Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1297
Viruses as a Model System forStudies of Eukaryotic mRNA
Processing
BY
ANETTE LINDBERG
ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2003
Dissertation for the degree of Doctor of Philosophy, Faculty of Medicine, in Medical Virology presented atUppsala University in 2003
ABSTRACTLindberg, A. 2003. Viruses as a model system for studies of eukaryotic mRNA processing. Acta UniversitatisUpsaliensis. Comprehensive summaries of Uppsala Dissertations from the Faculty of Medicine 1297. 52 pp.Uppsala ISBN 91-554-5775-4
Viruses depend on their hosts for the production and spread of new virus particles. For efficient virus replication,the viral genes have adapted the strategy of being recognized and processed by the cellular biosyntheticmachineries. Viruses therefore provide an important tool to study the cellular machinery regulating geneexpression. In this thesis, we have used two model DNA viruses; herpes simplex virus (HSV) and adenovirus, tostudy RNA processing at the level of pre-mRNA splicing in mammalian cells.
During a lytic infection, HSV causes an almost complete shut-off of host cell gene expression.Importantly, HSV infection causes inhibition of pre-mRNA splicing which is possibly advantageous to the virus,as only four HSV genes contain introns.
The HSV immediate early protein, ICP27, has been shown to modulate several post-transcriptionalprocesses such as polyadenylation and pre-mRNA splicing. We have studied the role of ICP27 as an inhibitor ofpre-mRNA splicing. We show that ICP27 inhibits pre-mRNA splicing in vitro in the absence of other HSVproteins. We further show that ICP27 inhibits splicing at the level of spliceosome assembly. Importantly, ICP27-induced inhibition of splicing can be reversed, either by the addition of purified SR proteins, which are essentialsplicing factors, or by the addition of an SR protein-specific kinase, SRPK1. We propose that SR proteins areprime candidates as mediators of the inhibitory effect of ICP27 on pre-mRNA splicing.
In order to learn more about how splicing is organized in the cell nucleus in vivo, we investigated howcellular splicing factors are recruited to sites of transcription and splicing in adenovirus-infected cells usingconfocal microscopy. Our results showed that the SR proteins, ASF/SF2 and SC35, are efficiently recruited tosites in the nucleus where adenovirus genes are transcribed and the resulting pre-mRNAs are processed. Ourresults demonstrate that only one of the two RNA recognition motifs (RRMs) present in the ASF/SF2 protein isrequired for its recruitment to active sites of splicing. The arginine/serine rich (RS) domain in ASF/SF2 isredundant for the translocation of the protein to active viral polymerase II genes in adenovirus-infected cells.
Keywords: ICP27; herpes simplex virus; mRNA; splicing; adenovirus; ASF/SF2
Anette Lindberg, Department of Medical Biochemistry and Microbiology,Uppsala University, Box 582, S-751 23 Uppsala, Sweden
ISSN 0282-7476ISBN 91-554-5775-4
Printed in Sweden by Eklundshofs Grafiska AB, Uppsala 2003
To my family
Main references
This thesis is based on the following articles, which will be referred to in the text by theirroman numerals.
I. Anette Lindberg and Jan-Peter Kreivi. 2002. Splicing Inhibition at theLevel of Spliceosome Assembly in the Presence of HerpesSimplex Virus Protein ICP27. Virology 294, 189-198
II. Anette Lindberg and Jan-Peter Kreivi. 2003. Herpes Simplex Virus proteinICP27 induced inhibition of splicing is reversed by SR-proteins. Manuscript
III. Anette Lindberg, Margarida Gama-Carvahlo, Maria Carmo-Fonseca andJan-Peter Kreivi. 2003. A single RNA recognition motif in SR proteinsdirects them to nuclear sites of adenovirus transcription. Manuscript
The reprint (Lindberg and Kreivi) was made with permission from the publisher.
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Table of contents
Page1. Abbreviations 2
2. Introduction 3
2.1 Co-transcriptional RNA processing 4
3. Introduction to pre-mRNA splicing 8
3.1 The mechanism of pre-mRNA splicing 8
3.2 The chemistry of the splicing reaction 9
3.3 Components of the splicing machinery 10
3.4 The splicing process 13
3.5 Exon definition 16
3.6 Splicing enhancers and silencers 16
3.7 Organization of splicing factors in the cell nucleus 17
4. Herpes simplex virus-1 (HSV-1) 20
4.1 Structure and genome organization 20
4.2 Herpes simplex virus infection 20
4.3 Herpes simplex virus lytic life cycle 21
5. The multifunctional HSV-1 protein ICP27 22
5.1 ICP27 interacting proteins 22
5.2 Functional domains within ICP27 23
6. The role of ICP27 at the post-transcriptional level 24
6.1 ICP27 promotes RNA 3’ end processing at weak viral poly(A) sites 24
6.2 ICP27 stabilizes and binds to 3´ ends of labile mRNA 24
6.3 ICP27 stimulates the nuclear to cytoplasmic export of intronless 24
viral mRNAs6.4 ICP27 inhibits pre-mRNA splicing 25
6.5 ICP27 homologues in other herpes viruses 25
7. Adenovirus 27
7.1 Introduction 27
7.2 Nuclear organization in adenovirus-infected cells 28
8. Present investigation 29
8.1 Project 1, Papers I & II 29
8.2 Project 2, Paper III 35
9. Acknowledgements 38
10. References 39
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1. Abbreviations
ASF/SF2 alternative splicing factor/splicing factor 2DNA deoxyribonucleic acidDBP adenovirus DNA binding proteinGFP green fluorescent proteinhnRNPs heterogenous nuclear ribonucleoprotein particlesh p i hours post infectionHSV herpes simplex virusICP27 infected cell protein 27, a herpes simplex virus proteinIGC interchromatin granule clusterskDa kilo DaltonmRNA messenger ribonucleic acidNE nuclear extracts prepared from HeLa cellsNLS nuclear localization signalRNA pol II RNA polymerase IIRRMs RNA Recognition MotifsRS-domain arginine- and serine-rich domain in the SR family of splicing factorssnRNPs small nuclear ribonucleoprotein particlesSRrp SR-related proteinSRs serine-arginine proteins, splicing factorsS100 cytoplasmic extract prepared from HeLa cellsU2AF U2 snRNP Auxiliary Factor
3
2. Introduction
The genetic information of eukaryotic cells is stored as double stranded DNA
(dsDNA). The DNA is transcribed into a precursor messenger RNA (pre-mRNA), which is
processed and subsequently translated into proteins. Most newly synthesised pre-mRNA
contains stretches of non-coding RNA (introns) that must be excised by splicing before the
RNA can be translated into a protein. Although many of the splicing factors and components
of the splicing machinery are well characterized today, there are still gaps in our knowledge
about the complex process of splicing. By alternative splicing, one gene can express more
than one mRNA thus generating functionally diverse isoforms. Since the vast majority of
human genes contain introns and most pre-mRNAs undergo alternative splicing, disruption of
the normal splicing pattern is a common cause of human disease (reviewed in (Faustino and
Cooper 2003) (Blencowe 2000)).
Viruses have been used as model systems to study eukaryotic gene regulation for
decades. When a virus infects a cell, viral mRNA production is reliant, to a varying degree, on
the cellular transcription machinery. In addition, the viral mRNAs must be translated by the
cellular protein-synthesising machinery. Since viral genes have adapted to be recognized and
processed by the cellular machinery, they represent an important tool to study cellular gene
expression and regulation.
In order to study mRNA processing in mammalian cells at the level of mRNA
splicing, we have used two model DNA viruses; herpes simplex virus-1 (HSV-1) and
adenovirus. During lytic HSV-1 and adenovirus infections, almost all host cell gene
expression is turned off. Results from different studies indicate that in the case of HSV-1, a
viral protein termed ICP27 (Infected Cell Protein 27) blocks cellular gene expression by
inhibiting pre-mRNA splicing (Sandri-Goldin and Mendoza 1992; Hardy and Sandri-Goldin
1994; Bryant, Wadd et al. 2001; Sciabica, Dai et al. 2003). As only four of approximately 80
HSV genes contain introns, the virus is relatively resistant to the inhibition of splicing.
Projects I & II were initiated to investigate the role of ICP27 in the inhibition of pre-mRNA
splicing. Our aims were to establish an in vitro splicing system that would allow for the
characterization of how ICP27 inhibits the splicing process and reveal which cellular splicing
factor(s) are targeted.
In project III, we used adenovirus as a model system to learn more about how splicing
factors are organized in the cell nucleus in vivo. Relatively little is known about how splicing
4
factors are recruited to sites of active RNA polymerase II transcription and pre-mRNA
splicing. Generally, splicing factors are found throughout the nucleoplasm, but many of them
are concentrated in speckles. Numerous studies have concluded that inactive splicing factors
are recycled and temporarily stored in speckles (Huang and Spector 1996; Gama-Carvalho,
Krauss et al. 1997; Misteli and Spector 1998). When transcription is initiated, the splicing
factors are suggested to be rapidly recruited from the speckles to nuclear regions where they
are needed (Misteli, Caceres et al. 1997). The recruitment of splicing factors seems to be
dependent upon transcription and protein phosphorylation, but other than that, little is known
(Kuroyanagi, Onogi et al. 1998; Misteli and Spector 1999). Previous microscopy studies of
adenovirus infected cells have shown that there is a dramatic reorganization of the nucleus
during the intermediate phase of a lytic infection (Bridge and Pettersson 1995). At this stage,
splicing factors are recruited to sites where viral genes are transcribed and processed (Bridge,
Carmo-Fonseca et al. 1993). In this project, we investigated how the SR proteins, ASF/SF2
and SC35 are recruited to sites of transcription and splicing in adenovirus-infected cells using
confocal microscopy.
2.1 Co-transcriptional RNA processing
This chapter will briefly summarize how proteins are expressed in eukaryotic cells, and
describe the link between transcription, pre-mRNA splicing, and other RNA processes (see
Fig. 1). Although capping, splicing and polyadenylation can occur as independent processes
in vitro, recent studies indicate that most pre-mRNA processing occurs co-transcriptionally
rather than post-transcriptionally in vivo (reviewed in(Daneholt 2001; Bentley 2002)). In this
thesis, the term post-transcriptional refers to processes that occur after the RNA transcript has
left the exit channel of the polymerase.
The DNA is transcribed in the nucleus by multi-subunit enzymes, the RNA
polymerases (reviewed in (Lee and Young 2000)). In eukaryotic cells, there are three different
RNA polymerases; I, II and III, which synthesise different classes of cellular RNA. RNA pol
II transcribes mRNA and some small nuclear RNAs. All genes have a promoter area from
which transcription of the gene starts. Most RNA pol II promoters contain the transcription
start site, a TATA box, and DNA sequence elements to which regulatory transcription factors
bind. The RNA pol II needs general transcription factors in order to assemble at the promoter
and initiate transcription. After initiation, phosphorylation of the C-terminal domain (CTD) of
RNA pol II switches the polymerase activity to elongation of transcription (reviewed in (Lee
and Young 2000)).
RNA pol II
ds DNA
pre-mRNA
m7GhnRNPs
snRNPs
exon-junctioncomponents
splicingpolyadenylationediting
spliceosome
m7GAAAAAA
EJC
mRNA
nucleus
cytoplasm
mRNA export
nuclear pore complex
m7GAAAAAA
EJC
mRNA
translation degradation
protein
capping
Figure 1. mRNA biogenesis (adapted from (Dreyfuss, Kim et al. 2002)).
When the pre-mRNA is about 25 nucleotides long, capping enzymes bound to the
phosphorylated CTD of RNA pol II add the cap, an inverted methylated guanosine analogue,
to the 5´end. Only RNAs transcribed by pol II are capped at the 5´ end, and this is due to
direct binding of the capping enzymes to pol II (reviewed in (Schroeder, Schwer et al. 2000)
(Shatkin and Manley 2000)). The 5´ cap modification is important for protection against 5´ to
3´ exonucleases, and for recognition by the translation machinery through the cap-binding
complex (CBC), and the translation initiaton factor eIF4E (McKendrick, Thompson et al.
2001). The capping reaction can be further linked to the splicing process, as CBC also plays a
role in splicing of the first intron and promotes the nucleocytoplasmic export of snRNAs
(Izaurralde, Lewis et al. 1994; Izaurralde, Lewis et al. 1995).
Splicing of the new transcript can begin as soon as splice sites have been transcribed.
A direct connection between RNA pol II transcription and splicing have been proposed but it
5
6
is still somewhat controversial (reviewed in (Hirose and Manley 2000; Neugebauer 2002)). In
vitro splicing is stimulated by phosphorylated RNA pol II or by CTD alone ((Hirose, Tacke et
al. 1999)) and different types of promoters appear to dispose a specific splicing pattern to
transcripts initiated on the promoter ((Cramer, Pesce et al. 1997; Cramer, Caceres et al.
1999)). So far, the yeast U1snRNP component, Prp40 and the SR-related proteins called
SCAFs have been shown to associate with the CTD ((Yuryev, Patturajan et al. 1996; Kim, Du
et al. 1997)).
All eukaryotic mRNAs with the exception of histone mRNAs contain poly(A) tails.
Polyadenylation occurs when the cleavage polyadenylation-specificity factor (CPSF),
together with the downstream cleavage stimulatory factor (CstF) recognizes the poly(A)
consensus sequence, AAUAAA, on the mRNA. Following endonucleolytic cleavage of the
mRNA, poly(A) synthesis takes place. The formation of the poly(A) tail and the poly(A) tail
itself are important for termination of transcription as well as for mRNA stability, splicing,
transport from the nucleus to the cytoplasm and for translation (reviewed in (Zhao, Hyman et
al. 1999)). Cleavage/polyadenylation factors have also been clearly associated with the CTD
during transcriptional elongation (Hirose and Manley 2000). Polyadenylation and splicing
appear to be connected events in the recognition of 3´ terminal exon. For example, the
poly(A) polymerase interacts with 65 kDa U2 snRNP Auxiliary Factor, U2AF65, and this
interaction probably assists in exon recognition of terminal exons and promotes the assembly
of the polyadenylation machinery within the exon (Vagner, Vagner et al. 2000).
RNA editing is a co- or post-transcriptional process that changes the nucleotide
sequence of the substrate (Schaub and Keller 2002). RNA editing occurs in many organisms,
and operates by many different molecular mechanisms; either by insertion or deletion of
nucleotides or by base modification. Adenosine to inosine conversion catalysed by adenosine
deaminases in pre-mRNA coding or non-coding regions (introns, untranslated regions) can
alter the specificity of a codon or can affect splicing by altering splice-site sequences to
produce alternative splicing (Rueter, Dawson et al. 1999).
During transcription, the pre-mRNA is bound not only by splicing factors but also by
heterogeneous ribonucleoprotein particles (hnRNPs) (reviewed in (Kim and Dreyfus 2001)).
These are thought to induce proper processing and folding of the pre-mRNAs. The hnRNPs
remain bound after splicing is completed. Nucleo-cytoplasmic export occurs through the
nuclear pore complex (NPC), which provides docking sites for transport complexes. During
the export to the cytoplasm, some hnRNPs are removed at the NPC while others follow the
mRNA to the cytoplasm and shuttle back to the nucleus.
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The composition of the proteins bound to the mRNA is altered near the exon-exon
junction following splicing. A protein complex is deposited, known as the exon-junction
complex, (EJC), which serves as a binding platform for export factors (Zhou, Luo et al. 2000).
This complex consists of at least DEK, SRm160, Y14 and RNPS1 (Kataoka, Yong et al.
2000; Le Hir, Izaurralde et al. 2000; Le Hir, Gatfield et al. 2001). Importantly, this complex
provides a link between RNA splicing and export of the RNA. Three classes of factors appear
to be involved in mRNA export; adapter proteins that bind directly to the mRNA, receptor
proteins that recognize and bind to adapter proteins, and NPC components that mediate the
export across the nuclear membrane (reviewed in (Komeili and O'Shea 2001)). So far, two
pathways for export of mRNAs have been uncovered, involving two different export
receptors, CRM1 and TAP (reviewed in (Cullen 2000)). In one of the pathways, an RNA
binding adaptor protein, REF (RNA and export factor-binding protein), interacts directly with
the export receptor, TAP (Kang and Cullen 1999) and is recruited to mRNA sites near exon-
junctions as part of the EJC (Fig. 1) (Le Hir, Gatfield et al. 2001). At the EJC, REF becomes
bound to the spliced mRNA, which is then efficiently exported to the cytoplasm. REF is most
efficiently recruited to mRNA during splicing (Luo and Reed 1999).
After reaching the cytoplasm, the mRNA is translated into a protein by the ribosome, a
multi-component complex consisting of RNA and proteins. The mRNAs in the cytoplasm are
eventually subjected to degradation (reviewed in (Guhaniyogi and Brewer 2001)). The
poly(A) tail is gradually shortened, which in turn causes decapping at the 5´ end followed by
a 5´ to 3´-exonucleolytic degradation. RNA stability can also be regulated through AU-rich
elements present in the 3´untranslated regions of short-lived mRNAs.
Even if mRNAs are exported to the cytoplasm, they may be defective, due to
incomplete splicing or splicing errors. These defective mRNAs often contain premature
translation termination stop codons, which trigger their rapid degradation by a process termed
nonsense-mediated mRNA decay (also called mRNA surveillance) (Kim and Dreyfus 2001).
3. Introduction to pre-mRNA splicing
3.1 The mechanism of pre-mRNA splicing
Most newly synthesised mRNAs contain stretches of non-coding RNA (introns) that must be
excised before the RNA can be translated into a protein (see Fig. 2). The introns are removed
from the pre-mRNA by a large macromolecular complex called the spliceosome. The
consensus sequence elements at the splice sites of the transcript direct the assembly of the
spliceosome. In mammals, the 5´splice site signal is AG/GURAGU (exon/intron), (R =
purine). At the 3´ splice site, three different signals are used; the branch point sequence
(BPS), YNYURAC, (Y = pyrimidine, N = variable nucleotides), a polypyrimidine tract (10-
20 nucleotides) and the actual 3´ splice site, YAG/N (intron/exon) (Burge, Tuschl et al. 1999).
There is also a minor class of introns that have dinucleotides AU and AC at their 5´ and 3´
splice site and they are most often spliced by the minor spliceosome (Tarn and Steitz 1996).
AG GU A Y(10-20) AG
3'splice site 5'splice site
pre-mRNA
branch point sequence
polypyrimidine tract
EXON 1 EXON 2
INTRON
Figure 2. Pre-mRNA with the classical splicing signals found in the major class of introns.
The specificity of the splicing reaction is also dependent on exonic or intronic
enhancer and repressor elements (reviewed in (Blencowe 2000; Smith and Valcarcel 2000;
Hastings and Krainer 2001)). Many transcripts have multiple consensuses splice site
sequences whose activities are regulated by transacting factors binding to the enhancer or
repressor element. Alternative choices of splice sites and alternative splicing creates different
transcripts and subsequently, multiple protein products (see example in Fig. 3) (reviewed in
(Lopez 1998)). Alternative splicing is perhaps the most important mechanism for increasing
protein diversity in vertebrates (reviewed in (Graveley 2001)).
8
EXON 1 EXON 2EXON 1 EXON 3
EXON 1 EXON 2 EXON 3
EXON 1 EXON 3
Figure 3. Example of alternative splicing of a pre-mRNA transcript.
3.2 The chemistry of the splicing reaction
The pre-mRNA is spliced by two trans-esterification reactions (see Fig. 4). In the first,
reaction the 2´ hydroxyl group of the conserved A residue in the BPS of the intron attacks the
3´-5´ phosphodiester bond at the 5´ splice site. This results in cleavage at the 5´ exon-intron
junction and generation of two splicing intermediates; a 5´ exon with a free hydroxyl group at
its 3´ terminus, and the lariat intermediate with the 5´ end of the intron joined with the branch
site adenosine via a 5´-2´ bond. In the second reaction, the free hydroxyl group of the 5´ exon
attacks the 3´-5´ phosphodiester bond at the 3´ splice site and displaces the intron lariat with a
3´ hydroxyl group. This results in the joining of the two exons via a 5´-3´ phosphodiester
bond generating the spliced product (reviewed in (Burge, Tuschl et al. 1999)).
GU A Y(n) AG
3' 5'
2'OH
AG
5'GpA Y(n)
5'
3'
3'OH
PA G
5'Gp
3'OH
Step 1
Step 2
Figure 4. The catalytic steps in pre-mRNA splicing (adapted from (Gesteland 1999)).
9
10
3.3 Components of the splicing machinery
The spliceosome
In the major spliceosome, five small nuclear RNAs participate in the splicing reaction; U1,
U2, U4, U5 and U6 RNAs. These RNAs vary in length from 100 to 200 nucleotides and
associate with proteins to form small nuclear ribonucleoprotein particles (snRNPs) (reviewed
in (Will and Luhrmann 2001)). The RNA and protein components of snRNPs have essential
functions in recognition, selection and regulation of spliceosome assembly and catalysis. The
minor spliceosome uses the same biochemical splicing mechanism as the major spliceosome
but consists of a different set of snRNPs; U11, U12, U4atac, U6atac and U5 (reviewed in (Wu
and Krainer 1999)). The proportion of introns that are spliced by the minor (U12)
spliceosome is only about one in a thousand. This thesis will focus on splicing by the major
(U2) spliceosome.
The snRNP biogenesis
All U snRNAs, with the exception of U6 and U6atac, are transcribed by RNA pol II, capped
and exported to the cytoplasm where the U snRNP assembly is initiated (reviewed in (Will
and Luhrmann 2001)). The export of the capped U snRNAs requires an export adapter, PHAX
(phosphorylated adapter for RNA export), to mediate the interaction with the export receptor
CRM1/RanGTP (Ohno, Segref et al. 2000). In the cytoplasm, the U snRNA interacts in an
ordered, stepwise manner with seven Sm proteins. The Sm proteins interact as pre-formed
complexes with the Sm site of the U snRNA. After methylation of the cap and 3´ end
trimming, the U snRNP Sm core is imported to the nucleus. The methylated cap and the Sm
core domain form a U snRNP nuclear localization signal (NLS) required for the import of the
U snRNP. Import is mediated by the import factor, Snurportin-1, which binds to the
methylated cap on the snRNAs and to the general import factor, Importin-β.
After import, these factors dissociate and the specific proteins for each U snRNP,
which appear to be imported separately, associate with the U snRNP Sm core. Biogenesis of
the U6 snRNP, and presumably the U6atac snRNP differ from that of the other U snRNPs.
The U6 snRNA is transcribed by RNA pol III and assembly of the U6 snRNP is thought to
take place entirely in the nucleus.
The SR protein family of splicing factors
The SR proteins are a family of highly conserved serine/arginine-rich RNA binding proteins.
They are essential for constitutive splicing and regulate alternative splice site selection in a
concentration-dependent manner both in vivo and in vitro (Ge and Manley 1990; Krainer,
Conway et al. 1990; Zahler, Lane et al. 1992; Zahler, Neugebauer et al. 1993; Caceres,
Stamm et al. 1994). Some SR protein functions are shared by all members, but some functions
are specific to certain family members (reviewed in (Tacke and Manley 1999; Graveley
2000). SR proteins have been identified in all metazoan species examined, and the family
currently contains 10 members (see Fig. 5). Any member of the SR protein family can
complement splicing-deficient S100 extracts (cytoplasmic extracts from HeLa cells that
contain all components necessary for in vitro splicing except a functional SR protein)
(reviwed in (Fu 1995)).
SRp55
SRp20 RRM RS
SC35 RRM RS
SRp46 RRM RS
SRp54 RRM RS
9G8 RRM RS
SRp30c RRM RSRRM
ASF/SF2 RRM RSRRM
SRp40 RRM RSRRM
RRM RSRRM
SRp75 RRM RSRRM
Figure 5. The human SR proteins (Graveley 2000).
The SR proteins have a modular structure with an N-terminal RNA binding domain
consisting of either one or two RRMs (RNA Recognition Motifs) and a C-terminal domain
containing repetitive arginine-serine dipeptides, a so called RS domain. The RNA binding
domain provides specificity for binding certain mRNAs, while the RS domain interacts with
other proteins (Wu and Maniatis 1993; Kohtz, Jamison et al. 1994; Xiao and Manley 1997)
(Caceres, Misteli et al. 1997; Mayeda, Screaton et al. 1999). The RNA binding domain can be
exchanged between SR proteins and can bind RNA in the absence of the RS domain
11
12
(Chandler, Mayeda et al. 1997; Mayeda, Screaton et al. 1999). The RS domain can also be
functionally exchanged between SR proteins and it can even function when fused to a
heterologous RNA-binding domain (Chandler, Mayeda et al. 1997; Graveley and Maniatis
1998; Wang, Xiao et al. 1998).
The SR proteins are phosphorylated at serine residues within the RS domain and their
activity is thus regulated by this phosphorylation (Kanopka, Muhlemann et al. 1998; Wang,
Lin et al. 1998), (Xiao and Manley 1997; Xiao and Manley 1998). The two most extensively
studied protein kinases that have been shown to phosphorylate SR proteins are SRPK1 and
Clk/Sty (Gui, Tronchere et al. 1994; Colwill, Feng et al. 1996). Phosphorylated SR proteins
are required for the assembly of the spliceosome and dephosphorylation of the same proteins
are required for catalysis of splicing in vitro (Cao, Jamison et al. 1997). However, both hyper-
and hypophosphorylation of SR proteins can reduce their splicing activity (Kanopka,
Muhlemann et al. 1998; Prasad, Colwill et al. 1999).
SR proteins are ubiquitously expressed, but there are cell-type specific differences in
abundance. This suggests that different cell types might have a distinct pattern of relative SR
protein concentrations, which could influence regulation of alternative splicing (Zahler,
Neugebauer et al. 1993; Screaton, Caceres et al. 1995). The function of SR proteins in
alternative 5´splice site selection can be antagonized by the heterogeneous nuclear
ribonucleoprotein particle A1, hnRNPA1 (Eperon, Makarova et al. 2000). hnRNPA1 can
prevent binding of ASF/SF2 to enhancer elements in a concentration dependent manner
(Mayeda and Krainer 1992).
Only a few of the SR proteins in metazoans have been shown to be essential. SRp55 is
essential for proper development in Drosophila (Ring and Lis 1994) and ASF/SF2
(Alternative Splicing Factor/Splicing Factor 2) is essential for viability of chicken DT40 cells
(Wang, Takagaki et al. 1996). When the ASF/SF2 homologue, rsp-3 in C.elegans was
targeted by RNAi, an embryonic lethal phenotype was observed (Longman, Johnstone et al.
2000). However, individual silencing of six other SR protein-encoding genes had no effect on
embryonic development, although simultaneous silencing of all six was lethal (Longman,
Johnstone et al. 2000). The reason why only some of the SR proteins are essential is
unknown, either, the non-essential SR proteins do not participate in splicing of essential genes
or, alternatively, other SR proteins functionally substitute for the missing protein.
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SR-related proteins
There are also a number of SR proteins distinct from the classical SR proteins that are
required for splicing. These are collectively referred to as SR-related proteins, SRrps
(reviewed in (Blencowe, Bowman et al. 1999). The SRrps have an RS domain but not
necessarily an RNA binding domain. Although many of them are essential splicing factors
they cannot complement splicing-deficient S100 extracts. Examples include both subunits of
U2AF (Zhang, Zamore et al. 1992) (Zamore, Patton et al. 1992), snRNP components (like
U1-70K (Spritz, Strunk et al. 1987)), splicing regulators (like hTra2 (Dauwalder, Amaya-
Manzanares et al. 1996)), splicing co activators (like SRm160 (Blencowe B.J. 2000)), RNA
helicases (like hPrp16 (Zhou and Reed 1998)) and protein kinases (like Clk/Sty (Johnson and
Smith 1991)).
One of the SR-related proteins, SRp38 (also known as NSSR-1 (Komatsu, Kominami
et al. 1999), TASR-2 (Yang, Embree et al. 2000) or SRrp40 (Cowper, Caceres et al. 2001)),
has a typical SR protein structure but very unusual properties. Phosphorylated SRp38, cannot
activate splicing and is essentially inactive in splicing assays. However, dephosphorylation
converts SRp38 to a potent general repressor of splicing (Shin and Manley 2002) and cell
cycle-specific dephosphorylation of SRp38 has been proposed to play a role in gene silencing
during mitosis.
3.4 The splicing process
During the stepwise assembly of the spliceosome, the splicing reaction takes place in two
catalytic steps (see Fig. 6) (reviewed in (Hastings and Krainer 2001) (Reed 2000)). The first
complex formed in this pathway is the E-complex.
(i), Formation of the E-complex, also called the commitment complex, involves the
initial recognition of splice sites and commits the pre-mRNA to spliceosome assembly and
splicing. Thus, assembly of the E-complex is likely to be the key step at which alternative
splicing is regulated. It is initiated by U1 snRNP binding to the 5´ splice site by direct base
pairing of U1 snRNA, SF1 (Splicing Factor 1) binding to the adenosine nucleotide within the
BPS, 65 kDa U2AF binding to the polypyrimidine tract and 35 kDa U2AF to the AG
dinucleotide at the 3´ splice site (Gaur, Valcarcel et al. 1995; Berglund, Abovich et al. 1998)
(Eperon, Ireland et al. 1993). SF1 directs the spliceosome to the intron, and when SF1 leaves
the BPS, it assists in the base pairing of U2 snRNP to the same sequence. U2AF promotes the
binding of U2 snRNP to the BPS by direct interaction between the 65 kDa subunit and
SAP155, a component of U2 snRNP (Ruskin, Zamore et al. 1988). SR proteins also promote
14
assembly of the E-complex by assisting binding of both the U1 70K protein of U1 snRNP and
U2AF35, thus contributing to the bridging of the splice sites at early stages of the reaction
(reviewed in (Graveley, Hertel et al. 2001). Selection of 3´ splice sites can be AG-dependent
or AG-independent. The 35 kDa U2AF is essential for splicing of AG-dependent introns
which require the AG dinucleotide for spliceosome assembly, but dispensible for AG-
independent introns, which have an extended pyrimidine tract (Wu, Romfo et al. 1999).
(ii), U2 snRNP is believed to bind loosely to the BPS in the E-complex through its
subunits, SF3a and SF3b. During the subsequent formation of the A-complex, an ATP-
dependent process leads to stable U2 snRNP binding to the BPS (Das, Zhou et al. 2000). The
stable U2 snRNP binding require U2AF65-associated DEAH helicase, UAP56 (U2AF65
associated protein of 56 kDa) which is believed to facilitate the RNA-RNA rearrangements
(Fleckner, Zhang et al. 1997).
(iii), Subsequent binding of U5-U4/U6 allows the transition from A-complex to B-
complex. U5 binds to sequences in both the 5´ and 3´ exons and base pairs with the 5´ end of
U1 snRNA. These interactions bring the two splice sites together.
(iiii), The last complex, the C-complex, is formed when U6 snRNP dissociates from
U4, forms base pairing with U2 snRNP and replaces U1 snRNP as the factor interacting with
the 5´ end of the intron (reviewed in (Murray and Jarrell 1999)). It is believed that RNA
rearrangements activate the spliceosome for catalysis of splicing. The binding of U2 to U6
snRNA, positions the branch point and the 5´ splice site sequence for the first catalytic step,
generating the lariat intermediate and the free 5´ exon. Before the second catalytic step, the
spliceosome undergoes additional conformational changes, creating new RNA-RNA
interactions. The U2-U6 snRNP interaction is rearranged, and it makes contact with the 5’
splice site in the intron-lariat. Finally, the 5´ splice site in the free exon is joined together with
the 3´ exon of the lariat intermediate (Burge, Tuschl et al. 1999).
The splicing factor, hSlu7 is required to hold exon 1 tightly within the spliceosome in
order to select the correct AG at the 3´ splice site (Chua and Reed 1999). All steps in the
spliceosome assembly require ATP hydrolysis, except formation of the E-complex. It is
believed that the spliceosome consumes ATP to rearrange RNA-RNA and RNA-protein
interactions during assembly and the catalytic steps of splicing (reviewed in (Staley and
Guthrie 1998)).
3' splice site 5' splice sitepolypyrimidine tract
branch point
U1
U1
70K
70K
SR
SRSR
SR
U2AF35 U2
SF1
U2AF35
SF1
SR
SRSR
U2SF1 U2AF35
70KSR
SRSR
U2
U1
U1
70K
U2AF35
U4U5 U6
U6U4
U5
70KU1
U4U2
U5 U6SR
U2U5
U6
E-complex
A-complex
C-complex
B-complex
mRNA
ATP
ADP
ATP
ADP
ATP
ADP
Figure 6. The spliceosome assembly.
15
3.5 Exon definition
The average vertebrate gene consists of multiple internal exons (usually 300 nucleotides or
less) separated by introns that are considerably larger (possibly mega bases in length). Berget
and co-workers observed that a 5´ splice site on the downstream side of an exon promotes
splicing of the intron immediately upstream and proposed that initial recognition requires an
interaction between 5´ and 3´ splice sites across the short exon (<350 nucleotides) (see Fig. 7)
(Berget 1995). In this exon definition model, the binding of the U1 and U2 snRNPs and
associated factors, including U2AF65
, U2AF35
and SR proteins, define the exon. In the case of
terminal exons, it has been shown that the cap structure is essential for the recognition of the
first exon (Izaurralde, Lewis et al. 1994). Terminal exons that end with a poly(A) site use
polyadenylation factors for interactions across 3´ terminal exons (Berget 1995). More
recently, Lam et al demonstrated that a single exonic splicing enhancer element, ESE
promotes the recognition of both exon/intron junctions within the same step during exon
definition. They suggested that the exonic splicing enhancer element recruits a multi-
component complex that contains the minimal complement of components of the splicing
machinery required for 5´ and 3´ splice site selection (Lam and Hertel 2002).
ESE AG GU A Y(10-20) AG GUY(10-20) AG ESEU2AF 65 35 U2AF 65 35
SR70K
U170K
U1U2SR
SRSR
Figure 7. The exon definition model.
3.6 Splicing enhancers and silencers
Pre-mRNA splicing enhancers play a critical role in the regulation of alternative splicing and
in correct splice site recognition of constitutively spliced pre-mRNAs elements (reviewed in
(Blencowe 2000; Smith and Valcarcel 2000; Hastings and Krainer 2001)). Exonic splicing
enhancers, ESEs are required for excision of introns that contain splice sites that do not
conform to the consensus sequence (reviewed in (Varani G. 1998)). Although splicing
enhancers are usually located downstream of the affected introns, they are also found within
introns and upstream of regulated 5´ splice sites (Hastings, Wilson et al. 2001). Many of these
RNA elements are recognized by SR proteins, which are thought to function by recruiting
components of the general splicing machinery to the nearby splice sites (Sun, Mayeda et al.
16
17
1993; Liu, Zhang et al. 1998). The strength of splicing enhancers is determined by the relative
activities of the bound splicing factor, the number of proteins within the enhancer complex
and the distance between the enhancer and the intron (Graveley, Hertel et al. 1998a). The
enhancer elements are usually positioned less than 70 nucleotides from the 3´ splice site.
There is also increasing evidence that exons contain sequences that inhibit splicing, exonic
splicing silencers (Graveley, Hertel et al. 1998a). In at least some cases, silencing is mediated
by hnRNP proteins (heterogeneous nuclear ribonucleoproteins), such as hnRNPA/B and
hnRNP H (Del Gatto-Konczak, Olive et al. 1999). In vivo, hnRNP proteins are thought to
bind co-operatively to mRNA and form stable hnRNP complexes that in some cases can
compete with spliceosome assembly and with enhancer-bound SR proteins (Eperon,
Makarova et al. 2000; Zhu, Mayeda et al. 2001).
SR proteins may also mediate inhibition through the splicing silencers. Kanopka et al
found that SR proteins inhibit splicing of the regulated adenovirus L1 pre-mRNA by binding
an intronic repressor element, located immediately upstream of the IIIa branch site and
thereby preventing U2 snRNP recruitment to the spliceosome. They also demonstrated that
moving the IIIa repressor element to the second exon of the IIIa pre-mRNA converted the
repressor element to a classical splicing enhancer element (Kanopka, Muhlemann et al. 1996).
Thus, depending on where they bind (in the intron or exon), SR proteins may act as splicing
enhancers or silencers.
3.7 Organization of splicing factors in the cell nucleus
The splicing factors are diffusely distributed throughout the nucleus, with enrichment in
compartments commonly referred to as speckles (recently reviewed in (Misteli 2000; Lamond
and Spector 2003)). At the fluorescence-microscopic level, the speckles appear as irregular,
punctuated structures, which vary in size and shape and when examined by electron
microscopy they are seen as clusters of interchromatin granules (Thiry 1995).
Originally, speckles were characterized as sites having high concentrations of splicing
factors but more recently they have also been shown to contain transcription factors, 3´
processing factors and ribosomal proteins (Mortillaro, Blencowe et al. 1996; Schul, van Driel
et al. 1998; Mintz, Patterson et al. 1999; Dostie, Lejbkowicz et al. 2000). They are often
located close to highly transcribed genes but are not themselves sites of active transcription or
splicing (Misteli, Caceres et al. 1997; Cmarko, Verschure et al. 1999). Although several
proteins with possible structural roles in the nucleus, such as lamin A and snRNP-associated
18
actin, have been found in the speckles, no underlying scaffold has so far been identified
(Nakayasu and Ueda 1984; Jagatheesan, Thanumalayan et al. 1999).
One function of the speckles is thought to be recycling and/or acting as a temporal
storage and assembly site for splicing factors, but since they contain other RNA processing
factors and accumulate polyadenylated RNA (Carter, Taneja et al. 1991; Visa, Puvion-
Dutilleul et al. 1993), there are reasons to believe that the speckles could have more functions.
The speckles consist of numerous spherical sub domains referred to as sub speckles. Each
speckle is composed of 5-50 sub speckles. Spector and co-workers have proposed that the
compartmentalization into sub speckles may represent an efficient way of organizing these
factors for their subsequent transport to transcription/RNA processing sites (Mintz and
Spector 2000).
Splicing factors are also found in other nuclear structures such as Cajal bodies,
interchromatin-associated zones and perichromatin fibrils. The Cajal bodies contain newly
assembled snRNPs and small nucleolar ribonucleoproteins (snoRNPs), the latter being active
in ribosomal RNA processing. Recent evidence suggests that Cajal bodies may be involved in
coordinating the assembly and maturation of nuclear RNPs and possibly other
macromolecular complexes (reviewed in (Ogg and Lamond 2002)). The interchromatin-
associated zones are found adjacent to speckles and contain U1 snRNA (Visa, Puvion-
Dutilleul et al. 1993). The perichromatin fibrils can be observed by electron microscopy and
are believed to represent nascent transcripts (Fakan 1994).
The majority of pre-mRNA splicing probably occurs co-transcriptionally at the site of
transcription (Beyer and Osheim 1991; Xing, Johnson et al. 1993; Bauren and Wieslander
1994). The pre-mRNA is spliced, released from the site of transcription and exported to the
cytoplasm for translation. Transcripts that are not correctly spliced or cleaved at their 3´-ends,
accumulate at the site of transcription indicating that completion of splicing and 3´-end
processing is necessary for the release of transcripts (Custodio, Carmo-Fonseca et al. 1999).
However, highly expressed pre-mRNAs or pre-mRNAs with many introns, like the RNA for
the Balbiani ring of Chironomous tentants can be spliced after its release. Wetterberg et al
analyzed the splicing in the Balbiani Ring 3, (BR3), gene in Chironomous tentants and
showed in vivo that intron excision initiates co-transcripitonally in an overall 5´ to 3´ order.
The proximal 5´ introns of the BR3 transcript are excised co-transcripitonally while the most
distal 3´ introns are excised post-transcriptionally (Wetterberg, Bauren et al. 1996). This
suggests that complete splicing is not crucial for the BR3 mRNA to leave the site of
19
transcription, and that the timing of the release of the transcript depends more on the extent of
splicing that is necessary.
Changes in the overall trancriptional activity of a cell cause redistribution of splicing
factors. Inhibition of transcription results in immobilization of the splicing factors in enlarged
speckles (Misteli, Caceres et al. 1997). On the other hand, when transcription levels are high
during an adenovirus infection, the majority of the splicing factors are redistributed to viral
replication and transcription centres, where they accumulate in ring-like structures (Bridge
and Pettersson 1995). In contrast, during infection with herpes simplex virus, which only has
four genes containing introns, no obvious recruitment occurs, which suggests that the
accumulation of splicing factors at sites of active transcription is intron-dependent (Phelan,
Carmo-Fonseca et al. 1993). Similar results have been observed when transcribing artificially
intron-less genes (Huang and Spector 1996).
Several protein kinases and protein phosphatases are also localized to speckles
(Colwill, Pawson et al. 1996; Mintz, Patterson et al. 1999; Brede, Solheim et al. 2002).
Dephosphorylation is perhaps the major control mechanism for localization of at least the SR
protein family of splicing factors to speckles. Time-lapse microscopy experiments in living
cells show that release of splicing factors from speckles is inhibited in the presence of a
kinase inhibitor (Misteli, Caceres et al. 1997). Reassociation of splicing factors in speckles
requires the removal of one or more phosphate groups (Misteli and Spector 1996). This
supports the idea that speckles might be involved in regulating the pool of factors that are
accessible to the transcription/RNA processing machinery. Controlling the concentration of
splicing factors in the nucleoplasm might optimise the efficiency of splicing.
20
4. Herpes Simplex virus-1
4.1 Structure and genome organisation
Herpes simplex virus-1 (HSV-1) is a member of the Herpesviridae family. Herpes viruses
infect members of all groups of vertebrates and the same host can be infected with multiple
distinct types. To date, eight human herpes viruses have been described and each causes a
characteristic disease. HSV-1, for instance, causes oral cold sores and ocular lesions whereas
infections by two other human herpes viruses, Epstein-Barr virus and human herpes virus-8,
are linked to human cancer (reviewed in (Roizman 1996) (S.J Flint 2000)).
HSV-1 is a double stranded DNA virus with an icosahedral capsid surrounded by an
envelope. The virions of the herpes viruses, contain far more proteins than any other virus
described so far. Over half of the proteins encoded by the HSV genome are present in the
virion. The virion proteins are found in the nucleocapsid, in the layer between the envelope
and the nucleocapsid (the tegument layer) and embedded in the envelope. The diameter of the
virion is approximately 200 nm. The linear genome is organised into two unique segments;
one long region of 126 kb (UL) and one short region of 26 kb (US). Both the long and the
short region have inverted repeat sequences and terminal repeats. The HSV-1 can recombine
via the inverted repeats. The viral genome contains three origins of replication, one within the
UL and two within the US (reviewed in (Roizman 1996) (S.J Flint 2000)).
4.2 Herpes simplex virus-1 infection
HSV-1 infection occurs via skin or mucosa. The virus starts a primary replication in the
infected cells immediately after infection. During this phase, the virus travels via an axonal
transport mechanism into the nervous system and infects peripheral neurones. In the neurons,
the virus establishes a latent infection and hides in dorsal root ganglia. The nervous system is
an ideal hiding site for the virus since it is not patrolled by the immune system. However, the
major factor for latency is a restricted gene expression (reviewed in (Whitley 1996) (S.J Flint
2000)).
During a latent infection, normal viral transcription is blocked by an unknown
mechanism. The terminally differentiated neurones neither replicate nor divide, so once a
viral genome is established, it does not need to replicate to persist for the life of the neuron.
When reactivated, HSV-1 travels down the sensory nerves to mucosal surfaces where it
restarts the lytic life cycle, which causes the pathologic manifestation of cold sores. It is not
21
clear what causes the reactivation but there are many possible alternatives, including both
physical and psychological factors e.g. sunburn, stress and nerve damage. Some individuals
experience reactivation every two to three weeks while others experience no reactivation
(reviewed in (Whitley 1996; S.J Flint 2000)).
4.3 Herpes simplex virus-1 lytic life cycle
Herpes simplex virions attach to the cell when the viral envelope proteins bind to cellular
receptors, such as heparan sulphate, and to the extra cellular matrix. Thereafter, the virion
enters the cell by fusion of the viral envelope with the plasma membrane. The tegument
proteins are released into the cytoplasm and the uncoated virion is transported via
microtubules to the nucleus, where it docks at the nuclear pore and releases the viral DNA
into the nucleus. Some of the tegument proteins like vhs (virion host cell shut-off factor) stay
and act in the cytoplasm, while others are transported into the nucleus (reviewed in (Roizman
1996) (S.J Flint 2000).
During a productive infection, the HSV-1 genes are expressed in a temporal cascade.
After expression of the immediate-early genes (IE), the IE-gene products, in turn activates
transcription of the early genes (E) and auto regulate transcription of IE genes. The E proteins
function mainly in viral DNA replication and the production of substrates for DNA synthesis.
When sufficient levels of the viral replication proteins have accumulated in the cell, the
replication of viral genome starts and the expression of the late genes (L) starts. L proteins are
primarily structural proteins of the virion and proteins needed for virus assembly (reviewed in
(Weir 2001).
Newly replicated viral DNA is packaged into preformed capsids in the nucleus. Some
tegument proteins are added in the nucleus and others in the cytoplasm. It is not known if the
membrane with embedded glycoproteins of mature virions originates from the inner nuclear
membrane or are derived from the Golgi or the endoplasmatic reticulum. How the virion is
subsequently transported to the plasma membrane is also currently unknown. The enveloped
virion can either remain cell-associated or it can be released from the cell by exocytosis
for re-infection (reviewed in (Roizman 1996; S.J Flint 2000).
5. The multifunctional HSV-1 protein, ICP27
Herpes simplex virus-1 infected cell protein, ICP27 is a 63 kDa phosphoprotein, which is
essential for lytic infection and is the only IE protein that is conserved among all known
herpes viruses. ICP27 is a multifunctional protein: it regulates expression of both viral and
cellular genes at both the transcriptional and post-transcriptional levels (reviewed in
(Clements 1998)).
5.1 ICP27-interacting proteins
ICP27 has been shown to interact with a number of proteins (Table 1).
Table 1.
Protein Activity Method Reference
hnRNP K transcription activator, regulate
translation
yeast-two hybrid screen
in vivo binding studies
(Wadd, Bryant et al.
1999)
casein kinase
2
serine/threonine protein kinase yeast-two hybrid screen
in vivo binding studies
(Wadd, Bryant et al.
1999)
common
Sm proteins
protein components of
snRNP
in vivo binding studies
with anti-Sm serum
(Sandri-Goldin and
Hibbard 1996)
SAP 145 protein component
of U2 snRNP
yeast-two hybrid screen
in vivo binding studies
(Bryant, Wadd et al.
2001)
TAP cellular export receptor yeast-two hybrid screen
in vivo binding studies
(Chen, Sciabica et
al. 2002)
REF cellular RNA export factor yeast-two hybrid screen
in vivo binding studies
(Koffa, Clements et
al. 2001)
RNA pol II
holoenzyme
catalyses synthesis of
RNA
in vivo binding studies (Zhou and Knipe
2002)
P32 Cellular
(mainly mitochondrial )
multifunctional protein
yeast-two hybrid screen
in vivo binding studies
(Bryant, Matthews et
al. 2000)
SRp20 member of the SR protein
family of splicing factors
yeast-two hybrid screen
in vivo binding studies
(Sciabica, Dai et al.
2003)
SRPK1 SR protein kinase 1, highly
specific for serine/arginine
dipeptides
in vivo binding studies (Sciabica, Dai et al.
2003)
22
5.2 Functional domains within ICP27
ICP27 is an RNA binding protein that shuttles between the nucleus and the cytoplasm (see
Fig. 9). It has a nuclear export signal (Sandri-Goldin 1998) and nuclear and nucleolar
localization signals (Mears, Lam et al. 1995), that may control its sub-cellular localization.
The C-terminus of ICP27 is the most conserved part of the protein and is important for
its repressing and activating functions in viral transcription (Hardwicke, Vaughan et al. 1989;
Rice and Knipe 1990; Rice and Lam 1994). The extreme C-terminus of ICP27 contains an
Sm-homology domain, a structure that mediates protein-protein interactions between Sm
proteins associated with U snRNAs (Sandri-Goldin and Hibbard 1996). The amino-terminal
acidic region of ICP27 performs essential functions in viral DNA replication (Rice, Lam et al.
1993).
The structural domains in ICP27 involved in RNA binding are the RGG-box (Mears
and Rice 1996), and the KH-like domains, KH1, KH2 and KH3 (Soliman and Silverstein
2000). The RGG box is an arginine- and glycine-rich region similar to an RNA binding motif
found in a number of cellular proteins that are involved in nuclear RNA processing (Lengyel,
Guy et al. 2002). The KH-like domains bind single stranded RNA individually or in
combination. ICP27 has been shown to interact with a broad range of HSV-1 mRNAs through
its RGG box (Sokolowski, Scott et al. 2003).
NES 3-28
KH1288-350
KH2368-421
KH3451-512 Zink finger-like
407-512
Sm homology domain
262 327 512
repressor domain
activation domain
NLS109-138
RGG box138-152
nucleolarlocalisationsignal
CN
12 63
acidic domain
Figure 8. Schematic representation of the ICP27 protein (not to scale), (adapted from (Bryant,
Wadd et al. 2001)).
23
6. The role of ICP27 at the post-transcriptional level 6.1 ICP27 promotes RNA 3´ end processing at weak viral poly(A) sites
There are several well-documented examples in both mammalian and viral systems that
employ alternative or regulated usage of poly(A) sites as a mean of controlling gene
expression (Galli, Guise et al. 1988; Falck-Pedersen and Logan 1989). Poly(A) site usage is
dependent on the inherent poly(A) site efficiency and/or on the concentrations of components
of the 3´ processing complex. Some HSV-1 late mRNAs contain poly(A) addition signals that
function poorly in the absence of ICP27 (McLauchlan, Simpson et al. 1989; McLauchlan,
Phelan et al. 1992). UV cross linking experiments have shown that ICP27 enhances binding
of protein factors, including the 64 kDa subunit of CstF, to poly(A) sites of viral mRNAs
from viral mRNAs with weak poly(A) signals (McGregor, Phelan et al. 1996). The L poly(A)
sites, that responded to ICP27 were inherently less efficient than IE and E sites. This indicates
that their poly(A) site strength and their enhanced expression in the presence of ICP27 may
influence the temporal expression of the L genes. Whether cellular poly(A) site usage is
affected during an HSV infection is still unknown.
6.2 ICP27 stabilizes and binds to 3´ ends of labile mRNA
The intrinsic lifetime of an mRNA can be a critical parameter in the regulation of gene
expression. Within the 3´ untranslated regions of many mRNAs there are AU-rich sequences
that signal rapid turnover of the mRNA. Some proteins stabilize such mRNAs by binding to
these sequences whereas other factors can induce shortening of the poly(A) tail (reviewed in
(S.J Flint 2000)). ICP27 has been shown to bind and stabilize labile 3´ ends of mRNAs
(Brown, Nakamura et al. 1995). ICP27 was, by itself, able to increase the half-life and
stimulate the steady-state accumulation of reporter genes containing AU-rich untranslated
regions. However, the mechanism behind this stimulation is still unknown.
6.3 ICP27 stimulates the nuclear to cytoplasmic export of intronless viral mRNAs
All mRNAs made in the nucleus must be transported to the cytoplasm for translation. Cellular
pre-mRNAs that contain introns and splice sites are ordinarily retained in the nucleus until
they are completely spliced or degraded. Koffa et al recently demonstrated that ICP27
interacts with REF in order to stimulate the export of intronless viral mRNAs (Koffa,
Clements et al. 2001). Shortly after, Sandri-Goldin and co-workers confirmed that ICP27
24
interacts with REF for export of intronless viral mRNAs (Chen, Sciabica et al. 2002). They
also found an in vivo interaction between ICP27 and TAP but not with CRM1, demonstrating
that ICP27 interacts with REF to direct HSV-1 intronless mRNAs to the TAP export pathway.
6.4 ICP27 inhibits pre-mRNA splicing
In 1987, Martin et al demonstrated that an HSV-1 infection caused a redistribution of snRNPs
from a normal widespread speckled pattern to a highly punctuated organization in the nucleus
(Martin, Barghusen et al. 1987). It has subsequently been shown that ICP27 is necessary for
this event (Sandri-Goldin and Mendoza 1992; Phelan, Carmo-Fonseca et al. 1993; Hardy and
Sandri-Goldin 1994; Sandri-Goldin, Hibbard et al. 1995). Redistribution of splicing factors
like snRNP has also been shown during infection with adenovirus and influenza virus and
may reflect movement of the factors from active sites of splicing to inactive storage sites
(Bridge, Carmo-Fonseca et al. 1993; Fortes, Lamond et al. 1995). ICP27 was shown to
colocalize with the redistributed snRNPs at later time points of infection (Phelan, Carmo-
Fonseca et al. 1993) and Sandri-Goldin et al also demonstrated a physical interaction between
ICP27 and U1 snRNP (Sandri-Goldin and Hibbard 1996). In the same study it was shown that
pre-mRNAs with introns accumulated in the nucleus during wild type HSV infections but not
in cells infected with a mutant virus lacking ICP27. In a separate paper, it was shown how
splicing of a -globin transcript was inhibited in nuclear extracts prepared from HSV-1 wild-
type infected cells but not from cells infected with a mutant virus lacking ICP27 (Sandri-
Goldin 1994). These results suggested that ICP27 has a splicing inhibitory function, but it still
remained to be elucidated whether ICP27 is directly involved, or if another viral protein
whose production or activity is dependent on ICP27, causes the inhibition. More studies on
ICP27-induced splicing inhibition are presented and discussed in the section Present
investigation.
6.5 ICP27 homologues in other herpes viruses
ICP27 homologues have been identified in each of the herpes virus subfamilies (listed in
Table 2). The functional data that are available for ICP27 homologues indicate that although
these proteins show sequence similarity, they exhibit considerable functional diversity. For
example, the human cytomegalovirus (CMV) UL69 and varicella-zoster virus (VZV) ORF4
proteins primarily stimulate gene expression at the level of transcription (Moriuchi, Moriuchi
et al. 1994; Winkler, Rice et al. 1994; Defechereux, Debrus et al. 1997). In contrast,
functional studies of the Epstein-Barr virus (EBV) SM protein, herpes virus samiri (HVS)
25
ORF57, and human herpesvirus-8 (HHV-8) ORF57, indicate that these proteins function at
post-transcriptional steps of gene expression and that they shuttle between the nucleus and the
cytoplasm (Bello, Davison et al. 1999; Boyle, Ruvolo et al. 1999; Goodwin, Hall et al. 1999;
Gupta, Ruvolo et al. 2000; Kirshner, Lukac et al. 2000). The EBV SM protein activates
expression of intronless reporter gene constructs and inhibits expression of intron-containing
constructs (Ruvolo, Wang et al. 1998). SM also stimulates viral gene expression by enhancing
levels of cytoplasmic and nuclear EBV mRNAs and by promoting EBV mRNA export
(Semmes, Chen et al. 1998). Further, Boyer et al have shown that the SM protein has the
ability to stimulate growth of an HSV-1 ICP27-null virus, suggesting that SM and ICP27 may
regulate gene expression through a common pathway that is evolutionarily conserved between
these two herpes viruses (Boyer, Swaminathan et al. 2002).
Table 2.
Herpes virus Subfamily ICP27
homologue
Reference
herpes virus varicella-zoster virus (VZV)
equine herpes virus type 1 (EHV-
1)
ORF4
UL3
(Inchauspe, Nagpal et al.
1989)
(Zhao, Holden et al. 1992)
herpes virus human cytomegalovirus (CMV) UL69 (Chee, Bankier et al.
1990)
-herpes virus Epstein-Barr virus (EBV)
Herpes virus samiri (HVS)
human herpes virus 8 (HHV8-)/
Kaposi's sarcoma associated virus
(KSHV)
SM
ORF57
ORF57
(Cook, Shanahan et al.
1994)
(Nicholas, Gompels et al.
1988)
(Bello, Davison et al.
1999)
26
27
7. Adenovirus
7.1 Introduction
Over the years, many important discoveries have been accomplished by the use of adenovirus
as a model system. There are two genera of adenoviruses: Aviadenovirus (Avian) and
Mastadenovirus (mammals). The adenoviruses that infect humans include more than 50
immunologically distinct types, subdivided into six subgenera, A-F (reviewed in (Shenk
1996; S.J Flint 2000)). Adenoviruses most commonly cause respiratory tract infections, like
common cold, croup and bronchitis. Adenovirus has a linear double stranded DNA genome of
about 36 kb, which is encapsulated into a non-enveloped icosahedral capsid particle.
Adenovirus enters the cell by endocytosis, thereafter the virus particle is partially
dismantled, and the viral genome is deposited into the nucleus. The majority of the adenovirus
genes are transcribed by the host cell RNA polymerase II (reviewed in (Cann 1993; Shenk
1996; S.J Flint 2000)). In cell cultures, transcription of the viral genome starts immediately
upon entry into the nucleus and at 3-5 hours post-infection, approximately 15% of the mRNA
in the cytoplasm is virus-encoded (reviewed in (Pombo 1995)). The adenovirus genes are
classified into early and late genes. During the early phase, early region 1A (E1A) is the first
gene to be transcribed. E1A encodes for transacting factors required for the expression of
other early genes. The early phase of gene expression is followed by the onset of viral DNA
replication, which in cultured cells occurs approximately 6-8 h after infection (reviewed in
(Pombo 1995)). Replication of viral DNA is required for the transition to the late phase of
gene expression. Almost all late genes are transcribed from the major late transcription unit,
which by alternative splicing and alternative polyadenylation produces approximately 20
different mRNAs. Most of these mRNAs code for the structural components of the virion
(reviewed in (Cann 1993; Shenk 1996; S.J Flint 2000)). Each cell can produce up to 10 000
new virus particles. By 32 h after infection, the cellular content of protein and DNA is about
two fold higher than in normal cells (reviewed in (Pombo 1995)).
28
7.2 Nuclear organization in adenovirus infected cells
One of the first virus-induced structural changes observed in adenovirus-infected cells is
small inclusions in the nucleus that rapidly increase in size as viral DNA synthesis continues.
These inclusions are called viral replication centres and contain viral DNA and the viral 72K
DNA binding protein (Puvion-Dutilleul and Pichard 1992). The number of replication centres
is proportional to the multiplicity of the infection. Adenovirus DNA replication starts with a
protein priming mechanism at either end of the viral genome. At each replication fork, only
one of the two daughter strands is replicated producing a new duplex and a displaced single
strand of DNA (ssDNA). In a second round of replication the displaced ssDNA serves as a
template for second strand DNA synthesis (reviewed in (Cann 1993; Shenk 1996; S.J Flint
2000)). As the replication proceeds, the replication centres become larger and
compartmentalized, with the single stranded DNA in the core of the centre surrounded by
double stranded DNA that forms a network named the peripheral replication zone that is
active in both replication and transcription (Puvion-Dutilleul and Pichard 1992; Pombo,
Ferreira et al. 1994).
Intranuclear distribution of the splicing machinery varies during the course of an
adenovirus infection (Bridge and Pettersson 1996). During the early stages of infection,
shortly after the onset of viral replication, snRNPs and SR proteins are recruited to sites of
viral transcription. They accumulate into ring-like structures surrounding the viral replication
centres (Bridge, Carmo-Fonseca et al. 1993; Pombo, Ferreira et al. 1994; Bridge and
Pettersson 1995; Bridge, Riedel et al. 1996). As the adenovirus infection proceeds and more
late viral RNA is transcribed, splicing factors again accumulate in speckle-like structures that
become progressively enlarged (Bridge, Riedel et al. 1996). These enlarged speckles
concentrate splicing factors, poly(A) RNA and spliced viral mRNA (Bridge, Riedel et al.
1996), (Bridge, Xia et al. 1995). The localization of splicing factors in enlarged speckles has
been shown to correlate with late mRNA export to the cytoplasm and production of late
proteins (Aspegren, Rabino et al. 1998). This suggests that the enlarged speckles might have a
posttranscriptional role in the production of RNA.
8. Present investigation
8.1 Project 1, papers I & II
8.1.1 Introduction
This study was initiated in order to investigate how the HSV-1 ICP27 protein induces an
inhibition of pre-mRNA splicing and to increase our understanding of the basal mechanisms
behind splicing. HSV-1 appears to attack essential splicing factor(s) and thereby potentially
cause a complete shut-off of splicing. This is possible since the majority of HSV-1 genes are
intron-less. Only four of the approximately 80 HSV-1 genes contain introns, and only one is
expressed at late stages of infection. For this reason we believe that identification of the
factor(s) that are targeted by ICP27 could be of general importance since it might reveal
previously uncharacterized parts of the splicing machinery. Previously published in vivo and
in vitro studies have been based on analyses of the splicing phenotype in HSV-1 wild type or
ICP27 mutant virus infected cells. In this study, ICP27 was expressed in eukaryotic cells in
the absence of other HSV-1 proteins.
8.1.2 Results and discussion
Paper I
With the development of an ICP27-expressing adenovirus vector, we were able to produce
high amounts of the protein in eukaryotic cells in the absence of other HSV-1 proteins. The
target cells are co-infected with a viral vector that express the trans-gene ICP27 and a trans-
activator virus that constitutively produce an inducer-dependent trans-activator protein
(Edholm, Molin et al. 2001). Pre-mRNA splicing was studied in a cell-free in vitro system,
using two different 32
P-labeled exogenous pre-mRNA substrates: the cellular -globin pre-
mRNA and the standard adenovirus derived pre-mRNA splicing substrate. The labeled
exogenous pre-mRNA substrates were mixed with nuclear extracts and incubated under
optimal conditions for in vitro splicing. The splicing activity in ICP27 containing nuclear
extracts was compared with the activity in nuclear extracts prepared from cells infected with
AdICP27 and the trans-activator virus, but without addition of the inducer. Combined results
from these studies demonstrated that there is a drastic reduction of splicing products from
both the cellular and the viral pre-mRNA transcript in ICP27-containing extracts. These
results further suggest that the presence of ICP27 causes a general inhibition of splicing.
29
30
To determine at which stage of the splicing process the ICP27-mediated inhibition of
splicing was manifested, we analyzed the formation of spliceosomal complexes. The results
of this experiment showed a reduced rate of formation of the A-complex in ICP27-containing
extract, indicating that the splicing inhibition is already manifested at or before the formation
of the first energy-dependent spliceosomal A-complex. Further analysis of the conversion
rates of the complexes, from A-complex to B-complex, and B-complex to C-complex showed
that all complex conversion rates were reduced in ICP27-containing extracts. However, the
largest difference between ICP27-containing extracts and control extracts appeared to be the
formation of the mature spliceosomes, the C-complex. The different RNA-protein complexes
formed in ICP27 extracts also migrated notably faster during electrophoresis than their
counterparts formed in control extracts. This altered mobility might reflect a qualitative
and/or a quantitative difference between the complexes formed in the two types of extracts.
We also found that the splicing activity in the ICP27-containing extracts could be completely
recovered by addition of small amounts of competent control extracts. This suggesed that one
or more essential splicing factors are post-transcriptionally modified in ICP27-containing
extracts. When ICP27-containing extracts were added to the control extracts, inhibition of
splicing was not transferred unless the extracts were preincubated at 30°C. Longer incubation
times increased the level of splicing inhibition. These results could indicate that an enzymatic
activity in the ICP27-containing extracts targets an essential component(s) of the splicing
machinery.
To determine whether the splicing inhibition caused by ICP27 was specific, we
compared RNA polymerase II activity in ICP27 extracts and control extracts. A DNA
fragment containing the adenovirus major late promoter was incubated with 32P-labeled CTP
in ICP27-containing extracts or control extracts under conditions optimal for RNA
polymerase II transcription. The results demonstrated that there were no major differences in
RNA polymerase II activity between the ICP27-containing extracts relative to the controls.
This confirms that the splicing inhibition in ICP27 extracts is specific rather than a general
toxic effect of an overexpressed protein.
Our observation that splicing is inhibited at an early stage of spliceosome formation,
and that ICP27 causes a shift in mobility of the spliceosomal complexes is supported by
another study performed by Bryant et al, in which they showed that ICP27 interacts with
SAP145 during an HSV-1 infection (Bryant, Wadd et al. 2001). SAP145 is a component of
the SF3b particle, which is implicated in tethering U2snRNP binding to the branch site and is
therefore needed at an early stage of spliceosome assembly. Moreover, when ICP27 was
31
present, splicing in vitro was inhibited prior to the first catalytic step and B/C-complexes
formed during splicing increased in mobility and reduced in intensity.
Paper II
With the conclusions drawn from paper I, our next goal was to identify cellular splicing
factors that were targets for ICP27. We employed a biochemical complementation assay, in
which whole cell extracts were fractionated using ammonium sulfate, and magnesium
chloride precipitation steps. We found that several fractions from the ammonium sulfate
precipitation could restore the splicing activity in splicing incompetent ICP27 extracts. The
90% ammonium sulfate precipitate, however, was most efficient in restoring the splicing
activity. This fraction is highly enriched in the essential SR family of splicing factors. Since
previous studies have shown that SR proteins are prime targets for viral control (Kanopka,
Muhlemann et al. 1998; Estmer Nilsson, Petersen-Mahrt et al. 2001; Huang, Nilsson et al.
2002), we decided to continue with the 90% ammonium sulfate precipitate.
In the next step, we further purified the SR proteins present in the 90% ammonium
sulfate fraction using magnesium chloride precipitation. The highly purified SR proteins were
found to be very competent in activating splicing in ICP27-containing extracts. This suggest
that SR proteins on their own are capable of reversing ICP27-mediated splicing inhibition.
We also analyzed another essential splicing factor, U2AF65, for its capacity to restore splicing
activity in ICP27-containing extracts. In this case, we did not observe any activation of the
splicing activity, indicating that ICP27 specifically targets splicing factors of the SR protein
family.
Our next question was whether individual SR proteins are capable of restoring splicing
in ICP27 extracts. Also, it was important to exclude the possibility that the purified SR
protein fraction contained components other than SR proteins that were responsible for the
restoration of the splicing activity in ICP27-containing extracts. For this experiment we used a
bacterially produced ASF/SF2 protein. ASF/SF2 was co-expressed in E.coli together with SR
protein kinase 1, (SRPK1), which selectively phosphorylates serines in serine/arginine-rich
regions. Addition of increasing amounts of phosphorylated ASF/SF2 activated splicing in
ICP27-containing extracts. Collectively, these results suggest that ICP27 blocks splicing by
inhibiting the SR protein family of splicing factors. Thus, addition of the whole family of SR
proteins or a single SR protein is sufficient to reverse ICP27-mediated splicing inhibition.
The effects of SR proteins on splicing depend upon the phosphorylation status of the
protein. SR proteins in their active form are phosphorylated, whereas both
32
hypophosphorylation and hyperphosphorylation decrease their activity as splicing factors
(Kanopka, Muhlemann et al. 1998), (Xiao and Manley 1997). To investigate whether ICP27
inhibits splicing by changing the phosphorylation status of SR proteins, we analyzed different
ICP27-containing nuclear extracts. We found that the majority of the SR proteins were
dephosphorylated in extracts containing ICP27. Finally, we analysed whether phosphorylation
of SR proteins in the ICP27-containing extracts could restore the splicing activity. For this
experiment we added purified SRPK1 to ICP27 extracts. Although the effect was less
dramatic compared to addition of ASF/SF2, or purified SR proteins, addition of SRPK1 also
resulted in an increase in the splicing activity in ICP27 extracts.
The inactivation of SR proteins by ICP27-mediated dephosphorylation is thus perhaps
reversible, since addition of a kinase to the splicing reaction probably phosphorylates SR
proteins to the extent that they can activate splicing. Another indication that SR proteins are
reversibly dephosphorylated in ICP27-containing extracts comes from the observation that
addition of SR proteins purified from ICP27-expressing cells restores splicing activity to
ICP27 extracts. Western blot and coomassie analysis of the proteins in the ICP27 SR fraction
demonstrated that they were phosphorylated. It is most likely the SR proteins we purify from
the whole cell extract become phosphorylated during the purification process and regain their
activaty as splicing factors.
Phosphorylation of SR proteins also influences their sub cellular distribution:
dephosphorylated SR proteins are localized to nuclear speckles whereas phosphorylated SR
proteins are localized to sites of active transcription (Koizumi, Okamoto et al. 1999). Previous
studies have shown that essential splicing factors are redistributed in HSV-infected cells
(Phelan, Carmo-Fonseca et al. 1993; Sandri-Goldin, Hibbard et al. 1995). Since SR proteins
in nuclear extracts prepared from ICP27-expressing cells were found to be dephosphorylated,
we analysed the distribution of ASF/SF2 in ICP27-expressing cells by immunofluoresence.
HeLa cells were transfected with a plasmid expressing GFP-ICP27 and the distribution of
endogenous ASF/SF2 was monitored by monoclonal antibody 103. In cells expressing ICP27,
ASF/SF2 accumulated in speckles, which are noticeable larger than in control cells.
Moreover, ICP27 is not present in the enlarged speckles with ASF/SF2, and instead, often
appears to be surrounding them. This result showed that expression of ICP27 causes a
redistribution of ASF/SF2 in the nucleus, and that this may be a consequence of an ICP27-
mediated dephosphorylation of ASF/SF2.
33
Based on these results, we concluded that the SR protein family of splicing factors are
main targets for ICP27 and that the activity of SR proteins as regulatory splicing factors is
reduced in ICP27-expressing cells by dephosphorylation.
A major finding was recently published by Sciabica et al (Sciabica, Dai et al. 2003). In
this study they show that ICP27 interacts with SRPK1 to mediate splicing inhibition by
altering the SR protein phosphorylation. Sciabica et al also reported that during an HSV-1
infection, phosphorylation of several SR proteins was reduced and that this correlated with a
sub-nuclear redistribution of the SR proteins. Further, ICP27 was shown to interact
specifically with SRp20 both in vivo and in vitro. ICP27 interaction with SRPK1, alters
SRPK1’s activity and relocalizes it to the nucleus. In conclusion, they propose that ICP27
recruits SRPK1 to the nucleus and interacts with SR proteins at the site of spliceosome
assembly. Their finding thus supports our observation that SR proteins are dephosphorylated
in ICP27-expressing cells.
8.1.3 Perspectives
To date, two other DNA viruses, adenovirus and vaccinia virus have been reported to regulate
the cellular splicing machinery by inducing a dephosphorylation of the SR family of splicing
factors (Kanopka, Muhlemann et al. 1996; Estmer Nilsson, Petersen-Mahrt et al. 2001;
Huang, Nilsson et al. 2002). SR proteins prepared from late adenovirus-infected cells are
inactivated by a virus-induced partial de-phosphorylation. This modification enhances the
production of the alternatively spliced late mRNA IIIa, which otherwise is repressed by
phosphorylated SR proteins binding to an intronic repressor element, the 3RE (Kanopka,
Muhlemann et al. 1998). Presumably, splicing of most late stage-specific adenovirus mRNAs
is stimulated by this virus-induced dephosphorylation of SR proteins. Most adenovirus late
genes are expressed from the major late transcription unit, which by alternative splicing and
alternative polyadenylation, produce approximately 20 different mRNAs. Adenovirus is
therefore dependent on the cellular splicing machinery for processing its own pre-mRNAs.
During an adenovirus infection, the splicing machinery is not functionally blocked, and
instead, a partial dephosphorylation of SR proteins appears to promote a temporal shift into
the late stage of infection and production of late viral mRNAs.
Vaccinia virus, on the other hand, replicates in the cytoplasm and encodes for genes
lacking introns. This suggests that this virus could benefit from a total block of the splicing
machinery for maximal expression of its own genes. When SR proteins from vaccinia virus-
infected cells were analysed, they were indeed found to be hypophosphorylated and
34
functionally inactivated as splicing regulatory proteins. Re-phosphorylation of these SR
proteins only partially restored the splicing enhancer or splicing repressor activity (Huang,
Nilsson et al. 2002). It is possible that vaccinia viruses use an alternative, or additional
mechanism, to alter the phosphorylation status of the SR proteins compared to adenovirus.
Adenovirus has been shown to induce dephosphorylation of SR proteins by the virus-encoded
E4ORF4, which binds to the cellular protein phosphatase 2A, (PP2A), and directs it to SR
proteins for dephosphorylation. So far, it has not been elucidated how vaccinia virus
inactivates SR proteins as splicing regulatory proteins. However, vaccinia virus encode for its
own dual protein phosphatase, VH1, which is required for virus growth and gene expression
(Liu, Lemon et al. 1995). VH1 may be a possible candidate for causing SR protein
dephosphorylation.
In this study, we present a third DNA virus, HSV-1, which uses the SR protein family
of splicing factors to regulate the splicing machinery. HSV-1 only has four intron-containing
genes, so it would benefit from blocking most of the splicing activity in the cell. In the study
presented in paper II, the SR family of splicing factors appeared to be functionally inactivated
by a reversible dephosphorylation. One question that remains is how the SR proteins are
dephosphorylated and whether ICP27 targets more components of the splicing machinery to
induce splicing inhibition. The accumulation of hypophosphorylated SR proteins in HSV-1-
infected cells could be induced by at least three different mechanisms; i, by inhibiting a
protein kinase(s) responsible for SR protein phosphorylation; ii, by inducing
dephosphorylation by a phosphatase and iii, by inducing or encoding for a protein that blocks
SR protein phosphorylation.
Sciabica et al have proposed that ICP27 prevents SRPK1 phosphorylation of SR
proteins by interacting with and changing its activity and cellular localization (Sciabica, Dai
et al. 2003). ICP27 has been shown to interact with numerous proteins (listed in Table 1, page
25). The cellular p32 is another protein with which ICP27 interacts, and which could play a
role in the ICP27 induced inhibition of splicing (Bryant, Matthews et al. 2000). p32 has been
shown to interact with and inhibit ASF/SF2 as a splicing factor by blocking phosphorylation
of its RS domain (Petersen-Mahrt, Estmer et al. 1999). It is possible that the interaction of
ICP27 with p32 is used to direct p32 to SR proteins to inhibit their phosphorylation.
35
8.2 Project 2, paper III
8.2.1 Introduction
This study was undertaken in order to investigate how cellular splicing factors are recruited to
sites of splicing in the cell nucleus. We compared the recruitment of SR-proteins ASF/SF2
and SC35 to active sites of splicing with splicing factors U2AF65 and snRNPs, during an
adenovirus infection. We also analysed the contribution of the different domains of ASF/SF2
for the recruitment to active sites of viral transcription and splicing.
8.2.2 Results and discussion
In order to analyse the spatial distribution of ASF/SF2 during an adenovirus infection, HeLa
cells were infected with adenovirus and fixed and permeabilized after different times of
infection. The sub-nuclear distribution of ASF/SF2 was first determined by
immunofluorescence confocal microscopy using a monoclonal antibody directed against the
endogenous protein. The viral replication centres were visualized using a separate antibody
directed against the viral 72K DNA-binding protein. The results showed that the distribution
of ASF/SF2 during an adenovirus infection is dramatically altered. At an intermediate stage of
infection, at 12-16 hours post-infection (hpi), almost all detectable ASF/SF2 had accumulated
at sites surrounding the viral replication centres. It is in the immediate vicinity of these
structures that transcription and splicing is predominantly detected. This indicates that
ASF/SF2 is recruited to the nuclear sites where adenovirus genes are transcribed and
processed. At late stages of infection, 20 hpi, ASF/SF2 is found in so-called enlarged speckles
distant from the replication centres.
The staining pattern from endogenous ASF/SF2 detected with the monoclonal
antibody was confirmed by transient expression of a myc- or Green Fluorescent Protein-
tagged (GFP) version of the protein. The transfected cells were subsequently superinfected
with adenovirus. Both the myc- and the GFP-tagged ASF/SF2 proteins displayed similar, if
not identical, distribution pattern to endogenous ASF/SF2 during an adenovirus infection.
U2AF65 is similar to ASF/SF2 in that it contains an RNA-binding domain and an RS domain.
Previous studies have demonstrated that an adenovirus infection induces a redistribution of
U2AF65 into ring-like structures that colocalize with the sites of viral transcription (Gama-
Carvalho, Krauss et al. 1997). GFP-ASF/SF2 distribution was compared with U2AF65 in
order to examine if ASF/SF2 is recruited to the same ring-like structures as U2AF65 during an
36
adenovirus infection. Both proteins were present in ring-like structures at the intermediate
stage of infection and in enlarged speckles at the late phase of infection. When superimposing
the GFP-ASF/SF2 and U2AF65 signals, it is apparent that they colocalize during the
adenovirus infection. Although U2AF65 appears to be more evenly distributed throughout the
nucleus of an infected cell compared to ASF/SF2, they show a clear colocalization in the ring-
like structures. SC35 and ASF/SF2 are similar in size and both play a role in constitutive and
alternative splicing. However, SC35 has only one RRM and it does not shuttle between the
cytoplasm and the nucleus. When ASF/SF2 distribution is compared to that of SC35 during
an adenovirus infection, they display the same pattern of redistribution. Further, a confocal
overlay of the signals shows an almost identical distribution pattern. This demonstrates that
SC35, like ASF/SF2, accumulates in the ring-like structures in which adenovirus genes are
actively transcribed and processed.
Experiments with mutant proteins showed that the two RNA-binding domains present
in ASF/SF2 are sufficient for efficient recruitment of the protein to the ring-like structures
surrounding viral replication centres. The ASF mutant, in which the two RNA binding
domains were deleted (GFP-RS), was evenly distributed throughout the nucleus and was not
notably redistributed during an adenovirus infection. The protein did not accumulate in ring-
like structures or enlarged speckles during the infection.
These results indicate that ASF/SF2 is recruited to sites of viral transcription and
splicing by a different mechanism compared to that previously shown for the essential
splicing factor U2AF65 (Gama-Carvalho, Krauss et al. 1997). Localization of U2AF65 to these
sites is totally dependent upon an intact RS domain. The RS domains of both ASF/SF2 and
U2AF65 have been shown to mediate protein-protein interactions with other splicing factors.
In the case of U2AF65, the RS domain has also been shown to be necessary for efficient
binding of U2 snRNA to the branch site in pre-mRNA, perhaps by binding to one of the two
RNA components (Valcarcel, Gaur et al. 1996). Previous studies have shown that a virus-
induced dephosporylation of the majority of the SR proteins, including ASF/SF2, late during
an adenovirus infection reduces their activity as splicing enhancer or splicing repressor
proteins (Kanopka, Muhlemann et al. 1998). Despite this, we observed that ASF/SF2
accumulates in ring-like structures where late adenoviral genes are transcribed and their pre-
mRNAs processed. This may suggest that the hypophosphorylated SR proteins are also
needed for late viral mRNA processing or that the SR proteins are required for some process
other than splicing. ASF/SF2 has been shown to interact directly both with the general mRNA
export factor TAP (Huang, Gattoni et al. 2003) and with the transcriptional coactivator, p52
37
(Ge, Si et al. 1998). Thus, it is possible that ASF/SF2 may be required for efficient
transcription of adenovirus genes and/or in export of viral mRNAs.
8.2.3 Perspectives
Cellular compartments are important for functions of the cell and for the regulation of gene
expression (reviewed in (Carmo-Fonseca 2002)). Molecules can move between cellular
compartments in different ways. Nuclear compartments differ from most cytoplasmic
compartments in that they are not surrounded by membranes. Recent data from imaging of the
motion of macromolecules inside the living cells have shown that splicing factors are in
continuous flux between nuclear compartments which are not separated by a physical barriers
(Eils, Gerlich et al. 2000). As proteins diffuse through the nuclear space, they transiently
accumulate in a steady-state compartment by interactions with high-affinity binding sites.
According to this view, sequences and domains that are necessary for proper localization
within the cell, for example the nuclear retention signal within hnRNP C, would more likely
act as retention signals rather than as targeting or transport signals. Consequently, the
recruitment of splicing factors to sites of splicing would not be a directed process where the
factors are transported from storage in speckles to the active splice site. Instead, the steady-
state level of the factors would increase dynamically at the active splice site because of the
prolonged binding and interactions by the factors. The RS domain of ASF/SF2 has been
reported to function in the dissociation of the protein from speckles, and phosphorylation of
serine residues is a prerequisite for this event (Misteli, Caceres et al. 1998). It is possible that
the dephosphorylated RS domain has a higher binding affinity to the other components of the
speckles, which cause retention of the protein in speckles. The observation from this study
that the RS domain of ASF/SF2 is not needed for accumulation of the protein at sites of viral
transcription and splicing implies that interactions with the RS domain are not sufficient to
anchor the protein at the transcription/splice site. Instead it is the affinity of the RRMs of
ASF/SF2 that anchor the protein to the site of active splicing. Whether ASF/SF2 is anchored
to the activated viral gene by interaction with an RNA component and/or a protein and
whether this is unique for viral genes or is universal for metazoan genes remains to be
established.
9. Acknowledgements
First, I should like to thank my enthusiastic supervisor, Peter Kreivi for all the effort put in
supervision and for incredible patience and support over the years. Next, I would like to thank
Göran Akusjärvi and Catharina Svensson for guidance and making our Friday seminars into
scientific adventures. I am also grateful for all their support during the writing of this thesis.
Special thanks to:
My family, who has contributed to this thesis in many ways, you have always encouraged me
and made me believe that anything is possible.
I would like to thank:
My father, for sharing his interest in science and biology.
My mother, for giving me some of her determination and patience, which has been very
useful during these years.
My sister and her lovely family; Niklas, Emma-Lisa, Linnea och Isak, for always being there.
A visit at your place is always a real energizer!
Sven for all love and for being my best friend, no one is more alive than you! With you by my
side even difficult things are easy.
The Kilander family; Axel, Lennart, Elisabet, Sara, Emil, Helena, Lukas och Tova for all their
encouragement.
My godmother, Eva-Lotta for all her support.
All my friends and climbing partners, you are all included, to write about all of you would fill
up another, much longer thesis. You have given me the best of times!!
I look forward to more adventures with you!
I should also like to thank:
Everyone in the lab for all the good fun, help and discussions,
Christina (I will miss our chats and good discussions in the office), Tanel (collaborations in
the future?!), Cecilia (thank you for all fun and for correcting my English), Saideh, Anette,
Josef (I liked all the “fika” you brought), Gunnar, Ellenor, Ning, Anders, Vita, Raj, Margret
(meet again in Norway?), Daniel and Shaomin……I will miss you!
Erica, for answering all my questions at any time
Maud, Kerstin, Ylva and Tony for all assistance with administration, computer service and
technical matters
Lena Möller, always there to cheer us up
Göran Magnusson and Stefan Schwartz
All colleagues at IMBIM
Former members of the group; Elisabet Boija, Camilla Estmer-Nilsson, Anders Sundkvist,
Magnus Molin, Brian Collier and Lisa Wiklund (my personal running-trainer, I will miss all
the training, talking and laughing).
38
39
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Acta Universitatis UpsaliensisComprehensive Summaries of Uppsala Dissertations
from the Faculty of Medicine
Editor: The Dean of the Faculty of Medicine
Distribution:Uppsala University Library
Box 510, SE-751 20 Uppsala, Swedenwww.uu.se, [email protected]
ISSN 0282-7476ISBN 91-554-5775-4
A doctoral dissertation from the Faculty of Medicine, Uppsala University,is usually a summary of a number of papers. A few copies of the completedissertation are kept at major Swedish research libraries, while the sum-mary alone is distributed internationally through the series Comprehen-
sive Summaries of Uppsala Dissertations from the Faculty of Medicine.
(Prior to October, 1985, the series was published under the title “Abstracts ofUppsala Dissertations from the Faculty of Medicine”.)
