Holdings: Regulation of mRNA stability during embryogenesis and cell differentiation

Regulation of mRNA stability during embryogenesis and cell differentiation:

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Bibliographic Details
Main Author:	Bruer, Marius 1988- (Author)
Format:	Thesis Book
Language:	English
Published:	Heidelberg [2019]
Subjects:	Hochschulschrift
Online Access:	Inhaltsverzeichnis Inhaltsverzeichnis
Physical Description:	v, 230 Seiten Illustrationen, Diagramme 30 cm

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MARC


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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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