Regulation of early and late HIV-1 gene expression by alternative pre-mRNA splicing [Elektronische Ressource] = Die Regulation der frühen und der späten HIV-1 Genexpression durch alternatives prä-mRNA-Spleißen / vorgelegt von Corinna Asang

De
Publié par

Regulation of early and late HIV-1 gene expression by alternative pre-mRNA splicing Die Regulation der frühen und der späten HIV-1 Genexpression durch alternatives prä-mRNA SpleißenInaugural-Dissertation zurErlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf vorgelegt von Corinna Asang aus Düsseldorf April 2010 aus dem Institut für Virologie der Heinrich-Heine-Universität Düsseldorf Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Referent: Prof. Dr. H. Schaal Koreferent: Prof. Dr. D. Willbold Tag der mündlichen Prüfung: 27.10.2010 Zusammenfassung ______________________________________________________________________________ZusammenfassungDas alternative Spleißen stellt einen bedeutenden Mechanismus für die zeitlich regulierte HIV-1 Genexpression dar, der für die virale Replikation unentbehrlich ist. Zahlreiche cis-wirkendeElemente beeinflussen die Prozessierung der viralen prä-mRNA zu unterschiedlichen mRNA-Isoformen, die die frühe, intermediäre und späte virale Genexpression kennzeichnen. Die vorliegende Arbeit untersuchte spleiß-regulatorische Mechanismen, die entweder die Bildung früher mRNAs oder die Expression genomischer RNA steuern.
Publié le : vendredi 1 janvier 2010
Lecture(s) : 31
Tags :
Source : DOCSERV.UNI-DUESSELDORF.DE/SERVLETS/DERIVATESERVLET/DERIVATE-17820/DISSERTATION%20-%20CORINNA%20ASANG.PDF
Nombre de pages : 303
Voir plus Voir moins

Regulation of early and late HIV-1 gene expression by
alternative pre-mRNA splicing
Die Regulation der frühen und der späten HIV-1
Genexpression durch alternatives prä-mRNA Spleißen
Inaugural-Dissertation
zur
Erlangung des Doktorgrades der
Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf
vorgelegt von
Corinna Asang
aus Düsseldorf
April 2010 aus dem Institut für Virologie
der Heinrich-Heine-Universität Düsseldorf
Gedruckt mit der Genehmigung der
Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf
Referent: Prof. Dr. H. Schaal
Koreferent: Prof. Dr. D. Willbold
Tag der mündlichen Prüfung: 27.10.2010 Zusammenfassung
______________________________________________________________________________
Zusammenfassung
Das alternative Spleißen stellt einen bedeutenden Mechanismus für die zeitlich regulierte HIV-1
Genexpression dar, der für die virale Replikation unentbehrlich ist. Zahlreiche cis-wirkende
Elemente beeinflussen die Prozessierung der viralen prä-mRNA zu unterschiedlichen mRNA-
Isoformen, die die frühe, intermediäre und späte virale Genexpression kennzeichnen. Die
vorliegende Arbeit untersuchte spleiß-regulatorische Mechanismen, die entweder die Bildung
früher mRNAs oder die Expression genomischer RNA steuern.
Im ersten Teil der Arbeit wird gezeigt, dass ein zuvor identifizierter, mehrere Bindestellen
umfassender, purin-reicher exonischer Spleiß-Enhancer (GAR ESE) im Exon 5 durch die
bidirektionale Aktivierung der jeweiligen 3’ ss und der 5’ ss für den Einschluss der internen
alternativen Exons 4c, 4a, 4b und 5 in frühe rev- und nef-mRNAs entscheidend ist. Es wurde
erkannt, dass der GAR ESE eine doppelte spleiß-regulatorische Funktion ausübt, indem er (i)
den internen Exon-Einschluss durch alle identifizierten Bindestellen für die SR-Proteine
SF2/ASF und SRp40 synergistisch steigert und (ii) innerhalb der 3’ ss-Gruppe stromaufwärts
des Exons 5 die 3’ ss A5 ausschließlich durch die proximalen SF2/ASF-Bindestellen spezifisch
aktiviert. Die GAR ESE-vermittelte 3’ ss Selektivität wird vermutlich durch die
65phosphorylierungs-abhängige Stabilisierung von U2AF stromaufwärts der 3’ ss A5 ausgeübt.
Als weitere mögliche Faktoren für den GAR ESE-abhängigen Exon-Einschluss wurden die
spleiß-regulatorischen Proteine hTra2- , hnRNP H und hnRNP Q identifiziert. Es wurde
beobachtet, dass der interne Exon-Einschluss zusätzlich auf stromabwärts gelegene Elemente
des Exons 5, der 5’ ss D4 und der E42-Sequenz, angewiesen ist. Dies spricht für ein
regulatorisches Netzwerk, das sich über das Exon 5 spannt. Diese Interaktionen sind auch für
die Prozessierung intron-haltiger vpu/env-mRNAs essentiell. Daher reguliert der GAR ESE das
Spleißen der viralen prä-mRNA sowohl in der frühen als auch in der intermediären Phase der
HIV-1 Genexpression.
Im zweiten Teil dieser Arbeit offenbarte die Suche nach spleiß-regulatorischen Elementen, die
für die Expression genomischer HIV-1 RNA verantwortlich sind, dass die mit der 5’ ss D1
überlappenden Bindestellen für die SR-Proteine SC35 und SRp55, die Nutzung dieser
Spleißstelle reduzieren. Im Gegensatz dazu verstärkte die Bindung der RS-Domäne von SC35
in einer nicht-überlappenden stromaufwärts gelegenen Position das Spleißen an D1. Dies
deutet darauf hin, dass eine Konkurrenz zwischen SR-Proteinen und spleißosomalen
Komponenten um die Bindung an den D1 besteht. Dieses Modell wurde durch die Beobachtung
erhärtet, dass die weiter stromaufwärts des D1 liegenden Bindestellen für SR-Proteine und
hnRNP H die Nutzung der 5’ ss nicht beeinflussen. Darüber hinaus wurde beobachtet, dass D1-
flankierende Sequenzen die interne 3’ ss Auswahl und das Spleißen des distalen Introns
beeinflussen. Jedoch reicht der regulatorische Einfluss der dem D1 benachbarten Sequenzen
I
EZusammenfassung
______________________________________________________________________________
vermutlich nicht aus, um den beachtlichen Anstieg der Menge vollständig ungespleißter RNA in
der späten Phase der viralen Genexpression herbeizuführen. Ein wesentlicher spleiß-
regulatorischer Effekt des viralen Proteins Gag auf das Spleißen der HIV-1 mRNA konnte nicht
bestätigt werden. Hingegen wurde erkannt, dass das p17-ins Element stromabwärts des D1
durch die Wirkung als intronischer Spleiß-Enhancer das Spleißen an dieser Stelle kontrolliert.
Zugleich ist dieses Element für die Rev-abhängige Expression vollständig ungespleißter RNA,
aber nicht intermediärer vpu/env-mRNAs, entscheidend. Daher scheinen sich intron-haltige
intermediäre und späte virale mRNAs in ihren Sequenzvoraussetzungen für die Rev-
Abhängigkeit zu unterscheiden. Dies könnte die molekulare Ursache für die verzögerte
Expression der vollständig ungespleißten genomischen HIV-1 mRNA sein.
IISummary
______________________________________________________________________________
Summary
Alternative splicing is a major mechanism for temporally regulated HIV-1 gene expression
essential for viral replication. Numerous cis-acting elements affect viral pre-mRNA processing
into distinct mRNA isoforms characterising early, intermediate and late viral gene expression.
This thesis investigated splicing regulatory mechanisms controlling either the generation of early
mRNAs or the expression of genomic RNA.
In the first part of this work a previously identified purine-rich multisite exonic splicing enhancer
(GAR ESE) located in exon 5 was shown to be crucial for inclusion of the alternative exons 4c,
4a, 4b and 5 into early rev- and nef-mRNAs by bidirectionally activating the respective 3’ ss and
the 5’ ss. The GAR ESE was found to perform a dual splicing regulatory function (i) by
synergistically enhancing internal exon inclusion through all identified binding sites for the SR
proteins SF2/ASF and SRp40, and (ii) by specifically activating A5 of the 3’ ss cluster upstream
of exon 5 solely by the proximal SF2/ASF binding sites. GAR ESE-mediated 3’ ss selectivity is
65likely exerted by phosphorylation-dependent stabilisation of U2AF upstream of 3’ ss A5. For
GAR ESE-dependent exon inclusion the splicing regulatory proteins hTra2- , hnRNP H and
hnRNP Q were identified as further candidate factors. Internal exon inclusion was observed to
additionally depend on downstream elements of exon 5, i.e. 5’ ss D4 and the E42 sequence
indicating a regulatory network spanning across exon 5. These interactions are also essential
for processing of intron-containing vpu/env-mRNAs. Therefore, the GAR ESE regulates viral
pre-mRNA splicing in the early as well as in the intermediate phase of HIV-1 gene expression.
In the second part of this thesis, searching for splicing regulatory elements responsible for the
expression of genomic HIV-1 RNA revealed that binding sites for the SR proteins SC35 and
SRp55 partly overlapping D1 reduce 5’ ss efficiency. In contrast, tethering the SC35 RS domain
into a non-overlapping upstream position enhanced splicing at D1 indicating a competition of
SR proteins and spliceosomal components for binding to D1. This model was substantiated by
the finding that binding sites for SR proteins and hnRNP H further upstream of D1 did not affect
5’ ss usage. In addition, sequences flanking D1 were found to affect internal 3’ ss selection and
distal intron removal. However, the regulatory impact of the sequences neighbouring D1 is likely
not sufficient to mediate the striking increase in the level of completely unspliced RNA in the late
phase of viral gene expression. A significant effect of the viral protein Gag on splicing of the
HIV-1 mRNA could not be confirmed. In contrast, the p17-ins element downstream of D1 was
found to control splicing at D1 by acting as intronic splicing enhancer. At the same time, this
element is essential for Rev-dependent expression of completely unspliced mRNA, but not for
intermediate vpu/env-mRNAs. Therefore, intron-containing intermediate and late viral mRNAs
appear to differ in their sequence requirements for Rev-dependency. This might represent the
molecular basis for the delayed expression of completely unspliced genomic HIV-1 mRNA.
III
ETable of contents
______________________________________________________________________________
Table of contents
Zusammenfassung ........................................................................................................ I
Summary ...................................................................................................................... III
A. Introduction ................................................................................................... 1
A.1 The Human Immunodeficiency Virus Type 1 (HIV-1) – persisting a global health
challenge ...................................................................................................................................1
A.2 HIV-1 replication critically depends on RNA processing ..................................................... 2
A.2.1 Gene expression of HIV-1 ..........................................................................................................3
A.2.2 Alternative splicing of the HIV-1 pre-mRNA ensures complete expression of the viral
proteome ....................................................................................................................................6
A.2.3 Exploiting and finally overriding the cellular splicing pathway regulates temporal HIV-1
gene expression .........................................................................................................................8
A.3 Mechanism of pre-mRNA splicing .......................................................................................... 9
A.3.1 Spliceosome assembly and catalysis ........................................................................................ 9
A.3.2 Splice site-bridging interactions ...............................................................................................15
A.3.3 Splice site recognition – finding a needle in the haystack ....................................................... 17
A.3.3.1 5’ss recognition ..................................................................................................................18
A.3.3.2 3’ss recognition ..................................................................................................................20
A.3.4 Alternative splice site selection ................................................................................................27
A.3.5 Cis-acting splicing regulatory elements ................................................................................... 31
A.3.6 Splicing regulatory proteins ......................................................................................................37
A.3.6.1 SR proteins ........................................................................................................................37
A.3.6.2 Heterogeneous nuclear ribonucleoparticle (hnRNP) proteins ........................................... 43
A.3.6.3 Combinatorial control of alternative splicing by splicing regulatory proteins ..................... 47
A.4 Exploitation of cellular pre-mRNA splicing by HIV-1 ......................................................... 48
A.4.1 Cis-regulatory elements control splice site recognition in the HIV-1 pre-mRNA ..................... 48
A.4.2 Modulation of cellular splicing regulatory protein expression and activity after HIV-1
infection ....................................................................................................................................51
A.5 Expression of intron-containing HIV-1 mRNAs .................................................................. 52
A.5.1 Rev-mediated expression of intron-containing HIV-1 mRNAs ................................................. 53
A.5.2 Inhibitory elements interfere with the expression of intron-containing HIV-1 mRNAs ............. 58
A.6 Aim of this work .....................................................................................................................62
B. Materials and Methods ............................................................................... 63
B.1 Materials ..................................................................................................................................63
B.1.1 Chemicals, culture media and solvents ................................................................................... 63
B.1.2 Enzymes...................................................................................................................................65
B.1.3 Cells .........................................................................................................................................65
B.1.3.1 Prokaryotic cells.................................................................................................................65
B.1.3.2 Eukaryotic cells ..................................................................................................................66
B.1.4 Oligonucleotides .......................................................................................................................66
B.1.4.1 Cloning primer ...................................................................................................................66
B.1.4.2 Sequencing primer ............................................................................................................ 68
B.1.4.3 RT-PCR primer ..................................................................................................................68
B.1.4.4 shRNA ...............................................................................................................................70
B.1.4.5 In vitro transcription primer ................................................................................................ 70
B.1.5 Recombinant plasmids .............................................................................................................72
B.1.5.1 HIV-1 derived minigene plasmids ...................................................................................... 72
B.1.5.2 Plasmids for protein expression ........................................................................................ 79
B.1.5.3 Plasmids for recombinant U1snRNA expression .............................................................. 80
B.1.5.4 Control plasmids ................................................................................................................80
B.1.6 Antibodies ................................................................................................................................80
B.1.7 Algorithms and databases........................................................................................................82
B.1.7.1 Evaluation of splice site strength ....................................................................................... 82
B.1.7.2 Prediction of SR protein binding sites ............................................................................... 82
B.1.7.3 2D protein separation and identification ............................................................................ 82
B.2 Methods ...................................................................................................................................84
B.2.1 Cloning .....................................................................................................................................84
B.2.1.1 Polymerase-Chain-Reaction (PCR)................................................................................... 84
IVTable of contents
______________________________________________________________________________
B.2.1.2 Ligation .............................................................................................................................. 84
B.2.1.3 Transformation .................................................................................................................. 85
B.2.1.4 Analytical plasmid DNA isolation ....................................................................................... 85
B.2.1.5 Preparative plasmid DNA isolation .................................................................................... 86
B.2.1.6 DNA sequencing ................................................................................................................ 86
B.2.2 Expression and purification of recombinant T7 RNA polymerase ........................................... 87
B.2.3 Eukaryotic cell culture .............................................................................................................. 88
B.2.3.1 Maintenance of eukaryotic cells ........................................................................................ 88
B.2.3.2 Transfection of eukaryotic cells using FuGene 6 .............................................................. 88
B.2.3.3 Viral infection ..................................................................................................................... 89
B.2.3.4 shRNA-mediated knockdown of hnRNP H expression ..................................................... 89
B.2.3.5 Inhibition of SR protein phosphorylation ........................................................................... 90
B.2.4 Immunofluorescence ................................................................................................................ 91
B.2.5 Western Blot Analysis .............................................................................................................. 92
B.2.5.1 Cell harvesting ................................................................................................................... 92
B.2.5.2 Determination of protein concentration ............................................................................. 92
B.2.5.3 Normalisation of transfection efficiency using a Luciferase assay .................................... 92
B.2.5.4 Sodium Dodecyl Sulfate-Polyacrylamide gel electrophoresis (SDS-PAGE) ..................... 93
B.2.5.5 Western Blot ...................................................................................................................... 93
B.2.6 RNA isolation ........................................................................................................................... 93
B.2.6.1 Phenol/chloroform-based isolation of total RNA ............................................................... 94
B.2.6.2 Isolation of total RNA using anionic exchange columns ................................................... 94
B.2.6.3 Fractionation of cytoplasmic and nuclear RNA ................................................................. 94
B.2.7 RT-PCR .................................................................................................................................... 95
B.2.7.1 Two-step RT-PCR ............................................................................................................. 95
B.2.7.2 One-Step RT-PCR ............................................................................................................. 96
B.2.7.3 Native gel electrophoresis followed by EtBr staining ........................................................ 96
B.2.7.4 Denaturating gel electrophoresis of fluorescently labelled cDNA ..................................... 97
B.2.7.5 Isolation of cDNA from native polyacrylamide gels ........................................................... 97
B.2.8 RNA affinity chromatography ................................................................................................... 98
B.2.8.1 In vitro transcription ........................................................................................................... 98
B.2.8.2 Protein isolation by RNA affinity chromatography ............................................................. 99
B.2.8.3 In vitro dephosphorylation of HeLa nuclear extract ........................................................... 99
B.2.8.4 2D gel electrophoresis ..................................................................................................... 101
B.2.9 Protein sequencing by mass spectrometry ............................................................................ 102
B.2.9.1 In gel digestion and sample preparation ......................................................................... 102
B.2.9.2 Mass spectrometry .......................................................................................................... 102
B.2.10 Microfluidics analyses using Lab-on-a-chip assays ............................................................... 103
B.2.10.1 Protein analyses .............................................................................................................. 103
B.2.10.2 RNA analyses .................................................................................................................. 103
C. Results ....................................................................................................... 104
C.1 Splice site activation of the alternative exons 4c, 4a, 4b, and 5 during early and
intermediate HIV-1 gene expression .................................................................................. 106
C.1.1 Characterisation of the GAR ESE within exon 5 .................................................................... 110
C.1.1.1 The GAR enhancer is essential for inclusion of exon 5 into early mRNA isoforms. ....... 110
C.1.1.2 Individual SR protein binding sites of the GAR ESE synergistically increase
recognition of the alternative exons 4a and 5. ................................................................ 114
C.1.1.3 The proximal SF2/ASF binding site specifically contributes to A5 usage. ...................... 116
C.1.1.4 All SR protein binding sites of the GAR ESE are required to efficiently activate A5 for
the generation of intron-containing vpu/env-mRNAs. ..................................................... 117
C.1.1.5 A predicted third SF2/ASF binding site covering the intron/exon 5 border ensures
preferential activation of A5. ............................................................................................ 118
C.1.1.6 The GAR ESE is essential for activation of each of the competing 3’ ss clustering
upstream of exon 5. ......................................................................................................... 120
C.1.2 The GAR ESE – a key player in a network of regulatory elements across exon 5. .............. 124
C.1.2.1 GAR ESE mediated 3’ ss selectivity is augmented by an efficient downstream 5’ ss .... 124
C.1.2.2 Binding of U1 snRNP at D4 is necessary to activate the 3’ ss cluster in Rev-
dependent, intron-containing mRNAs ............................................................................. 128
C.1.2.3 The 3’ region of exon 5 contains an additional splicing enhancer. ................................. 128
C.1.3 The mechanism of GAR ESE enhancer function ................................................................... 132
65
C.1.3.1 The GAR ESE stabilises binding of U2AF at 3’ ss A5. ................................................. 132
VTable of contents
______________________________________________________________________________
C.1.3.2 The GAR ESE does not stabilise spliceosomal components SF1/mBBP and SAP155. .135
C.1.3.3 The GAR ESE promotes binding of the splicing regulatory protein hTra2- . .................. 136
C.1.3.4 Identification of additional proteins stabilised by the GAR ESE ...................................... 140
C.1.3.5 U2AF heterodimer binding to the 3’ ss (“complex formation”) requires a
phosphorylated factor. ..................................................................................................... 145
C.1.3.6 The impact of splicing regulatory protein phosphorylation on the function of the GAR
ESE complex in vivo ........................................................................................................ 148
C.1.4 Summary: A functional network emanating from exon 5 and flanking splice sites
regulates inclusion of exon 4c, a, b and 5. .............................................................................151
C.2 Expression of unspliced RNA during late HIV-1 gene expression ................................. 153
C.2.1 Identification of cis-acting splicing regulatory elements in the proximity of D1 ...................... 154
C.2.1.1 Individual SR protein binding sites overlapping D1 differentially affect D1 efficiency
and alternative 3’ ss selection. ........................................................................................ 154
C.2.1.2 Tethering SC35 into an upstream exonic position enhances D1 usage. ........................ 162
C.2.1.3 hnRNP H binding at exon 1 does not influence splicing at D1. ....................................... 165
C.2.1.4 Identification of additional proteins binding to exon 1 and D1 ......................................... 168
C.2.1.5 Sequences surrounding D1 do not affect D1 activation, but modulate downstream
3’ ss selection and distal intron removal. ......................................................................... 170
C.2.1.6 The viral Gag protein does not affect D1 activation. ....................................................... 172
C.2.2 The p17-inhibitory element in the viral pre-mRNA regulates temporal HIV-1 gene
expression. .............................................................................................................................178
C.2.2.1 The proximal 202 nt of the Gag-ORF are required for Rev-reactivity in the presence
of the p17-inhibitory element. .......................................................................................... 178
C.2.2.2 The Rev binding site in stem loop 1 does not contribute to Rev-mediated increase of
unspliced RNA. ................................................................................................................184
C.2.2.3 The first 202 nt of the p17-ins element contain an intronic splicing enhancer. ............... 187
C.2.3 Summary: Splicing at D1 is oppositely regulated by SR protein binding sites overlapping
the 5’ss and the downstream located intronic p17-ins element. ............................................ 189
D. Discussion................................................................................................. 192
D.1 The GAR ESE constitutes a main regulatory molecular hub for central splice site
activation of the HIV-1 pre-mRNA. ..................................................................................... 192
D.1.1 The GAR ESE is essential for exon recognition. ................................................................... 193
D.1.2 Exon recognition is synergistically activated by the SR protein binding sites of the GAR
ESE. .......................................................................................................................................194
D.1.3 The GAR ESE mediates 3’ ss selectivity by its proximal SF2/ASF binding sites. ................. 198
D.1.4 Mechanism of GAR ESE function .......................................................................................... 203
D.1.5 A functional network of exon-crossing interactions regulates splicing of the alternative
exons 4c, 4a, 4b and 5. ..........................................................................................................220
D.1.6 Future perspectives: Internal exon recognition mediated by the GAR ESE .......................... 224
D.2 Expression of genomic HIV-1 RNA in the late phase of viral replication ....................... 225
D.2.1 The p17-inhibitory element – a Janus regulator for the expression of genomic HIV-1 RNA . 227
D.2.2 D1 usage is suppressed by SR proteins binding to the 5’ ss. ................................................237
D.2.3 Sequences neighbouring D1 affect far distant splice site usage – implications for splicing
kinetics. ..................................................................................................................................239
D.2.4 Future perspectives: Regulation of D1 usage ........................................................................ 243
E. Conclusions .............................................................................................. 245
F. References ................................................................................................ 246
G. Appendix ................................................................................................... 284
G.1 Experimental data ................................................................................................................284
G.2 Abbreviations and units ...................................................................................................... 288
G.2.1 Abbreviations .........................................................................................................................288
G.2.2 Units .......................................................................................................................................289
G.3 Publications ..........................................................................................................................290
G.4 Curriculum Vitae ..................................................................................................................291
G.5 Erklärung ...............................................................................................................................292
Danksagung .............................................................................................................. 293
VI
E

Soyez le premier à déposer un commentaire !

17/1000 caractères maximum.