Epstein-Barr Virus SM Protein Utilizes Cellular Splicing Factor SRp20
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JOURNAL OF VIROLOGY, Nov. 2010, p. 11781–11789 Vol. 84, No. 22
0022-538X/10/$12.00 doi:10.1128/JVI.01359-10
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Epstein-Barr Virus SM Protein Utilizes Cellular Splicing Factor SRp20
To Mediate Alternative Splicing䌤
Dinesh Verma, Swarna Bais, Melusine Gaillard, and Sankar Swaminathan*
Division of Infectious Diseases, Department of Medicine, and Shands Cancer Center, University of Florida, Gainesville, Florida 32610
Received 28 June 2010/Accepted 24 August 2010
Epstein-Barr virus (EBV) SM protein is an essential nuclear protein produced during the lytic cycle of EBV
replication. SM is an RNA-binding protein with multiple mechanisms of action. SM enhances the expression
of EBV genes by stabilizing mRNA and facilitating nuclear export. SM also influences splicing of both EBV and
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cellular pre-mRNAs. SM modulates splice site selection of the host cell STAT1 pre-mRNA, directing utilization
of a novel 5ⴕ splice site that is used only in the presence of SM. SM activates splicing in the manner of SR
proteins but does not contain the canonical RS domains typical of cellular splicing factors. Affinity purification
and mass spectrometry of SM complexes from SM-transfected cells led to the identification of the cellular SR
splicing factor SRp20 as an SM-interacting protein. The regions of SM and SRp20 required for interaction
were mapped by in vitro and in vivo assays. The SRp20 interaction was shown to be important for the effects
of SM on alternative splicing by the use of STAT1 splicing assays. Overexpression of SRp20 enhanced
SM-mediated alternative splicing and knockdown of SRp20 inhibited the SM effect on splicing. These data
suggest a model whereby SM, a viral protein, recruits and co-opts the function of cellular SRp20 in alternative
splicing.
SM protein (EB2, Mta, and BMLF1) is a nuclear phospho- pathways (for a review, see reference 7). STAT1 is expressed
protein synthesized by Epstein-Barr virus (EBV) during the as two isoforms, STAT1␣ and STAT1. STAT1 mRNA is
early stage of lytic replication (for a review, see reference 38). generated by cleavage and polyadenylation at an alternative
SM has multiple functions in enhancing EBV gene expression site in the last intron of the STAT1 pre-mRNA, leading to
posttranscriptionally, binds target gene mRNA, enhances nu- production of a protein which lacks the transactivating do-
clear mRNA export and stability, and modulates cellular and main encoded in the last exon of the STAT1 gene (see Fig.
EBV RNA splicing (2, 9, 17, 18, 23, 31, 35, 39, 40). SM is 5A). STAT1 homodimers are not capable of activating
essential for EBV replication and EBV recombinants with GAS sequences, and STAT1 may therefore act as a dom-
insertional deletion of the SM gene are defective for virus inant-negative repressor of STAT1␣ (3, 27, 41). Consistent
production (12). SM is required for the efficient accumulation with a role for STAT1 as an antagonist of STAT1␣, the
of ca. 60% of EBV lytic transcripts (13). SM is required for ratio of STAT1␣ and - isoforms has been shown to affect
efficient expression of both EBV DNA primase (BSLF1) and cellular apoptosis and resistance to viral infection (1, 26).
EBV DNA polymerase (BALF5) mRNAs, leading to severely Interestingly, SM disproportionately increases the relative
impaired lytic EBV DNA replication in the absence of SM amounts of STAT1 mRNA.
(13). SM also directly enhances accumulation of specific late Further investigation of previous findings that SM changed
gene mRNAs in addition to enabling DNA replication (13). the ratio of two functionally distinct STAT1 isoforms gener-
This combination of effects on DNA replication and late gene ated by alternative processing (30) led to the finding that SM
mRNAs leads to a global deficiency of late gene expression in directed splicing of STAT1 to an alternative 5⬘ splice site with
the absence of SM. high efficiency and specificity (40). This activity was based on
We recently demonstrated that SM acts as an alternative preferential binding of SM to specific regions of the pre-
splicing factor and modulates cellular splicing (40). The effects mRNA, indicating that SM may function in a manner similar to
of SM on host cellular gene expression during lytic EBV cellular splicing factors. Although SM does bind to RNA di-
replication remain to be fully characterized. When inducibly rectly (14, 29), it does not possess arginine-serine (RS) repeats
expressed in EBV-negative cells, SM has a broadly inhibi- typically found in cellular SR proteins that act as alternative
tory effect on cellular mRNA accumulation (30). Neverthe- splicing factors (11). We report here the interaction of SM with
less, SM causes several cellular transcripts to accumulate at SRp20, a cellular SR protein, and its role in modulation of
higher levels (30). These transcripts include STAT1 and splicing by SM.
several interferon-stimulated genes. The STAT1 protein is
an integral mediator of both type I (alpha/beta interferon MATERIALS AND METHODS
[IFN-␣/]) and type II (IFN-␥) IFN signal transduction Cell lines and transfections. 293 is a cell line derived from human embryonic
kidney cells (10). 293T and HeLa cells were maintained in Dulbecco modified
Eagle medium containing 10% fetal calf serum supplemented with Glutamax
* Corresponding author. Mailing address: 2033 Mowry Road, (Invitrogen). HeLa cell transfections were performed with Lipofectamine Plus
Gainesville, FL 32610-3633. Phone: (352) 273-8206. Fax: (352) 273- (Invitrogen) in six-well plates using 1 g of DNA per transfection, according to
8299. E-mail: sswamina@ufl.edu. the manufacturer’s protocols. 293T cells transfections were performed with
䌤
Published ahead of print on 1 September 2010. TransIT293 reagent according to the manufacturer’s protocols (Mirus). Cells
1178111782 VERMA ET AL. J. VIROL.
were harvested 48 h after transfection. P3HR1/ZHT cell line was derived from buffered saline [pH 7.4], 500 mM NaCl, 1% Triton X-100) and once in low-salt
P3HR-1 (28) by transfection with a plasmid expressing a tamoxifen-inducible wash buffer (Tris-buffered saline [pH 7.4], 150 mM NaCl, 1% Triton X-100) and
EBV Zta activator of lytic gene expression (pCDNA3-ZHT), followed by selec- then boiled for 5 min in 1⫻ SDS-PAGE loading buffer. After electrophoresis,
tion in G418 (20). P3HR1/ZHT cells were cultured in RPMI supplemented with proteins were transferred to nitrocellulose membranes (Bio-Rad) and probed
10% estrogen-free fetal bovine serum and 0.8 mg of G418 (AG Scientific)/ml. with specific antibodies. The following conditions were used for immunoblotting:
EBV lytic replication was induced by adding 100 nM 4-hydroxytamoxifen for SM, a 1:500 dilution of rabbit polyclonal antibody; for HA, a 1:600 dilution
(Sigma) to the culture medium as previously described (30). of mouse monoclonal antibody (16B12; Covance); for SRp20 antibody, a neat
Plasmids. To generate the STAT1 minigene construct, pDV1, 1.088 kb of the hybridoma supernatant was used (CRL-2384; American Type Culture Collec-
STAT1 gene (AY865620, nucleotides 40293 to 41381), which contains exon 23, tion). After washing, secondary antibodies were then applied to primary anti-
exon 24, and the intron between exons 23 and 24, was amplified by high-fidelity bodies using horseradish peroxidase-conjugated sheep anti-mouse or goat anti-
PCR and cloned in mammalian expression vector pCDNA3 (Invitrogen) at the rabbit antibodies (Amersham) diluted to 1:3,000 for monoclonal antibodies and
EcoRV site (40). SRp20 expression vector (amino acids [aa] 1 to 164) and 1:7,500 for polyclonal antibodies. Antigen and antibody complexes were then
various truncated mutants of SRp20 (aa 1 to 104,1 to 83, 1 to 50, and 1 to 25) detected with enhanced chemiluminescence detection reagents (Pierce). Quan-
were generated by PCR using AccuPrime Pfx DNA polymerase (Invitrogen) and titation of the band intensity was performed by using ImageQuant software and
cloned into pcDNA3 expression vector. All SRp20 constructs were tagged with a Photoshop.
hemagglutinin (HA) epitope at N terminus by cloning into pCDNA3 with an HA GST pull-down assays. Cell-free coupled transcription-translations were per-
epitope inserted between the HindIII and BamHI sites. DV30 contains the SM formed according to the manufacturer’s protocols (TnT-Quick kit; Promega).
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response element cloned in the intron of the splicing reporter plasmid pI-12 (40). Full-length SRp20 mRNA was transcribed from 1 g of linearized plasmid DNA
The PI-12 plasmid provided by Mariano Garcia-Blanco has been previously and translated in the presence of [35S]methionine (Perkin-Elmer). Fusion pro-
described (5). The SRp20 expression plasmid was kindly provided by H. Lou teins consisting of GST fused to SM were synthesized in bacteria (E. coli DH5␣).
(24). The SM expression plasmid was constructed by PCR amplification from Portions (25 g) of GST or GST fusion proteins were incubated with glutathi-
B95-8 EBV DNA as previously described (31). Glutathione S-transferase one-Sepharose beads in binding buffer (1⫻ PBS, 0.1% NP-40, 0.5 mM dithio-
(GST)-SM fusion plasmids were generated by cloning various PCR-generated threitol, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride) in the presence of
fragments of SM into pGEX plasmid and have been reported previously (29). A bacterial protease inhibitors (P8465; Sigma). Then, 25 l of [35S]methionine-
FLAG-calmodulin-binding peptide (CBP)-tagged SM expression plasmid was labeled SRp20 protein was incubated with each fusion protein bound to Sepha-
constructed by sequential cloning of PCR-amplified fragments into pCDNA3. rose beads for 2 h at 4°C. The beads were washed five times with binding buffer,
The FLAG, CBP, and tobacco etch virus (TEV) protease sites were amplified and bound proteins were treated for 5 min in 1⫻ SDS-protein loading buffer at
from pMZ1 (42) and inserted with the three FLAG epitopes, followed by a TEV 95°C and analyzed by SDS-PAGE and autoradiography.
protease site and the CBP epitope located upstream of the amino terminus of
SM. All plasmids constructed in our laboratory were verified by DNA sequenc-
ing. RESULTS
SM complex affinity purification. SM complexes were purified from cell lysates
48 h after transfection. 293T cells were transfected with FLAG-tagged SM SM associates with SRp20 in vivo. In order to gain insight
expression vector or empty vector. Cells were detached from plates with phos- into the mechanism of SM function in enhancing gene expres-
phate-buffered saline (PBS) containing EDTA, washed, and lysed in lysis buffer sion and regulating splicing, we performed a series of purifi-
(50 mM Tris-HCl [pH 7.4], 1 mM EDTA, 1% Triton X-100) with mammalian
cations to isolate cellular protein complexes containing SM. To
protease inhibitor cocktail (Sigma), followed by incubation at 4°C with occasional
mixing for 30 min, followed by sonication at 4°C. Lysates were clarified by identify SM-interacting proteins, we isolated protein com-
centrifugation for 10 min at 12,000 ⫻ g and incubated for 2 h with anti-FLAG M2 plexes from cells transfected with an epitope-tagged SM ex-
antibody-conjugated affinity gel. The agarose matrix was washed extensively and pression vector. We constructed a version of SM tagged with
SM complexes were eluted with 150 ng of FLAG peptide/l in Tris-buffered three FLAG epitopes and a CBP epitope separated by a TEV
saline with shaking for 30 min at room temperature. Vector-transfected cells and
SM-transfected cells were processed in parallel, and eluates were analyzed by
protease site (Fig. 1A). Epitope tagging was performed by the
SDS-PAGE and silver staining to assess recovery and purity. Tandem mass fusion of PCR-generated epitopes to the amino terminus of
spectrometry (MS/MS) analysis of protein complexes in solution was performed SM. We confirmed that the epitope-tagged SM retained func-
at the University of Florida ICBR Proteomics facility, and the data were analyzed tion in enhancing reporter gene expression by performing co-
by using Scaffold software.
transfection assays (data not shown). Although tandem affinity
In vitro splicing assays. RNA from transfected HeLa cells were harvested 48 h
after transfection. Cells were washed with PBS and lysed in 750 l of QIAzol purification has been used successfully to isolate complexes
reagent (Qiagen), and RNA was isolated by using miRNeasy mini columns from a variety of cell types, we found that the yield of SM was
according to the manufacturer’s protocol (Qiagen). Reverse transcription-PCR severely reduced after binding to calmodulin-Sepharose and
(RT-PCR) was performed with a One-Step Access Quick RT-PCR kit (Pro- TEV protease cleavage. We therefore utilized a single-step
mega) according to the manufacturer’s instructions. The primers used for am-
purification with FLAG antibody-conjugated matrix alone.
plification of the STAT1 splice products were previously reported (40). RT-PCR
products were analyzed by electrophoresis in 1.5% agarose gels, followed by Optimization of washing conditions and elution with a triple
ethidium bromide staining. The sequence of the spliced products was verified by FLAG peptide resulted in adequate reduction of background
DNA sequencing as necessary. The relative intensity of the bands in ethidium- to permit analysis of SM complexes by MS. Verification of the
stained agarose gels was quantified by using both ImageQuant and Photoshop presence of SM and its associated proteins in complexes puri-
software.
Immunoprecipitation and immunoblotting. 293T cells were harvested 48 h
fied from SM-transfected cell lysates was performed by silver
after transfection and lysed in ice-cold lysis buffer (Tris-buffered saline [pH 7.4], staining of polyacrylamide gels (Fig. 1B). Negligible back-
1% Triton X-100, protease inhibitor cocktail [Sigma-Aldrich], phosphatase in- ground was observed in eluates from vector-transfected cells
hibitor cocktail [Roche]). The cells were lysed in 200 l of lysis buffer for 10 min (Fig. 1B). The identity of the prominent band on the silver-
on ice with frequent pipetting and were also sonicated for maximum lysis. The
stained gel was confirmed to be SM by immunoblotting with
lysed cell suspension was centrifuged for 30 min at 10,000 ⫻ g at 4°C. The cleared
supernatant was precleared with 1.0 g of rabbit immunoglobulin G (Bethyl), anti-FLAG antibody (Fig. 1C). Liquid eluate samples were
followed by incubation with protein A-conjugated agarose beads. The precleared digested with trypsin and subjected to a solid-phase extraction
samples were used for immunoprecipitation. Mouse monoclonal HA antibody and Nanoflow liquid chromatography-MS/MS on an ABI
(Covance), control IgG (Bethyl), or rabbit polyclonal anti-SM antibody (31) was QSTAR XL.
added to the lysate, followed by incubation for 1 h at 4°C. Immune complexes
were incubated with either protein A-conjugated beads or protein G-conjugated
One of the proteins identified in this manner was SRp20
beads for 2 h at 4°C. RNase treatment was performed with 100 g of RNase A/ml (SFRS3), a nucleocytoplasmic shuttling cellular SR protein
for 30 min at 37°C. The beads were washed four times in wash buffer (Tris- involved in both splicing and RNA export (6, 19). SRp20 has aVOL. 84, 2010 EBV SM PROTEIN UTILIZES SRp20 IN ALTERNATIVE SPLICING 11783
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FIG. 1. Affinity purification of SM protein complexes. (A) Diagram
of epitope-tagged SM gene used for affinity purification of SM com-
plexes. A triple FLAG epitope was fused in frame to a TEV protease
site and a CBP epitope at the amino terminus of SM cDNA.
(B) Epitope-tagged SM was transfected into 293T cells, and SM was
isolated by affinity purification with M2 anti-FLAG antibody-conju-
gated beads, followed by elution with triple FLAG peptide. Eluates of
lysates from vector-transfected (C) or SM-transfected cells (SM) were
analyzed in parallel by SDS-PAGE and silver staining. The major SM
band is shown at right. (C) Immunoblotting of unfractionated cell
lysate and affinity-purified FLAG-SM. 293T cells transfected with FIG. 2. Interaction of SM with SRp20. (A) Diagram of SRp20
FLAG-SM plasmid or empty vector (C) were lysed, clarified by cen- structure. The amino-terminal RBD and the RS region comprised of
trifugation, and analyzed by immunoblotting with anti-FLAG antibody arginine-serine dipeptide repeats is shown. The domain that is re-
in parallel with eluates from the M2 FLAG antibody-Sepharose col- quired for interaction with SM lies between aa 83 and 105. The amino
umn shown in panel B above. acid numbering is indicated below. (B) Coimmunoprecipitation of
transfected SM and SRp20. Lysates of cells transfected with HA-
tagged SRp20 and either empty vector (C) or SM were immunopre-
cipitated with either preimmune serum (PI) or anti-SM antibody (Ab).
structure typical of highly conserved SR proteins that play Input lysates used in the immunoprecipitations are shown at left.
multiple roles in constitutive and regulated splicing and con- Immunoblotting was done with anti-HA monoclonal antibody. (C) Co-
immunoprecipitation of cellular SRp20 with SM. 293T cells were trans-
tain one or two RNA-binding domains (RBDs) and a carboxy- fected with SM or empty vector (C) and immunoprecipitation was
terminal RS domain with various numbers of RS repeats (Fig. performed with preimmune serum (PI) or anti-SM antibody (Ab).
2A). To confirm the interaction of SM and SRp20, we per- Endogenous cellular SRp20 coimmunoprecipitating with SM was de-
formed cotransfections of HA-tagged SRp20 and SM in 293T tected by anti-SRp20 monoclonal antibody (upper panel). P3HR1/
ZHT Burkitt lymphoma cells were induced to permit lytic EBV rep-
cells and immunoprecipitated the cell lysates with either anti-
lication and immunoprecipitation was performed with either normal
SM antibody or preimmune serum. Immunoprecipitates were rabbit serum (IgG) or anti-SM antibody (Ab), and immunoblotting
analyzed by SDS-PAGE, followed by immunoblotting with was performed with anti-SRp20 monoclonal antibody (lower panel).
anti-HA monoclonal antibody to detect SRp20 (Fig. 2B). (D) Resistance of SRp20-SM interaction to RNase. Immunoprecipi-
SRp20 was coimmunoprecipitated by anti-SM antibody but not tation of lysates from cells transfected with HA-tagged SRp20, and
either empty vector (C) or SM, was performed with anti-SM antibody,
by preimmune serum. We next sought to determine whether as in panel B above. Immunoprecipitates were treated with RNase A
SM could be shown to interact with endogenous cellular (⫹) or mock treated (⫺) prior to immunoblotting with anti-HA anti-
SRp20 in EBV-infected cells. 293T cells were transfected with body to detect SRp20.
either SM or empty vector, and SM was immunoprecipitated
with anti-SM antibody. The immunoprecipitates were analyzed
by immunoblotting with anti-SRp20 monoclonal antibody. As and the hormone-binding domain of estrogen receptor, which
shown in Fig. 2C, SRp20 was specifically immunoprecipitated allows nuclear translocation of Zta and triggering of EBV lytic
with SM. In order to determine whether SM interacts with replication when tamoxifen is added to the culture medium
SRp20 during the course of EBV lytic replication, we per- (20). SM was immunoprecipitated from tamoxifen-treated
formed the analogous experiment in an EBV-infected Burkitt P3HR1/ZHT cells, and the coimmunoprecipitated SRp20 was
lymphoma cell line. P3HR1/ZHT is a derivative of the human detected by immunoblotting with anti-SRp20 monoclonal an-
Burkitt lymphoma P3HR1 cell line engineered to allow effi- tibody. As shown in Fig. 2C, endogenous SRp20 and SM were
cient induction of EBV replication by tamoxifen. P3HR1/ZHT coimmunoprecipitated from P3HR1/ZHT lysates (lower
was generated by stable transfection of a fusion between Zta panel).11784 VERMA ET AL. J. VIROL.
RNA-binding proteins may coimmunoprecipitate because
they both bind to the same RNA molecules, rather than due to
direct protein-protein interactions. Since SRp20 and SM are
both RNA-binding proteins, it was possible that the SM-SRp20
interaction was stabilized by RNA binding. To determine
whether such RNA bridging was required for SM-SRp20 in-
teraction, we transfected cells with SM and either empty vector
or HA-SRp20, as in Fig. 2B above, and performed immuno-
precipitations with anti-SM antibody. Prior to SDS-PAGE and
immunoblotting, the immunoprecipitates were either treated
or mock treated with RNase A. As shown in Fig. 2D, coim-
munoprecipitation of SRp20 and SM was resistant to RNase
treatment, indicating that the SM-SRp20 interaction is not
RNA dependent.
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In order to map the region of SRp20 required for SM bind-
ing, we generated an influenza virus HA epitope-tagged series
of SRp20 deletions by PCR and cloning in an expression vector
(Fig. 3A). These SRp20 expression vectors were cotransfected
with SM, and immunoprecipitation with anti-HA monoclonal
antibody was performed, followed by immunoblotting with
anti-SM antibody. As shown in Fig. 3B, deletion of aa 105 to
164 of SRp20 did not reduce SM binding. The first 104 aa of
SRp20 contained the SM interacting motif, and the deletion of
additional amino acids eliminated SM association, locating the
SM interaction motif between aa 84 and 104. To confirm these
findings, we performed the reciprocal experiment, immunopre-
cipitating with anti-SM antibody and immunoblotting with
anti-HA antibody to detect SRp20 peptides. As shown in Fig.
3C, aa 84 to 104 of SRp20 were required for binding to SM.
SM interacts with SRp20 at a region overlapping the puta-
tive SM RNA-binding motif. In order to map the region of
SM that binds SRp20, we used bacterially derived GST-SM
fusion proteins encompassing defined portions of the SM
protein (Fig. 4A) (29). We then performed “pulldown” as-
says with 35S-labeled SRp20 that was synthesized in vitro. It
should be noted that we used SM fusion proteins with an
internal deletion of aa 149 to 185 that consisted of RXP
tripeptide repeats. Deletion of the RXP motif allows more
efficient production of intact SM protein in bacteria (29).
The expression of all GST-SM fusion proteins was verified
FIG. 3. Mapping of the SM interaction site in SRp20. (A) Sche-
by Coomassie blue staining of the bacterial lysates used in matic representation of HA-tagged SRp20 constructs used in immu-
the SRp20 pulldown assays (Fig. 4B). The full-length noprecipitation experiments to map the SM binding region in SRp20
GST-SM fusion protein with the internal RXP deletion in- protein. Amino acids at sites of truncation are shown. (B) 293T cells
teracted with SRp20, demonstrating that aa 149 to 185 are were transfected with empty vector (⫺SM), or SM, in combination
with either full-length HA-tagged SRp20 or the truncated HA-tagged
not required for SRp20 binding (Fig. 4C). The carboxy- SRp20 plasmid DNAs diagrammed in 3A above. Cells were lysed and
terminal amino acids of SM from aa 281 to 479 were also immunoprecipitated with anti-HA antibody. Bound proteins were im-
dispensable for SRp20 interaction. Although SM aa 1 to 281 munoblotted with anti-SM polyclonal antibody. The amount of input
bound SRp20, the deletion of aa 133 to 281 eliminated SM and SRp20 in each immunoprecipitation was measured by immu-
noblotting with anti-SM and anti-HA antibodies, respectively, and is
binding. Thus, the major SRp20 interacting region lies
shown below. (C) 293T cells were transfected with empty vector
within aa 185 to 281. This portion of SM also contains the (⫺SM), or SM, and either empty vector or HA-tagged SRp20 plas-
putative Ref interaction domain and RNA-binding regions mids. Lysates were immunoprecipitated with anti-SM antibody and
(17, 18). immunoblotted with anti-HA antibody to detect coimmunoprecipi-
SRp20 synergizes with SM to direct alternative splicing. SM tated SRp20. The amount of input SM and SRp20 in each immuno-
precipitation was measured by immunoblotting with SM and HA an-
increases total cellular STAT1 expression, a central mediator tibodies, respectively, and is shown below.
of IFN signal transduction, but disproportionately increases
the abundance of the STAT1 isoform, which can act as a
dominant-negative suppressor of STAT1 (1). Constitutive age and polyadenylation occurring in the intron between exons
splicing of STAT1 joins exons 23 and 24, the latter exon en- 23 and 24 (Fig. 5A). We have shown that SM induces bypassing
coding the STAT1␣ transactivation domain. STAT1 is gen- of the constitutive (␣) 5⬘ donor splice site of exon 23 and
erated by alternative processing of the pre-mRNA, with cleav- instead induces utilization of a cryptic alternative 5⬘ splice siteVOL. 84, 2010 EBV SM PROTEIN UTILIZES SRp20 IN ALTERNATIVE SPLICING 11785
analyzed by RT-PCR and sequencing. Using our previously
published alternative splicing assay (40), we transfected cells
with pDV1, a STAT1 pre-mRNA construct, and an SM plas-
mid, with or without SRp20. As shown in Fig. 5B, SRp20
enhanced the SM effect on STAT1 splicing by promoting ␣⬘ 5⬘
splice-site utilization and inclusion of the alternative, longer
exon 23. Importantly, overexpression of SRp20 alone did not
induce alternative splicing at the SM-directed site, indicating
that SRp20 itself does not induce alternative STAT1 splicing,
and exerts its effect via SM.
RS domain-truncated SRp20 (⌬RS-SRp20) acts as a dom-
inant-negative mutant when expressed in the presence of
wild-type SRp20, because the RS domain is required for
interaction with other splicing factors and with specific re-
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gions of the RNA undergoing splicing (24). If SM were
recruiting SRp20 to promote alternative splicing, we ex-
pected that ⌬RS-SRp20, which still binds SM strongly,
would repress alternative splicing induced by SM, similar to
the manner in which it represses the splicing factor activity
of full-length SRp20 on SRp20 substrates. The effect of
⌬RS-SRp20 on SM-induced alternative splicing was there-
fore tested by substituting the truncated SRp20 for full-
length SRp20 in the splicing assay. Cotransfection of ⌬RS-
SRp20 with did indeed inhibit SM-directed alternative
splicing of the STAT1 minigene, indicating that the effect of
SRp20 on SM splicing requires the RS domain (Fig. 5B).
FIG. 4. Mapping of the SRp20 binding region in SM protein. We have also shown that the effect of SM on splicing of
(A) Schematic diagram of GST-SM fusion constructs used in GST- STAT1 is mediated via nucleotides in the distal STAT1
pulldown experiments to map the SRp20 binding region in the SM
protein. The arginine-proline-rich repeat domain (RXP) was de- RNA alternative exon, i.e., between exon-intron boundary
leted in all constructs. Additional deleted amino acids are shown at of exon 23 and the alternative 5⬘ donor splice site (40). This
the left of each construct. (B) Coomassie blue-stained gel of SM response element (SMRE), when placed between two
GST-SM fusion proteins used in the pulldown assay. Washed beads adenovirus-derived exons in a minigene directs alternative
were boiled in loading buffer prior to SDS-PAGE. The molecular
splicing at the STAT1 alternative 5⬘ splice site, but only in
mass in kilodaltons is indicated on the left (lane M). (C) [35S]me-
thionine-labeled SRp20 protein was incubated with GST or the presence of SM (Fig. 5C) (40). This additional assay thus
GST-SM fusion proteins as shown in panel B and bound to gluta- serves as a confirmation of the specificity of SM activity as
thione-Sepharose beads. Interacting proteins were analyzed by an alternative splicing factor. In order to confirm that the
SDS-PAGE and autoradiography. synergy of SRp20 with SM did not require additional STAT1
sequences besides the SMRE, we transfected HeLa cells
with the minigene containing the SM response element and
(␣⬘). Utilization of ␣⬘ results in a STAT1 mRNA incapable of either empty vector or SM plasmid, with or without SRp20.
producing STAT1␣ protein due to inclusion of an in-frame Parallel transfections were also performed with ⌬RS-
stop codon in between exons 23 and 24 (Fig. 5A) (40). SM- SRp20. As shown in Fig. 5D, SM leads to usage of the
induced alternative splicing is dependent on the presence of an alternative 5⬘ SS, and SRp20 overexpression synergizes with
RNA sequence in the STAT1 pre-mRNA to which SM binds SM, increasing the proportion of RNA spliced at the alter-
directly and which can confer SM-dependent splicing on het- native 5⬘ SS. As was seen when STAT1 was used as the
erologous RNA (40). Based on a series of mutational analyses, target pre-mRNA, ⌬RS-SRp20 antagonizes SM activity in
we also showed that the effect of SM was not due to suppres- directing alternative splicing.
sion of the constitutive splice site and unmasking of the cryptic In order to confirm the involvement of SRp20 in SM func-
splice site. SM therefore acts like an SR protein, promoting tion, we performed siRNA knockdown of SRp20 prior to per-
utilization of an alternative splice site by binding to exonic forming STAT1 splicing assays. First, efficacy of SRp20 knock-
splicing enhancers (6). However, SM does not possess any of down was assessed by immunoblotting cell lysates from small
the motifs typically associated with such functions, such as RS interfering RNA (siRNA)-transfected cells. HeLa cells were
repeats. The association of SM with SRp20 suggested the pos- transfected with either a non-target control siRNA or SRp20-
sibility that SM was recruiting SRp20 to pre-mRNA, allowing specific siRNA. Efficiency of knockdown was assessed by pre-
SRp20 to perform its function as a splicing enhancer. We paring cell lysates and immunoblotting for SRp20. SRp20
therefore tested the effect of overexpressing SRp20 on SM- siRNA reduced SRp20 expression by ca. 60% as quantified by
directed alternative splicing of STAT1. The effect of SM on densitometry of the immunoblot (Fig. 6A). We then examined
alternative splicing of STAT1 can be assessed by means of a the effect of SRp20 knockdown on constitutive STAT1 splicing
splicing assay in which splicing substrates are transfected along by using the previously described splicing assay with the
with putative splicing factors and the products of splicing are STAT1 minigene. As seen in Fig. 6B, SRp20 knockdown did11786 VERMA ET AL. J. VIROL.
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FIG. 5. Effect of SRp20 on SM-induced alternative splicing of STAT1. (A) Schematic representation of STAT1 minigene (pDV1), used in
splicing assay. The minigene contains STAT1 exon 23, exon 24, and the intervening sequence cloned into pCDNA3. Alternative splicing events
generate either the STAT1␣ spliced form (exons 23 and 24) or the SM-directed alternative ␣⬘ form, in which the ␣⬘ donor 5⬘ SS site is used, leading
to a spliced product with an additional 210 nucleotides (nt) in the spliced RNA. The STAT1 form is generated by cleavage and polyadenylation
at an alternative processing site in the intron (CP). Constitutive splicing leading to the ␣ isoform generates STAT1␣ protein, which includes the
transactivating domain in exon 24. The stop codon in the intron is retained in the ␣⬘ and  isoforms, leading to production of STAT1  protein,
which lacks amino acids encoded in exon 24. (B) The pDV1 minigene construct was transfected into HeLa cells with various plasmids DNA as
shown. RNA was isolated 48 h after transfection and analyzed by RT-PCR using STAT1 primers. The positions of the constitutive ␣ splice product
and the expected position of the SM-induced alternative ␣⬘ splice product are shown with arrows. Both amplification products are slightly larger
than the expected 179 nt (␣ isoform) and 389 nt (␣⬘ isoform) due to the inclusion of primer sequences. The effect of SRp20 or ⌬RS-SRp20 on
SM-mediated alternative splicing was quantified by densitometry and is shown below each lane as the percentage of alternatively spliced product.
(C) Schematic diagram of a plasmid containing the SM response element cloned in the intron of a heterologous splicing reporter plasmid composed
of upstream (U) and downstream (D) exons from adenovirus. Constitutive splicing excises the intron and the SMRE. SM induced alternative
splicing generates the alternative splice product in which the 5⬘ SS in the U exon is bypassed and the alternative donor 5⬘ SS in the SMRE is utilized.
Constitutively spliced (␣) and SM-induced alternative spliced (␣⬘) RT-PCR products are shown at right. (D) The heterologous minigene splicing
target shown in panel C above was transfected into HeLa cells with various plasmid DNAs as shown. RNA was isolated and analyzed by RT-PCR
using U and D exon primers. The effect of SRp20 or ⌬RS-SRp20 on SM-mediated alternative splicing was quantified by densitometry and is shown
below each lane as the percentage of alternatively spliced product.
not affect constitutive STAT1 splicing. The effect of SRp20 cantly reduced SM-directed alternative splicing compared to
knockdown on SM-induced alternative STAT1 splicing was negative control siRNA (Fig. 6C). These data thus indicate
similarly assessed by transfecting SM with the STAT1 mini- that endogenous SRp20 plays a role in the ability of SM to
gene after SRp20 knockdown. Knockdown of SRp20 signifi- direct alternative splicing.VOL. 84, 2010 EBV SM PROTEIN UTILIZES SRp20 IN ALTERNATIVE SPLICING 11787
FIG. 7. SM recruitment of SRp20 to pre-mRNA. SM is shown
binding to pre-mRNA at an SM binding site near a 5⬘ exon to facilitate
splicing at a potential alternative splice site. The SM response element
(RNA binding site) is shown as a cross-hatched box. SM is shown
interacting with SRp20 at the binding site in the central portion of
SRp20 between the RBD and RS domain. SRp20 is shown interacting
with one component of the splicing machinery (U2AF) via RS-RS
interactions although other interactions are possible. SRp20 is also
shown interacting with the pre-mRNA branchpoint (star).
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18, 22). SM also enhances the nuclear accumulation of target
mRNAs posttranscriptionally, suggesting that SM stabilizes
mRNA, similar to its KSHV homolog ORF57 (25, 31, 32).
SM also has distinct effects on spliced mRNA transcripts.
SM inhibits the expression of spliced cellular targets in trans-
fection assays (31). Recently, we demonstrated that SM inhib-
its expression of the spliced immediate-early EBV transactiva-
tor Rta (39). Inhibitory effects of SM on spliced gene
expression have been attributed to premature export of un-
FIG. 6. Effect of inhibiting SRp20 expression with siRNA on SM-
mediated alternative splicing. (A) Knockdown of SRp20. HeLa cells spliced pre-mRNAs by SM, analogous to the mechanism of
were transfected with nontarget siRNA (Control) or siRNA specific HIV Rev protein (4). However, the inhibitory effect of SM on
for SRp20. Cell lysates were made 48 h after transfection and analyzed spliced gene expression is target specific, suggesting that SM
by immunoblotting with anti-SRp20 antibody. Blot was stripped and interacts specifically with the process of splicing (25, 30, 39).
reprobed with anti-actin antibody as a loading control. (B) Effect of
Our recent demonstration that SM can affect splice-site selec-
SRp20 knockdown on constitutive STAT1 splicing. HeLa cells were
transfected with control siRNA or SRp20-specific siRNA and 48 h tion lends further support to the hypothesis that SM exerts
later transfected with STAT1 minigene plasmid DNA and empty direct effects on splicing in the nucleus (40). The interaction of
pCDNA3 vector. RNA was analyzed by RT-PCR using STAT1 prim- SM with SRp20 and the involvement of this association in
ers. The constitutively spliced ␣ isoform generated by splicing of alternative splice site selection suggest a model whereby SM
STAT1 exons 23 and 24 is shown and is not affected by SRp20 knock-
down. (C) Effect of SRp20 knockdown on SM-induced alternative binds to specific pre-mRNA sites and recruits SRp20 via pro-
STAT1 splicing. HeLa cells were transfected as in panel B above to tein-protein interactions (Fig. 7). The RS domain of SRp20
decrease SRp20 expression and transfected with pDV1 (STAT1 mini- would then direct the formation of spliceosome assembly by
gene) and SM plasmids. RNA was analyzed by RT-PCR as described recruiting snRNPs and potentially by contacting RNA at dis-
above. Both constitutive (␣) and SM-induced alternative (␣⬘) splice
crete sites in the intron (33, 36, 37). In this manner, SM could
products are shown. The extent of alternative splicing was quantified
by densitometry and expressed as the percentage of product in the utilize SRp20 and co-opt its functions of directing splice-site
alternatively spliced (␣⬘) form. selection even at sites of mRNA devoid of SRp20 binding sites.
Recruitment of SRp20 by SM would thereby enable usage of a
set of novel splice sites to generate mRNA isoforms unique to
DISCUSSION cells in which EBV is replicating lytically.
SRp20 is important for export of intronless histone mRNAs
We and others have previously shown that SM plays multiple and is thought to act as an export factor by virtue of its ability
roles in posttranscriptional processing of EBV mRNA and to bind RNA via its RBD and undergo nucleocytoplasmic
cellular mRNA (2, 9, 17, 18, 23, 31, 35, 39, 40). Although the shuttling (19, 20). SRp20 also interacts with the export medi-
target sequences for SM binding have not been biochemically ator TAP via a region in the central portion of the SRp20
defined, abundant evidence indicates that SM contacts RNA molecule (15). Interestingly, this region overlaps with the SM-
directly. SM can be covalently cross-linked to mRNAs in vitro binding region of SRp20. Thus, it is possible that binding to
and exhibits RNA-binding specificity when immunoprecipi- SM may interfere with SRp20’s ability to enhance export of its
tated from EBV-infected cells undergoing lytic replication (14, own RNA cargoes. Alternatively, SRp20 binding to SM may be
40). Further, SM enhances the expression of EBV mRNAs compatible with TAP recruitment in a manner analogous to
preferentially, with some genes being SM dependent for ex- the process whereby Ref/Aly, a cellular RNA-binding protein,
pression, while others are SM independent (13). The posttran- is thought to bind mRNA and then hand off the mRNA to TAP
scriptional effect of SM in enhancing mRNA accumulation has (16). The net effect of the interaction of SM with SRp20 on
been attributed to an ability to act as a nuclear export factor. export of cellular histone mRNAs and other RNAs remains to
According to this model, SM binds to RNA via an RBD and be determined. Herpes simplex virus (HSV) ICP27, a homolog
recruits Ref/Aly, TAP, or CRM1 cellular export proteins (2, of EBV SM, has been shown to inhibit splicing by altering the11788 VERMA ET AL. J. VIROL.
function of SRPK1, an SR protein kinase, leading to hypo- 13. Han, Z., E. Marendy, Y. D. Wang, J. Yuan, J. T. Sample, and S. Swami-
nathan. 2007. Multiple roles of Epstein-Barr virus SM protein in lytic rep-
phosphorylation of SRp20 and other SR proteins (34). Re- lication. J. Virol. 81:4058–4069.
cently, SRp20 has been implicated in the export of HSV in- 14. Han, Z., D. Verma, C. Hilscher, D. P. Dittmer, and S. Swaminathan. 2009.
tronless mRNAs (8). Because hypophosphorylation of SRp20 General and target-specific RNA binding properties of Epstein-Barr virus
SM posttranscriptional regulatory protein. J. Virol. 83:11635–11644.
is linked to its ability to export RNA (19), ICP27 may facilitate 15. Hargous, Y., G. M. Hautbergue, A. M. Tintaru, L. Skrisovska, A. P.
HSV RNA export by SRp20. Whether SRp20 plays a role in Golovanov, J. Stevenin, L. Y. Lian, S. A. Wilson, and F. H. Allain. 2006.
enhancing intronless EBV mRNA stabilization and export is Molecular basis of RNA recognition and TAP binding by the SR proteins
SRp20 and 9G8. EMBO J. 25:5126–5137.
another important question that is raised by these findings. The 16. Hautbergue, G. M., M. L. Hung, A. P. Golovanov, L. Y. Lian, and S. A.
net effect of the SRp20-SM interaction on EBV mRNA ex- Wilson. 2008. Mutually exclusive interactions drive handover of mRNA from
pression is likely to be complex, since the SRp20 effects may export adaptors to TAP. Proc. Natl. Acad. Sci. U. S. A. 105:5154–5159.
17. Hiriart, E., L. Bardouillet, E. Manet, H. Gruffat, F. Penin, R. Montserret, G.
also be RNA target dependent. Farjot, and A. Sergeant. 2003. A region of the Epstein-Barr virus (EBV)
In summary, we have shown that SM protein binds SRp20 mRNA export factor EB2 containing an arginine-rich motif mediates direct
independently of RNA, via discrete protein-protein interac- binding to RNA. J. Biol. Chem. 278:37790–37798.
18. Hiriart, E., G. Farjot, H. Gruffat, M. V. Nguyen, A. Sergeant, and E. Manet.
tions, and that the interaction is important for the ability of SM 2003. A novel nuclear export signal and a REF interaction domain both
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to direct specific alternative splice-site selection. Further, re- promote mRNA export by the Epstein-Barr virus EB2 protein. J. Biol.
Chem. 278:335–342.
cruitment of SRp20 by SM suggests a model by which SM may 19. Huang, Y., T. A. Yario, and J. A. Steitz. 2004. A molecular link between SR
utilize the RNA and spliceosome-recruiting properties of SR protein dephosphorylation and mRNA export. Proc. Natl. Acad. Sci. U. S. A.
proteins to redirect these proteins to convert SM-binding sites 101:9666–9670.
20. Johannsen, E., M. Luftig, M. R. Chase, S. Weicksel, E. Cahir-McFarland, D.
to splicing enhancers. These findings implicate mRNA splicing Illanes, D. Sarracino, and E. Kieff. 2004. Proteins of purified Epstein-Barr
as another target pathway by which EBV modulates the cellu- virus. Proc. Natl. Acad. Sci. U. S. A. 101:16286–16291.
lar milieu during lytic replication. Inasmuch as ⬎70% of cel- 21. Johnson, J. M., J. Castle, P. Garrett-Engele, Z. Kan, P. M. Loerch, C. D.
Armour, R. Santos, E. E. Schadt, R. Stoughton, and D. D. Shoemaker. 2003.
lular genes undergo alternative splicing (21), it is likely that Genome-wide survey of human alternative pre-mRNA splicing with exon
EBV affects a number of genes in addition to those currently junction microarrays. Science 302:2141–2144.
described, thereby altering the “splicing code” of the cell to 22. Juillard, F., E. Hiriart, N. Sergeant, V. Vingtdeux-Didier, H. Drobecq, A. Ser-
geant, E. Manet, and H. Gruffat. 2009. Epstein-Barr virus protein EB2 contains
facilitate EBV replication. an N-terminal transferable nuclear export signal that promotes nucleocytoplas-
mic export by directly binding TAP/NXF1. J. Virol. 83:12759–12768.
ACKNOWLEDGMENTS 23. Key, S. C., T. Yoshizaki, and J. S. Pagano. 1998. The Epstein-Barr virus
(EBV) SM protein enhances pre-mRNA processing of the EBV DNA poly-
This study was supported by grant RO1 CA81133 from the NCI, merase transcript. J. Virol. 72:8485–8492.
National Institutes of Health. 24. Lou, H., K. M. Neugebauer, R. F. Gagel, and S. M. Berget. 1998. Regulation
Proteomic analyses were carried out by the Proteomics Center at the of alternative polyadenylation by U1 snRNPs and SRp20. Mol. Cell. Biol.
ICBR, University of Florida. 18:4977–4985.
25. Nicewonger, J., G. Suck, D. Bloch, and S. Swaminathan. 2004. Epstein-Barr
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