Regulation of mRNA stability during embryogenesis and cell differentiation:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
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[2019]
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| Beschreibung: | v, 230 Seiten Illustrationen, Diagramme 30 cm |
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| 100 | 1 | |a Bruer, Marius |d 1988- |e Verfasser |0 (DE-588)1176432370 |4 aut | |
| 245 | 1 | 0 | |a Regulation of mRNA stability during embryogenesis and cell differentiation |c presented by Marius Bruer, M.Sc. Biochemie |
| 264 | 1 | |a Heidelberg |c [2019] | |
| 300 | |a v, 230 Seiten |b Illustrationen, Diagramme |c 30 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Ruperto Carola University Heidelberg |d 2019 | ||
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Datensatz im Suchindex
| _version_ | 1804180589993000960 |
|---|---|
| adam_text | CONTENTS
ABSTRACT 1
LIST O F FIGURES 5
LIST OF TABLES 7
1 INTRODUCTION 9
1.1 REGULATION OF GENE EXPRESSION IN EUKARYOTIC C E LLS
........................... 9
1.2 TRANSCRIPTIONAL CONTROL OF GENE
EXPRESSION............................................11
1.2.1 INITIATION OF TRANSCRIPTION AND THE ROLE OF RNA POLY
MERASE II C-TERMINAL D O M AIN
....................................................
11
1.2.2 HISTONE MODIFICATIONS AND NUCLEOSOME REMODELING .... 12
1.2.3 ENHANCERS, TRANSCRIPTION FACTORS, COREGULATORS, AND TOPO
LOGICALLY ASSOCIATING DOMAINS (TADS)
.................................
16
1.2.4 HISTONE AND PROTEIN LYSINE ACETYLATION: MOLECULAR MA
CHINERY
..........................................................................................
20
1.3 POST-TRANSCRIPTIONAL CONTROL OF GENE EXPRESSION BY CYTOPLASMIC
MRNA D E G RA D A TIO N
.................................................................................25
1.3.1 5* CAP & 3* POLY(A) TAIL: INTRINSIC STABILITY DETERMINANTS
O FM R N A S
....................................................................................25
1.3.2 INFLUENCE OF TRANSLATION AND CODON USAGE ON MRNA STA
BILITY
.............................................................................................
26
1.3.3 MODULATION OF MRNA TRANSLATION AND DECAY BY N6-METHYL-
ADENOSINE AND 3* OLIGO-URIDYLATION
..........................................
28
1.3.4 CYTOPLASMIC MRNA DEGRADATION PATHWAYS & MOLECULAR
M A O H IN E RIE S
................................................................................
29
1.3.5 PROTEIN ACETYLATION AND ITS INFLUENCE ON GLOBAL MRNA
DECAY RATES
................................................................................
35
1.3.6 POTENTIAL CROSS-TALK BETWEEN TRANSCRIPTIONAL ACTIVITY AND
MRNA D E C A Y
.............................................................................
36
1.4 BIOLOGICAL PROCESSES TH AT REQUIRE CELLS TO IMPLEMENT GLOBAL CHANGES
TO THEIR TRA N SCRIP TO M
ES..........................................................................
38
1.4.1 EXAMPLES FOR MODULATION OF MRNA TURNOVER UNDER STRESS
C O N D ITIO N S
....................................................................................39
1.4.2 GLOBAL TURNOVER OF TRANSCRIPT POOLS DURING EMBRYOGENESIS 40
1.4.3 MOUSE EMBRYONIC STEM CELL DIFFERENTIATION AS A MODEL TO
INVESTIGATE GLOBAL CHANGES IN MRNA STA B ILITY
...................
44
1.5 AIMS OF STUDY AND WORKING H Y P O TH E S E S
..........................................48
RESULTS 51
2.1 MILD FORMALDEHYDE CROSSLINKING FACILITATES DETECTION OF NUCLEAR
LOCALIZED PROTEINS IN CELL FRACTIONATION EX P ERIM EN TS
..........................
51
2.2 MATERNAL MRNA TURNOVER DURING DROSOPHILA MELANOGASTER MZT 54
2.2.1 POLY(A) RNA TURNOVER IN DROSOPHILA S-2 CELLS IS REGU
LATED THROUGH CHANGES IN INTRACELLULAR ACETYLATION .... 54
2.2.2 ANALYSIS OF MATERNAL MRNA TURNOVER RATES IN DROSOPHILA
EMBRYOS BY Q R T - P C R
..............................................................54
2.2.3 MATERNAL KNOCK-DOWN OF RPDS (HDACL) DOES NOT ACCEL
ERATE TURNOVER OF MATERNAL MRNAS DURING THE MZT . . . 56
2.2.4 EFFICIENT MATERNAL KNOCK-DOWN OF NEJ (CBP) DISRUPTS
OVARY MORPHOLOGY AND PREVENTS OOGENESIS..............................58
2.2.5 ALTERNATIVE EXPERIMENTAL APPROACHES DO NOT YIELD EM
BRYOS WITH REDUCED NEJ LEVELS
.................................................60
2.2.6 PARTIAL KNOCK-DOWN OF NEJ (CBP) DOES NOT DELAY MATER
NAL MRNA DEGRAD ATIO N
..............................................................61
2.2.7 MATERNAL KNOCK-DOWN OF POP2 (CAFLA) INDUCES OVARY
PHENOTYPES THAT ARE DETRIMENTAL TO OOGENESIS
.......................
63
2.2.8 S U M M A R Y
....................................................................................
63
2.3 DEVELOPMENT OF AN EIF4E-BASED RNA IMMUNOPRECIPITATION (RNA-
IP) METHOD FOR SELECTIVE ENRICHMENT OF CAPPED M R N A S
.............
64
2.3.1 EIF4E AS A TOOL FOR THE SELECTIVE ENRICHMENT OF CAPPED
MRNAS FROM CELLS OR TIS S U E S
....................................................
64
2.3.2 GENERATION OF HELA CELL LINES STABLY EXPRESSING TAGGED
E I F 4 E
..........................................................................................
64
2.3.3 CHARACTERIZATION OF GENERATED STABLE CELL L I N E S
.....................
67
2.3.4 TESTING AND OPTIMIZATION OF AN RNA-IP PROTOCOL FOR
EIF4E-MEDIATED MRNA E N RIC H M E N T
.......................................
69
2.3.5 S U M M A R Y
....................................................................................
72
2.4 CHANGES IN MRNA TURNOVER DURING NEURONAL DIFFERENTIATION OF
MOUSE EMBRYONIC STEM CELLS (M E S C
S)................................................. 73
2.4.1 CHOICE OF DIFFERENTIATION S Y S TE M
..............................................73
2.4.2 MESCS TRANSIENTLY UPREGULATE P300 AND HISTONE ACETYLA
TION LEVELS AND INDUCE CN OTL EXPRESSION DURING DIFFER
ENTIATION IN THE NEURONAL P RO TO C O L
..........................................
74
2.4.3 BULK POLY(A) RNA STABILITY AND TRANSCRIPT-SPECIFIC MRNA
HALF-LIVES GRADUALLY INCREASE DURING DIRECTED MESC DIF
FERENTIATION
.................................................................................76
2.4.4 QRT-PCR AND RNASEQ REVEAL FAILURE OF MESCS TO UN
DERGO NEURONAL D IFFE RE N TIA TIO N
.................................................81
2.4.5 REPETITION OF NEURONAL DIFFERENTIATION WITH ANOTHER MESC
BATCH YIELDS ONLY SMALL NUMBERS OF N EU RO N
S............................82
2.4.6 S U M M A R Y
....................................................................................87
2.5 CHANGES IN MRNA STABILITY AND H3K27AC DURING UNDIRECTED
DIFFERENTIATION OF MESCS INTO EMBRYOID B O D IE S
..................................
88
2.5.1 NEURONAL AND UNDIRECTED EB DIFFERENTIATION ARE ACCOM
PANIED BY SIMILAR CHANGES IN CN OTL PROTEIN EXPRESSION
AND HISTONE H3 A CETY LATIO N
....................................................... 88
2.5.2 BULK POLY(A) MRNA AND INDIVIDUAL TRANSCRIPTS ARE GLOB
ALLY STABILIZED DURING UNDIRECTED EB DIFFERENTIATION . . . 91
2.5.3 DESEQ2 ANALYSIS REVEALS REDUCED EXPRESSION OF SEVERAL
GENES INVOLVED IN CONSTITUTIVE MRNA TURNOVER UPON DIF
FERENTIATION
.................................................................................96
2.5.4 CHROMATIN IP (CHIP) REVEALS NO CORRELATION OF MRNA
STABILITY CHANGES WITH CHANGES IN H3K27AC ON A GENE-
BY-GENE B A S I S
............................................................................100
2.5.5 S U M M A R Y
..................................................................................
103
DISCUSSION 107
3.1 THE ROLE OF PROTEIN ACETYLATION DURING MATERNAL MRNA DEGRA
DATION IN DROSOPHILA MELANOGASTER
.....................................................
107
3.1.1 REGULATION OF THE DROSOPHILA DEADENYLASE MACHINERY BY
ACETYLATION..................................................................................
108
3.1.2 KNOCK-DOWN OF MATERNAL RPD3 (H D A C
L)............................108
3.1.3 KNOCK-DOWN OF MATERNAL NEJ (C B P )
.....................................
109
3.1.4 ALTERNATIVE STRATEGIES FOR THE REDUCTION OF NEJ ACTIVITY IN
E M B RY O S
.....................................................................................
110
3.1.5 KNOCK-DOWN OF MATERNAL POP2 ( C A F L )
..................................
I L L
3.2 EIF4E-BASED RNA-IP FOR THE ENRICHMENT OF CAPPED MRNAS . . . 113
3.2.1 EXPRESSION OF TAGGED EIF4E IN HELA C E LLS
............................113
3.2.2 BIOCHEMICAL CHARACTERIZATION OF 4E-NFTG AND 4EK2WA-
N F T G
........................................................................................
114
3.2.3 ENRICHMENT OF CELLULAR MRNAS BY 4E-NFTG-RNA-IP . .115
3.2.4 CROSSLINKING STRATEGIES TO AVOID POST-LYSIS RE-ASSOCIATION
OF 4E-NFTG WITH M R N A S
.....................................................
116
3.2.5 STRATEGIES TO ENSURE CONTINUED 4E-NFTG EXPRESSION . . .118
3.3 REGULATION OF MRNA STABILITY AND TRANSCRIPTIONAL ACTIVITY DUR
ING MESC
DIFFERENTIATION.....................................................................
119
3.3.1 MRNAS ARE GLOBALLY STABILIZED UPON MESC DIFFERENTIATION 119
3.3.2 GROUPS OF MRNAS SHOW DISTINCT HALF-LIFE PATTERNS .... 122
3.3.3 IDENTITY OF DIFFERENTIATING CELLS AND TECHNICAL ISSUES IN THE
NEURONAL DIFFERENTIATION SYSTEM
............................................124
3.3.4 CHANGES IN GENE EXPRESSION TH AT COULD EXPLAIN GLOBAL
MRNA
STABILIZATION..................................................................
126
3.3.5 CHIPSEQ DOES NOT REVEAL EVIDENCE FOR COUPLING OF MRNA
DECAY AND TRANSCRIPTIONAL ACTIVITY ON A GENE-BY-GENE BASIS 130
3.4 S U M M A R Y
............................................
133
3.5 O U TLO O K
..................................................................................................
135
4 MATERIALS AND METHODS 139
4.1 FLY STOCK MAINTENANCE, CROSSES, EMBRYO C O LLE C TIO N
........................
139
4.2 CLONING OF E IF 4 E -N F T G
.....................................................................
140
4.3 CELL C U LTU R E
............................................................................................141
4.4 ACTINOMYCIN D RNA DECAY A S S A Y S
.....................................................
143
4.5 HELA CELL FRA C TIO N A TIO N
........................................................................
144
4.6 C O-IM M UNOPRECIPITATION
.....................................................................
144
4.7 C A P - I P
..................................................................................................
145
4.8 EIF4E-RNA-IP
.....................................................................................
145
4.9 CHROMATIN IP (C H IP
)............................................................................146
4.10 POLYSOME FRA C TIO N A TIO N
.........................................................................148
4.11 IMMUNOFLUORESCENCE STAINING OF DROSOPHILA O V A RIE S
........................
149
4.12 IMMUNOFLUORESCENCE STAINING OF HELA CELLS, MESCS, NEURONAL
C U L T U R E S
..................................................................................................
149
4.13 FACS STAINING OF MESCS AND DIFFERENTIATING C E L L S
........................
150
4.14 RNA E X TRA C TIO N
.....................................................................................
151
4.15 NORTHERN B L O T
........................................................................................
151
4.16 RT-QPCR AND CHIP-QPCR
...............................................................152
4.17 WESTERN B LO T T I N G
..................................................................................
153
4.18 R N A S E Q
..................................................................................................
154
4.19 C H IP S E Q
..................................................................................................
155
ABBREVIATIONS 157
ACKNOWLEDGMENTS 161
BIBLIOGRAPHY 163
5 APPENDIX 203
5.1 MATERIALS USED IN THIS S T U D Y
...............................................................203
5.1.1 TECHNICAL EQUIPMENT & SO FTW A RE
...........................................
203
5.1.2 CONSUMABLES & COMMERCIAL K I T S
...........................................
205
5.1.3 CHEMICALS, ENZYMES & INHIBITORS
........................................
207
5.1.4 TISSUE CULTURE MEDIA C O M P O N E N TS
........................................
209
5.1.5 A
NTIBODIES..................................................................................211
5.1.6 OLIGONUCLEOTIDES
........................................................................
213
5.1.7 DROSOPHILA S TO C K S
.....................................................................
218
5.2 SUPPLEMENTARY F IG U RE S
.......................................................................219
|
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| spelling | Bruer, Marius 1988- Verfasser (DE-588)1176432370 aut Regulation of mRNA stability during embryogenesis and cell differentiation presented by Marius Bruer, M.Sc. Biochemie Heidelberg [2019] v, 230 Seiten Illustrationen, Diagramme 30 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Ruperto Carola University Heidelberg 2019 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf http://d-nb.info/1192845269/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=031583749&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Bruer, Marius 1988- Regulation of mRNA stability during embryogenesis and cell differentiation |
| subject_GND | (DE-588)4113937-9 |
| title | Regulation of mRNA stability during embryogenesis and cell differentiation |
| title_auth | Regulation of mRNA stability during embryogenesis and cell differentiation |
| title_exact_search | Regulation of mRNA stability during embryogenesis and cell differentiation |
| title_full | Regulation of mRNA stability during embryogenesis and cell differentiation presented by Marius Bruer, M.Sc. Biochemie |
| title_fullStr | Regulation of mRNA stability during embryogenesis and cell differentiation presented by Marius Bruer, M.Sc. Biochemie |
| title_full_unstemmed | Regulation of mRNA stability during embryogenesis and cell differentiation presented by Marius Bruer, M.Sc. Biochemie |
| title_short | Regulation of mRNA stability during embryogenesis and cell differentiation |
| title_sort | regulation of mrna stability during embryogenesis and cell differentiation |
| topic_facet | Hochschulschrift |
| url | http://d-nb.info/1192845269/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=031583749&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT bruermarius regulationofmrnastabilityduringembryogenesisandcelldifferentiation |
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