RNA-binding proteins in the early Drosophila embryo:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
Heidelberg
2016
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| Online-Zugang: | Inhaltsverzeichnis Inhaltsverzeichnis |
| Beschreibung: | 150 Seiten Illustrationen, Diagramme 30 cm |
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| 502 | |b Dissertation |c Ruperto-Carola University of Heidelberg |d 2016 | ||
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Datensatz im Suchindex
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|---|---|
| adam_text | RNA-BINDING PROTEINS IN THE EARLY
DROSOPHILA EMBRYO
EXAMINERS:
DR. ALEXANDER AULEHLA
DR. MICHAEL BOUTROS
TABLE OF CONTENTS
ABSTRACT.....................................................................................................................5
ZUSAMMENFASSUNG.................................................................................................7
ACKNOWLEDGEMENTS...............................................................................................9
TABLE OF
CONTENTS................................................................................................11
LIST OF
FIGURES.......................................................................................................15
1
INTRODUCTION..................................................................................................17
1.1 THE EARLY DROSOPHILA EMBRYO IS A SELF-ORGANIZING SYSTEM IN WHICH
RNA-PROTEIN INTERACTIONS PLAY AN IMPORTANT ROLE
....................................................
19
1.2 RBPS IN MRNA LOCALIZATION AND TRANSLATIONAL
CONTROL......................20
1.3 RBPS DRIVE THE MATERNAL-TO-ZYGOTIC
TRANSITION..................................23
1.4 DIVERSITY OF RNA-BINDING
PROTEINS.......................................................26
2 GOALS OF THE
PROJECT.....................................................................................33
3 RNA INTERACTOME OF A DROSOPHILA EMBRYONIC CELL
LINE.....................37
3.1 BACKGROUND AND
AIMS.............................................................................39
3.2
CONTRIBUTIONS............................................................................................40
3.3 OPTIMIZATION OF RNA INTERACTOME CAPTURE FOR THE DROSOPHILA
EMBRYO-DERIVED 82 CELL
LINE..........................................................................................40
3.4 DETERMINATION OF OPTIMAL CROSS-LINKING CONDITIONS IN S2R+ CELLS. 43
3.5 THE INTERACTOME CAPTURE METHOD ALLOWS SELECTIVE PRECIPITATION OF
RNA RATHER THAN
DNA.....................................................................................................47
3.6 THE CO-PRECIPITATED RRNA IS NOT
POLYADENYLATED..............................48
3.7 PRELIMINARY MASS-SPECTROMETRY ANALYSIS OF SAMPLES CAPTURED FROM
S2R+ CELLS 50
3.8 TWO ROUNDS OF OLIGO(DT) PULL-DOWN IMPROVES SAMPLE PURITY
.......
52
3.9 RNA INTERACTOME OF THE DROSOPHILA S2R+ CELL LINE
............................
55
3.10
CONCLUSIONS.............................................................................................57
3.11 MATERIALS AND METHODS
........................................................................
58
CELL
CULTURE........................................................................................................58
UV IRRADIATION OF S2 AND S2R+
CELLS................................................................58
OLIGO(DT)
PULL-DOWN........................................................................................58
RNA AND PROTEIN
ANALYSIS................................................................................59
RT-QPCR............................................................................................................60
SEQUENCING OF 185 3
END.................................................................................60
MASS-SPECTROMETIY..........................................................................................61
3.12 SUPPLEMENTARY DATASET: RNA INTERACTOME OF S2R+ CELLS
...............
61
4 RNA INTERACTOME OF THE EARLY DROSOPHILA
EMBRYO...........................63
4.1 BACKGROUND AND
AIMS.............................................................................65
4.2
CONTRIBUTIONS...........................................................................................
65
4.3 DETERMINATION OF OPTIMAL CROSS-LINKING CONDITIONS IN DROSOPHILA
EMBRYOS 65
4.4 APPROACHES USED TO REDUCE RRNA CONTAMINATION DURING RNA
INTERACTOME CAPTURE IN EARLY DROSOPHILA
EMBRYOS.................................................67
4.5 LYSIS AT ELEVATED TEMPERATURE AND UNDER REDUCTIVE CONDITIONS
IMPROVES QUALITY OF CAPTURED
SAMPLES......................................................................70
4.6 SAMPLES USED FOR DETERMINATION OF HIGH CONFIDENCE EMBRYO RNA
BINDING
PROTEINS............................................................................................................
74
4.7 MASS-SPECTROMETRY AND DETERMINATION OF THE EMBRYO RNA
INTERACTOME 76
4.8 OVERVIEW OF THE DROSOPHILA EMBRYO RNA INTERACTOME
....................
79
4.9 FEATURES OF NOVEL AND PREVIOUSLY KNOWN DROSOPHILA RBPS............82
4.10 COMPARISON OF FIVE EUKARYOTIC RNA INTERACTOMES
.........................
87
4.11 NOVEL DROSOPHILA RNA-BINDING PROTEINS ARE LINKED TO
DEVELOPMENT 88
4.12 VALIDATION OF RNA-BINDING CAPACITY OF INTERACTOME PROTEINS BY
PNK ASSAY 91
4.13 CONCLUSIONS AND OUTLOOK
.....................................................................
97
4.14 MATERIALS AND
METHODS........................................................................98
EMBRYO COLLECTION AND UV
IRRADIATION............................................................98
OLIGO(DT)
PULL-DOWN........................................................................................99
PRECIPITATION OF RIBOSOMES PRIOR TO OLIGO(DT)
PULL-DOWN.............................99
RNA AND PROTEIN
ANALYSIS................................................................................99
RT-QPCR..........................................................................................................100
STAGING OF
EMBRYOS.........................................................................................100
VALIDATION OF RNA-BINDING ABILITY BY PNK ASSAY
.........................................
100
SAMPLE PREPARATION FOR
MS...........................................................................101
LC-MS/MS.......................................................................................................102
PEPTIDE IDENTIFICATION AND
QUANTIFICATION....................................................102
5 RNA-BOUND PROTEOME DYNAMICITY DURING THE MATERNAL-TO-ZYGOTIC
TRANSITION 105
5.1 BACKGROUND AND
GOALS..........................................................................107
5.2
CONTRIBUTIONS.........................................................................................107
5.3 EXPERIMENTAL DESIGN AND SAMPLES USED IN THE
ANALYSIS.................107
5.4 PROTEIN ABUNDANCE CHANGES IN THE TOTAL EMBRYO AND IN THE RNA
BOUND
FRACTIONS.............................................................................................................108
5.5 COMPARISON OF TOTAL PROTEOMES AND RNA INTERACTOMES IN PRE- AND
POST-MZT EMBRYOS REVEALS GLOBAL
CHANGES.............................................................109
5.6 CLASSIFICATION OF RBPS BASED ON THEIR DYNAMIC BEHAVIOR DURING THE
MZT 112
5.7 POTENTIAL ROLES OF ALTERNATIVE SPLICING IN REGULATION OF RNA-BINDING
ACTIVITY 114
5.8 EVIDENCE FOR ADDITIONAL MECHANISMS AFFECTING OBSERVED RNA
BINDING ACTIVITY OF SOME PROTEINS
.............................................................................
120
5.9 CONCLUSIONS AND
OUTLOOK......................................................................122
5.10 MATERIALS AND
METHODS......................................................................123
6 CONCLUSIONS AND OUTLOOK
.............................................................
125
6.1
BACKGROUND............................................................................................127
6.2 OPTIMIZATION OF THE RNA INTERACTOME CAPTURE
................................
127
6.3 RNA INTERACTOME OF THE EARLY DROSOPHILA EMBRYO
..........................
128
6.4 NEW LINKS BETWEEN DEVELOPMENT AND RNA METABOLISM
................
129
6.5 DYNAMICITY OF THE RNA BOUND PROTEOME DURING THE MZT
.............
129
6.6 ROLES OF DISORDERED REGIONS IN
RBPS..................................................130
6.7 FUTURE
DIRECTIONS...................................................................................131
APPENDIX............................................................................................133
TABLE AL. ANTIBODIES USED FOR
IMMUNOBLOTTING.........................................133
TABLE A2. OLIGONUCLEOTIDES USED FOR
QPCR..................................................134
LIST OF
ABBREVIATIONS.........................................................................135
REFERENCES..........................................................................................137
|
| any_adam_object | 1 |
| author | Sysoev, Vasily |
| author_facet | Sysoev, Vasily |
| author_role | aut |
| author_sort | Sysoev, Vasily |
| author_variant | v s vs |
| building | Verbundindex |
| bvnumber | BV044316956 |
| ctrlnum | (OCoLC)969453147 (DE-599)DNB1119086531 |
| discipline | Biologie |
| format | Thesis Book |
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| indexdate | 2024-07-10T07:49:35Z |
| institution | BVB |
| language | English |
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| physical | 150 Seiten Illustrationen, Diagramme 30 cm |
| publishDate | 2016 |
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| spelling | Sysoev, Vasily Verfasser aut RNA-binding proteins in the early Drosophila embryo presented by Vasily Sysoev Heidelberg 2016 150 Seiten Illustrationen, Diagramme 30 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Ruperto-Carola University of Heidelberg 2016 Drosophila (DE-588)4150708-3 gnd rswk-swf RNS-Bindungsproteine (DE-588)4178253-7 gnd rswk-swf Embryonalentwicklung (DE-588)4070792-1 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Drosophila (DE-588)4150708-3 s Embryonalentwicklung (DE-588)4070792-1 s RNS-Bindungsproteine (DE-588)4178253-7 s DE-604 B:DE-101 application/pdf http://d-nb.info/1119086531/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029720506&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Sysoev, Vasily RNA-binding proteins in the early Drosophila embryo Drosophila (DE-588)4150708-3 gnd RNS-Bindungsproteine (DE-588)4178253-7 gnd Embryonalentwicklung (DE-588)4070792-1 gnd |
| subject_GND | (DE-588)4150708-3 (DE-588)4178253-7 (DE-588)4070792-1 (DE-588)4113937-9 |
| title | RNA-binding proteins in the early Drosophila embryo |
| title_auth | RNA-binding proteins in the early Drosophila embryo |
| title_exact_search | RNA-binding proteins in the early Drosophila embryo |
| title_full | RNA-binding proteins in the early Drosophila embryo presented by Vasily Sysoev |
| title_fullStr | RNA-binding proteins in the early Drosophila embryo presented by Vasily Sysoev |
| title_full_unstemmed | RNA-binding proteins in the early Drosophila embryo presented by Vasily Sysoev |
| title_short | RNA-binding proteins in the early Drosophila embryo |
| title_sort | rna binding proteins in the early drosophila embryo |
| topic | Drosophila (DE-588)4150708-3 gnd RNS-Bindungsproteine (DE-588)4178253-7 gnd Embryonalentwicklung (DE-588)4070792-1 gnd |
| topic_facet | Drosophila RNS-Bindungsproteine Embryonalentwicklung Hochschulschrift |
| url | http://d-nb.info/1119086531/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029720506&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT sysoevvasily rnabindingproteinsintheearlydrosophilaembryo |
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