65
4.1.7. Oligonucleotides ..................................................................... 65
4.1.8. Plasmids .................. 69
4.1.9. Strains ..................................................................................................................... 72
4.2. METHODS ................ 76
4.2.1. Standard methods .................................................................................................. 76
4.2.2. Yeast specific techniques ......