Role of nuclear RNP assembly in cytoplasmic mRNA localization [Elektronische Ressource] / Tung-Gia Du

Tung-Gia Du

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 5
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

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




Role of nuclear RNP assembly in
cytoplasmic mRNA localization






Tung-Gia Du
aus
Saigon


München
2007

Ehrenwörtliche Versicherung

Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe erarbeitet.

München, am ……………………….



………………………………………..
(Tung-Gia Du)








Dissertation eingereicht am 6.9.2007
1. Gutachter Prof. Dr. Ralf-Peter Jansen
2. Gutachter oland Beckmann
Mündliche Prüfung am 31.10.2007 Table of contents

1 Introduction ....................................................................................................... 5
1.1 The yeast Saccharomyces cerevisiae ......................................................................5
1.2 The yeast life cycle ...................................................................................................5
1.3 Mating type switching...............................................................................................6
1.4 Control of HO expression .........................................................................................8
1.5 Localization of ASH1 mRNA in S. cerevisiae .........................................................11
1.5.1 SHE genes......................................................................................................11
1.5.2 ASH1 mRNA – the cargo................................................................................12
1.5.3 Other localized mRNAs ..................................................................................13
1.5.4 She1/Myo4p – a yeast class V myosin motor.................................................14
1.5.5 The adapter protein She3 ...............................................................................15
1.5.6 She2 – the RNA binding protein .....................................................................15
1.5.7 She4 – a putative myosin chaperone .............................................................16
1.5.8 She5/Bni1p and Bud6p...................................................................................17
1.6 Trans-acting factors of ASH1 mRNA......................................................................17
1.6.1 Khd1p.............................................................................................................18
1.6.2 Puf6p..............................................................................................................18
1.6.3 Loc1p19
1.7 The biological functions of mRNA localization........................................................20
1.8 Initiation of mRNA localization................................................................................24
1.9 Aim of this work ......................................................................................................26

2 Results ..............................................................................................................27
2.1 Purification of a specific antibody directed against She2p......................................27
2.2 She2 is a nucleo-cytoplasmic shuttling protein.......................................................29
2.3 A subpopulation of She2p is nuclear ......................................................................32
2.4 Subnuclear accumulation of She2p upon inhibition of mRNA export .....................34
2.5 The export of She2p is dependent on the binding to its target mRNA....................35
2.6 Following She2p in vivo..........................................................................................37
2.7 Binding of She2p to ASH1 mRNA occurs at early stages of mRNA maturation.....38
2.8 She2p dimerisation is necessary for localization....................................................40
ts2.9 Inhibition of mRNA export in a mex67-5 / ∆rrp6 mutant leads to accumulation of
ASH1 mRNA in the nucleolus ............................................................................................41
2.10 Trans-acting factors Puf6 and Loc1 are nucleolar proteins ....................................44
2.11 Khd1p does not accumulate in the nucleolus upon block of mRNA export ............47
2.12 She2p does not physically interact with other RNA localization factors .................48

12.13 Nuclear Export of ASH1 mRNA does not require She2p........................................49
2.14 Cytoplasmic retention of She2 protein....................................................................50
2.15 She2p artificially tethered to cytoplasmic She3p leads to its nuclear exclusion .....52
2.16 Cells expressing She3N-She2 fusion protein are able to localize ASH1 mRNA ....54
2.17 Localization mediated by She3N-She2p leads to ineffective sorting of Ash1p into
daughter cells .....................................................................................................................55
2.18 The absence of a nucleolar RNA localization factor leads to an increased rate of
Ash1p synthesis .................................................................................................................57
2.19 Loc1p binding to ASH1 mRNA is dependent on the delivery of She2p..................59
2.20 A direct binding of She3p to ASH1 may be involved in cytoplasmic tethering to the
motor complex....................................................................................................................61

3 Discussion ........................................................................................................64
3.1 Nuclear factors involved in cytoplasmic RNA localization ......................................64
3.2 The Nucleolus, a multifunctional compartment.......................................................68
3.2.1 Ribosome Biogenesis.....................................................................................68
3.2.2 Assembly of non-ribosomal RNPs ..................................................................69
3.2.3 Post-transcriptional modifications...................................................................71
3.2.4 Transit of ASH1 mRNA through the nucleolus ...............................................71
3.2.5 Assembly of localized RNPs...........................................................................73
3.3 She2p’s ‘nuclear history’ is required for efficient asymmetric sorting of Ash1p......76

4 Materials............................................................................................................80
4.1 Consumables..........................................................................................................80
4.2 Commercially available kits ....................................................................................80
4.3 Enzymes.................................................................................................................81
4.4 Antibodies...............................................................................................................81
4.5 Oligonucleotides.....................................................................................................82
4.5.1 Primer for she2::KANMX4 gene deletion........................................................82
4.5.2 Primer for she2::HISMX6 gene deletion .........................................................82
4.5.3 Primer for rrp6::natNT2 gene deletion ............................................................82
4.5.4 Primer for Puf6p epitope tagging ....................................................................83
4.5.5 Primer for Khd1p epitope ...................................................................83
4.5.6 Primer for Loc1p epitope tagging83
4.5.7 Primer for cloning of YCplac111-She2 ...........................................................83
4.5.8 e284
4.5.9 Primer for cloning of YCplac111- She3N-She2 ..............................................84

24.5.10 Primer for cloning of p413-HA -She3C...........................................................84 6
4.5.11 Primer for ASH1 RT-PCR ...............................................................................84
4.5.12 Primer for ASH1 probe (Northern blot)84
4.5.13 She2-N36S mutagenesis primer.....................................................................85
4.5.14 She2-R63K primer85
4.5.15 She2-S120Y mutagenesis primer...................................................................85
4.5.16 Sequencing primer..........................................................................................85
4.6 Vectors and Plasmids.............................................................................................86
4.6.1 Vectors............................................................................................................86
4.6.2 Plasmids.........................................................................................................86
4.7 Bacterial strains......................................................................................................87
4.8 Yeast strains.................................................