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INTRODUCTION |
The papillomaviruses are a family of small, nonenveloped,
double-stranded DNA viruses that cause epithelial and fibroepithelial lesions (1). With few exceptions, papillomaviruses are highly species-
and lesion-specific. A large group (~20 types) of human papillomaviruses are specifically linked with epidermodysplasia verruciformis (EV),1 which is
a rare hereditary, lifelong disease characterized by the development of
multiple cutaneous warts (2). Infection by EV-associated HPV types 5 and 8 carries a high risk of developing squamous cell carcinoma,
implicating these viruses in oncogenicity (2).
Papillomavirus gene expression is controlled by the products encoded by
the viral E2 gene (3). The papillomavirus E2 proteins recognize the
palindromic sequence ACCN6GGT, which occurs in multiple copies within the long control region of viral genomes and function as
transcriptional activators. The E2 protein contains two defined functional domains that are relatively conserved among different types
of papillomaviruses. The N-terminal conserved domain, consisting of
approximately 200 amino acids, is crucial for transcriptional activation, whereas the domain at the C terminus consisting of approximately 100 amino acids contains the DNA binding and dimerization properties of the protein. These two domains are linked by a hinge region that lacks conservation in amino acid sequence and varies in
length among papillomaviruses. The hinge region of the bovine papillomavirus (BPV) E2 protein was predicted to adopt a random coil
structure and could confer flexibility to the E2 molecule (3). Unlike
the two well defined terminal domains, the function of the hinge region
remains largely unclear.
In comparison with several well studied E2 gene products, the E2
protein of EV-associated HPVs harbors a relatively long hinge region
(~200 amino acid residues) and the hinge sequence is rich in
arginine, serine, and glycine residues (Fig. 1). The arginine/serine (RS) dipeptide repeat in the hinge is characteristic of a superfamily of proteins, which are primarily involved in the splicing of precursor mRNA (4-6). A group of prototypical SR proteins containing an extensive RS domain at the C terminus can be recognized by monoclonal antibody (mAb) 104, which stains lateral loops corresponding to sites
of RNA pol II transcription on amphibian lampbrush chromosomes (7).
Individual SR proteins can complement splicing deficient cytoplasmic
S100 extract, indicating that they are essential splicing factors but
have redundant functions in splicing (8). SR proteins are also crucial
players in alternative splicing by modulating splice site choice;
different SR proteins affect splice site selection with different
efficiencies and with different mechanisms (4-6). In addition to the
prototypical SR proteins, the RS domain or its analogous arginine
alternating domain (9) is also present in other essential splicing
factors and regulators including U2AF subunits, U1 and U5
snRNP-associated SR proteins, and Drosophila Tra and Tra-2
proteins (4). Using a variety of methods, some RS domains have been
shown to mediate protein-protein interaction between SR proteins (10).
Some RS domains can influence RNA binding (5) or promote RNA-RNA
annealing (11, 12).
In addition, short stretches of RG or RGG repeats scatter within the
hinge region of EV-HPV E2 proteins; some of these repeats neighbor a
serine residue as SRG or SRGG (Fig. 1). The RG-rich sequence of the E2
hinge is reminiscent of the RGG box that is present in a number of
RNA-binding proteins, including some nucleolar proteins, the ICP27
protein of herpes simplex virus, and the EBNA1 protein of Epstein-Barr
virus (13).
The realization that EV-HPV E2 proteins contain an RS-rich hinge domain
suggests that they may exert a function similar to that of cellular SR
proteins or interact with SR proteins through their RS repeat sequence.
In the present study, we show that the HPV type-5 (an EV-HPV) E2
protein can specifically interact with several cellular splicing
factors of the SR protein family. Our indirect immunofluorescent study
revealed that transiently expressed HPV-5 E2 protein in transfected
HeLa cells colocalize with a splicing factor in nuclear matrix
speckles. More importantly, we provide evidence suggesting that the
HPV-5 E2 protein likely participates in pre-mRNA splicing as well
as in transcriptional regulation.
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EXPERIMENTAL PROCEDURES |
Plasmid Construction--
Plasmids HPV5/9 and pSV2-neo HPV-16
were obtained from E.-M. de Villiers (Deutsches Krebsforschungszentrum,
Heidelberg, Germany) and W. C. Y. Yu (Academia Sinica,
Taipei, Taiwan), respectively, and they were used as template for
polymerase chain reaction (PCR) amplification of the HPV-5 or HPV-16 E2
coding region. The PCR product containing HPV-5 E2 ORF was cloned into
pGEM-1 (Promega), generating plasmid pSP5E2. Likewise, plasmid pSP16E2
was constructed. These two plasmids were used as template for in
vitro synthesis of HPV-5 and HPV-16 E2 proteins, respectively.
Bacterial expression vector pAR5E2
H was constructed as follows. The
PCR product containing HPV-5 E2 ORF was inserted into pAR3930-1 (14)
to generate pAR5E2. The restricted fragment of pAR5E2 coding for amino
acid residues 228-515 in full-length HPV-5 E2 was replaced by the PCR
product spanning amino acid residues 397-515, generating pAR5E2
H.
The pAR5E2
H plasmid was used to overproduce the hinge-deleted E2 protein.
Plasmids pAD5E2 and pAD5E2
H used for yeast two-hybrid assays were
constructed as follows. The coding sequences for full-length and
hinge-deleted HPV-5 E2 were in-frame placed downstream of the GAL4
activation domain in pACT2 (CLONTECH) using
restriction enzymes BamHI and EcoRI. To construct
pEGU5-100kD, the DNA fragment encoding the U5-100kD protein was
generated by PCR using pBluescriptSK-100KHeLa (gift of R. Luhrmann,
Philipps-Universität, Marburg, Germany) as template and then
inserted into pEG202 (15). The resulting plasmid produced the U5-100kD
protein fusion with the LexA DNA binding domain. The remaining
pEG202-derived plasmids including pEGASF, pEGSC35, and pEGU1-70K were
generous gifts of J. Y. Wu (Washington University, St. Louis, MO).
Mammalian expression vectors for transient expression of the
full-length and hinge-deleted HPV-5 E2 proteins were constructed as
follows. The DNA fragment containing E2 ORF amplified by PCR was
inserted into pCEP4 (Invitrogen), creating the pC5E2 plasmid. Similarly, plasmid pC5E2
H was constructed in which amino acid residues 228-396 were deleted from the HPV-5 E2 protein and plasmid pC5E2.HA was constructed to express the E2 protein containing the HA
epitope at the C terminus.
The reporter plasmid pE2Sp1-CAT, which was previously named as
pE2x2-Sp1x2-tk(-38)-cat (16) was a kind gift of
T. H. Haugen (University of Iowa, Iowa City, IA). Plasmid
pE2Sp1-CAT(In1) was constructed by insertion of a 130-base pair intron
into the PvuII site of the chloramphenicol acetyltransferase
(CAT) ORF. The intron was derived from the human
-globin gene and
slightly modified to confer the 5' splice site consensus GTAAGT.
Likewise, the
-globin intron was inserted into the filled-in
HindIII site, upstream of the CAT ORF (In2) or the
ScaI site near the 3' end of the CAT ORF (In3), or the
HincII site upstream of the polyadenylation signal (In4),
resulting in three other intron-containing reporters. The pE2Sp1-
gal
plasmid was made by replacing the CAT gene with the
-galactosidase
gene derived from pCH110 (Amersham Pharmacia Biotech). The
pSV40-CAT(In1) plasmid was constructed by placing a fragment containing
the CAT gene with the
-globin intron insertion derived from
pE2SP1-CAT(In1) into pCH110 using restriction enzymes HindIII and BamHI.
In this study, the sequences of DNA fragments amplified by PCR were
verified by sequencing using the dideoxy method.
Nuclear Extracts, snRNP Proteins, and SR Proteins--
To
fractionate extracts by (NH4)2SO4
precipitations, HeLa cell nuclear extracts were prepared from frozen
nuclei (Cell Culture Center) according to Dignam et al.
(17), except that glycerol was omitted from the dialysis buffer D. After dialysis, the extract was subjected to centrifugation at 4 °C
in a JA-20 rotor (Beckman) at 35,000 × g for 30 min.
Solid (NH4)2SO4 was added to the
supernatant to yield 65% saturated solution. The precipitate was
collected by centrifugation and then resuspended in buffer D, followed
by dialysis against buffer D. The concentration of the
(NH4)2SO4-precipitated nuclear
protein fraction (NE65P) was ~12 mg/ml.
To enrich snRNP-associated proteins, 20 µl of anti-Sm monoclonal
antibody (gift of J. A. Steitz, Yale University, New Haven, CT)
was coupled to 5 mg of protein A-Sepharose (PAS) (Amersham Pharmacia
Biotech) in 0.5 ml of NET-2 buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.05% Nonidet P-40.
Two hundred microliters of NE65P were incubated with antibody-coupled
PAS at 4 °C for 1 h. After removing unbound proteins, the resin
was washed four times with 1 ml of NET-2 buffer. Bound proteins were recovered and subjected to Western and Far-Western blot analyses.
A mixture of SR proteins was prepared from HeLa cell nuclei according
to the method described by Zahler et al. (8). SR proteins
were examined by complementation of the splicing defect of the HeLa
cell S100 extract.
Preparation of Anti-E2 Antibodies--
Plasmid pAR5E2
H was
transformed into Escherichia coli BL21(DE3); overproduction
of the hinge-deleted HPV-5 E2 (E2
H) protein was induced by
isopropyl-1-thio-
-D-galactopyranoside. Overproduced E2
H protein was solubilized from inclusion bodies with 8 M urea followed by SDS-polyacrylamide gel purification and
then used to immunize rabbits. To purify antibodies, recombinant E2
H
was coupled to Sepharose according to the method provided by Amersham Pharmacia Biotech. Anti-HPV-5 E2 sera were subjected to chromatography on E2
H-coupled Sepharose essentially according to Harlow and Lane
(18). The concentration of affinity-purified antibodies was ~5
mg/ml.
In Vitro Transcription-Translation--
In vitro
transcription-translation-coupled reactions were performed according to
the manufacturer's instruction (Promega). Plasmids used in this
experiment were all derived from pGEM-1 (Promega); each contained the
coding region for HPV-5 E2, HPV-16 E2, ASF/SF2, SC35, U1-70K,
U5-100kD, and GAL4-VP16 (gift of Y.-S. Lin, Academia Sinica, Taipei,
Taiwan), respectively. When used as probes, unincorporated labels were
removed by chromatography on a NAP column (Amersham Pharmacia Biotech).
Coimmunoprecipitation--
To co-immunoprecipitate HPV-5 E2 and
SR proteins, 25 µl each of in vitro
transcription-translation mixtures containing unlabeled HPV-5 E2 and
35S-labeled SR protein were incubated for 30 min at
4 °C. Ten micrograms of affinity-purified anti-E2 antibodies were
coupled to 2.5 mg of PAS in NET-2 buffer as described above. The
mixture containing HPV-5 E2 and an SR protein was then incubated with
anti-E2 coupled PAS at 4 °C for 1 h. Unbound proteins were
removed, and the resin was then washed four times with 1 ml of NET-2
buffer. Bound proteins were recovered, resolved on SDS-PAGE and
visualized by autoradiography.
Far-Western and Western Blot Analyses--
To analyze
protein-protein interaction by Far-Western blotting, SR and snRNP
proteins were fractionated on 10% SDS-PAGE and then transferred onto
polyvinylidene difluoride membranes (Bio-Rad). The blot was incubated
with binding buffer (25 mM HEPES (pH 7.9), 3 mM
MgCl2, 4 mM KCl, and 1 mM DTT)
containing 6 M guanidine hydrochloride for 10 min at
4 °C. The blot was subsequently incubated with binding buffers
containing decreasing concentrations of guanidine hydrochloride for 5 min per change. Blockage of the blot was performed by incubation with
5% skim milk and 1 mM DTT in TBST (100 mM
Tris-HCl (pH 7.6), 150 mM NaCl, and 0.05% Tween 20) for
1 h, followed by 1% skim milk and 1 mM DTT in TBST
for 1 h. The blot was incubated with 0.5-1 × 106 cpm/ml of 35S-labeled E2 protein in TBST
containing 1% skim milk and 1 mM DTT at 4 °C overnight.
To remove unbound proteins, the blot was washed with TBST containing
1% skim milk twice and then with TBST once for 10 min per wash. The
blot was subjected to autoradiography.
For Western blotting analysis, proteins were transferred onto
nitrocellulose (Schleicher & Schuell) or polyvinylidene difluoride membranes. The blot was probed with anti-U1-70K antibody or
anti-U5-100kD anti-serum (generous gifts of R. Luhrmann), and signals
were detected by using the enhanced chemiluminescence detection system
(Amersham Pharmacia Biotech).
Yeast Two-hybrid Assays--
A pACT2-derived bait plasmid and a
pEG202-derived prey plasmid were co-transformed with the LacZ reporter
pSH18.34 into Saccharomyces. cerevisiae EGY48
(Mata trp1 ura3 his3
LEU::pLexop6-LEU2) using the modified lithium acetate
transformation protocol provided by CLONTECH.
Plasmid DNA was recovered from the transformants and verified by
Southern blot analysis. Five independent clones of each transformants
were subjected to the liquid
-galactosidase assay using
o-nitrophenyl-
-D-galactopyranoside as
substrate according to the protocol recommended by
CLONTECH; each clone was assayed three times.
Indirect Immunofluorescence--
HeLa cells were grown at
37 °C in Dulbecco's modified Eagle's medium supplemented with
L-glutamine and 10% fetal bovine serum. Cells were
transfected using calcium phosphate. Twenty-four hours after
transfection, cells were plated onto Lab-Tek chamber slides (Nunc Inc.)
and incubated for another 24 h. For double-label
immunofluorescence experiments, cells grown on chamber slides were
rinsed twice with PBS and fixed with 2% formaldehyde for 30 min
followed by permeabilization with 0.5% Triton X-100 for 10 min. After
washing with PBS, cells were blocked by 3% skim milk in PBS for 30 min
and then incubated with primary antibodies in PBS for 1 h at room
temperature. The primary antibodies used were affinity-purified anti-E2
antibodies (5 µg/ml), mAb B4A11 (1 µg/ml; Calbiochem), and anti-HA
antibody (1:20 dilution from the supernatant of hybridoma culture
medium; gift of S.-C. Cheng, Academia Sinica, Taipei, Taiwan). Cells
were then washed with PBS, followed by incubation with appropriate secondary antibodies, fluorescein-conjugated anti-mouse IgG (7.5 µg/ml; Cappel Laboratories) or rhodamine-conjugated anti-rabbit IgG
(12 µg/ml, Cappel Laboratories), in PBS for 1 h at room
temperature. After extensive washing with PBS, cells on slides were
mounted immediately with mounting medium (Biomeda). The specimens were observed using a laser confocal microscope (MRC 600 model; Bio-Rad) coupled with an image analysis system.
Transient Transfection and CAT and
-Galactosidase Activity
Assays--
HeLa cells were grown in Dulbecco's modified Eagle's
medium as described above. Cells were seeded 18 h before
transfection and transfection using LipofectAMINE (Life Technologies,
Inc.) was performed essentially according to the manufacturer's
instructions. For transfection, the amount of plasmids used was
indicated in each figure legend. Cells were collected 48 h after
transfection. One tenth of the cells were lysed in a denaturing buffer
and subjected to Western blot analysis, and the remaining cells were
lysed in 120 µl of lysis buffer containing 0.25 M
Tris-HCl (pH 7.5) and 0.5% Triton X-100 (~2.5 × 104 cells/µl of buffer) to obtain the cell extract for
CAT assay or for both CAT and
-galactosidase activity assays. The
method for the CAT assay is described by Carey et al. (19).
For the
-galactosidase activity assay, the reaction was performed
using the chemiluminescent substrate Galacton-Star
(CLONTECH) according to the manufacturer's
instructions, and the luminescent enzyme activity was measured
according to the method described by Schoneich et al. (20)
with minor modifications.
RNase Protection and Northern Blot Analysis--
For RNase
protection assays, all RNA samples were prepared from 60-mm dish
cultures 24 h after transfection. Total RNA was prepared using
Trizol reagent (Life Technologies, Inc.) and then treated with DNase I
(1 unit/1 µg of RNA; Roche Molecular Biochemicals). Five (pSV40
plasmid) or 15 (pE2Sp1 plasmids) micrograms of DNase-treated RNA were
combined with 2 × 105 cpm anti-CAT probe (specific
activity 3.4 × 108 cpm/µg) in a 30-µl mixture
containing 40 mM PIPES (pH 6.7), 0.4 M NaCl, 1 mM EDTA, and 80% formamide. The RNA-probe mixture was heated at 85 °C for 5 min and then incubated at 45 °C for 5 h to allow annealing. The digestion was performed by adding 300 µl of
a buffer containing 10 mM Tris-HCl (pH7.4), 0.3 M NaCl, 5 mM EDTA, and RNase T1 (0.6 unit/µl
of reaction; Roche Molecular Biochemicals), and the reaction mixture
was incubated at 30 °C for 1 h. The samples were analyzed by
electrophoresis on a 6% polyacrylamide denaturing gel.
Northern blot analysis was performed essentially as described by Tarn
et al. (21) except that each sample contained 0.5 µg of
RNA and anti-U1 riboprobe was used.
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RESULTS |
EV-HPV E2 Proteins Contain RS Dipeptide Repeats in the
Hinge--
As described above, the E2 protein of EV-HPVs has an
unusual feature in its primary structure, i.e. a long hinge
region containing multiple RS dipeptide repeats and RG or RGG repeats
(for HPV type-5, see Fig. 1). A
phylogenetic analysis by comparing the hinge amino acid sequences
revealed distinct groupings of E2 proteins encoded by EV-HPVs (data not
shown). In one group of EV-HPVs (including HPV-5), the hinge region of
E2 is the longest (~200 aa) among all papillomaviruses and contains
most RS repeats (20 in average), whereas the E2 hinge in another group
of EV-HPVs is moderate in length (~150 aa) and contains fewer RS
repeats (12 in average). In sharp contrast, the hinge of the E2 protein
encoded by non-EV-HPVs is relatively short in length (~60 aa) and
lacks repeated RS or RG sequences (Fig. 1).

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Fig. 1.
Domain structure of the human EV-associated
papillomavirus E2 proteins. The E2 protein consists of two defined
functional domains, i.e. the transactivation domain and the
DNA binding/dimerization domain. These two domains are linked by a
non-conserved hinge region, which varies in length among the E2
proteins of different types of HPV. The hinge of EV-HPV E2 proteins is
longer than that of non-EV-HPVs and contains RS dipeptide repeats and
RG or RGG repeats. Hinge-deleted HPV-5 E2 protein used in this study
lacks aa residues 228-396, which are corresponding to the RS-rich
sequence.
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In Vitro Interactions of the HPV-5 E2 Protein with RS
Domain-containing Splicing Factors--
RS domains are known to be
involved in protein-protein interaction between pre-mRNA splicing
factors (4-6). This notion prompted us to ask whether an E2 protein
containing the RS-rich hinge could specifically interact with cellular
splicing factors that also harbor an RS domain. A mixture of mAb104
reactive SR proteins was isolated from HeLa cells by a two-step salt
precipitation as described by Zahler et al. (8). The SR
protein preparation contained six major polypeptides, two of which are
of ~30 kDa (Fig. 2A,
lane 2), as reported (8). For Far-Western blot analysis, SR
proteins were fractionated by SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane, followed by incubation with
in vitro translated, 35S-labeled HPV-5 or HPV-16
E2 protein. The HPV-5 E2 protein appeared to interact with all members
of this set of SR proteins (lane 3), but no significant
interaction was detected with the labeled HPV-16 E2 protein (lane
4) or with the HPV-5 E2 deletion mutant lacking the hinge region
(data not shown). This result suggests that interactions occur between
the HPV-5 E2 protein and SR proteins, likely via their RS domain.

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Fig. 2.
The HPV-5 E2 protein interacts with
prototypical SR proteins and two snRNP-associated proteins.
Panel A, SR proteins were isolated from HeLa cell nuclei,
fractionated on SDS-PAGE and detected with Coomassie Blue (lane
2). Purified SR proteins were subjected to Far-Western blot
analysis using 35S-labeled HPV-5 (lane 3) or
HPV-16 (lane 4) E2 protein as a probe. Lane 1 shows the molecular size marker. Panel B, snRNPs were
enriched from the HeLa nuclear extract by immunoprecipitation with
anti-Sm monoclonal antibody. The mock immunoprecipitate with PAS and
snRNP proteins ( Sm) were fractionated on SDS-PAGE and
stained with Coomassie Blue (lanes 1 and 2), or
subjected to Western blot analysis by probing with U1-70K antibody
(lanes 3 and 4) or U5-100kD anti-serum
(lanes 7 and 8), or subjected to Far-Western blot
analysis by incubation with 35S-labeled HPV-5 E2 protein
(lanes 5, 6, 9, and 10).
Ig(H) and Ig(L) represent mouse immunoglobin heavy and light chains,
respectively. The asterisk represents a nonspecific band
cross-reacted with U5-100kD anti-serum.
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We next examined whether the HPV-5 E2 protein could interact with
components of spliceosomal snRNPs. Proteins associated with Sm snRNPs
were enriched from HeLa cell nuclear extracts by precipitation with
65% saturated (NH4)2SO4, followed
by immunoprecipitation with anti-Sm antibody. snRNP-associated proteins
are shown in Fig. 2B (lane 2). Using
35S-labeled HPV-5 E2 protein as a probe, snRNP-associated
polypeptides of approximately 100 and 70 kDa were reproducibly detected
on Far-Western blots (lanes 6 and 10). The
triplet 70-kDa bands probably correspond to U1-70K (lane
4), which is an integral component of the U1 snRNP and contains an
arginine alternating domain at the C terminus. Recently, two additional
snRNP-associated SR proteins were identified: the 27-kDa protein
specific to the U4/U6.U5 tri-snRNP and the 100-kDa U5 snRNP protein,
which is also present in the tri-snRNP complex (22, 23). Both proteins
contain an RS or an arginine alternating domain at their N terminus.
Antiserum against the U5-100kD protein specifically recognized a
doublet band in the snRNP protein enriched fraction (lane
8), but not in the mock immunoprecipitate (lane 7). The
doublet comigrated with the ~100-kDa HPV-5 E2-interacting protein
(lane 10), suggesting that the U5-100kD protein is possibly
the other snRNP-associated protein that can interact with the HPV-5 E2
protein (see below for further analyses). This result therefore
suggests that the HPV-5 E2 protein can interact with snRNP-associated
proteins, which contain an arginine alternating domain.
To examine whether interactions between HPV-5 E2 and SR proteins could
occur in solution, we performed in vitro association assay
by coimmunoprecipitation. The immunoprecipitation efficiency was first
evaluated by incubation of the reticulolysate mixture containing
in vitro synthesized, 35S-labeled HPV-5 E2
protein (~0.7 pmol) with anti-E2 antibody-coupled protein
A-Sepharose. Approximately 10% of loaded E2 protein was immunoprecipitated (Fig. 3, top
panel). For coimmunoprecipitation experiments, HPV-5 E2 and
individual SR proteins were synthesized separately by in
vitro translation; only SR proteins but not the E2 protein were
labeled with [35S]methionine in order to avoid confusing
data resulted from comigration of U1-70K and E2 on SDS-PAGE. The E2
protein was then mixed with an SR protein in the reticulocyte lysate,
followed by immunoprecipitation. As shown in Fig. 3 (bottom
panel), all four SR proteins were coimmunoprecipitated with the
HPV-5 E2 protein by anti-E2 antibodies, but not by control antibodies.
However, SR proteins alone failed to be precipitated by anti-E2
antibodies, and the control protein, GAL4-VP16, had no detectable
interaction with the E2 protein (Fig. 3, bottom panel),
suggesting that the immunoprecipitation of SR proteins can be
attributed to their specific association with the HPV-5 E2 protein.
Consistent with the results of Far-Western blot analysis, the E2
protein lacking the RS-rich hinge had no significant interaction with
ASF/SF2 (data not shown). In summary, the result of the association assay provides additional evidence for in vitro interactions
of the HPV-5 E2 protein with at least four cellular RS
domain-containing splicing factors.

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Fig. 3.
The HPV-5 E2 protein interacts with ASF/SF2,
SC35, U1-70K, and U5-100kD in solution. Top panel, 25 µl of in vitro translation reaction mixture containing
35S-labeled HPV-5 E2 were immunoprecipitated with purified
anti-E2 antibodies ( E2) or rabbit anti-mouse Ig
antibodies (cont.). Bottom panel, 25 µl of
translation reaction mixture containing 35S-labeled
individual SR protein or GAL4-VP16 were incubated with the same volume
of the mixture containing or not containing unlabeled HPV-5 E2 protein.
The resulting 50-µl mixture was subjected to immunoprecipitation with
purified anti-E2 antibodies ( E2) or anti-mouse Ig
antibodies (cont.). Numbers represent that
fraction of input protein directly analyzed on SDS-PAGE.
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Detection of Specific Interactions between HPV-5 E2 and SR Proteins
in Vivo by the Yeast Two-hybrid Assay--
We next examined whether
the HPV-5 E2 protein interacts with SR proteins in vivo by
the yeast two-hybrid assay. The full-length or hinge-deleted HPV-5 E2
(Fig. 1) was fused to the GAL4 transactivation domain, and SR proteins
were fused to the LexA DNA binding domain. Recombinant plasmids as well
as the Lac Z reporter were transformed into the yeast strain EGY48 and
the transformants were assayed for the
-galactosidase activity. As
shown in Table I, the full-length HPV-5
E2 protein interacted with all the tested SR proteins, whereas deletion
of the hinge in the E2 protein dramatically reduced its interaction
with SR proteins. Western blot analysis using anti-E2 antibodies
revealed that the level of the GAL4-E2
H fusion protein was
severalfold higher than that of the full-length E2 fusion protein (data
not shown). Thus, we conclude that the RS-rich hinge region of the
HPV-5 E2 protein is essential for E2 interactions with RS
domain-containing splicing factors. However, in the two-hybrid assay,
the two snRNP proteins appeared to have only weak interaction with the
E2 protein. This may in part be attributed to the low amount of U1-70K
and U5-100kD fusion proteins yielded in yeast (data not shown).
Moreover, it is important to note that interactions between SR proteins
can be influenced by phosphorylation of RS domains (24).
Phosphorylation of SR proteins in the reticulocyte lysate may differ
from that in yeast, thus resulting in different relative strength of
interaction observed in the various assays. Nevertheless, both in
vitro and in vivo analyses demonstrated the importance
of the hinge domain for the interactions of the HPV-5 E2 protein with
SR proteins.
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Table I
Pairwise protein-protein interactions detected by the yeast
two-hybrid assay
Bait (pACT-2 derivative) and prey (pEG202 derivative) plasmids were
co-transformed with the LacZ reporter pSH18.34 into yeast strain EGY48
using lithium acetate. Quantitative liquid -galactosidase assays
were performed on five independent isolates for each combination.
Mean ± standard deviation represents data derived from three
independent experiments and is expressed as -fold activation above
background. Background refers to the amount of -galactosidase
activity observed with cotransformation of empty bait and prey plasmids
and the LacZ reporter.
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Localization of Transiently Expressed HPV-5 E2 Protein in Nuclear
Speckles--
Localization studies using antibodies specific for
snRNPs and non-snRNP SR proteins revealed that these nuclear factors
are concentrated in nuclear speckles, the interchromatin
granule-related clusters (4). Since the HPV-5 E2 protein contains an
RS-rich sequence and can interact with several splicing factors, we
examined whether the E2 protein behaves similarly to cellular SR
proteins with regard to the cellular localization. HeLa cells were
transfected with a vector that expressed the HPV-5 E2 protein under the
control of the human cytomegalovirus enhancer-promoter. Double
immunofluorescent staining was then performed using anti-E2
antibodies and mAb B4A11, which was selected because it gives prominent
punctate immunofluorescent staining pattern in interphase nuclei and
its antigen is colocalized with Sm antigens and SC35 in nuclear speckle
domains (25). Polyclonal anti-E2 antibodies were raised against the
hinge-deleted E2 protein expressed in E. coli; thus, the
antibodies should not cross-react with any cellular SR proteins. In
untransfected HeLa cells, immunofluorescent staining using purified
anti-E2 antibodies revealed only very weak, diffuse signals throughout
the whole cell (data not shown). Fig.
4a shows that transiently
expressed HPV-5 E2 protein, like cellular SR proteins, displayed a
speckled staining pattern in the nucleus. Colocalization experiments
revealed that the majority of punctate spots of the E2 protein appears
to overlap with foci labeled with mAb B4A11 within the optical section
(panels a-c). At least 50 transfected cells scored from
several separate experiments were analyzed. Although E2 and B4A11
fluorescent signals displayed a high degree of overlap in the overlaid
image shown in panel c, 10-30% of speckles were stained
either by anti-E2 or by B4A11 antibodies in a small fraction of
transfected cells (data not shown). To exclude the possibility that
overexpressed E2 protein aggregated to form a speckled pattern, a
vector that does not replicate to high copy number was used to express
the HPV-5 E2 protein in HeLa cells. A similar result was obtained (data
not shown). Moreover, a similar staining pattern was also observed in
cells expressing the HA epitope-tagged HPV-5 E2 protein (panel i). Thus, it appears very unlikely that a cellular protein
cross-reacting with anti-E2 antibodies was induced and accumulated in
nuclear speckles upon overexpression of E2. Deletion of the hinge
prevented the E2 protein from accumulating in nuclear speckles, and
instead resulted in uniform nucleoplasmic distribution but with
exclusion of nucleoli (panel e). Localization of the
hinge-deleted HPV-5 E2 protein is therefore coincident with that of the
BPV-1 E2 protein (Ref. 26 and data not shown). In summary, the HPV-5 E2
protein is localized in nuclear speckle domains where an SR-related
splicing coactivator is concentrated, and the RS-rich hinge is critical for targeting the E2 protein to such domains.

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Fig. 4.
Confocal microscopy analysis of full-length
and hinge-deleted HPV-5 E2 proteins in transfected HeLa cells.
HeLa cells were transiently transfected with pC5E2 (panels
a-d), pC5E2 H (panels e-h), or pC5E2.HA
(panels i and j) for ~40 h. Transfected cells
were fixed and double-labeled with purified anti-E2 antibodies
(panels a and e) and mAb B4A11 (panels
b and f) or labeled with anti-HA antibody alone
(panel i). The secondary antibody for E2 was coupled to
rhodamine (red), and that for mAb B4A11 and HA was
coupled to fluorescein (green). Merged images are shown in
panels c and g; colocalization appears as a
yellow coloration. Cells visualized by phase contrast
microscopy are shown in panels d, h, and
j.
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Dramatic changes in nuclear ultrastructure have been observed upon
infection of a variety of viruses or upon transient expression of a
viral protein (27-29). Distribution of nuclear speckles reacting with
mAb B4A11 was apparently not affected by overexpression of the HPV-5 E2
protein (Fig. 4, panels a-c, and data not shown). Nor was a
change in the number or size of coiled bodies observed under the same
conditions (data not shown). Nevertheless, it is still possible that
infection of EV-HPV could cause reorganization of splicing factors in
the nucleus of host cells.
The HPV-5 E2 Protein Transactivates an Intron-containing Reporter
More Efficiently than Its Hinge-deleted Mutant--
The various assays
described above indicated that the HPV-5 E2 protein could interact with
several RS domain-containing splicing factors through its hinge. We
therefore hypothesized that the E2 protein functions not only in
transcriptional activation but also in the splicing of primary
transcripts. To test this possibility, we first determined the ability
of the HPV-5 E2 protein to transactivate its responsive promoter and
further examined whether deletion of the hinge would have any effect on
E2's transactivation ability. Previously, BPV-1 and HPV-16 E2
proteins were shown to transactivate an E2-dependent
reporter, pE2x2-Sp1x2-tk(-38)-cat (abbreviated herein as pE2Sp1-CAT; Ref. 16), which contains two consensus E2
sites and two Sp1 sites in the HSV-1 thymidine kinase (tk) gene minimal promoter upstream of the CAT reporter gene (Fig. 5A). Like those two E2
proteins, HPV-5 E2 activated transcription at such a defined promoter
in a concentration-dependent fashion and at a comparable
level as HPV-16 E2 when the same amounts of DNA per culture were
employed (data not shown). Fig. 5B, upper panel,
shows that the transactivation activity of the hinge-deleted E2 protein
(lane 3) was similar to that of full-length E2 (lane 2), suggesting that the RS-rich hinge domain apparently makes no
significant contribution to transcriptional activation.

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Fig. 5.
Transactivation assays of the HPV-5
full-length and hinge-deleted E2 proteins. Panel A,
schematic diagrams for three E2-responsive reporters, CAT, CAT(In1),
and -gal, driven by a chimeric promoter containing E2 and Sp1 sites
in the HSV-1 tk gene promoter, and reporter pSV40-CAT(In1),
driven by the SV40 early promoter. An Sp6 antisense probe and RNase T1
protected fragments for CAT pre-mRNA and mRNA are 408, 347, and
223 nucleotides in length, respectively. Panel B, top
panel, HeLa cells in 35-mm culture dishes were transfected with 1 µg of CAT (lanes 1-3) or CAT(In1) (lanes 4-6)
reporter and 0.2 µg of control vector (lanes 1 and
4) or E2 (lanes 2 and 5) or E2 H
(lanes 3 and 6) expression vector. Representative
CAT assay was performed with extracts from each transfectant; the assay
with the CAT(In1) reporter was carried out using 5 times more extract
than that with the CAT reporter. The reporter, activator, percentage of
substrate conversion, and -fold activation are indicated
above each track of the autoradiogram. Bottom
panel, Western blot analysis. Total proteins extracted from the
transfectants as shown above were detected with anti-E2 antiserum. The
asterisks represent cross-reacted proteins. Panel
C, E2 H transactivation activity relative to E2 transactivation
activity on various reporters. HeLa cells in 60-mm culture dishes were
transfected essentially as in panel B except that 1.8 µg
of CAT or CAT(In1) reporter and 0.6 µg of transactivator were used
and 1.8 µg of -gal reporter was included in all samples. The
values represent E2 H/E2 relative transactivation activity on the CAT
(lane 1), CAT(In1) (lane 3), and -gal
(lanes 2 and 4) reporters; average and standard
deviation were obtained from three independent experiments.
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Next, to test whether the E2 hinge plays some role in the splicing of
pre-mRNA, we inserted a modified human
-globin intron 1 into the
CAT coding region (Fig. 5A). The resulting plasmid is
referred to as pE2Sp1-CAT(In1). The presence of an in-frame stop codon
within the
-globin intron would impede production of active enzyme
without splicing of the CAT pre-mRNA. When the pE2Sp1-CAT(In1)
construct was used as a reporter, 5-fold more extract was required in
the assay to achieve levels of CAT activity comparable to those of the
reporter lacking the intron. This indicated that CAT mRNA
production was suppressed in cis by the insertion of the
-globin intron. Nevertheless, Fig. 5B shows that the
transactivation activity of full-length E2 was ~4-fold higher than
that of hinge-deleted E2 (upper panel, lanes 5 and 6). Expression of the transactivators was examined by
Western blot analysis using anti-E2 antiserum and showed that the level
of E2 or E2
H protein was similar in transfectants with different
reporters (Fig. 5B, bottom panel, compare
lanes 2 and 5 for E2, and lanes 3 and
6 for E2
H). Moreover, transactivation activity was
monitored by cotransfection of either of the CAT reporters with a
-galactosidase construct that did not contain the intron. Expression
of the
-galactosidase gene was driven by the E2-responsive promoter,
the same as that used in the CAT reporter constructs (Fig.
5A). Regardless of whether the CAT reporter contained the
-globin intron, extracts from E2 and E2
H transfectants exhibited
similar levels of
-galactosidase activity (data not shown). The
results from three cotransfection experiments are summarized in Fig.
5C, emphasizing that the transactivation activity of
hinge-deleted E2 became significantly lower than that of full-length E2
only when the E2-responsive reporter contained the intron. It is most
noteworthy that the level of the CAT activity transactivated by E2 was
only ~10-fold higher than the background when pE2Sp1-CAT was used as
a reporter (Fig. 5B, upper panel, lane
2); however, transactivation by E2 appeared to increase
synergistically to 30-fold when the reporter used contained the
-globin intron (lane 5). This result indicated that,
besides transcriptional activation, at least another mechanism involves
to achieve the synergistic effect of the full-length E2 protein on the
transactivation of an intron-containing reporter, which is thus likely
the splicing of pre-mRNA.
The HPV-5 E2 Protein Facilitates the Splicing of Pre-mRNA
Transactivated by E2 Itself--
We then analyzed the splicing of the
CAT primary transcript transactivated by full-length and hinge-deleted
E2. Duplicate transfections were performed. Total RNA from one dish of
transfected cells was collected and subjected to RNase T1 protection
assay using the probe spanning the entire
-globin intron and its
downstream CAT gene (Fig. 5A). Transfected cells in the
duplicate dish were assayed for CAT activity in order to evaluate CAT
production. No significant difference was observed between the amounts
of the CAT mRNA transactivated by E2 and E2
H while normalized by the level of U1 small nuclear RNA (Fig.
6A, lanes 2 and
3). This confirmed that transcriptional activation was of
nearly equal efficiency with these two transactivators when pE2Sp1-CAT
was used. Examination of the CAT pre-mRNA splicing from three
independent experiments revealed that a 2-fold difference in the ratio
of spliced to unspliced CAT transcript between full-length and
hinge-deleted E2 (Fig. 6A, lanes 5 and
6, and 6B, lane 1). CAT production at the protein level was simply represented by CAT activity,
i.e. percentage of chloramphenicol conversion without
subtraction of the background obtained from transfection with the empty
expression vector. The level of the CAT protein in E2
H transfected
cells was ~36% that of the CAT protein produced by full-length E2
when the intron-containing reporter was used (Fig. 6B,
lane 4). A greater difference between E2 and E2
H in
producing CAT protein than in promoting splicing can be rationalized by
the fact that the transactivation activity of hinge-deleted E2 reduced
slightly by the presence of the
-globin intron in the reporter (Fig.
5B, compare lane 6 to lane 3 for the
activation (n-fold) of E2
H). Nevertheless, all above
results can support our previous speculation that the E2 transactivator
plays a role in assisting pre-mRNA splicing through its RS domain.
However, RS domain-containing proteins may have some other biological
activities (30, 31); thus, the possibility that HPV-5 E2 can act
through other mechanisms to regulate gene expression cannot be
excluded.

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Fig. 6.
RNase protection assays. Panel
A, HeLa cells in 60-mm culture dishes were transfected with 3 µg
of pE2Sp1-CAT (lanes 1-3), pE2Sp1-CAT(In1) (lanes
4-6), or pSV40-CAT(In1) (lanes 7 and 8) and
0.6 µg of control vector (lanes 1 and 4), E2
(lanes 2, 5, and 7), or E2 H
(lanes 3, 6, and 8) expression vector.
Transfections were performed in duplicate. Total RNA was isolated from
one dish of transfectants 24 h after transfection for analysis.
Representative RNase protection assay and Northern blotting analysis
are shown. The left two lanes are DNA
marker and the probe, respectively; the lengths in nucleotides of DNA
marker are given at left of the autoradiogram. Panel
B, lanes 1 and 2, quantification of the
effect of E2 on CAT pre-mRNA splicing. The values represent the
splicing efficiency of CAT pre-mRNA in E2 H transfected cells
relative to that in E2 transfected cells. Splicing efficiency was
measured by the arbitrary unit of spliced CAT mRNA, which was
quantified by PhosphorImager scanning and normalized with the CAT
pre-mRNA. Lanes 3-5, CAT assay was performed with
extracts from the duplicate dish of transfectants 48 h after
transfection. The values represent CAT activity (production at the
protein level) transactivated by E2 H relative to that transactivated
by full-length E2 with the reporters as indicated. Average and standard
deviation were obtained from three independent experiments.
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Next, in order to examine whether the HPV-5 E2 protein can function in
trans to facilitate splicing, we replaced the promoter of
the pE2Sp1-CAT(In1) reporter with the SV40 enhancer-promoter (Fig.
5A), which should be independent of E2 transactivation. Cotransfection of full-length E2 or hinge-deleted E2 with the SV40-CAT(In1) reporter resulted in similar level of the CAT activity (Fig. 6B, lane 5). RNase protection assays
revealed that splicing of the CAT pre-mRNA transactivated through
the SV40 promoter was of similar efficiency in E2 and E2
H
transfected cells (Fig. 6A, lanes 7 and
8, and 6B, lane 2). Thus, the results
suggest that the E2 protein could not trans-activate
splicing without activation of gene transcription through its binding
to the promoter.
The HPV-5 E2 Protein Probably Assists Pre-mRNA Splicing in a
Distance-dependent Manner--
As described above, by
insertion of the
-globin intron into a E2-responsive CAT reporter at
the position 209 base pairs downstream of the transcription start site,
we observed that the RS-rich hinge-containing E2 protein transactivated
more efficiently than hinge-deleted E2 (Fig. 5), apparently by
facilitating the splicing of the CAT transcript (Fig. 6). We next made
three other constructs in which the
-globin intron was placed at
different positions in the transcription unit of the CAT gene in order
to examine whether the intron's position influences the efficacy of
the E2 protein in assisting pre-mRNA splicing (Fig.
7A). For each
intron-containing reporter, transactivation activity of hinge-deleted
E2 relative to full-length E2 was measured (Fig. 7B,
lanes 2, 4, 6, and 8) and
compared with the control reporter lacking the
-globin intron (lanes 1, 3, 5, and 7).
When the intron was inserted in the 5'-untranslated region of the CAT
gene, the activity ratio of E2
H to E2 was about 0.5, i.e.
~2-fold-decreased transactivation by E2
H compared with transactivation by full-length E2 (Fig. 7B, lanes
1 and 2). When the intron was located near the 3' end
of the CAT coding region, the E2
H to E2 activity ratio slightly
increased to ~0.7, indicating less difference between E2 and E2
H
in their transactivation activity (lanes 5 and
6). In contrast to the above results, the transactivation activity of E2
H was no longer lower than that of E2 when the intron
was moved near to the 3' end of the transcription unit of the reporter
(lanes 7 and 8). This result suggests that the splicing activation effect mediated by the RS-rich hinge of the E2
transactivator was negligible when the intron was located far away from
the promoter. To conclude, E2 protein's function in the activation of
pre-mRNA splicing is in part determined by the distance of the
intron from the 5' end of the transcript, indicating that a geometric
factor may confine the action of the E2 protein in promoting
pre-mRNA splicing.

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Fig. 7.
Transactivation assays with four
-globin intron-containing CAT reporters.
Panel A, schematic diagrams of four -globin
intron-containing CAT reporters. Each intron-containing reporter
contains one -globin intron in the transcription unit of the
E2-responsive CAT reporter. The positions of -globin intron
insertion are indicated as black arrows, and the
distances from the transcription start site to each intron insertion
are given in parentheses. Panel B, relative
transactivation activity of E2 H/E2 on various reporters. HeLa cells
in 35-mm culture dishes were transfected with 1 µg of pE2Sp1-CAT
(control) or -globin intron-containing CAT reporter and 0.2 µg of
E2 or E2 H expression vector. Relative CAT activity of each
transactivator was obtained by subtraction of the background, which was
observed with cotransfection of the empty transactivator vector and a
reporter. E2 H/E2 relative transactivation activity on each control
and -globin intron-containing CAT reporters was obtained from
dividing relative CAT activity of E2 H by that of E2 while they were
assayed with the same reporter. Average and standard deviation were
obtained from three (lanes 1, 2, 7,
and 8) or four (lanes 3-6) independent
experiments.
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DISCUSSION |
The E2 protein encoded by EV-associated HPVs contains an RS-rich
sequence in the central hinge region, implying its role in pre-mRNA
splicing. As predicted, we demonstrate here that the RS-rich hinge is
essential for HPV-5 E2's interaction with cellular splicing factors
and also for its colocalization with SR proteins in nuclear speckles.
More importantly, functional evidence showed that the RS-rich hinge of
the HPV-5 E2 transactivator can facilitate the splicing of a primary
transcript transactivated by E2 itself probably in a
distance-dependent manner.
The various assays showed that the HPV-5 E2 protein can interact with
at least four SR proteins (Figs. 2 and 3 and Table I). Although it is
presently unclear whether all these proteins represent physiologically
relevant targets for E2 in vivo, the following reasons may
argue against that the E2-SR protein interactions detected in this
study lacked specificity. First, SR proteins used in our in
vitro protein-protein interaction assays were either isolated from
HeLa cells or synthesized in the reticulocyte lysate, thus preventing
nonspecific interaction between unphosphorylated RS domains as reported
previously (32). Second, in the presence of RNase, the interactions
between E2 and SR proteins on Far-Western blots were still detectable,
albeit relatively weak (data not shown). This rules out the possibility
that E2 and SR proteins only nonspecifically anchor an RNA molecule.
Third, only two SR proteins from a pool of snRNP proteins were
identified on Far-Western blots by using E2 as a probe, indicating the
specificity of the interactions. Moreover, the recombinant HPV-5 E2
protein purified from the baculovirus system can be detected by mAb 104 (data not shown), suggesting that E2, like SR proteins, is
phosphorylated in vivo. Phosphorylated RS domain may thus
mediate specific interactions between E2 and cellular SR proteins under
physiological conditions (32, 33). Finally, since in this study we
examined the interactions of the HPV-5 E2 protein with subsets of SR
proteins, which are either recognized by mAb104 or associated with the
Sm snRNPs, it is still possible that the E2 protein can interact
through its hinge with other cellular RS domain-containing proteins,
for example, SR-like CTD-associated factors (30, 34), SR-related splicing coactivators (35), or a set of high molecular weight SR
proteins recognized by mAb 16H3 (36).
Both in vitro (Fig. 3) and in vivo (Table I)
protein-protein interaction assays provided an indication that the E2
protein has higher affinity to ASF/SF2 than to SC35. Both these two SR proteins can activate constitutive splicing and influence
selection of alternative splice sites (4-6). However, their
distinct RNA binding specificity suggests that they behave differently
by binding to specific RNA elements (37). It is also noteworthy
that ASF/SF2 can assist the recruitment or stabilization of the U1
snRNP to the functional 5' splice site (38). Thus, E2's apparent
preference for ASF/SF2 may suggest that E2 can collaborate with ASF/SF2
in recognizing intron's 5' splice site or any other specific RNA elements such as splicing enhancers. In addition, we observed from the
in vitro association experiment that the HPV-5 E2 protein interacted significantly with two snRNP-associated SR proteins, U1-70K
and U5-100kD; the former appeared to be the most preferred protein
bound by E2 among all those tested (Fig. 3). Conceivably, the U1-70K
protein becomes near the 5' splice site of the intron in the early
splicing complex. The U5-100kD protein has been speculated to replace
U1-70K in its interactions with other SR proteins when U5 and U6
snRNPs take over the 5' splice site following destabilization of U1,
because it, like U1-70K, possesses an alternating charge domain
instead of a typical RS domain (23). Our results may therefore indicate
that the HPV-5 E2 protein is capable of interacting with the SR
proteins, which favor association with the 5' splice site of
pre-mRNA intron. Next, it will be of interest to test whether the
E2 protein can direct SR proteins to modulate 5' splice site selection
and thus participate in alternative splicing.
Here, we demonstrate that the RS-rich hinge is required for E2
protein's colocalization with splicing factors in nuclear speckles (Fig. 4) and also for its function in promoting pre-mRNA splicing (Fig. 5). However, questions still remain as to whether the E2 transactivator functions within nuclear speckles and whether the speckle pattern of E2 expression is critical for E2's function in
facilitating splicing. Recent data have revealed that precursor mRNA is transcribed and processed at foci dispersed throughout the nucleoplasms, supporting the idea that speckles may function as
reservoirs that supply factors for gene expression (39, 40). Therefore,
it is reasonable to predict that the HPV-5 E2 transactivator may leave
speckles, coordinately with splicing factors, and accumulate at the
sites of transcription for function. Moreover, phosphorylation of the
RS-rich hinge region may regulate the subnuclear localization of the E2
protein, thereby modulating its function. All these hypotheses remain
to be tested.
This study provides evidence that the HPV-5 E2 protein can facilitate
the splicing of CAT primary transcripts containing the
-globin
intron; however, the intron's distance relative to the 5' end of the
transcript can determine the efficacy of the RS-rich hinge domain in
assisting splicing. In fact, we observed in this study that insertion
of the
-globin intron at different positions of the CAT reporter
resulted in different degrees of reduction in transactivation by E2,
and among all the constructs transactivation of pE2Sp1-CAT(In1) (the
intron at position 209) was affected most severely. Accordingly, the
synergistic effect of E2 in transactivation appeared to be most
pronounced with this reporter. This is also partially in concert with
our another observation that the insertion of an adenovirus intron (41)
at position 209 markedly activated expression of the CAT reporter but
completely neglected E2' effect in splicing (data not shown). We
therefore conclude from the apparent results that the HPV-5 E2
transactivator may conditionally facilitate the splicing of precursor
mRNA in which the intron is inefficiently spliced, or located close
to the 5' end of the transcript. However, the possibility that the E2
protein controls the nuclear export of incompletely spliced mRNA or
even the translation cannot be excluded. Although an artificial system
was used, this study provides some clues to the mechanistic action of
the HPV-5 E2 transactivator on assisting the splicing of
pre-mRNA.
Recently, increasing evidence indicates previously unexpected roles for
transcription factors in linking transcription and pre-mRNA
processing (42, 43). The largest subunit of RNA pol II was first
implicated in pre-mRNA splicing and polyadenylation through its
C-terminal domain (44-46). Many lines of evidence suggest that the
interaction of splicing factors with phosphorylated RNA pol II CTD is
probably essential for spliceosome function (30, 47). Moreover,
polyadenylation factors and the capping enzymes are also targeted onto
pre-mRNA by binding to the phosphorylated CTD of RNA pol II (46,
48, 49). More recently, a holo-TFIID complex containing cleavage
poly(A) specificity factor (CPSF) was found, indicating that TFIID not
only nucleates transcription initiation but also assists
polyadenylation by recruiting the CPSF complex to the promoter (50). In
this study, we showed that pre-mRNA splicing may be mechanistically
coupled to transcription through an RS-rich sequence-containing
transactivator. Therefore, besides RNA pol II and TFIID, another
category of transcription factors, DNA binding transactivators, can
play a role in coordinating transcription and RNA processing.
Conceivably, RNA pol II is physically proximal to the growing nascent
transcript while RNA synthesis proceeds, whereas TFIID only binds to
the core promoter during transcription initiation (51). Thus, CPSF
recruited by TFIID prior to the elongation step of transcription may
need to be delivered to the nascent RNA through phosphorylated RNA pol
II (50). More like TFIID, transactivators bind to a sequence-specific
element nearby the promoter and primarily play a role in
initiation/reinitiation of transcription. The mechanism whereby the E2
transactivator functions post-transcriptionally needs further
investigation. At present, a plausible mechanism is that the RS-rich
hinge of the E2 transactivator recruits essential splicing factors
including non-snRNP SR proteins and spliceosomal snRNPs, thereby
increasing their concentration surrounding the promoter of an actively
transcribed gene (Fig. 8). Whether the
HPV-5 E2 transactivator can pass SR proteins to the RNA polymerase
or even become incorporated into the spliceosome remains to be
examined.

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Fig. 8.
A model for the mechanistic action of the
HPV-5 E2 protein in assisting pre-mRNA splicing. The HPV-5 E2
transcriptional activator binds to its cognate DNA elements as a dimer
and interacts with RS domain containing-splicing factors through the
RS-rich hinge, thereby increasing the concentration of splicing factors
near the active promoter. Assuming that E2 does not leave from the
binding sites onto the RNA nor pass the bound SR protein to RNA pol II,
it may thus only facilitate the splicing of transcripts in which the
intron is located closely to the promoter. Such transcripts can be
spliced, albeit less efficiently, when they are transactivated by the
E2 protein lacking the RS domain, since splicing factors are recruited
to the transcription sites in response to the initiation of active
transcription of intron-containing genes (see Ref. 40 and references
therein).
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