dc.contributor.author
Wang, Yongbo
dc.date.accessioned
2018-06-08T00:18:49Z
dc.date.available
2013-01-25T14:09:53.521Z
dc.identifier.uri
https://refubium.fu-berlin.de/handle/fub188/11770
dc.identifier.uri
http://dx.doi.org/10.17169/refubium-15968
dc.description
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 Cis-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 of gene expression control
9 1.2.2 Types of alternative splicing 9 1.2.3 General mechanisms of
alternative splicing 11 1.3. Alternative splicing and human diseases 15 1.3.1
Cis-effects: disruption of splicing code 15 1.3.2 Tans-effects: defects of
splicing machinery or splicing regulators 16 1.3.3 Splicing and disease
diagnosis, prognosis and targeted therapies 18 1.4 Technologies for global
mapping of RBP-RNA interactions and analysis of alternative splicing 19 1.4.1.
Technologies for global mapping of RBP-RNA interactions 20 1.4.2. Technologies
for global analysis of alternative splicing 22 1.5 RBM10 in human diseases 23
1.6 Previous studies on RBM10 24 1.7 Objective of this project 25 2 MATERIALS
AND METHODS 27 2.1 Materials 27 2.1.1 Cell lines 27 2.1.2 Vectors 27 2.1.3
Cell culture mediums 27 2.1.4 Enzymes 28 2.1.5 Kits 28 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 of RBM10 into Gateway expression vector 30 2.2.1.1 Total RNA
extraction 30 2.2.1.2 Reverse transcription (RT) and PCR 31 2.2.1.3 Cloning of
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 of RNA 36 2.2.3.4 RNA cloning and sequencing 36
2.2.4 RBM10 knockdown 38 2.2.5 RBM10 overexpression 38 2.2.6 mRNA sequencing
39 2.2.7 qRT-PCR 40 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 44 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 of gene expression 46 2.3.2.2
Quantification of alternative splicing 46 3 RESULTS 47 3.1 Identification of
an in-frame deletion within RBM10 in patients afflicted with an X-linked
recessive disorder 47 3.2 Establishment of stable HEK293 cell lines 48 3.3
PAR-CLIP reproducibly identifies RBM10 Binding Sites 49 3.4 RBM10 binds
preferentially in the vicinity of splice sites 52 3.5 RBM10 binds to splicing
snRNAs 54 3.6 Gene expression changes upon RBM10 depletion or overexpression
56 3.7 Splicing changes upon RBM10 depletion and overexpression 60 3.8 RBM10
autoregulation 64 3.9 RBM10 mutant changes subcellular localization and
patient derived lymphoblast cell line showed splicing changes resembling the
changes upon RBM10 knockdown 65 4 DISCUSSIONS 68 4.1 PAR-CLIP recovers
transcriptome wide RNA binding sites of RBM10 68 4.2 Putative sequence motif
of RBM10 71 4.3 RNA-Seq reveals splicing changes induced by RBM10 72 4.4
Autoregulation of RBM10 73 4.5 RBM10 regulates splicing of disease associated
genes 73 4.6 Conclusion and perspective remarks 75 5 REFERENCE 76 6 APPENDIX
93 7 ZUSAMMENFASSUNG 118 8 PUBILCATIONS 119 DECLARATION 120 CURRICULUM VITAE
121
dc.description.abstract
Defects in RBM10 have been identified as the cause of TARP syndrome and other
less severe developmental disorders. Although the RNA-binding protein encoded
by RBM10 has been identified as a component of spliceosome, very little is
known so far about the target genes as well as the molecular mechanism
underlying the regulation mediated by RBM10. Here, we used photoactivatable-
ribonucleoside-enhanced crosslinking and immunoprecipitation to identify
89,247 RBM10 binding sites in 6,396 target genes. Those binding sites are
enriched in the vicinity of both 5’ and 3’ splice sites in introns and in
exons, which implicated the potential role of RBM10 in splicing regulation. In
consistent with this, we also found that RBM10 binds specifically to U2 snRNA.
Based on RNA sequencing, we determined 281 and 356 alternative splicing events
following RBM10 depletion or overexpression respectively. Notably, the
splicing changes upon RBM10 knockdown and overexpression are largely anti-
correlated, indicating these alternative splicing were sensitive to cellular
RBM10 abundance and were probably under its direct regulation. Finally, we
showed that an in-frame deletion of RBM10 identified in the patient changed
its normal nuclear localization and thereby disrupted its function in splicing
regulation. Taken together, our extensive genome-wide datasets demonstrate
that RBM10 functions as a novel splicing regulator via its direct binding to
pre-mRNA substrates and potentially coordinates interplays between core
splicing machinery and other splicing regulators.
de
dc.description.abstract
Defekte in RBM10 sind die Ursache für das TARP-Syndrom und für weitere,
weniger schwere Entwicklungsstörungen. Man hat herausgefunden, dass das durch
RBM10 kodierte RNA-Bindeprotein Teil des Splicosoms ist, doch ist ansonsten
nur wenig bekannt über seine Zielgene oder darüber, welche molekularen
Mechanismen der Regulation durch RBM10 zugrunde liegen. In dieser Arbeit
konnten wir durch photoactivatable-ribonucleoside-enhanced Crosslinking und
Immunoprezipitation 89.247 Bindestellen von RBM10 in 6.396 Zielgenen
bestimmen. Diese Bindestellen finden sich vermehrt in der Nähe von 5’ und 3’
Splice-Sites in Introns und in Exons, was vermuten lässt, dass RBM10 eine
Rolle bei der Splicing-Regulation spielen könnte. Dazu passend konnten wir
auch feststellen, dass RBM10 speziell an U2 snRNA bindet. Durch RNA-
Sequenzierung konnten wir 281 Änderungen des Splicings nach Abreicherung und
356 nach Anreicherung von RBM10 bestimmen. Dabei sind die Änderung des
Splicings durch An- und Abreicherung von RBM10 größtenteils anti-korreliert.
Folglich hängt alternatives Splicing von der zellularen RBM10-Konzentration ab
und wird vermutlich direkt von dieser reguliert. Schließlich konnten wir
zeigen, dass die bei einem Patienten vorliegende in-frame Deletion von RBM10
dessen normale Lokalisierung im Zellkern verändert und auf diese Weise seine
Funktion bei der Splicing-Regulation stört. Insgesamt zeigen unsere
umfangreichen, Genom-weiten Daten, dass RBM10 ein neuer Splicing-Regulator
ist, der durch direktes Binden an pre-mRNA-Materiel wirkt und möglicherweise
das Zusammenspiel zwischen dem zentralen Splicingmechanismus und anderen
Splicing-Regulatoren koordiniert.
de
dc.format.extent
VI, 123 S.
dc.rights.uri
http://www.fu-berlin.de/sites/refubium/rechtliches/Nutzungsbedingungen
dc.subject.ddc
500 Naturwissenschaften und Mathematik::570 Biowissenschaften; Biologie
dc.title
Transcriptome wide characterization of target sites and alternative splicing
regulation mediated by RBM10
dc.contributor.contact
yongbo.wang@mdc-berlin.de
dc.contributor.firstReferee
Prof. Dr. Constance Scharff
dc.contributor.furtherReferee
Prof. Dr. Markus Wahl
dc.date.accepted
2012-12-20
dc.identifier.urn
urn:nbn:de:kobv:188-fudissthesis000000044808-7
dc.title.translated
Transkriptombreite Charakterisierung von Zielstellen und alternative, durch
RBM10 vermittelte Spleiß-Regulierung
de
refubium.affiliation
Biologie, Chemie, Pharmazie
de
refubium.mycore.fudocsId
FUDISS_thesis_000000044808
refubium.mycore.derivateId
FUDISS_derivate_000000012916
dcterms.accessRights.dnb
free
dcterms.accessRights.openaire
open access
