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Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 139 Functional Characterization of the Cellular Protein p32 A Protein Regulating Adenovirus Transcription and Splicing Through Targeting of Phosphorylation CHRISTINA ÖHRMALM ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2006 ISSN 1651-6206 ISBN 91-554-6533-1 urn:nbn:se:uu:diva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ill min älskade familj Maja, Saga och Johan Mamma och Pappa Annika Members of the committee Opponent Associate Professor Neus Visa Department of Molecular Biology and Functional Genomics The Wenner-Gren Institute Stockholm University Stockholm Members of the committee Professor Anders Virtanen Department of Cell and Molecular Biology Biomedical Center Uppsala University Uppsala Professor Stefan Schwartz Department of Medical Biochemistry and Microbiology Biomedical Center Uppsala University Uppsala Associate professor Marie Öhman Department of Molecular Biology and Functional Genomics The Wenner-Gren Institute Stockholm University Stockholm List of manuscript I. Kanopka, A., Mühlemann, O., Petersen-Mahrt, S., Estmer, C., Öhrmalm, C., Akusjärvi, G. (1998) Regulation of adenovirus alternative RNA splicing by dephosphorylation of SR proteins, Nature 393:185-187 II. Petersen-Mahrt, S. K., Estmer, C., Öhrmalm, C., Matthews, D. A., Russell, W. C., Akusjärvi, G. (1999) The splicing factor-associated protein, p32, regulates RNA splicing by inhibiting ASF/SF2 RNA binding and phosphorylation, EMBO J 18:1014-1024 III. Öhrmalm, C. and Akusjärvi, G (2006) Identification of a carboxy-terminal sequence in the splicing factor-associated protein, p32, necessary for p32-mediated inhibition of ASF/SF2 RNA binding, Manuscript IV. Öhrmalm, C. and Akusjärvi, G. (2006) Cellular splicing and transcription regulatory protein p32 represses adenovirus major late transcription and causes hyperphosphorylation of RNA polymerase II, In press Journal of Virology Contents Introduction...................................................................................................11 From gene to protein ................................................................................11 Transcription ............................................................................................12 Basal transcription of mRNA encoding genes.....................................12 Transcription cycle of RNA Pol II.......................................................12 Coupling of transcription and splicing ................................................15 The CTD kinases and phosphatases ....................................................15 Proteins involved in regulating the activity of CTD kinases and phosphatases ........................................................................................16 Activation and Repression of Transcription ........................................17 Splicing ....................................................................................................19 Constitutive splicing ............................................................................19 Alternative splicing..............................................................................19 Spliceosome assembly .........................................................................20 SR proteins ..........................................................................................21 SR-related proteins ..............................................................................24 Phosphorylation and dephosphorylation of SR proteins......................24 Proteins regulating SR protein activity................................................24 p32............................................................................................................27 Structure of p32 ...................................................................................28 p32 protein interactions .......................................................................29 p32 and protein modifications .............................................................31 Adenovirus ...............................................................................................32 Virus life cycle.....................................................................................32 Early genes ..........................................................................................34 The Major Late Transcription Unit......................................................36 Present Investigation and discussion.............................................................39 Paper I ......................................................................................................39 Regulation of adenovirus alternative RNA splicing by dephosphorylation of SR proteins .......................................................39 Paper II .....................................................................................................41 p32 regulates RNA splicing by inhibiting ASF/SF2 RNA binding and phosphorylation ...................................................................................41 Paper III....................................................................................................44 Identification of a carboxy-terminal sequence in p32 necessary for p32-mediated inhibition of ASF/SF2 RNA binding ............................44 Paper IV ...................................................................................................46 p32 represses the adenovirus major late transcription and causes hyperphosphorylation of RNA polymerase II .....................................46 Conclusions...................................................................................................52 Paper I ......................................................................................................52 Paper II .....................................................................................................52 Paper III....................................................................................................52 Paper IV ...................................................................................................52 Acknowledgements.......................................................................................53 References.....................................................................................................55 Abbreviations aa AdTTflag-p32 Ad5 ASF/SF2 bp CDK7 CDK9 CTD C-terminal DNA Dox ESE E4-ORF4 FCP1 HeLa-NE hpi kDa L1 MLP MLTU mRNA NE N-terminal PIC Pol II Pol IIA Pol IIO PP2A Pre-mRNA P-TEFb p(Y) tract p32 RNA amino acid recombinant inducible adenovirus expressing flag-tagged p32 Adenovirus 5 Alternative splicing factor/splicing factor 2 branch point cycline-dependent kinase 7 cycline-dependent kinase 9 carboxy-terminal domain of the large subunit of RNA Pol II carboxy-terminal deoxyribonucleic acid Doxycyclin exonic splicing enhancer Protein of open reading frame 4 of early region 4 TFIIF- associating RNA polymerase C-terminal domain phosphatase Nuclear extract from HeLa cells Hours post infection kilo Dalton, molecular weight Late region 1 of MLTU of adenovirus Major late promoter Major late transcription unit messenger ribonucleic acid Nuclear extract amino-terminal pre-initiation complex of transcription RNA polymerase II Hypophosphorylated RNA polymerase II Hyperphosphorylated RNA polymerase II Protein phosphatase 2A Precursor mRNA positive transcription elongation factor b polypyrimidine rich sequence upstream of 3´ss cellular protein with molecular weight of 32 kDa ribonucleic acid RRM RS-domain Ser 2 Ser 5 snRNA SR-Ad SR-HeLa snRNP ss TF 3RE RNA recognition motif Arginine (R)- and Serine (S)-rich domain in SR proteins Serine at position 2 Serine at position 5 small nuclear RNA SR proteins from adenovirus infected nuclear extract SR proteins purified from HeLa nuclear extract small nuclear ribonucleoprotein particle splice site transcription factor IIIa repressor element Introduction From gene to protein All complex organisms, like an animal or a plant, are built up of small specified cells forming different tissues that together create the organism. Within its cell nucleus, each cell contains identical genomic material that describes the construction of different proteins performing different functions or acting as building blocks in the cell. For a cell to become specialized, also called differentiated, only some genes of the genome are activated and transcribed into pre-mRNAs, which after being processed and transported from the cell nucleus to the cytoplasm further are translated into proteins. All these steps are highly regulated at many different levels. A virus consists of genetic material, DNA or RNA, and a capsid of proteins, which is enveloped or non-enveloped with membrane from the cell the virus was produced in. The shell of proteins functions both to protect the viral genome and to provide molecules to target cell surface receptors during infection. The genome of a virus must be small enough to fit into the viral capsid and consequently only contains a small number of genes. These encode the structural proteins of the virus and proteins needed to reprogram the cell to efficiently produce new viral particles. Both eukaryotic cells and some virus compact their genome by utilizing one gene to produce many diverse proteins. This is achieved by a process called alternative splicing. The pre-mRNA, which is produced during transcription of the gene, is cleaved and different fragments are ligated in a highly regulated manner by the spliceosome to create different mRNAs, giving the ability for one gene to encode for many different proteins. Both transcription and splicing are regulated by cellular as well as viral proteins. The activity of these regulatory proteins is often regulated by phosphorylation and dephosphorylation, enabling the virus or the cell to efficiently control a particular process. In this thesis we describe how an adenoviral protein, the E4-ORF4 protein, and a cellular protein, the p32 protein, regulate the splicing event by taking control over the activity of ASF/SF2, which is an important splicing regulatory protein (Paper I and Paper II, respectively). Moreover we have analyzed which parts of p32 and ASF/SF2 that interact with each other (Paper III). In paper IV we show that p32 also harbors the function as a transcription regulatory protein (Paper IV). 11 Transcription Transcription of genes can be preformed by three different multi-subunit complexes containing specific RNA polymerases, Pol II: x Pol I transcribes the ribosomal genes to produce 28S, 18S and 5.8S ribosomal RNA, rRNA x Pol II transcribes protein encoding genes to produce messenger RNA, mRNA x Pol III transcribes genes to produce transfere RNA, tRNA, and 5S rRNA The polymerase is binding to the promoter, mostly situated just upstream of the initiator site of transcription, +1, although some RNA Pol III genes have internal promoters. After polymerase complex assembly transcription can be further activated or repressed by different gene specific transcription coactivators and co-repressors. Basal transcription of mRNA encoding genes Most Pol II promoters contain a TATA-element and an initiator site, INR. The TATA-element is situated approximately 25-30 base pairs upstream of the transcription start site and is the binding site for the TATA-binding protein, TBP, whereas the INR is a pyrimidine rich sequence surrounding the +1 start site (-3 to +5) [181]. The INR constitutes the basal element for initiation of transcription in promoters lacking a TATA-box [180, 219] The general transcription factors are recruited to the promoter in a specific order (for review [147]). The first transcription factor, TF, to bind to the promoter is TFIID, followed by TFIIB. The TATA-binding protein, TBP, is a subunit of TFIID and binds to the minor groove of the DNA in the TATAsequence [88, 183]. The co-crystal structure of TBP binding to the TATA-box of the adenovirus major late promoter, MLP, has been described [88]. The additional subunits of TFIID are the TBP associated factors, TAFIIS [56]. The Pol II and TFIIF associate and bind as a complex to the promoter/TFIID/TFIIB. Subsequently, TFIIH and TFIIE follow and the complex formed is called the pre-initiation complex, PIC. TFIIA can join the complex at any time after TFIID has bound to DNA. The PIC causes the two DNA strands the promoter region to melt and the first phosphodiester bond is produced to form the protruding pre-mRNA. Thereafter, the Pol IIcomplex leaves the promoter, promoter clearance, and starts the elongation phase of transcription. Transcription cycle of RNA Pol II The metazoan large subunit of Pol II has a carboxy-terminal domain, CTD, consisting of a heptapeptide sequence (YSPTSPS) repeated 52 times. This 12 motif undergoes extensive serine phosphorylation and dephosphorylation during the transcription cycle, especially at positions 2 and 5, and works as a platform for the recruitment of factors involved in co-transcriptional events like capping, splicing, and poly-adenylation of the nascent pre-mRNA [15]. The length of the CTD varies in different organisms: x x x x Homo Sapiens the heptad motif is repeated 52 times Drosophila melanogaster 45 times Caenorhabditis elegans 37 times Saccharomyces cerevisiae 27 times It is thought that the length of the CTD increases with the complexity of the organism, indicating that complex organisms need for a more intricate system to recruit pre-mRNA processing factors [38]. The N-terminal part of the CTD consists of perfect YSPTSPS repeats and has been shown to support RNA synthesis and capping, while the heptad repeats towards the C-terminal end of the CTD deviate from the consensus sequence and has been shown to support splicing and poly-adenylation of the 3’end of the transcript [49]. In vertebrates the C-terminus of the CTD also contains ten conserved amino acids, aa,: ISPDDSDEEN, which are essential for high level of transcription, splicing and poly(A) site cleavage [50]. Transcription is initiated by hypophosphorylated Pol II, Pol IIA, binding to the promoter to form the pre-initiation complex, PIC (Figure 1). After PIC formation the CTD is phosphorylated on Ser 5 by CDK7/TFIIH. The hyperphosphorylation on Ser 5 is necessary for promoter clearance and the transition from the initiation to the elongation stage of transcription [94]. Soon after PIC formation the DRB sensitivity-inducing factor, DSIF, is recruited, which in turn recruits the negative elongation factor, NELF, to the Pol II resulting in an arrest of transcription allowing time for the Ser 5 hyperphosphorylated Pol II, Pol IIO, to recruit capping enzymes to the protruding RNA molecule [94, 168, 212]. Capping guanylyltransferase, Cgt1, and RNA guanine-7-methyltransferease bind directly to phosphorylated CTD [87]. Cgt1 is specifically stimulated by Ser 5 phosphorylation [70, 126] and deletion studies have shown that Pol II without a CTD produces transcripts with a lower proportion of capped 5’ ends [126]. The capping enzymes are also suggested to play a role in the promoter-proximal checkpoint to ensure that only correctly capped transcripts are allowed to be further transcribed [87, 120]. The CDK9 kinase of the positive elongating factor b, P-TEFb, phosphorylates the CTD on Ser 2 residues leading to a relief of the DSIF/NELF mediated arrest of transcription elongation. The increase in Ser 2 phosphorylation renders the Pol II to be more processive during the elongation phase of transcription [35]. Phosphorylation of Ser 2 is also important for the coupling of transcription and mRNA poly-adenylation. Thus, deletion of the yeast Ser 2 kinase, Ctk1, or inhibition of CDK9/P-TEFb causes defects in poly13 adenylation [4, 141]. Poly-adenylation factors CPSF, CstF, Ocfl 1, and Pta 1 bind preferentially to phosphorylated CTD [126]. The phosphatase FCP1 is counteracting CDK9/P-TEFb during the elongation by causing Ser 2 dephosphorylation [35]. Mutations of FCP1 cause a decrease in the number of polymerases initiating at a promoter and an increase in the amount of Ser 2 phosphorylation during elongation, while the level of Ser 5 phosphorylation seems to be unaffected [35]. FCP1 has been shown to be responsible to recycle Pol II [36, 92]. FCP1 PIC formation Elongation Initiation Termination RNA pol II CTD CDK7 CDK9 mRNA pre-mRNA Capping AA AA AA A DNA Splicing Poly-adenylation Splicing factors Exon Ser 5 phosphorylation Intron lariat Cap Ser 2 phosphorylation Figure 1. The transcription cycle of the Pol II and the coupling of transcription and splicing. The CTD of Pol II is phosphorylated at Ser 5 by CDK7 during initiation and at Ser 2 by CDK9 during the elongation phase of transcription. FCP1 dephosphorylates the CTD to recycle the Pol II. 14 Coupling of transcription and splicing During the last years many reports have provided evidence that splicing occurs co-transcriptionally (Figure 1) [reviewed in 95, 139, 148]. Truncation of the CTD in in vivo experiments shows a failure to recruit splicing factors to transcription start sites and inefficient pre-mRNA splicing [127, 129]. Further, in vitro splicing experiments have demonstrated that addition of purified Pol IIO stimulates splicing, whereas Pol IIA inhibits the splicing reaction [69]. Promoter proximal splice sites have shown to enhance transcription [52]. The strength of a splice site can also effect the Pol II processivity [144], and stimulation of Pol II processivity affects splice site choice in alternative splicing [143]. Different transcriptional factors and splicing proteins can interact with each other. The subunit Prp40 of the spliceosomal U1 small nuclear ribonucleoprotein particle, U1 snRNP, has also been shown to interact directly with the CTD of Pol II [84, 138] and furthermore the human transcription elongation factor TAT-SF1 interacts with U snRNPs resulting in stimulated Pol II elongation and an increase in splicing efficiency in in vitro splicing assays [51]. Table 1. CTD kinases and phosphatases Human Yeast Main target Action Ref. Kin28-Ccl1 Ser 5 Facilitates promoter clearance and mRNA capping [157, 163] Ser 2 Promotes elongation and recruitment of poly(A)factors Promotes elongation [157] CTD kinases CDK7/cyclinH in TFIIH CDK9/cyclinT in P-TEFb CDK8/cyclin8 in mediator NAT CTDK-1 Ser 2 [163] Bur1-Bur2 Ser 5 Srb10-Srb11 Ser 5, cyclin H Inhibits PIC formation [157] Ser 5 and Ser 2 Recycling of Pol II [109] [163] CTD Phosphatases FCP1 Ssu72 SCP1 Ssu72 Ser 5 [99] Ser 5 [214] The CTD kinases and phosphatases The kinases and phosphatases known to have a regulatory role in controlling the phosphorylation status of the CTD are summaried in Table 1. As described above, phosphorylation of Ser 5 and Ser 2 of CTD by CDK7/TFIIH and CDK9/P-TEFb, respectively, and dephosphorylation by FCP1 create a 15 phosphorylation/dephosphorylation cycle for the Pol II (Figure 1). Another kinase, CDK8/cyclin C, found in the mediator complex NAT, is thought to repress transcription by reducing the number of Pol II molecules capable of initiating transcription by phosphorylating the CTD prior to DNA binding and also by phosphorylating cyclinH in the CDK7/cyclinH complex of the general transcription factor TFIIH [5, 185]. Recently, the SCP1 phosphatase was shown to dephosphorylate Ser 5 and it is thought to play a key role in the transition from initiation to elongation. Both FCP1 and SCP1 activities are enhanced by the RAP74 subunit of TFIIF [214]. Proteins involved in regulating the activity of CTD kinases and phosphatases In all cellular processes, for example transcription and splicing, a tight control of different protein activities is of a great importance. The transcriptional activity of different gene families varies between cell types and transcription is strictly regulated during cell differentiation and the cell cycle. Transcription is also regulated at different stages, both before initiation and during the elongation phase of transcription. As an example, transcriptional activators and repressors can act by recruiting histone acetyl transferases, HATs, or histone deacetylaces, HDACs, to enhance or inhibit transcription, respectively. The importance of the phosphorylation status of the CTD in different phases of transcription makes the CTD a great target for proteins that regulate transcription. The variable phosphorylation pattern of the CTD attracts different proteins important for post- or co-transcriptional processes. By enhancing or inhibiting phosphorylation of specific CTD residues the recruitment of these factors can be affected as well as the activity and the processivity of Pol II. Only a few proteins have so far been reported to stimulate or inhibit kinases and phosphatases involved in CTD phosphorylation (see Table 2 and section regulating the activities of RNA Pol II kinases and phosphatases in Paper IV). The Pin 1, as well as its yeast homologue Essp1, binds directly to the CTD, while others like RAP74 is part of a classical transcriptional factor, TFIIF. Paper IV demonstrates that the p32 protein now can be added to this list of regulatory proteins controlling the activity of CTD kinases/phosphatases (Table 2). 16 Table 2. Proteins affecting the activity of Pol II phosphatases and kinases Protein Effect on phophatase Effect on kinase Comments Ref. - + cdc2/cyclinB Mitotic genes [211] + Mitotic genes [206] Binds all four yeast CTD kinases Cellular Pin 1 FCP1 Ess1p RAP74 + FCP1 and SCP1 Hce1 - FCP1 p32 Not known Not known HIV-1 Tat - + EBNA2 Not known BRCA1 [32, 93, 214] - CDK7/TFIIH [133] Capping enzyme [149] Ser 5 and Ser 2 hyperphosphorylation Paper IV viral FCP1 CDK9/P-TEFb Not known [1, 89] Ser 5 hyperphosphorylation [9] Activation and Repression of Transcription To enhance or inhibit the basal transcription machinery different transcriptional co-activators or co-repressors bind to enhancer elements or repressor elements. These can be placed within the promoter region or at a very long distance from the gene. The co-activators and co-repressors can have enzymatic activities themselves or they can recruit other proteins that introduce different modifications like phosphorylation, acetylation, sumolylation, ubiquitination, or methylation, on either the TFs, the co-factors or on the DNA, resulting in changes of the transcriptional activity of the gene. CBF/NF-Y and the CAAT-box motif CBF/NF-Y is a heterotrimeric transcription factor consisting of three subunits; NF-YA, NF-YB and NF-YC. The CBF/NF-Y protein is evolutionary conserved and shows more then 95% aa sequence identity between mouse, rat and human isoforms [105]. All three subunits are needed for DNA binding to the CAAT-box motif, one of the most common sequence elements in eukaryotic promoters. The consensus binding site for CBP/NF-Y is defined as 5´-(T/C) (A/G) (A/G) CCAAT (C/G) (A/G)-3´ [18]. Mutational studies have 17 shown that CBF/NF-Y requires all five nucleotides in the core motif for efficient binding [116]. Not all promoters that contain the CAAT-box are activated by CBF/NF-Y demonstrating that the neighboring sequences of the motif are also important for high affinity binding of CBF/NF-Y. There are many different transcription factors that bind to the CCAAT-box motifs, like CTF/NF1, a 47 kDA protein that recognizes the sequence GCCAAT [166] and the family of CCAAT/enhancer binding proteins, C/EBP, which has the (A/G)TTGCG(C/T)AA(C/T) as recognition sequence [158]. In contrast to CBF/NF-Y they do not require an intact sequence of the CCAAT-motif. The CAAT-box exists in both forward and reverse direction and a comparative investigation of 96 genes containing 178 CAAT-boxes demonstrated that CAAT-boxes are common in both TATA-containing and TATA-less promoters [121]. In TATA-containing promoters the CAAT-box is often located at position -100/-80 and in TATA-less promoters it is located closer to the transcription start site, at position -60, and often in a reversed orientation [121]. 18 Splicing The pre-mRNA, produced by Pol II during transcription, consists of both protein encoding and non-encoding ribonucleotide sequences, called exons and introns, respectively. In the nucleus a large protein complex, the spliceosome, removes introns and ligates selected exons of the pre-mRNA in a two step trans-esterification reaction, called splicing, creating an mRNA. After transport into the cytoplasm the ribosome will read the code of the mRNA and produce the described protein, in a process called translation. Constitutive splicing The consensus sequence of a splice site, ss, in mammals is defined as AG/GURAGU at the 5’ ss and YAG/N at the 3’ ss, where / indicates the boundary between the exon and the intron. In the intron, close to the 3’ ss, there are two important sequence elements: the branch point, YNYURAC (placed at 18-40 nt upstream of the 3’ ss), and the polypyrimidine p(Y) tract, the which is a stretch of uridine residues of variable length (Figure 2). A long p(Y) tract is efficiently recruiting the splicing factor U2AF in the early steps of spliceosome assembly, resulting in that 3’ ss usage, and is therefore referred to as a strong splicing signal. Two different models to define splice sites exist, the exon definition model and the intron definition model, where of the first model is the predominant in higher eukaryotes. Alternative splicing Approximately 30 to 50 % of the genes in higher eukaryotes are alternatively spliced [131, 161]. The pattern of alternative splicing varies in different cell types and tissues, during cell differentiation and embryonic development, and under certain physiological conditions [112]. A pre-mRNA that can be alternative spliced contains introns that are defined by alternative 5’ ss and/or 3’ ss and this process can also lead to inclusion or exclusion of whole exons. Alternative splicing is highly regulated and the SR family of splicing factors plays an important role in defining the ss selection. Cis-elements in the pre-mRNA, like exonic splicing enhancers, ESE, exonic splicing silencers, ESS, intronic splicing enhancers, ISE, and intronic splicing silencers, ISS, are targeted by different trans-acting factors to activate or repress splice signals in the surrounding sequence. A typical illustration of a regulated alternative splicing event is when an SR protein by binding to an ESE recruits the U2 auxiliary factor, U2AF, to a 3’ ss with a weak p(Y) tract and 19 thereby causing the weak 3’ ss to be activated by helping in the recruitment of U2 snRNP to the branch point. Spliceosome assembly The spliceosome is assembled in a sequential manner involving almost 200 proteins associated with U small nuclear RNA, U snRNA, forming nuclear ribonucleoprotein particles, snRNPs, or acting as splicing cofactors with different functions. In the first step, formation of the early (E) complex, U1-snRNP binds to the 5’ ss through RNA-RNA interaction, which is stabilized by a set of co-factors (Figure 2). 5’ pre-mRNA Exon 1 GU AG 3’ BP p(Y)tract Intron Exon 2 U1 SF1 E U2AF 35 65 BP U1 U2 A U2AF BP U4 U1 U6 U5 U2 B U2AF BP Exo n1 U2 U6 C BP mRNA Exon 1 Exon 2 Exon 2 Figure 2. A simplified overview of spliceosome assembly. The different complexes are indicated on the left side. U1,U2, and U4/U5/U6 represent the UsnRNPs. Further, the SR proteins (not present in the picture) bind to regulatory elements on the RNA and by different protein-protein interactions they bring the two ss together. 20 The branch point binding protein, SF1/mBBP, binds to the branch point with a binding that is stabilized by an interaction with U2AF, which in turn binds to the p(Y) tract. The following A-complex formation is characterized by the ATP-dependent recruitment of the U2 snRNP to the branch point. The binding of the U4, U5 and U6 snRNPs, together called the tri-snRNP, create the B complex and is followed by a massive rearrangement of the spliceosome in which U6 replaces U1 at the 5’ ss and interacts with U2, U5 forms a bridge between the 5’ ss and the 3’ ss, and U1 and U4 become destabilized and leaves the complex. The new complex is called the C complex and is the catalytically active spliceosome [reviewed in 66]. SR proteins SR proteins regulate splicing by binding to splicing repressor or splicing enhancer elements, which exist both in exons and introns and mediate protein-protein interaction with other SR proteins, SR-related proteins, and U snRNPs. The SR protein induced formation of RNA-protein and proteinprotein interactions helps to bring the splice sites together and is essential for the catalytic trans-esterification event to occur. SR proteins are binding very early during spliceosome assembly and are required for E complex formation. They help in recruitment and stabilization of U1 snRNP and U2AF to the 5’ ss and the 3’ ss, respectively, and play an important role in recruitment of the U4/U6.U5 tri-snRNP. Interestingly, Valcarcel et al. has demonstrated that the RS-domain of U2AF65, a subunit of U2AF, interacts directly with the branch point, and recently, Shen and Green confirmed these results by showing that the RSdomain of U2AF65 binds to the branchpoint in the E complex [reviewed in 66, 173, 174, 196]. They also demonstrated that the RS-domain of an ESE bound SR protein interacts with the branch point in the following A complex, and the RS-domain of a second SR protein interacts with the 5’ ss in the B complex [173, 174]. The function of SR protein can also be divided into exon dependent (binding of ESEs) and exon independent (recruitment of the U4/U6.U5 tri-snRNP, bridging between the 5’ ss and 3’ ss) activities. The activity of SR proteins is antagonized by members in the heterogeneous nuclear ribonucleoprotein A/B, hnRNP A/B, family of proteins (see section Proteins regulating SR protein activity). The classical SR proteins consists of around ten phosphoproteins which all have one or two N-terminal RNA recognition motifs, RRMs, and a C-terminal domain rich in arginine and serine, RS, dipeptide repeats (Figure 3). The length of the RS domain varies between 24 and 316 residues and the molecular weight of these proteins ranges from 20 to 75 kDa [reviewed in 58]. The RRMs consist of two conserved motifs, one octamer and one hexamer, called RNP-1 and RNP-2 respectively, which form four antiparallel E-strands packed against two D-helices (E1-D1-E2-E3-D2-E4). 21 SRp75 RRM1 GR RRM2 SRp55 RRM1 GR RRM2 SRp54 RRM1 SRp46 RRM1 SRp40 RRM1 RS RS RS RS R RRM2 ASF/SF2 RRM1 G RRM2 9G8 RRM1 RP Z SC35 RRM1 PG SCp30c RRM1 G SRp20 RRM1 RP RS RS RS RS RRM2 RS RS Figure 3. Human classical SR proteins. RRM, RNA recognition motif; RS, arginine/serine rich domain; Z, zinc knuckle; G, glycine, P, proline, and R, arginine, rich domains. (Modified from [6]) The criteria for an SR protein are defined as: x Containing one or two RRMs and an RS-domain x Ability to restore full splicing activity in an extract deficient in SR proteins, like cytoplasmic S100 extract x Redundant roles in constitutive splicing x Ability to regulate alternative splicing The SR proteins are concentrated in 20-40 distinct sub-organellar domains called speckles in the nucleus and are translocated to transcription sites upon gene activation [128]. Phosphorylation/dephosphorylation of the SR proteins has been suggested to play an important role in this translocationr [26, 128]. ASF/SF2, as well as SRp20 and 9G8, are shuttling between the nucleus and cytoplasm. The presence of the RS-domain has been shown to be required and its phosphorylation status regulates the shuttling of the SR proteins [27]. It has been suggested that the shuttling SR proteins in some cases are helping mRNA export since they have the ability to associate with the RNA export factor TAP [77]. SR proteins have essential splicing factor redundant functions. Individual targeting of six different SR proteins with dsRNA interference, RNAi, in Caenorhabditis elegans showed no effect on the phenotype, with the exception of CeASF/SF2, which downregulation caused lethality in late embry22 onic development. However, combinations of two or several simultaneous SR protein deletions displayed defects or lethality [111]. SR proteins have been identified in all metazoan species that have been analyzed, but they do not exist in all eukaryotic species, like for example Saccharomyces cerevisiae which lacks a recognizable SR protein. ASF/SF2 The SR protein ASF/SF2 was purified to almost homogeneity from HeLa cells and was shown to be able to complement S100 extract in an in vitro splicing assay [98]. At the same time it was identified as an activity regulating large and small T alternative splicing in 293 cells [53]. ASF/SF2 consists of 248 amino acids and the molecular weight is approximately 30 kDa. It has two RNA recognition motifs RRM1 (aa 1-98) and RRM2 (aa 106-201), and a C-terminal RS-domain (aa 201-248) containing arginine-serine dipeptides repeat in which the 20 serine residues are potential phosphorylation sites. Phosphorylation of ASF/SF2 is required for its specific interaction with U170K and U1 snRNP [209]. Table 3. Human SR-related proteins [modification of tables21] Protein name U2AF35 Number Number of of RRMs SR domains 1 Other domains Function 1 U2 auxiliary factor U2AF65 3 1 U2 auxiliary factor U1-70K 1 2 snRNP component U5-100K 1 U4/U5.U6-27K 1 hLuc7p 1 hTra2a 1 2 hTra2b 1 2 RSF1 1 SRrp40/SRp38 SRrp86 1 DEXD/H Box snRNP component snRNP component Two Zn-domains snRNP component Splicing regulator Splicing regulator GRS-domain Splicing regulator 1 K, E/D and Q/N in RSdomain Splicing regulator 2 EK/R-rich region Splicing regulator SRm160 2 SRm300 1 RS/P Splicing coactivator hPrp16 1 DEXD/H Box RNA helicase HRH1 1 DEXD/H Box RNA helicase Clk/Sty 1 Kinase Protein kinase Splicing coactivator 23 SR-related proteins SR-related proteins, SRrp, contain an RS-domain and sometimes also an RRM, but do not fulfill the rest of the criteria required for an SR protein. They can be divided in groups dependent on their function in RNA processing, chromatin association and transcription, but also their enzymatic activities. About 50 SR-related proteins have been defined in humans with a bioinformatic approach, while 80 SRrps was found in C. elegans and 110 in Drosophila [20]. SRrps involved in splicing are often interacting with the classical SR proteins and are typically involved in spliceosome assembly (U1-70k, U2AF65, and U2AF35) or splicing regulation (like SRrp86, SRp38/SRrp40, and SRm160/300) [10]. Phosphorylation and dephosphorylation of SR proteins Phosphorylation activates SR proteins through modulation of protein-protein interactions but also prevents SR proteins from non-specific protein-RNA interaction [29, 188, 209, 210]. Both hypo- and hyperphosphorylation can reduce the activity of SR proteins [154]. Phosphorylation of SR proteins are required for the translocation from the cytoplasm into the nucleus [85, 103] and influences the intranuclear localization [37, 60]. SR proteins are phosphorylated by a number of kinases whereof the phosphorylation preformed by Clk/Sty, SRPK (SR protein kinase) family members, as well as DNA topoisomerase I, have been shown to regulate their activity [7, 154, 155, 164, 203]. The Clk/Sty kinase has been shown to be a nuclear kinase in several cell lines [155], whereas SRPK is predominantly cytoplasmic in interphase cells [201]. The SRPK family, consisting of SRPK1 and SRPK2, phosphorylates serines in the RS-domain preferentially in the context of the RSR motif [201]. The Clk/Sty family, which belongs to the LAMMER kinases, consists of Clk/Sty, Clk/Sty-2, -3 and -4, and phosphorylates both serine/threonine (S/T) and tyrosine (Y) residues [13, 74, 104]. Further, Prasad and Manley have shown that Clk/Sty is autophosphorylated and its phosphorylation status modulates its kinase activity in vitro in a substrate specific manner [155]. Proteins regulating SR protein activity To regulate the activity of SR proteins, cofactors make new contact between the SR proteins and other splicing components and thereby change the ability of the SR protein to recognize its pre-mRNA target. These cofactors regulate the function of SR proteins by acting as antagonizing factors or by inhibiting their RNA binding either by sequestering the SR protein into new complexes or affecting its phosphorylation pattern. SR protein phosphorylation is regulated by different mechanisms, for example by sterical hindrance, 24 recruitment of phosphatase or inhibition of kinase activity. Some of the regulatory proteins are RNA-binding proteins while other interacts directly with the SR protein. Table 4. Proteins regulating SR protein activity Protein name RNA SR protein ininteraction teraction Function Ref. - Antagonist of ASF/SF2 [8, 28] Cellular proteins hnRNP A1 + RBM4 + RSF1 + + Antagonist of ASF/SF2 hTra2-D + + Complex with SR pro[189] tein to optimize splicing hTra2-E + + Complex with SR pro[189] tein to optimize splicing SRrp86 + + Inhibit or activate specific SR proteins [10] p32 - + Block phosphorylation of ASF/SF2 [152] Ad E4-ORF4 + + Inactivates SR proteins by bringing PP2A [44, 82] HSV-1 ICP27 - - Inhibits SRPK1 [169] Antagonist of SR protein [102] [101] Viral proteins The hnRNP A1 protein has an antagonizing effect on ASF/SF2 function by competing for RNA binding. ASF/SF2 promotes proximal 5’ ss selection in vitro and in vivo, while hnRNP A1 activates distal 5’ ss usage [28]. Bai et al. later demonstrated that hnRNP A1 also antogonize ASF/SF2 in 3’ ss choice, by enhancing the usage of distal 3’ ss instead of the ASF/SF2 induced utilisation of the proximal 3’ ss [8]. Recently, another non-SR protein named RNA-binding motif protein 4, RBM4, was shown to use the same nuclear import pathway as SR proteins (Transportin-SR2 import factor pathway) and further to act antagonistically against SR proteins in ss selection [102]. The human homologues of Drosophila Transformer 2, Tra2, are called hTra2-D and hTra2-E and function as regulators of alternative splicing both by binding to specific RNA sequences and interacting with SR proteins [189, 190]. The Tra2 protein forms a complex with Tra onto six repeats of an enhancer element called the doublesex repeat element, dsxRE, in the Drosophila double-sex, dsx, gene and recruits an SR protein for optimal splicing. This SR protein has been shown to be the SRp20 homologue RBP1 in Drosophila and 9G8 in mammalias [115]. The current model is that Tra2 is alter25 ing the conformation and thereby the RNA recognition specificity of certain SR proteins. The Drosophila splicing repressor factor, RSF1, is an RNA binding protein which has been shown to antagonize ASF/SF2 in vitro. RSF1 contains an N-terminal RRM and a C-terminal region rich in glycine (G), arginine (R), and serine (S) amino acids, a GRS-domain. SFR1 inhibits an early step in spliceosome assembly by inhibiting ASF/SF2 stabilization of U1 snRNP binding to the 5’ ss by interacting via its GRS domain with the RS-domain of ASF/SF2. Further, expression of RSF1 has been shown to rescue developmental defects caused by p55/SRp55 overexpression in Drosophila [101]. RSF1 has also been shown to compete in RNA binding with the SR proteins [100]. SRp86 contains both an RRM and two separated regions rich in RSresidues, but can not complement S100 extract. It inhibits splicing activated by SC35, ASF/SF2, and SRp55. Further, SRp86 can modestly increase splicing activated by SRp20 in vitro. Transient transfections have also shown that SRp86 affects alternative splicing in vivo [10]. Another protein which affects the activity of SR proteins is the herpes simplex virus ICP27. It binds to SRPK1 and inhibits its kinase activity causing SR protein hypophosphorylation [169] (see PaperI). The proteins described above are all able to affect the activity of SR proteins (Table 4). Two of the papers (Paper I and II) in this thesis add to this list of reports by describing that adenovirus E4-ORF4 and the human protein p32 regulate alternative splicing by interfering with SR protein activity [82, 152]. Both E4-ORF4 and p32 affect the phosphorylation of ASF/SF2, but they utilize different strategies (Figure 4). E4-ORF4 associates with protein phosphatase 2A, PP2A, which interacts with the RRMs of ASF/SF2, resulting in hypophosphorylation and inactivation of ASF/SF2 as both enhancer and repressor of splicing (Paper I and [44]). p32 blocks phosphorylation of ASF/SF2 through sterical hinderance by binding to the RRMs of ASF/SF2, leading to ASF/SF2 hypophosphorylation and loss of the RNA binding capacity (Paper II and paper III). 26 5’ ASF/SF2 3’ BP Exon 1 Intron Exon 2 3RE E4-ORF4 p32 5’ PP2A 3’ 3’ Figure 4. p32 and E4-ORF4 regulate the RNA binding capacity of ASF/SF2 by different mechanisms. p32 block phosphorylation and sequesters ASF/SF2 into an inactive complex, while E4-ORF4 recruits PP2A to dephosphorylate ASF/SF2. p32 The ubiquitous cellular protein p32/HABP1/gC1q-R is a multifunctional protein that is localized at the cell surface, in the mitochondria, cytoplasm and nucleus of various cell types. p32 has been reported to be involved in several activities like regulation of transcription and splicing, and receptor binding. It was originally isolated tightly associated with ASF/SF2 during purification from HeLa cells [98]. Deb and Datta have demonstrated that the 34-kDa hyaluronic acid-binding protein, HABP1, is identical to the ASF/SF2 binding protein p32 [39] and Ghebrehiwet et al. cloned p32 as a 33 kDa surface glycoprotein, gC1q-R, which interacts with the “globular heads” of C1q [55]. It has also been shown that the lamin B receptor-associated protein p34 is identical to p32 [179]. The p32 gene is positioned on human chromosome 17p13.3 and on mouse chromosome 11. p32 is translated into a pre-protein 27 of 282 aa, in which the N-terminal sequence has the characteristic features of a mitochondrial import sequence and thus the full length protein is mainly localized in mitochondria. The mature form of p32 lacks the 73 N-terminal aa and is found both in the cytoplasm and in the nucleus [73, 125, 202]. Amino acids 222-282 in p32 are highly conserved in several species, ranging from yeast to human [73, 136]. The mature protein is highly acidic with a calculated pI of 4.15. Structure of p32 Crystallization of p32 reveals a non-covalently tight associated trimer, forming a dough-nut shape [80]. p32 can exist as a monomer, trimer or even as a covalently-linked hexamer, which is linked by thiol group oxidation of Cys186, under different environmental conditions [79]. The topology of the monomer reveals an N-terminal D-helix (A) followed by seven twisted antiparallel E–sheets, a short D-helix (B) and a c-terminal D-helix (C) (Figure 5). Two loops connecting E1 to E2 and E3 to E4, in monomer I interact with the connecting loop between E6 and E7 of the monomer II. The D-helices A and C in monomer I also interact with the E-sheet and the D-helix B in monomer II [80]. When binding to hyaluronic acid p32 forms a homodimer of 68 kDa [62]. 233 244 250 96 225 213 180 174 127 124 227 205 187 168 134 280 C 77 N 110 117 114 Figure 5.The secondary structure of p32. (Modified from [80]). Open thick line marks the loop within the region of amino acid 226-239 demonstrated to be important in ASF/SF2 interaction (see Paper III), while black thick lines marks the loops involved in trimer formation. Grey boxes represent D-helixes and white open arrows represent E-sheets. 28 p32 protein interactions The p32 protein binds to many different cellular, bacterial and viral proteins, probably depending on its maturation state, quaternary structure and localization. Viral proteins like Ad pV, HSV-1 ICP27, HVS ORF73, gammaHV68 M2 and EBV EBNA-1 induce an accumulation of the mature p32 protein in the nucleus [25, 63, 106, 125, 202]. Table 5. Proteins interacting with p32 Protein name aa of p32 interacting with indicated protein Reference 74-239 [98, 152], paper III Cellular proteins ASF/SF2 CBF/NF-Y Not known [33] Cdc25 1-104 [118] Factor XII Not known [81] Fibrillanin Not known [213] gC1q 76-94 [55] high molecular weight kininogen Not known [67] Hrk 221-282 [186] Hyaluronic acid Not known [39] lamin B receptor Not known [142] protein kinase C Not known [162] TFIIB 244-255, 260-279 [216] vitronectin 74-96 [108] Not known [125] Viral proteins Ad pV CMV pUL97 214-282, (243) [124] EBV EBNA-1 103-282, (244-282) [202] gamma-HV68 M2 Not known [106] Hepatitis C virus core protein Not known [90] HIV-1 Rev 196-208 [191] HIV-1 Tat 244-255 [17, 216, 217] HSV-1 ICP27 Not known [25] HSV-1 ORF P Not known [24] HVS ORF73 Not known [63] Rubella virus core protein 214-282 [11, 12, 132] Not known [23] Not known [140] Bacterial proteins Listeria monocytogenes protein In1B Staphylococcus aureus protein A 29 The interaction with the SR protein ASF/SF2 has been shown to play a regulatory role in splicing [152], while some p32 protein interactions, for example with TFIIB and HIV-1 Tat, EBV EBNA-1, HSV ORF73 or gamma-HV68 M2, have indicated a role for p32 in transcription activation [63, 106, 200, 216]. Recently, Chattopadhyay et al. described that p32 can repress transcription by interacting with the B-subunit of the transcription factor CBF/NF-Y bound to the CAAT-box in the promoter. It appears that the proteins interacting with p32 bind to different domains of p32. As shown in Table 5 the N-terminal D-helix A or the C-terminal D-helixes B and C of p32 are important for the different interactions, with an exception of HIV-1 REV which interacts with E-sheet 6 [191]. In this thesis we demonstrate that ASF/SF2 can interact in vitro with p32 lacking the C-terminal D-helices (p32aa 74-239) but this interaction is lost when further deletions are made (p32aa74-226) (see section Present Investigation and Discussion; paper III). Interestingly, it has been reported that many of the p32 interacting protein are rich in arginine residues in their interacting regions like HSV-1 ICP27 [25], HVS ORF73 [63], HIV-1 REV [114], Lamin B receptor (p58) [142], EBV EBNA-1 [202] and Rubella virus core protein [11]. Table 6. p32 homologues and their interacting proteins Homologues PreMature origin protein protein Reported interacting protein Interacting domain of p32 homologues p38 207 aa chicken myosin 1-28 and 187-207 [146] p22 227 aa p30/Mam33p 266 aa SUAPRGA1 303 aa YL2 181 aa 230 aa T. Brucei RBP16 S. cerevisiae Cytochrome b2 A. Nidulans murine HIV-1 REV 208 aa Ref. [65] [170] [198] [114] The p32 protein has homologues in all mammals and lower organisms investigated (Table 6). In S. cerevisiae the p30/Mam33p has a 53 % similarity and 26 % overall identity with p32. This protein was identified as a mitochondrial matrix protein binding to cytochrome b2 [170]. The murine protein YL2 shows 92 % identity with p32 and binds also to HIV-1 REV like human p32 does [114, 191]. Further, a mitochondrial guide-RNA binding protein named p22 in Trypanosome brucei has been identified as a homologue of p32 and p30/Mam33p [65]. 30 p32 and protein modifications A sequence analysis of p32 reveals three potential N-glycosylation sites (114N, 136N and 223N), one tyrosine sulfation site (188Y), one potential myristylation site (250N) and one tyrosine recognition site (268Y) [39, 54]. p32 has also several potential phosphorylation sites for protein kinases like: protein kinase C (205S), casein kinase II (76T, 205S, 213S, 251T and 261T), and protein kinase ERK and cdc2 (160PELTSTP166) [39]. Several kinases can phosphorylate p32 under specific circumstances. Recently, p32 was reported to interact with the human cytomegalovirus (CMV) protein kinase pUL97. The p32-pUL97 complex is recruited to the lamin B receptor at the nuclear membrane where p32 and lamin B becomes phosphorylated by pUL97 leading to a redistribution of the lamina and increased release of virus particles [124]. Furthermore, Hyaluronic acid (HA), PMA, calyculin A and Ca2+ ionophore cause an enhanced phosphorylation of threonine residues in p32 and MAP kinase phosphorylation of p32 causes a translocation of p32 into the nucleus [117]. Co-immunoprecipitation studies have demonstrated that p32 is phosphorylated by CK2 when present in a complex consisting of p32-hnRNP K-CK2. Formation of this trimeric complex requires HSV-1 ICP27 (IE63) [25]. The only reported kinase whose activity is affected by p32 is protein kinase CP, PKCP. p32 binds to the kinase domain of PKCP and inhibits PKCP phosphorylation of the substrate aldolase. In contrast, p32 enhances the autophosphorylation activity of PKCP [184]. 31 Adenovirus Adenoviruses have been isolated from every vertebrate species that have been investigated and the classification of the family Adenoviridae consists of the two genera Mastadenoviridae and Aviadenoviridae [14, 165]. The human adenoviruses (belonging to Mastadenoviridae) are divided into six subgroups, A-F, based on their ability to agglutinate red blood cells [68] and their oncogenic potential in rodent cells [175]. The most studied adenoviruses of the approximately 50 human serotypes that exist are the adenovirus type 2 and 5, Ad2 and Ad5, which both belong to subgroup C, and type 12, Ad12, which belongs to subgroup A. Ad 12 is highly oncogenic in rodents but no human adenovirus has so far been shown to cause any human cancers [195]. Adenovirus has a non-enveloped icosahedral capsid of 70-90 nm containing a double stranded DNA molecule of 30-38 kbp. Ad2 and Ad5 have genomes of approximately 35 kbp. The genome is divided into transcriptional units in which the pre-mRNAs are extensively alternatively spliced to generate mRNAs that are translated into approximately 30-40 proteins dependent on the serotype. Virus life cycle Dependent on the serotype human adenoviruses attach to the cell via either the cellular coxsackie and adenovirus receptor (CAR) or the MHC class I receptor through interaction with the viral fiber extending out from the virion [16, 72, 194]. Ad2 and Ad5 bind to the CAR protein. Two secondary receptors, integrin DvE3 and DvE5, are helping the virus to enter the cell by receptor-mediated endocytosis via formation of a coated pit that fuses to endosomes [205]. In the cytosol, the virion is disassembled in the endosome due to a decrease in pH. After transport to the nucleus, along the microtubuli, the membrane of the vacuole is disrupted and the DNA along with the DNA-associated protein VII enter through the nuclear pore complex into the nucleus, where the viral DNA can be transcribed and replicated. The genome contains early (E1A, E1B, E2, E3 and E4), intermediate (IX and IVa2) and late (MLTU) protein encoding genes and two genes for production of the highly structured VA RNA I and II (Figure 6). The first viral gene to be transcribed is E1A which occurs approximately 1 hour after viral attachment to the cell surface receptor. E1A activates transcription of all the early viral and some cellular genes leading to replication of the viral genome at approximately 6-8 hour post infection, hpi. The viral DNA replication separates the early and the late phase of infection. 32 Figure 6. The genome of Adenovirus 2 and 5. Kindly provided by Göran Akusjärvi, IMBIM, Uppsala University. Black arrows indicated early genes and open arrows indicate late genes. The major late transcription unit, MLTU, is activated in the late phase to produce mainly the viral structural proteins. During the late phase of infection adenoviral proteins are almost exclusively produced although the transcription rate of cellular genes is not reduced. The early viral proteins E1B-55K and E4-ORF6 are responsible for a selective transport of viral mRNAs from nucleus to cytoplasm. During infection the cap-binding subunit eIF4E of the cellular translation initiation factor eIF4F becomes dephosphorylated and its reduced activity causes an inhibition of cellular translation. The viral mRNAs expressed from the MLTU can still be translated because they all contain a 5’ leader sequence, the tripartite leader, which functions as an eIF4F-independent translational enhancer. The VA RNA I and II are highly structured RNA molecules that are important primarily in protecting the virus against the interferone induced cellular immune system by inactivating the cellular kinase PKR. As an antiviral defense, the cell tries to shut down the translation machinery by activating the cellular kinase PKR, which inhibits the translational initiation factor 33 eIF2a by phosphorylation. To be activated PKR binds as dimers onto the dsRNA produced by symmetrical transcription of the viral genome. The short viral VA RNA I and II can keep PKR in an inactive form since PKR binds VA RNA as monomers. The infections cycle is complete after approximately 30 h at optimal conditions. After virus assembly the cell lyses due to disruption of the cellular cytoskeleton and 104-105 new virus particles per cell are released. Early genes The proteins from the early regions are involved in different mechanisms to drive the infection towards production of new viral particles. This is achieved by: x Forcing the cell into S-phase x Production of proteins needed for replication of viral DNA x Production of proteins protecting the infected cell from the host cell antiviral response. The E1A transcript, produced from a constitutive promoter of the E1Aregion, can be alternative spliced into five different mRNAs; 13S and 12S, which are produced early after infection and the 11S, 10S, and 9S, which are produced late in infection. The shift in E1A pre-mRNA splicing is caused by the sequestering of the SR proteins needed for E1A 13S and 12S splicing. It is believed that the massive amount of transcripts generated from the MLTU at late times of infection titrates out the limited amounts of SR proteins present in the cell. The E1A functions as a transcription activator of regulating both viral and cellular genes. The E1A protein does not bind DNA directly and is targeted to promoters through the interaction with transcription factors bound to target promoters. By removing and inhibiting HATs, like p300/CBP from a promoter E1A can also repress transcription. To activate genes E1A can either phosphorylate transcription factors or bridge between transcriptional activators and the basal transcription machinery. Furthermore, E1A can activate transcription by displacing inhibitory corepressor complexes like binding to tumour suppressor protein RB which releases the transcription factor E2F to activate S phase genes. To summarize, the most important function of E1A is to force the cell into S-phase to allow for viral replication. E1A causes increased amount of p53 in the cell, which can lead to apoptosis [151]. The two proteins encoded by the E1B-region, E1B-55K and E1B-19K, prevent from apoptosis. The E1B-55K protein can bind directly to the p53 transcriptional activation domain and repress p53 transcriptional activation [215]. Further, E1B-55K can form a complex with E4-ORF6 and both proteins can inactivate p53 by targeting it for proteosomal degradation [41, 156]. The E1B-19K protein, a homolog to the cellular anti-apoptotic 34 Bcl-2 protein, sequesters the pro-apoptotic factors Bax and Bak by forming heterodimers and prevents them from activating caspase 3 and 9 [204]. The E2 transcription unit consists of two units: E2A, encoding E2A-72K (also called the DNA binding protein, DBP), and E2B, encoding the precursor terminal protein pTP and the viral DNA polymerase [207]. The E2 region has two promoters, an early promoter, which is active early in infection and a late promoter that is activated at the intermediate phase of infection. E1A activates the early promoter, while the late promoter is repressed by E1A [61]. The late promoter is activated by the CAAT-box binding protein YB-1, which is recruited to the cell nucleus by the viral E1B-55K protein [71]. The proteins encoded from the E3-region are involved in counteracting the host immune response and are therefore dispensable in in vitro cell cultures [reviewed in 107]. Virus infections often induce a release of TNF-D and Fas causing induction of apoptosis. The E3-14.7 kD and the E3-10.4K/14.5K proteins can inhibit apoptosis and reduce the amount of Fas receptor by internalization [43]. The E3-19K protein can in turn reduce the amount of MHC I receptor on the cell surface rescuing cells from death by cytotoxic Tlymphocytes [159]. The adenovirus death protein (ADP) E3-11.6K promotes virus release by disrupting the cytoskeleton of the cell [192, 193]. The E4-region encodes for at least seven proteins which all have different functions during the life cycle of the virus. As mentioned above, E4-ORF6 binds to E1B-55K and acts anti-apoptotic, and this complex are also responsible for the selective transport of viral mRNAs into cytoplasm. E4-ORF3 and E4-ORF6 has showed to have opposite affect on alternative splicing, where E4-ORF3 facilitates i-leader exon inclusion, while E4-ORF6 preferentially favours i-leader exon skipping of the tripartite leader sequence of late transcripts from MLTU [145]. E4-ORF3 and E4-ORF6 can both inhibit cellular double-strand break repair system and the purpose seems to be to prevent the adenoviral genome from forming concatemers [22, 45]. E4-ORF6/7 acts as a transcription co-activator by stabilizing and promoting dimerization of the E2F transcription factor on the E2 early promoter [187]. E4-ORF4 protein The E4-ORF4 protein is a multifunctional protein that can affect different cellular processes like transcription and splicing. It can block E1A induced transcription [19, 91, 135], induce p53-independent apoptosis [122, 123, 176, 177], and cause G2/M arrest in yeast and mammalian cells [96]. In paper I we demonstrate that E4-ORF4 causes SR protein dephosphorylation during an adenovirus infection, reducing the SR protein RNA binding capacity and affecting alternative splicing [82]. It has been demonstrated that E4-ORF4 binds to the B-subunit of serine/threonine phosphatase PP2A [44, 91] and the current model is that E4-ORF4 performs different functions in the cell by bringing PP2A to dephosphorylate different target proteins. Interestingly, other viral proteins have been reported to interact with PP2A, like SV40 small T, 35 polyoma virus small T and middle T, and HIV-1 Vpr [75, 150, 197]. It is assumed that different viruses use the phosphatase to target the same cellular key proteins controlling cellular growth [48]. The Major Late Transcription Unit The late genes of adenovirus have a common promoter, the major late promoter, MLP, which produces one long capped pre-mRNA transcript of about 28,000 nucleotides. The late transcript can be poly-adenylated at five different poly-adenylation sites and the lates genes are therefore divided into five 3’-coterminal units, L1-L5 (Figure 6). Within each unit different mRNAs are produced by alternative splicing. The late mRNAs encode primarily for the late structural proteins required for capsid formation but also a few regulatory proteins [171]. All late transcripts receive through alternative splicing a common 5’ leader sequence, the tripartite leader sequence, which functions as a translational enhancer in late infected cells. The 201 nt long tripartite leader is made out of three exons: 1, 2, and 3, with one exception. The L1 transcript: 52,55K+i mRNA has an extra i-leader exon inserted between exons 2 and 3. The 52,55K and the i-leader protein are the only proteins produced from the MLTU during the early to intermediate phase of infection. The Major Late Promoter The major late promoter has both a TATA-box, 5’-TATAAAAG-3’ at position -31 to -24, and an initiator element, INR, 5’-TCACTCT-3’ at position -2 to +5 [180]. Several proteins have been reported to bind to the INR of MLP, like the different TBP-associated factors, TAFIIS, [86], but mutational studies have revealed that the INR of MLP is not necessary for activity in vivo unless another MLP element is disrupted at the same time [113]. The transcription factors DEF-A and DEF-B bind to three different elements within the DNA encoding the first exon of the tripartite leader; DE1, DE2b and DE2a [78]. There they form homo- or heterodimers with the adenovirus IVa2 protein. Further, two important upstream activating elements have also been identified, the upstream promoter element, UPE, 5’-GCCACGTGA-3’ at position -62 to -53, [59, 167] and a reversed CAATbox, 5’-TGATTGGTTT-3’ at position -82 to -73 [116]. The transcription factor binding to the UPE-motif was simultaneously identified by three different groups, which resulted in the names: MLTF (major late transcription factor) [30] UEF (upstream element factor) [130], and USF (upstream stimulatory factor) [167]. The heterotrimeric transcription factor CBF/NF-Y binds to the CAAT-box of MLP and stimulates MLP transcription in vitro [116]. Although the mechanism is unknown it is presumed that CBF/NF-Y must interact with one or more proteins in the preinitiation complex of the MLP [182]. The TATA-box and the CAAT-box are absolutely conserved in differ36 ent serotypes in contrast to the UPE motif, the downstream elements and the INR [182]. Analysis of the different promoter elements in the MLP has demonstrated that mutation of the UPE alone does not cause any severe effects on transcription from the MLP [160] and the same is noticed when a single nucleotide mutation is introduced into the CAAT-box. The two upstream activating elements are redundant, but the combination of these mutations causes the transcription from the MLP to decrease fifteen-fold [160]. An introduction of five nucleotide mutations either in the CAAT-box or in neighboring sequence together with the UPE mutant has an even more severe effect on viral replication [182]. The L1-region The L1 pre-mRNA is alternatively spliced during the viral life cycle, resulting in the accumulation of different mRNAs of the early and late phase of infection. A common 5’ ss is joined to either the proximal 3’ ss, giving raise to the 52,55K mRNA, or to the distal 3’ ss, creating the IIIa mRNA. The 52/55K protein is a scaffolding protein whereas the IIIa protein is a capsid protein. The exclusive L1 mRNA produced early in infection is the 52,55K mRNA due to a repression of the distal 3’ ss by SR proteins binding to an intronic repressor element, called the IIIa repressor element, the 3RE [83]. At late times of infection the IIIa mRNA becomes the predominant L1 mRNA expressed from the L1 unit. As we describe in Paper I, SR proteins become hypophosphorylated in late-infected cells resulting in a weakning of their RNA binding capacities [82]. The release of SR proteins from the 3RE element is important to induce the switch towards the distal IIIa 3’ splice site usage. Subsequently, it was shown that the IIIa branch point/pyrimidine tract acts as an inhibitory element in uninfected extract and as an enhancer element in infected extract. This element, which is the major element controlling IIIa 3’ ss activation, was named the IIIa virus infection-dependent splicing enhancer, 3VDE [137]. The L2-, L3-, L4- and L5-regions The L2-region encodes for five proteins where the core proteins VII and V are histone-like proteins that bind the viral DNA to form a structural core of the virus. pX is cleaved into two structural core proteins: X and P. Polypeptide III makes up a pentamer, called the penton base, which together with the fiber are assembled into the structural component penton [199]. Twelve fiber molecules are protruding out from the viral capsid and each are made up of a homo-trimer of polypeptide IV from the L5-region. The L3-region encodes for capsid protein VI and the homotrimeric hexon, II, which is the major coat protein. Each penton base is surrounded by five hexon proteins and polypeptide IX encoded from an intermediate gene function as a glue protein stabilizing the hexon proteins. The L3-region also en37 codes for the viral protease. Approximately 10-30 molecules of the L3 protease are included into each virus particle, which are needed for the uncoating of the virus. The viral protease is also active during virus maturation when it cleaves most of the minor capsid and the core polypeptides, including the terminal protein. Binding to DNA or an 11 aa peptide cleaved from pVI increases its activity thousand-fold [40]. One of the first proteins to be produced in the late phase of infection is the L4-100K protein. The L4-100K protein is needed for an efficient production of viral particles and one of its functions is to assemble the hexon protein into trimers in the cytoplasm [31]. It is tightly bound to mRNA [3] and a temperature sensitive L4-100K mutant displays reduced translation of viral mRNAs [64]. Studies have shown that the L4-100K protein stimulates translation of tripartite leader containing mRNAs by functionally inactivating the cellular cap-binding translation initiation factor eIF4F [42]. Viral factors, like E4-ORF4, are needed to affect alternative splicing within L1 and to trigger the shift towards production of proteins from the L2-L5 units. Recently, it was shown that a transfected MLP construct only containing L1-L3 was unable to shift from early to late protein expression. Addition of L4-33K in trans resulted in expression of L1-IIIa, L2 and L3 proteins suggesting that L4-33K might be important for the early-late protein expression shift from the MLTU during infection [46]. Table 7. Proteins present in the adenovirus particle Protein Transcription unit Copy number TP, terminal protein P V VII X L3 protease Capsid element E2B L2 L2 L2 L2 L3 2 ~104 Not known Not known Not known ~30-40 IIIa VI VIII IX Capsid L1 L3 L4 intermediate 60 monomers 60 hexamers Not known 80 trimers II, hexon III, penton base IV, fiber L3 L2 L5 240 trimers 12 pentamers 12 trimers Core 38 Present Investigation and discussion Paper I Regulation of adenovirus alternative RNA splicing by dephosphorylation of SR proteins In paper I we found a difference in splicing activity of SR proteins from uninfected and adenovirus infected cells and by further investigation we identified the adenoviral protein E4-ORF4 as a protein that can induce SR protein dephosphorylation. Previously, Kanopka et al. demonstrated that SR proteins like ASF/SF2 can repress splicing of the L1 IIIa pre-mRNA in an in vitro splicing assay. ASF/SF2 binds to an intronic repressor element, named 3RE, situated just upstream of the IIIa branch site, and blocks the formation of the A-complex. By moving the element it was shown that SR proteins could act as repressors or enhancers of splicing depending on where on the pre-mRNA the SR protein binding site was situated [83]. To extend the study on how SR proteins regulate splicing we compared the properties of SR proteins purified from HeLa nuclear extract, SR-HeLa, with SR proteins from adenovirus infected nuclear extract, SR-Ad. Interestingly, the SR-Ad proteins were inactive in in vitro splicing, both as splicing enhancer and splicing repressor proteins. Using an UV cross-linking assay we could also demonstrate that the SR-Ad proteins had a reduced RNA binding capacity. Normally, in in vivo splicing, SR proteins are highly phosphorylated, while SR proteins purified from adenovirus infected nuclear extract were shown to be hypophosphorylated, with the exception of SRp20. This was demonstrated by two-dimensional gel electrophoresis of purified SR proteins which had been 32P-labelled in vivo. A previous study had demonstrated that during infection with an adenovirus mutant lacking a functional E4-ORF4 protein hyperphosphorylated forms of c-Fos and E1A proteins are accumulating, by an unknown mechanism [135]. Soon after, E4-ORF4 was shown to interact with the B subunit of serine and threonine specific protein phosphatase 2A, PP2A, and the E4ORF4/PP2A complex possessed phosphatase activity which seemed to have a role in transcription regulation [19, 91]. In Paper I we demonstrate that a recombinant E4-ORF4 protein is able to induce SR-HeLa dephosphorylation when incubated in HeLa-NE. Addition of okadaic acid, which inhibits PP2A, 39 resulted in a loss of E4-ORF4 induced SR protein dephosphorylation. Normally, addition of SR-HeLa proteins to an in vitro splicing reaction programmed with the wild type IIIa transcript causes an inhibition of splicing. Addition of recombinant E4-ORF4 protein rescued IIIa splicing illustrating that dephosphorylation of SR-HeLa proteins reverses the SR protein mediated repression of IIIa splicing and that incubation with E4-ORF4 gives SRHeLa the properties of SR-Ad. Transient transfection experiments were utilized to investigate the effect of E4-ORF4 in vivo. Comparing different the IIIa constructs with or without the 3RE confirmed the result that E4-ORF4 can activate a repressed splicing also under in vivo conditions. When cotransfecting E4-ORF4 with a mini-construct containing both the 52,55K and the IIIa 3’ ss with the 3RE element a shift towards IIIa splicing was seen. This demonstrates that E4-ORF4 can reverse the SR protein mediated block in IIIa 3’ ss also during alternative splicing conditions. This paper was the first to show that alternative splicing is regulated by reversible protein phosphorylation. In an extension, Estmer Nilsson et al. demonstrated that E4-ORF4 binds both ASF/SF2 and SRp30c, but not the other SR proteins. E4-ORF4 binds preferentially to the hyperphosphorylated form of the SR proteins and interacts with ASF/SF2 through its RRMs [44]. In Paper I adenovirus infection caused a hypophosphorylation of all the classical SR proteins, with the possible exception of SRp20. In conclusion, some other viral factor, besides E4ORF4, is also needed for the SR protein hypophosphorylation. Other viral proteins affecting the activity of SR proteins Some viruses utilize alternative splicing to compress more information into their genomes. It ought to be of interest for such viruses to have the ability to regulate splicing by for example controlling the activity of the SR protein family of splicing factors. Viral proteins known to affect the activity of SR proteins are listed in Table 4. The herpes simplex virus ICP27 protein has been shown to bind SRPK1 and thereby cause a translocation of SRPK1 into the nucleus. ICP27 inhibits the activity of SRPK1 and SR proteins purified from HSV infected cells are hypophosphorylated [169]. Why is HSV-1 inactivating the SR family of splicing factors? It seems that the purpose for HSV-1 is somewhat different from the adenovirus induced SR protein dephosphorylation since HSV-1 contains very few introns. Another example of a virus that inhibits SR protein phosphorylation is the vaccinia virus [76]. The precise mechanism is unknown, but interestingly vaccinia virus genes do not have any introns. Both vaccinia and HSV-1 might cause hypophosphorylation of SR proteins as a strategy to inhibit the post-transcriptional processing of cellular transcript and thereby reduce the production of cellular proteins. In the case of adenovirus it seems that SR protein dephosphorylation gives the virus the 40 ability to regulate the shift in alternative splicing necessary to produce the viral proteins needed late in infection. Paper II p32 regulates RNA splicing by inhibiting ASF/SF2 RNA binding and phosphorylation No splicing activity was assigned to p32 in the first study when p32 was identified as a ASF/SF2 associated protein in HeLa cells [98]. In paper II we therefore decided to investigate whether p32 had a regulatory role on ASF/SF2 function rather than being a bona fide splicing factor itself. The important finding from this paper was that p32 indeed has a function in splicing regulation by sequestering ASF/SF2 into an inactive complex. p32 inactivates SR proteins as splicing regulators As expected, an in vitro splicing assay showed that splicing of a IIIa+3RE transcript in HeLa-NE was repressed due to the binding of SR proteins to the repressor element 3RE. Interestingly, addition of increasing amounts of recombinant p32 alleviated the repressive effect of SR protein on IIIa splicing. As a control for the specificity of the splicing enhancer effect of p32, another acidic protein was used, the glucose oxidase, GOD, showing no effect on the spliced products. Complex assembly assays showed that p32 acts at an early step of spliceosome assembly by enhancing the A complex formation. The 3RE element was then transferred to the intron of a E-globin transcript and the same splicing activation was noticed in the presence of p32. Previous experiments have shown that the position of an SR protein binding site in a pre-mRNA determines if the element acts as enhancer or a repressor element [83]. The transfer of 3RE from an intronic position to the 3’ end of a 52,55K transcript transformed the 3RE from a repressor element to an enhancer element of splicing [218]. In such a transcript addition of p32 repressed splicing by blocking A-complex formation. Altogether, these results demonstrated that p32 inactivates SR proteins both as repressor and enhancer proteins of splicing. To test if p32 could affect splicing under in vivo conditions we utilized transient transfection with plasmids expressing the p32 protein and the IIIa transcripts in HeLa cells. As expected, p32 activated IIIa 3’ ss usage. The in vitro and the in vivo data somewhat differed in the aspect that large amounts of p32 inhibited splicing. There is a possibility of aggregation of p32 in the cell due to overexpression of the protein, which could lead to p32 loosing its regulatory role in splicing. However, this is probably not the explanation, since p32 also inhibited IIIa-3RE when expressed in excess. More likely, p32 is sequestering ASF/SF2 in the cytoplasm and thereby disturbing 41 the normal balance of ASF/SF2 between the cytoplasm and the nucleus. Potentially, p32 may also affect the sub-cellular distribution of other SR proteins as well. p32 inactivates ASF/SF2 as a splicing regulator by blocking its RNA binding Further, our in vitro splicing results revealed that when the 3RE was exchanged with a consensus ASF/SF2 binding site in a IIIa transcript, p32 could still activate splicing. The addition of recombinant ASF/SF2 blocked A complex formation on a IIIa 3’ ss transcript, and p32 was able to release that repression. Krainer et al. have demonstrated that ASF/SF2 can influence alternative splicing by causing a shift from distal to proximal 5’ ss usage in a E-globin construct [97]. In agreement with their result ASF/SF2 promoted the proximal 5’ ss in our in vitro splicing assay, and importantly p32 had the opposite effect causing a shift from proximal towards distal 5’ ss usage. It should be noted that p32 affects splicing not only on adenoviral transcripts, but also on a cellular transcript indicating that it has a general role in splicing regulation. p32 does not bind RNA and therefore our hypothesis was that p32 directly regulated the RNA binding capacity of ASF/SF2, rather than competing for its RNA binding site like an antagonistic protein like hnRNP A1. To test this hypothesis we used an UV cross-linking assay. As predicted, addition of p32 caused a reduction in ASF/SF2 binding to RNA. By including hnRNP A1 in the reaction we could demonstrate that RNA was now available for the antagonist protein to bind. To demonstrate the specificity of p32’s action the acidic protein GOD was used as a control and it did not have any effect on the RNA binding capacity of ASF/SF2 in the UV crosslinking assay. In paper III we show additional results that p32 can reduce the RNA binding capacity of ASF/SF2 by using two additional constructs, IIIa+3RE and E-globin+3RE, in the UV cross-linking assay. Furthermore, in Paper II we used a recombinant ASF/SF2 protein lacking the RS-domain, ASF/SF2-'RS, in the UV cross-linking assay. Interestingly, p32 could also block the RNA binding capacity of ASF/SF2-'RS. Based on these observations we draw the conclusion that p32, at least in part, binds to the RNA-binding domain of ASF/SF2. This result is in contrast with the speculation of Yu et al. implying that the interaction of p32 with ASF/SF2 is due to ionic interactions between the highly basic RS domain in ASF/SF2 and the acidic C-terminus in p32 [216, 217]. In a later study (Paper III) we demonstrate that p32 in fact binds stronger to ASF/SF2 lacking the RS domain than full-length ASF/SF2, confirming that p32 interacts mainly with the RRMs of ASF/SF2. 42 Notably, the homologue of p32 in T.brucei, called p22, has been shown to stimulate the RNA binding capacity of the guide RNA (gRNA)-binding protein RBP16 [65]. Recently, Zheng et al. published a paper supporting our findings that p32 inhibits the function of ASF/SF2 [220]. Murine cells are unable to support HIV-1 replication. The unspliced 9kb HIV-1 transcript functions both as HIV-1 genome that should be incorporated into new viral particles and as an mRNA encoding the structural Gag-protein and the polymerase Pol. In murine cells, the 9 kb genomic transcript was excessively spliced and the amount of viral genomic and unspliced mRNA reduced, causing a reduction of Gagprotein and blockage of viral assembly. Introduction of human chromosome 11 in these transgenic cells had in a previous report been shown to complement these defects [178]. ASF/SF2 is known to enhance HIV-1 splicing by binding to ESEs in the exons of Tat [153, 191]. Given that human p32 is located on chromosome 11 and that we had shown that human p32 could inhibit ASF/SF2 as a splicing factor [152], Zheng et al. tested if human p32 could rescue HIV-1 infection. Indeed, p32 inhibited ASF/SF2 and prevented the excessive splicing of the HIV-1 genome, which relieved the posttranscriptional block and allowed HIV-1 to replicate in the mouse cells. The mouse p32 (called mp32 or YL2) and human p32 have a sequence homology of 92 % [114]. The difference in activity between human and mouse p32 was mapped to a single amino acid, aa 35 in human p32. Thus, changing aa 35 from glycine to aspartic acid abrogated the capacity of human p32 to block the excessive splicing of the HIV-1 genome. p32 blocks phosphorylation of ASF/SF2 Hypophosphorylated SR proteins have a reduced capacity to bind to RNA. Could p32 affect the phosphorylation status of ASF/SF2? By analyzing the phosphorylation of ASF/SF2 in the presence of different recombinant kinases in an in vitro kinase assay and then investigate the ability of p32 to bind to ASF/SF2 we observed that the phosphorylation status of ASF/SF2 affected the interaction between p32 and ASF/SF2. Even though SRPK1 phosphorylated ASF/SF2 more strongly than Clk/Sty, the Clk/Sty phosphorylation of ASF/SF2 was more efficient in preventing p32 from binding to ASF/SF2 than the SRPK1 phosphorylation indicating that Clk/Sty phosphorylates sites that are of more importance for p32 interaction with ASF/SF2 compared to SRPK1. Although phosphorylation of ASF/SF2 could reduce the interaction with p32, an even more interesting finding was that p32 could actually inhibit ASF/SF2 phosphorylation. By pre-incubating recombinant unphosphorylated ASF/SF2 and p32 before the addition of SRPK1, Clk/Sty, or HeLa-NE the inhibitory effect of p32 on ASF/SF2 phosphorylation was enhanced. Interestingly, p32 inhibition of ASF/SF2 phosphorylation was most effective against Clk/Sty phosphorylation. A dot-blot far-western approach also dem43 onstrated that phosphorylation by SRPK1 probably affected the conformation of ASF/SF2 causing an indirect effect on the interaction between p32 and ASF/SF2, while phosphorylation by Clk/Sty at the specific site(s) was directly inhibited p32 interaction. These findings also support the hypothesis that the amino acids in ASF/SF2 targeted by Clk/Sty are of greater importance for p32-ASF/SF2 interaction. The SRPK and Clk/Sty families of protein kinases have distinct enzymatic properties. Recently, Velazquez-Dones et al. preformed a mass spectrometric and kinetic analysis of SRPK1 and Clk/Sty phosphorylation of ASF/SF2. They demonstrated that SRPK1 efficiently phosphorylated a short stretch of amino acids near the N-terminal end of the RS-domain, while Clk/Sty was able to phosphorylate all 20 serine residues in the RS-domain. Current data indicate that the two kinases can phosphorylate ASF/SF2 sequentially, but not simultaneously and that they antagonize each other [203]. Further, Clk/Sty was shown to be a nuclear kinase in several cell lines [155]), whereas SRPK1 is predominantly cytoplasmic in interphase cells [201]. The current model that cytoplasmic SRPK family members are involved in the transport of SR proteins into the nucleus, while the nuclear Clk/Sty family of kinases are involved in regulating splicing [203] is in line with our finding that p32 efficiently inhibited Clk/Sty phosphorylation to regulate the RNA binding capacity of ASF/SF2. Other cellular proteins affecting the activity of SR proteins Not many cellular proteins have been reported to be able to affect the activity of SR proteins (see section Proteins regulating SR protein activity and Table 4). Some proteins compete with RS-proteins in binding to a particular RNA binding site, like hnRNP A1 and RBM4 [102]. The Tra2 proteins need SR proteins to be able to enhance splicing [189], while others like SRr86 can both enhance and repress SR protein induced splicing, through mechanism that currently is unknown [10]. So far, the only cellular protein known to affect the activity of a SR protein by controlling the phosphorylation and thereby the RNA binding capacity of an SR protein is the cellular protein p32 as reported in this thesis. Paper III Identification of a carboxy-terminal sequence in p32 necessary for p32-mediated inhibition of ASF/SF2 RNA binding In paper III the interaction between ASF/SF2 and p32 was studied in more detail. We chose to study the direct binding of the two proteins by using pull down assays with recombinant proteins and 35S-methionine labelled proteins produced by coupled transcription/translation in wheat germ extracts. 44 Results in Paper II demonstrated that p32 could prevent ASF/SF2-'RS from becoming phosphorylated in HeLa-NE and further that p32 could inhibit the RNA binding capacity of ASF/SF2-'RS. In paper III we demonstrated a direct interaction between ASF/SF2 lacking the RS-domain and recombinant p32 and found that the binding of p32 to ASF/SF2-'RS was much stronger than to full length ASF/SF2. Since the ASF/SF2 and the ASF/SF2-'RS proteins used in this study were produced in wheat germ extract this led to the production of phosphorylated proteins. A Calf Intestine Alkaline Phosphatase, CIAP, treatment of full length ASF/SF2 increased the binding to p32 to the same strength as for ASF/SF2-'RS, with or without CIAP treatment. The conclusion from this experiment was that p32 interacts primarily with the N-terminal RRMs in ASF/SF2. Further, reduction of the number of phosphorylated residues either by deletion or dephosphorylation of the RS-domain strengthened the interaction between p32 and ASF/SF2. p32 is known to bind to the lamin B receptor, p58, which is localized at the nuclear membrane. The p58 protein contains an N-terminal RS-rich region similar to the C-terminal RS-domain found in ASF/SF2. Due to this similarity Simos and Georgatos proposed that the RS-domain of p58 and ASF/SF2 would provide binding sites for p32 [179]. Later, it was shown that the N-terminal domain of p58 binds to p32 while a p58 mutant lacking the RS-domain does not bind. Interestingly, phosphorylation of p58 by a nuclear membrane RS kinase completely abolished binding to p32 [142]. Even though there are similarities in the interaction properties there are also differences since we have demonstrated that p32 binds directly to the N-terminal RRMs in ASF/SF2 and cause an inhibition of phosphorylation of the C-terminal RS-domain in ASF/SF2. Further, phosphorylation of the C-terminal RS-domain of ASF/SF2 prevents the p32-ASF/SF2 interaction (Paper II and III). We also wanted to determine which part of p32 that was important for the p32-ASF/SF2 interaction. The RNA binding capacity of recombinant ASF/SF2 was investigated in a UV-cross linking assay together with either recombinant full length p32 or a C-terminal deletion mutant of p32, p32-'C (amino acid 74-193). The recombinant p32-'C protein showed a dramatically reduced capacity to block ASF/SF2 RNA binding compared to the full length protein. In line with this result, pull down assays demonstrated that p32-'C protein, produced in wheat germ extract, could not bind to ASF/SF2. Analysis of other p32 C-terminal deletion mutants in pull down assays demonstrated that the region between amino acid 226-239 in p32 is critical for the interaction with ASF/SF2 (Figure 5). However, a GST-fusion protein containing the residues of 226-239 of p32 was unable to bind to ASF/SF2 suggesting that aa 226-239 in p32 are necessary but not sufficient for binding to ASF/SF2. The change of amino acid 35 from glycine to aspartic acid (G35D) in human p32 has a negative effect on HIV-1 replication in murine cells due to 45 excessive splicing of the HIV-1 transcript similarly to what have been shown with the murine p32 protein [220] (see section Paper II). This finding indicates that amino acid 35 is important for the ability of p32 to inhibit ASF/SF2. Zheng et al. speculated that introduction of the G35D mutation into human p32 disrupted the asymmetrically distributed negative charges in the doughnut shaped p32 trimer and thereby attenuated the ability of human p32G35D to inhibit ASF/SF2 activity [220]. Our results shows that a deletion of the C-terminal amino acids 239-282, as well as a internal deletion of amino acids 192-211, of p32 did not affect the interaction with ASF/SF2 indicating that the N-terminus of p32 might be involved in the direct binding. Although, a protein consisting of the Nterminal part of p32, p32-'C, consisting of aa74-193, and even p32(aa74226), could not bind to ASF/SF2 and p32-'C could not affect ASF/SF2 binding to RNA. Altogether, amino acids 226-239 in human p32 are important for the interaction with ASF/SF2, but this region is not sufficient for the direct interaction. The conclusion from our study is that it might be important for p32 to form a trimer to be able to bind to ASF/SF2. With these findings in mind it would be interesting to see if the G35D mutation of p32 is enough to prevent p32 from directly bind to ASF/SF2. Paper IV p32 represses the adenovirus major late transcription and causes hyperphosphorylation of RNA polymerase II In Paper IV we used recombinant adenovirus vector to overexpress the cellular protein p32 from an inducible promoter. Further, we used adenovirus as a model organism to analyze what effect p32 has on the protein expression from different types of viral genes. This in vivo study demonstrated that p32 has a promoter specific repressive effect on transcription which is manifested through regulation of Pol II phophorylation. Overexpression of p32 during an adenovirus infection causes a reduction of mRNA expression from MLP To further investigate how p32 affects splicing in vivo we constructed a recombinant adenovirus overexpressing p32 from an inducible promoter. The recombinant adenovirus Ad-TetTrip(flag-)p32, referred to as AdTT(flag-)p32 in paper IV, lacks the whole E1A and E1B-region, where the gene encoding an N-terminal flag-epitope tagged p32 fusion protein was inserted. The p32 gene is under the transcriptional control of an inducible promoter which has seven binding sites for the reverse tetracycline repressor, rtetR, and a minimal MLP. The Tet-ON system is based on a double infection strategy where the recombinant adenovirus with the gene of interest (in 46 this case the flag-p32 gene) and a recombinant helper adenovirus, AdCMVrtTA, expressing a fusion protein consisting of the DNA binding rtetR and the transcriptional activating domain of VP16 (Herpes simplex virus protein) under the constitutive Cytomegalovirus (CMV) promoter, are infecting the same cell. The rtetR protein, which is a mutant of the E.coli tetrepressor, tetR, binds to DNA in the presence of the antibiotic doxycyclin, dox. The purpose of the experiment was to investigate if p32 could affect the splicing pattern of the L1 mRNAs also under a lytic infection. To be able to analyze the late phase of infection the virus has to be able to replicate which was why the 293 cell line was chosen for infection. 293 cells are human embryonic neuronal cells from kindney [172] transformed with the E1A and E1B region of adenovirus type 5 (base pair 1-4033) and therefore provide the transcriptional activator E1A in trans [57]. Surprisingly, overexpression of p32 resulted in reduction of the total amount of mRNA expressed from the MLP and this led us to further analyze wether p32 had an effect on transcription. To investigate the specificity of p32 repression of transcription the mRNA amounts from the E2A- and the E4-region were also measured. In a wild type infection the early E2A promoter is activated by E1A, while the late E2A promoter is activated by E1B-55K and the CAAT-box binding protein YB-1 (see section Early genes) [71]. Northern blot of E2A revealed that the adenoviral E2A promoter is not affected by p32 overexpression. This result was also confirmed by Western blot assay demonstrating a stable expression of the E2A-72K protein. Similarly the adenoviral E4 promoter was not repressed by p32. Interestingly, the abundance of the largest E4 mRNA species was increased and we suggested that p32 might cause an affect on E4 alternative splicing in p32 overexpressing cells, similarly to what we reported in paper II. It has previously been demonstrated that inhibition of MLP by mutation analysis can affect early promoter usage and affect the splicing pattern of early genes [47]. Thus, since p32 represses MLP this might lead to an indirect effect on the E4 expression pattern. In conclusion, transcription of the MLP was severely inhibited by overexpressed p32, while the E2A and E4 promoters were not repressed. p32 repression of transcription is dependent on the presence of CAATbox In order to investigate the MLP promoter architecture important for p32 induced repression of transcription we utilized a transient transfection strategy with different CAT reporter constructs together with a plasmid constitutively expressing p32, pCMV-p32. Transcription of the wild type MLP-CAT construct is dramatically reduced with increasing amounts p32. The CAATbox (CCAAT) of MLP is one of two redundant upstream activating elements 47 important for MLP activity (CAAT-box and UPE element) and a mutation of the CAAT-box causes a reduction in virus replication especially if it is combined with mutation of the UPE element (see section Major late promoter; [160, 182]). As expected, introduction of a point mutation in the CAAT-box (CCCAT) in the MLP-CAT construct resulted in a reduced basal expression and destroyed the ability of p32 to further repress CAT-protein expression. Thus, p32 mediated transcriptional inhibition seems to need an intact CAATbox in the MLP. Maity et al. has reported that the CAAT-box binding transcription factor CBF/NF-Y is able to transcriptionally activate MLP [116]. Interestingly, p32 was recently shown to interact both in vitro and in vivo with CBF/NF-Y and further inhibit CBF/NF-Y activated transcription of the D2(1) collagen promoter [33]. The human tripeptidyl-peptidase II promoter contains two CAAT-boxes and has previously been demonstrated to bind CBF/NF-Y protein [110]. By utilizing different deletion mutants of the tripeptidyl-peptidase II promoter, lacking one or two CAAT-boxes, in front of the luciferase gene we could confirm that the inhibitory effect of p32 overexpression on transcription was dependent on the presence of intact CAATboxes. In conclusion, transient transfections show that transcription from promoters containing CAAT-boxes known to be stimulated by the CBF/NF-Y transcription factor, like the adenoviral MLP and the human tripeptidylpeptidase II promoter is inhibited by p32. p32 causes hyperphosphorylation of RNA Pol II CTD and affects the processivity of transcription elongation In order to analyse the effect of p32 on Pol II, chromatin immunoprecipitation assays, ChIP assays, were used to measure the presence of Pol II on the MLP and its coding region. Cells infected with the recombinant virus were induced to express p32. The cells were cross-linked with formaldehyde at 22 hpi forming covalent bonds between interacting proteins and protein-DNA. The DNA was fragmentized by sonication, immunoprecipitated and PCR amplified in order to analyse the gene of interest at the moment of fixation. Using an antibody that targets the N-terminus of the large subunit of Pol II we analysed the polymerase distribution on the L1 region of MLTU. PCR was used to amplify fragments from the MLP, the middle and at the end of the L1 gene. We detected a large fraction of Pol II at the promoter region suggesting pausing of the polymerase and also some accumulation towards the end of the gene. Though, the pausing effect on the promoter was not at the same extent as previously reported on eukaryotic genes [34]. As expected, hypophosphorylated polymerase, Pol IIA, was primarily detected at the promoter region. ChIP assays have showed that phosphoserine 5 specific antibody H14 cross-links primarily to Pol II at the promoter proximal region, while the phosphoserine 2 specific antibody H5 cross-links primarily to the 48 coding region of the gene [94]. This pattern was also found in the L1-region of wild-type adenovirus in our ChIP assays with the H14 and H5 antibodies. Using the Ad-TT(flag-)p32 virus and comparing the result between doxycyclin induced and uninduced samples we demonstrated that p32 caused a reduction of polymerase molecules during the elongation phase. The ChIP assays using H14 and H5 antibodies detecting Pol II IIO demonstrated that the polymerases that still were transcribing had an increased amount of both Ser 5 and Ser 2 phosphorylation, respectively. Incubation of GST-CTD protein together with increasing amount of recombinant p32 in vitro confirmed that p32 enhances the phosphorylation of the polymerase. Analysis of nuclear extract from p32 overexpressing cells revealed that a large fraction of the endogenous Pol II was hyperphosphorylated at both Ser 5 and Ser 2 positions. Regulating the activities of RNA Pol II phosphatases and kinases Overexpression of p32 during transcription of the MLTU L1-region causes both Ser 5 and Ser 2 hyperphosphorylation, which represses the processivity of the Pol II. Other proteins have been reported to affect the phosphorylation/dephosphorylation cycle of Pol II (Table 2). Interestingly, some of these proteins are causing a hyperphosphorylation of the CTD resulting in transcriptional inhibition similar to that by p32. The peptidyl-propyl isomerase Pin 1 causes hyper-hyperphosphorylation of CTD in vitro by both inhibiting FCP1 and stimulating the kinase cdc2/cyclin B, a mitotic kinase which is able to phosphorylate CTD at both Ser 5 and Ser 2 [211]. By constructing an inducible stable Pin 1 cell line, Xu et al. showed that overexpression of Pin 1 inhibited transcription in vivo. Further, Pin 1 caused a Pol II dependent inhibition of pre-mRNA splicing in vitro [211]. The yeast homologue of Pin 1, Ess1p, binds to the CTD both in vivo and in vitro and has been shown to interact with all four yeast CTD kinases. The present model is that Essp1 affects transcription of mitotic genes by coordinating multiple steps in transcription involving regulation of both isomerisation and phosphorylation of the CTD [134, 206, 208]. Further, BRCA1, a DNA binding protein which has shown to be a component of the Pol II holoenzyme, can block the ATP binding site of CDK7/TFIIH and thereby prevent the phosphorylation of Ser 5 [133]. The FCP1 phosphatase is regulated by HIV-1 Tat, CK2, TFIIB and the large subunit RAP74 of TFIIH. Phosphorylation of FCP1 by CK2 enhances FCP1 activity and strengthens its binding to RAP74, which even further stimulates the phosphatase activity [2]. The HIV-1 Tat protein inhibits both the binding of RAP74 to FCP1 and CK2 phosphorylation, resulting in an inhibition of FCP1 [1]. During HIV-1 Tat transcriptional activation Tat is on the other hand stimulating P-TEFb to phosphorylate Ser 5 and Ser 2 [89, 221]. Recently, Epsteinn-Barr virus, EBV, EBNA 2 was shown to stimulate transcription by hyperphosphorylating Ser 5 [9]. The recruitment of Pol II to 49 the promoter region was increased in presence of EBNA 2 and showed normal level of Ser 5 phosphorylation. During the elongation phase of transcription this increased number of Pol II showed an increasing level of Ser 5 phosphorylation but no further stimulation on Ser 2 phosphorylation [9]. The regulatory proteins of CTD kinases and phosphatases are of different origin and further the recruitment to their target enzymes differ; binding to the DNA, to transcription activators/repressors, to classical transcription factors, or via the CTD of Pol II. Some of the proteins in Table 2 seem to be acting gene specific or having a cell cycle specific function, like HIV-1 Tat and Pin1/Ess1p, while others might have a more general function since they interact with subunits of the Pol II complex, like RAP74. PIC formation Initiation Elongation Termination RNA pol II CTD CBF/NF-Y p32 Ser 5 phosphorylation Ser 2 phosphorylation Figure 7. Model on p32 repression of a CBF/NF-Y activated promoter. Overexpression of p32 causes hyperphosphorylation of both Ser 5 and Ser2 in the Pol II CTD and the processivity during the elongation phase of elongation is reduced. 50 Our data and the report of Chattopadhyay et al. support a model where p32 is recruited to promoters containing binding sites for the transcription factor CBF/NF-Y (paper IV and [33]). It is possible that CBF/NF-Y recruitment places p32 proximal to the initiation site leading to a direct association to the CTD. p32 might even travel along with the elongating complex and act on kinases/phosphatases during the elongation phase. Unfortunately, in our study we did not succeed in demonstrating a direct interaction between p32 and the Pol II or the MLP during elongation. This is due to low specificity of the flag epitope antibody used. Another hypothesis is that p32, bound to CBF/NF-Y on the MLP promoter, prevents the recruitment of FCP1 to the PIC during the transition from the initiation phase to the elongation phase of transcription. FCP1 has been shown to have a stimulatory effect on transcription elongation and that activity is independent on its phosphatase activity [36, 119]. ChIP experiments have also showed that FCP1 remains associated with Pol II during elongation [35]. Prevention of binding or sequestering of FPC1 would most likely lead to loss of FCP1 mediated stimulation of elongation. This hypothesis is in line with our results since p32 causes an increase of phosphorylation of both Ser 5 and Ser 2 and a reduced processivity of the Pol II during elongation. p32 might also recruit new CTD kinases or stimulate the activity of protein kinases like CDK7 and CDK9. In the hyperphosphorylated Pol II not all Ser 5 and Ser 2 residues in the 52 heptad repeats are phosphorylated at the same time and we speculate that p32 might stimulate phosphorylation of novel Ser 5 and/or Ser 2 residues, which might be more difficult to dephosphorylate. Interestingly, both Pin 1 and HIV-1 Tat inhibit the dephosphorylation activity of FCP1 and activate phosphorylation by specific kinases. This finding opens up the possibility for a dual function for p32 as well. To further dissect the repressive role of p32 on transcription of CAATbox containing genes it would be interesting to analyse the position of the p32 protein during the transcription process, as well as doing direct interaction studies between p32 and possible p32 interacting candidates like the kinases CDK7 and CDK9, the phosphatase FCP1, and different subunits of the Pol II would be of interest. 51 Conclusions Paper I x SR proteins inactivated in the late pase of adenovirus infection due to hypophosphorylation x E4-ORF4 and phosphatase PP2A cause the dephosphorylation of SR protein during the infection x Alternative splicing is regulated by reversible protein phosphorylation Paper II x p32 inactivates ASF/SF2 both as a splicing repressor and a splicing enhancer protein x p32 blocks the phosphorylation of ASF/SF2 and reduces the RNA binding capacity of ASF/SF2 Paper III x P32 binds to the RBD of ASF/SF2 and dephosphorylation of ASF/SF2 strengths the binding to p32 x The region of aa 226-239 in p32 is important, but not sufficient itself for binding to ASF/SF2 Paper IV x p32 inhibits transcription of genes with CAAT-box containing promoters. Further the results suggest that p32 is recruited to the promoter through the interaction with the transcription factor CBF/NF-Y. x p32 causes hyperphosphorylation of both Ser 5 and Ser 2 on the CTD of the large subunit of Pol II, both in vitro and in vivo. x p32 induced hyperphosphorylation causes a reduction in the processivity of Pol II elongation. 52 Acknowledgements First of all I would like to thank my supervisor Professor Göran Akusjärvi for all patience you have had with me during all these years. I am so grateful for the all your support and help, especially during these last interesting years. You have taught me a lot and you have encouraged me to think how to always try to take the project to a higher level. I also appreciate that you always have had time for me to discuss my projects. Thank you for allowing me to proceed with the thesis, even though the years started to fly away… I would also like to give my warmest thanks to Docent Catharina Svensson for your contribution to my development as a scientist. Thanks for all feed-back on my research, and all the pep-up talks you have been given me through the years! For the nice lunch talks about everything in life out on the sunny balcony! To my examiner Professor Göran Magnusson and to Professor Stefan Schwartz, for taking time to share your virology knowledge. All the virology PhD students, post-docs and scientists in the Adeno-group: Anders Sundqvist, Anne-Christine Ström, Anette Carlsson, Anette Lindberg , Arvydas Kanopka, Baigong Yue, Bin Wang, Camilla Estmer-Nilsson, Cecilia Johansson, Dan Edholm, Daniel Öberg, David Huang, Edyta Bajak, Ellenor Bäckström, Elisabeth Boija, Gunnar Andersson, Heidi Törminen, Josef Seibth, Kerstin Sollerbrant, Karin Öhman-Forslund, Lamine Bouakaz, Magnus Molin, Maria Bondesson, Mattias Mannervik, Martin Lützelberger, Niel Portwood, Ning Xu, Oliver Mühlemann, Petra Olsson, Peter Kreivi, Raj Aravelli, Saideh Berenjian, Shaoan Fan, Svend Petersen-Mahrt, Tanel Punga, Vita Dauksaite, Xiaofu Zhou and Öjar Melefors. Without all the fun that we have had together I probably wouldn’t have made it to the very end. I’m grateful for all the incredible memories in which you are included! The Friday meetings-members! All of you mentioned above and Anna, Anton, Emilio, Hongxing, Marcin, Margaret, Monika, Ulrika, Xiaomin and everyone who has attended at theses meetings have been to so much help. Thanks for all input you gave me on my projects and my literature seminars. It has been a tough, but great, school to discuss experiment failures and nice data, to handle criticism and encouragement, in front of such a very competent audience. For the staff that have always been there for me no matter if it had to do with broken machines, material supply, administration help, computer problems or a nice talk over a cup of coffée: Barbro Lowisin, Erika Enström, Kerstin Lidholt, Lena Möller, Lilian Ekström, Maud Pettersson, Olav Nordli, Tony Grundin and Ylva Jansson. 53 Cecilia- There would not be a thesis without you, at least not one in this nice shape! It has been fantastically fun to have you as my travel company the last years of this bumpy journey! You mean so much to me and I’m not sure that I will allow you to move abroad! Saideh- For being my friend and for all our talks about science and more importantly about the world “outside”. It has been invaluable for me. Tanel- For being such a friendly person and always helping out and sharing both your ideas and the lab stuff. Without the ChIPs, no thesis… Svend- for your enthusiasm and for introducing me to the multifunctional p32. Camilla- your friendship + our great cooperation during the struggling with the S1s. Ellenor- for being my exercise-friend and still pushing me to go there… Heidi- for sharing office and spreading a nice atmosphere. Marika and Andrzej- For the fun we have had before the kids and after! For our very important loooong phone calls! Marika, I know that you are always there for me and that means a lot to me… Anne-Christine- for your energy and never ending good mood! For cheering me up and having pep-talks about what to really focus on. Karin och Arne- for your friendship and support from day one, and all nice occasions at Bönan and in your home. Micke och Anna-Karin- for all memorable vacations and weekends we have spent together during these years, Idre, Skåne, Båven… J2+E2+W- for your friendship and fantastic dinners. For believing in me when I have said that “There will be a party soon…” and now: Dissertation party without you!!! Jonas and Johanna, I promise I will report everything to the Black Book. Ida, Lars and Wilhelm- for your support and to our future to come with CDF! Frida och Calle- for all cosy times together. Eva and Kurt- for all love that I get from you! For taking such good care of all of us and our home! My extra-parents Kerstin och Janne- for showing an endless support not only for me but also for the rest of my family. Annika and Tobias- for opening my eyes to another world! Annika, for being min älskade Pyss! Mamma och Pappa, What can I say! You did it! Your support in every level of my life and your endless belief in me made this thesis to come true. Thanks for allowing us to “refuel” at Tegelön during all of these summers and for all help during the years. Jag älskar er! Min älskade Maja och min älskade Saga! Ni är mitt allt! Johan- Without you, no list of references! Honestly, no thesis either. Thanks for taking care of me during all the ups and downs… You are always there for me no matter what and I am so grateful for that. I want to thank you for making my life to what it is. You give me so much love och jag älskar dig så… 54 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Abbott, K.L., Archambault, J., Xiao, H., Nguyen, B.D., Roeder, R.G., Greenblatt, J., Omichinski, J.G., and Legault, P., (2005) Interactions of the HIV-1 Tat and RAP74 proteins with the RNA polymerase II CTD phosphatase FCP1, Biochemistry. 44(8): p. 2716-2731. 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