Transcriptome wide characterization of target sites and alternative splicing regulation mediated by RBM10:
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2012
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| Schlagworte: | |
| Online-Zugang: | kostenfrei Inhaltsverzeichnis |
| Beschreibung: | Nebentitel: Transkriptombreite Charakterisierung von Zielstellen und alternative, durch RBM10 vermittelte Spleiß-Regulierung |
| Beschreibung: | VI, 123 Bl. |
Internformat
MARC
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| 245 | 1 | 0 | |a Transcriptome wide characterization of target sites and alternative splicing regulation mediated by RBM10 |c vorgelegt von Yongbo Wang |
| 246 | 1 | 3 | |a Transkriptombreite Charakterisierung von Zielstellen und alternative, durch RBM10 vermittelte Spleiß-Regulierung |
| 264 | 1 | |c 2012 | |
| 300 | |a VI, 123 Bl. | ||
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| 500 | |a Nebentitel: Transkriptombreite Charakterisierung von Zielstellen und alternative, durch RBM10 vermittelte Spleiß-Regulierung | ||
| 502 | |a Berlin, Freie Univ., Diss., 2012 | ||
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| 912 | |a ebook | ||
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Datensatz im Suchindex
| _version_ | 1804150016108920832 |
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| adam_text | IMAGE 1
TABLE OF CONTENTS
ACKNOLEDGEMENTS 1
SUMMARY 3
1 INTRODUCTION 4
1.1 PRE-MRNA SPLICING AND ITS REGULATION 4
1.1.1 PRE-MRNA SPLICING 4
1.1.2 CW-REGULATORY ELEMENTS IN PRE-MRNA SPLICING 5
1.1.3 SPLICEOSOME ASSEMBLY PATHWAYS 6
1.2 ALTERNATIVE SPLICING 9
1.2.1 ALTERNATIVE SPLICING IS AN IMPORTANT LAYER O F GENE EXPRESSION
CONTROL 9
1.2.2 TYPES O F ALTERNATIVE SPLICING 9
1.2.3 GENERAL MECHANISMS O F ALTERNATIVE SPLICING 11
1.3. ALTERNATIVE SPLICING AND HUMAN DISEASES 15
1.3.1 CW-EFFECTS: DISRUPTION O F SPLICING CODE 15
1.3.2 7AJ-EFFECTS: DEFECTS O F SPLICING MACHINERY OR SPLICING
REGULATORS 16
1.3.3 SPLICING AND DISEASE DIAGNOSIS, PROGNOSIS AND TARGETED THERAPIES
18
1.4 TECHNOLOGIES FOR GLOBAL MAPPING O F RBP-RNA INTERACTIONS AND
ANALYSIS O F ALTERNATIVE SPLICING 19
1.4.1. TECHNOLOGIES FOR GLOBAL MAPPING O F RBP-RNA INTERACTIONS 20
1.4.2. TECHNOLOGIES FOR GLOBAL ANALYSIS O F ALTERNATIVE SPLICING 22
1.5 RBM10 IN HUMAN DISEASES 23
1.6 PREVIOUS STUDIES ON RBM10 24
1.7 OBJECTIVE O F THIS PROJECT 25
2 MATERIALS AND METHODS 27
2.1 MATERIALS 27
2.1.1 CELL LINES 2 7
2.1.2 VECTORS 2 7
2.1.3 CELL CULTURE MEDIUMS 2 7
2.1.4 ENZYMES 2 8
2.1.5 KITS 2 8
HTTP://D-NB.INFO/1032011440
IMAGE 2
2.1.6 CHEMICALS 29
2.1.7 OTHER REAGENTS 29
2.1.8 MAJOR EQUIPMENTS 29
2.2 EXPERIMENTAL METHODS 30
2.2.1 CLONING O F R B M L O INTO GATEWAY EXPRESSION VECTOR 30
2.2.1.1 TOTAL R N A EXTRACTION 30
2.2.1.2 REVERSE TRANSCRIPTION (RT) AND P C R 31
2.2.1.3 CLONING O F RBM10 INTO GATEWAY EXPRESSION VECTOR 32
2.2.2 ESTABLISHING STABLE CELL LINES 33
2.2.3 PAR-CLIP 34
2.2.3.1 4-THIOURIDINE LABELING, DOXYCYCLINE INDUCTION AND CROSSLINKING
34
2.2.3.2 CELL LYSIS AND IMMUNOPRECIPITATION (IP) 35
2.2.3.3 SDS-PAGE AND ELECTROELUTION O F R N A 36
2.2.3.4 R N A CLONING AND SEQUENCING 36
2.2.4 RBM10 KNOCKDOWN 38
2.2.5 RBM10 OVEREXPRESSION 38
2.2.6 M R N A SEQUENCING 39
2.2.7 QRT-PCR 4 0
2.2.8 WESTERN BLOT 42
2.2.9 IMMUNOFLUORESCENCE 43
2.3 COMPUTATIONAL METHODS 44
2.3.1 PAR-CLIP ANALYSIS 44
2.3.1.1 READS MAPPING AND CLUSTER IDENTIFICATION 4 4
2.3.1.2 OVERLAPPED CLUSTER DEFINITION 45
2.3.1.3 BINDING SITES ANNOTATION AND TARGET TRANSCRIPT IDENTIFICATION 45
2.3.1.4 MOTIF ANALYSIS 45
2.3.1.5 ANALYSIS FOR SNRNA BINDING ANALYSIS 45
2.3.2 RNA-SEQUENCING ANALYSIS 46
2.3.2.1 QUANTIFICATION O F GENE EXPRESSION 4 6
2.3.2.2 QUANTIFICATION O F ALTERNATIVE SPLICING 4 6
3 RESULTS 48
3.1 IDENTIFICATION OF AN IN-FRAME DELETION WITHIN RBM10 IN PATIENTS
AFFLICTED WITH AN X-LINKED RECESSIVE DISORDER 48
IMAGE 3
3.2 ESTABLISHMENT OF STABLE HEK293 CELL LINES 49
3.3 PAR-CLIP REPRODUCIBLY IDENTIFIES RBM10 BINDING SITES 51
3.4 RBM10 BINDS PREFERENTIALLY IN THE VICINITY O F SPLICE SITES 53
3.5 RBM10 BINDS TO SPLICING SNRNAS 55
3.6 GENE EXPRESSION CHANGES UPON RBM10 DEPLETION OR
OVEREXPRESSION 57
3.7 SPLICING CHANGES UPON RBM10 DEPLETION AND OVEREXPRESSION .61
3.8 RBM10 AUTOREGULATION 65
3.9 RBM10 MUTANT CHANGES SUBCELLULAR LOCALIZATION AND PATIENT
DERIVED LYMPHOBLAST CELL LINE SHOWED SPLICING CHANGES RESEMBLING
THE CHANGES UPON RBM10 KNOCKDOWN 66
4 DISCUSSIONS 69
4.1 PAR-CLIP RECOVERS TRANSCRIPTOME WIDE RNA BINDING SITES OF
RBM10 69
4.2 PUTATIVE SEQUENCE MOTIF OF RBM10 72
4.3 RNA-SEQ REVEALS SPLICING CHANGES INDUCED BY RBM10 73
4.4 AUTOREGULATION OF RBM10 74
4.5 RBM10 REGULATES SPLICING OF DISEASE ASSOCIATED GENES 74
4.6 CONCLUSION AND PERSPECTIVE REMARKS 76
5 REFERENCE 77
6 APPENDIX 94
7 ZUSAMMENFASSUNG 119
8 PUBILCATIONS 120
DECLARATION 121
CURRICULUM VITAE 122
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| spelling | Wang, Yongbo Verfasser aut Transcriptome wide characterization of target sites and alternative splicing regulation mediated by RBM10 vorgelegt von Yongbo Wang Transkriptombreite Charakterisierung von Zielstellen und alternative, durch RBM10 vermittelte Spleiß-Regulierung 2012 VI, 123 Bl. txt rdacontent n rdamedia nc rdacarrier Nebentitel: Transkriptombreite Charakterisierung von Zielstellen und alternative, durch RBM10 vermittelte Spleiß-Regulierung Berlin, Freie Univ., Diss., 2012 (DE-588)4113937-9 Hochschulschrift gnd-content Erscheint auch als Online-Ausgabe urn:nbn:de:kobv:188-fudissthesis000000044808-7 http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000044808 kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025690155&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Wang, Yongbo Transcriptome wide characterization of target sites and alternative splicing regulation mediated by RBM10 |
| subject_GND | (DE-588)4113937-9 |
| title | Transcriptome wide characterization of target sites and alternative splicing regulation mediated by RBM10 |
| title_alt | Transkriptombreite Charakterisierung von Zielstellen und alternative, durch RBM10 vermittelte Spleiß-Regulierung |
| title_auth | Transcriptome wide characterization of target sites and alternative splicing regulation mediated by RBM10 |
| title_exact_search | Transcriptome wide characterization of target sites and alternative splicing regulation mediated by RBM10 |
| title_full | Transcriptome wide characterization of target sites and alternative splicing regulation mediated by RBM10 vorgelegt von Yongbo Wang |
| title_fullStr | Transcriptome wide characterization of target sites and alternative splicing regulation mediated by RBM10 vorgelegt von Yongbo Wang |
| title_full_unstemmed | Transcriptome wide characterization of target sites and alternative splicing regulation mediated by RBM10 vorgelegt von Yongbo Wang |
| title_short | Transcriptome wide characterization of target sites and alternative splicing regulation mediated by RBM10 |
| title_sort | transcriptome wide characterization of target sites and alternative splicing regulation mediated by rbm10 |
| topic_facet | Hochschulschrift |
| url | http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000044808 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025690155&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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