Binding Sites for Rev and ASF/SF2 Map to a 55-Nucleotide
Purine-rich Exonic Element in Equine Infectious Anemia Virus RNA*
Hye-kyung
Chung and
David
Derse
From the Basic Research Laboratory, NCI-Frederick, National
Institutes of Health, Frederick, Maryland 21702
Received for publication, October 2, 2000, and in revised form, February 12, 2001
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ABSTRACT |
The equine infectious anemia virus (EIAV) Rev
protein (ERev) negatively regulates its own synthesis by inducing
alternative splicing of its mRNA. This bicistronic mRNA
contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode
Rev. When Rev is expressed, exon 3 is skipped to produce an mRNA
that contains only exons 1, 2, and 4. The interaction of ERev with its
cis-acting RNA response element, the RRE, is also essential
for nuclear export of intron-containing viral mRNAs that encode
structural and enzymatic gene products. The primary ERev binding site
and the manner in which ERev interacts with RNA or cellular proteins to
exert its regulatory function have not been defined. We have performed
in vitro RNA binding experiments to show that recombinant
ERev binds to a 55-nucleotide, purine-rich tract proximal to the 5'
splice site of exon 3. Because of its proximity to the 5' splice site and since it contains elements related to consensus exonic splicing enhancer sequences, we asked whether cellular proteins recognize the
EIAV RRE. The cellular protein, ASF/SF2, a member of the serine- and
arginine-rich family of splicing factors (SR proteins) bound to
repeated sequences within the 55-nucleotide RRE region. Electrophoretic mobility shift and UV cross-linking experiments indicated that ERev and
SR proteins bind simultaneously to the RRE. Furthermore, in
vitro protein-protein interaction studies revealed an association between ERev and SR proteins. These data suggest that EIAV Rev-induced exon skipping observed in vivo may be initiated by
simultaneous binding of Rev and SR proteins to the RRE that alter the
subsequent assembly or catalytic activity of the spliceosomal complex.
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INTRODUCTION |
The Rev and Rex proteins encoded by lentiviruses and other complex
retroviruses mediate the nucleocytoplasmic transport of intron-containing, viral pre-mRNAs that would otherwise be retained in the nucleus (1-4). In addition to their roles in RNA transport, some members of this protein family have been shown to affect pre-mRNA splicing (5-8). Our current understanding of Rev function and mechanism derives largely from the
HIV1 type 1 (HIV-1) system;
Rex proteins encoded by the human T-cell leukemia virus group of
retroviruses (HTLV-I, HTLV-II, and bovine leukemia virus) have been
extensively studied as well (9-16). These and other retroviruses share
conserved mechanisms to regulate RNA transport, but the sequences and
structures of viral cis- and trans-acting
components display considerable differences. Rev and Rex proteins
contain a nuclear localization signal and a leucine-rich nuclear export
signal, as well as an arginine-rich RNA binding domain, that together
allow the proteins to shuttle between the nucleus and cytoplasm and
tether viral pre-mRNAs to a cellular protein-transport pathway (11,
17-22). The specificity of RNA recognition by the proteins is
determined, in part, by cis-acting RNA sequences called the
Rev- or Rex-responsive elements (RRE/RxRE) (23). Even though HIV-1 RRE
and HTLV-1 RxRE do not show significant sequence homology, both form
large, stable secondary structures (24). The HIV-1 RRE is a complex
234-nucleotide stem-loop structure located in the envelope gene, which
contains a 30-nucleotide high affinity binding site for Rev (25, 26). A
255-nucleotide stem-loop structure in the 3' long terminal repeat of
HTLV-1 functions as the RxRE and contains a 33-nucleotide element,
which represents the primary binding site for Rex (24). In these
viruses and others, there are secondary, lower affinity Rev/Rex binding
sites that cooperate with the primary binding elements to effect
Rev/Rex response (25, 27, 28).
Equine infectious anemia virus (EIAV) is a lentivirus distantly related
to HIV-1. As in other Rev/Rex systems, EIAV Rev (ERev) was shown to
mediate the nuclear export of unspliced viral RNAs and to be essential
for virus structural protein synthesis (20). Our previous studies
showed that, in addition to regulating viral mRNA transport, ERev
induced alternative splicing of EIAV mRNA in vivo (6,
8). The ERev protein has little sequence similarity with other Rev or
Rex proteins and does not recognize heterologous response elements
(20). The EIAV RRE is also distinct in that computer-assisted RNA
folding does not predict the formation of a large, stable secondary
structure (6). Previous results indicated that the EIAV RRE was located
in an exon near the 5' end of the env gene (8, 29), but the
primary ERev binding site and the manner in which ERev interacts with
RNA have not yet been defined.
In addition to the viral trans-regulatory proteins, a
variety of cellular proteins bind to retrovirus RNA sequences and
thereby control RNA processing and transport. These protein-RNA
associations can also influence the outcome of Rev-RRE interactions.
For example, cellular protein interactions with cis-acting
repressor elements and 5' splice sites (11, 30, 31) act to retain
pre-mRNAs in the nucleus, which can be circumvented by Rev
(32-34). Furthermore, cellular protein interactions with splice sites,
exonic splicing enhancer (ESE) and exonic splicing silencer elements
affect the efficiency and rate of viral pre-mRNA splicing (33,
35-37). Cellular proteins have been identified that bind directly to
RRE of HIV-1 and RxRE of HTLV-II (32). A purine-rich ESE was previously
identified in EIAV, which binds members of the SR-family of splicing
factors (6). In this report, we show that a 55-nucleotide region at the
3' end of exon 3 contains both an ESE, which binds ASF/SF2, and an RRE,
which binds ERev. ERev and ASF/SF2 interactions with this composite RNA
element were not mutually exclusive, but rather cell and virus proteins
appeared to bind simultaneously.
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EXPERIMENTAL PROCEDURES |
Plasmids--
The plasmids, pEX312 and pEX322, were constructed
by PCR amplification of EIAV exon 3 sequences and contain all of exon 3 (positions 5435-5541) or the 3' half of exon 3 (positions 5487-5541), respectively. PCR primers used to amplify the exon 3 and 3' half of
exon 3 (3'Ex3) fragments were: exon 3, 5' primer
(5'-CGGAGCTCGTAAGAACAGCATGGCAG-3'); 3'Ex3, 5' primer
(5'-CGCGGAGCTCGAAAGAAGAATCTAAAGAAG-3'); and exon 3, 3' primer
(5'-GCGGGATCCACATACCTATTTTCCACC-3'). The PCR fragments were cloned into
SacI and BamHI sites of pBluscript KS II (+). The
plasmids, pEXB1, pEXL21, pEXL22, pEXS11, and pEXS12, which contain
mutated versions of the 3' half of exon 3, were constructed by PCR and
cloned into SacI and BamHI site of pBluscript KS
II (+). In vitro transcription of plasmids with T7 RNA
polymerase yields RNAs of 139 and 88 base pairs for exon 3 and 3'Ex 3, respectively; both RNAs contain 33 nucleotides derived from plasmid
sequences. All plasmids were confirmed by DNA sequence analysis.
Bacterial Expression and Purification of ERev--
ERev coding
sequences were PCR amplified from pRS-ERev (8) with oligonucleotide
primers. The PCR product was cloned as an NcoI to
SmaI fragment into pTYB4 (New England Biolabs) to yield pERev-Intein. Escherichia coli strain ER2566, containing
pERev-Intein, was induced with
isopropyl-1-thio-
-D-galactopyranoside at 15 °C
overnight. The bacterial pellet was suspended in column bufffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.1 mM EDTA, 0.1% Triton X-100, and Boehringer protease
inhibitor mixture). ERev protein was purified by the manufacturer's
protocol (New England Biolabs). A His-tagged ERev protein was also
produced by cloning ERev cDNA, as a NdeI to
BamHI fragment, into pET-19b (Novagen). E. coli
strain BL21 DE3(Lys) codon plus containing pET-19b-ERev was induced
with isopropyl-1-thio-
-D-galactopyranoside at 37 °C
for 3 h. The bacterial pellet was suspended in lysis buffer (1 M NaCl, 6 M urea, 20 mM phosphate
buffer, 0.5% Triton X-100, 10 mM imidazole, and Sigma protease inhibitor mixture) and the His-ERev protein was purified by
the manufacturer's protocol (Amersham Pharmacia Biotech, His-trap column); His-ERev was eluted and stored in buffer containing 500 mM imidazole, 6 M urea, 1 M NaCl,
10% glycerol.
Synthesis of Labeled RNA--
The plasmids pEX312, pEX322, and
pEX322 mutants were linearized with SmaI and incubated with
T7 RNA polymerase and [32P]GTP as described previously
(6). The 139- and 88-nucleotide RNA products were purified by
polyacrylamide gel electrophoresis under denaturing conditions, eluted,
and ethanol-precipitated. T7-L2 synthetic oligonucleotide was incubated
with T7 RNA polymerase and [32P]GTP as prescribed
(Ambion). The 34-nucleotide RNA probe was purified by MicroSpin G-25
column. Synthetic RNA oligonucleotides (L2, A, SC35, SRp40, and KS)
were purchased from Cybersyn Inc.; 3 µg of L2 RNA, A RNA, and KS RNAs
were 5' end-labeled with [
-33P] ATP and T4
polynucleotide kinase (New England Biolabs).
RNA-binding Assays--
In vitro binding reactions
were carried out in a volume of 20 µl containing: 3.2 fmol of
32P-labeled RNA, 8.8 mM HEPES, pH 7.9, 10%
glycerol, 60 mM KCl, 3.2 mM MgCl2,
0.2 mM EDTA, 1 mM dithiothreitol, 1 mg/ml yeast RNA, 1 mg/ml heparin, and the indicated amounts of recombinant ERev or
purified HeLa SR proteins. HeLa SR proteins were prepared as described
previously (6, 38) and relative purity was assayed by Coomassie Blue
staining of proteins subjected to SDS-PAGE. For electrophoretic
mobility shift assays (EMSA), reaction mixtures were incubated for 15 min at room temperature and subjected to electrophoresis through 4%
nondenaturing polyacrylamide gels (1× Tris borate-EDTA, pH 8.3) at
4 °C overnight at 5 V/cm. UV cross-linking competition experiments
were carried out under binding condition with or without 5-50-fold
molar excess of unlabeled RNA competitors. Reaction mixtures were
transferred to parafilm on ice and exposed to UV light (254 nm) at a
distance of 4 cm for 10 min. RNase A (6 µg) was added into
competition experiment and the reactions were incubated for 15 min at
37 °C. The cross-linked products were analyzed by 4-12% SDS-PAGE,
followed by autoradiography.
Immunoprecipitations--
5' end-labeled A and KS RNAs were
incubated with SR proteins and then UV cross-linked as above. UV
cross-linked products were immunoprecipitated with anti-ASF/SF2
monoclonal antibody (generously provided by A. Krainer, Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY) and resolved by 4-12%
SDS-PAGE as described previously (6).
In Vitro Renaturation of Exon 3 RNA--
32P-Labeled Exon 3 and 3'Ex3 RNA (1 × 103 cpm) were heated for 2 min at 95 °C and gradually
cooled to 15 °C in the cold room for 1 h in binding buffer
condition (8.8 mM HEPES, pH 7.9, 3.2 mM
MgCl2, 0.2 mM dithiothreitol) containing varied
concentrations of KCl. The renaturation products were analyzed by
electrophoresis on native 8% polyacrylamide gel at 4 °C overnight
at 5 V/cm.
In Vitro Protein-Protein Interaction Assays--
Chelating
Sepharose high performance beads (Amersham Pharmacia Biotech) were
charged with Ni2+ and equilibrated in binding buffer
containing: 157 mM NaCl, 2.7 mM KCl, 4.3 mM
Na2HPO4·7H2O, 1.4 mM KH2PO4, 10 mM EDTA,
0.05% Triton X-100, and 10 mM imidazole. For each binding
reaction, 40 µl of a 50% slurry (v/v) of beads was used. ERev-coated
beads were prepared by incubating beads with 1 µg of purified
His-ERev in 250 µl of binding buffer for 1 h at 4 °C and then
washed four times in 500 µl of binding buffer. ERev-coated or
uncoated beads were incubated with 8 µg of purified HeLa SR proteins
in 500 µl of binding buffer at 4 °C for 3 h and then washed
four times with 500 µl of binding buffer. Bound proteins were
released from beads in SDS-sample loading buffer, and protein complexes
were separated by SDS-PAGE, transferred to the Immobilon-P membranes
(Millipore), and reacted with anti-ASF/SF2 antibody. Protein bands were
visualized using horseradish peroxidase-linked secondary antibody and
enhanced chemiluminescence detection reagents (New England Biolabs).
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RESULTS |
Rev Binds to the 3' Half of Exon 3 RNA in Vitro--
The genetic
organization and splicing pattern of the EIAV provirus and the exons
utilized to produce the bicistronic tat/rev mRNA are
shown in Fig. 1 (A and
B). Previous results suggested that sequences in exon 3 (Fig. 1C) were necessary for both Rev-dependent viral mRNA transport and alternative splicing in vivo
(6, 8). With respect to the latter, Rev was shown to induce a shift
from the multiply spliced, tat/rev mRNA to the synthesis
and accumulation of the singly spliced env mRNA and to
mRNAs in which exon 3 was skipped. In order to identify the minimal
sequences within exon 3 that are required for ERev binding,
32P-labeled RNA containing part of exon 3 was incubated
with recombinant ERev protein and the resulting complexes were examined
by EMSA. The 88-nucleotide 3' half of exon 3 RNA (3'EX3), which was
purified from a single band by denaturing gel electrophoresis, migrated as two bands under native conditions, designated as RNA I and RNA II
(Fig. 2A, lane
1). That these represent different structural isoforms was
shown in denaturation/renaturation experiments described below.
Addition of recombinant ERev protein to the RNA resulted in the
appearance of a protein-RNA complex, which migrated above the RNA II
band (Fig. 3A,
lanes 2-4). As the amount of ERev was increased,
there was an increase in the intensity of the ERev-RNA complex
accompanied by a reciprocal decrease in the RNA II band. At the highest
concentration of ERev, RNA II was quantitatively shifted into complexes
with ERev. Quantitation of the relative amounts of the RNAs and
protein-RNA complex by phosphorimage analysis (Fig. 2B)
revealed that complex formation occurred at the expense of RNA II with
no detectable change in the amounts of RNA I. These data suggested that
ERev binds to a unique structural element in form II RNA. Two different
antisera, raised against synthetic peptides representing ERev amino
acids 29-40 (
-Rev1) and amino acids 45-58 (
-Rev2), abolished
ERev/RNA complex formation (Fig. 2A, lanes
5 and 6) but pre-immune serum had no effect (data
not shown). That these antisera did not result in a supershifted
Rev/RNA complex was probably due to interference with the ability of
ERev to bind RNA.

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Fig. 1.
Genetic organization of EIAV and
post-transcriptional control of gene expression. A,
genetic organization of EIAV showing the long terminal repeats
(LTR); genes encoding structural and enzymatic proteins
(gag, pol, and env) and
trans-regulatory proteins (tat and
rev); and the S2 open reading frame. B, mRNAs
produced by alternative splicing of the genomic RNA. Nucleocytoplasmic
transport of the unspliced gag/pol mRNA and the singly
spliced env mRNA are dependent on ERev. A
multiply-spliced, bicistronic mRNA encodes tat on exons 1 and 2 and
rev on exons 3 and 4. A 3-exon mRNA, produced by
ERev-dependent exon 3 skipping, encodes tat only.
C, nucleotide sequence of exon 3, which contains
cis-acting regulatory elements that control Rev response and
alternative splicing. Regions of the exon that were used as RNA probes
in subsequent experiments (3'Ex3 and L2) are
indicated; slash marks designate splice
sites.
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Fig. 2.
EIAV Rev protein binds to exon 3 RNA in
vitro. Bacterially expressed ERev binding to 3'Ex3 RNA was
examined by EMSA. A, 3.2 fmol of 32P-labeled
3'Ex3 RNA (88 nucleotides) was incubated with 0 nM
(lane 1), 0.7 nM (lane
2), 1.4 nM (lane 3), or
2.8 nM (lane 4) of ERev protein.
3'Ex3 RNA migrated as two forms, designated RNA I and RNA II. In
lanes 5 and 6, 1 µl of rabbit
anti-Rev1 or anti-Rev2 antiserum, respectively, was added to the
binding reaction prior to addition of the RNA probe. The anti-Rev
antibodies inhibited complex formation. B, quantitations of
binding activity of 3'Ex3 RNA and ERev proteins were determined by
phosphorimage analysis of the gel shown in A and is
represented as percentage of total RNA (RNA I+RNA II+Rev/RNA complex)
plotted as binding activity of 100%.
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Fig. 3.
Exon 3 RNAs form a dimer-like structure which
binds ERev. A, heat denaturation of 3'Ex3 RNA converts
RNA II to RNA I. 32P-Labeled 3'Ex3 RNA probe (3.2 fmol) was
denatured by heating at 95 °C then quick-chilled on ice. Denatured
(D) or non-denatured (N) RNAs were incubated in
the absence of ERev (lanes 1 and 2) or
in the presence of 1.4 nM ERev (lanes
3 and 4), and complexes were resolved by
non-denaturing polyacrylamide gel electrophoresis. B, native
(N) and heat-denatured (D) RNAs were incubated in
the absence of SR proteins (lanes 1 and
3) or in the presence of 3.8 µg of SR proteins
(lanes 2 and 4). RNA probe preparation
and electrophoresis were carried out as in panel
A. C, 32P-labeled exon 3 RNA (139 nucleotides; 2 × 104 cpm) and 3'Ex3 RNA (88 nucleotides; 2 × 104 cpm) were denatured by heating
at 95 °C, mixed together, and slowly cooled to 4 °C in buffers
containing KCl at 60 mM (lane 1), 160 mM (lane 2), 260 mM
(lane 3), and 360 mM (lane
4). Non-denatured 3'Ex3 and exon 3 RNAs are shown in
lanes 5 and 6, respectively. The
reaction mixtures were subjected to electrophoresis on 8%
non-denaturing polyacrylamide gel. RNA bands corresponding to 3'Ex3 and
exon 3 RNA I and RNA II are designated as RNA I88, RNA
II88, RNA I139, and RNA II139,
respectively. A dimer-like structure of 3'Ex3 and exon 3 RNAs is
designated as RNA II 88/139. The molar ratio of 3'Ex3 RNA
to exon 3 RNA was 2:1.
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Exon 3 RNA Forms a Dimer-like Structure in Vitro--
The fact
that 3'Ex3 RNA migrated as two forms was unexpected since the 3'Ex3 RNA
was not predicted to form a stable, base-paired structure. Heat
denaturation and rapid cooling of the 3'Ex3 RNA prior to binding
reactions and native gel electrophoresis converted the slower migrating
RNA II to the RNA I form (Fig. 3A, lanes 1 and 2). ERev did not form a complex with
heat-denatured RNA nor did it induce a conformational change in RNA I
(lane 3) but, as before, it did bind to RNA II
(lane 4). In contrast to ERev binding, HeLa SR
proteins bound to both forms of RNA and bound to heat-denatured 3'Ex3
RNA (Fig. 3B, see below). Thus, the ERev binding site is
located in a 55-nucleotide region of exon 3 proximal to the 5' splice
site and ERev recognized an unusual structure in that region.
We next asked whether the form II RNA resulted from intramolecular or
intermolecular RNA interactions. A 139-nucleotide exon 3 RNA and the
88-nucleotide 3'Ex3 RNA were heat-denatured, mixed together, and slowly
renatured in buffer containing varied KCl concentrations. The size
difference between exon 3 and 3'Ex3 RNAs allowed each species to be
distinguished on non-denaturing polyacrylamide gels (Fig.
3C, lanes 5 and 6).
Conversion of denatured form I RNA to form II RNA was enhanced with
increasing concentrations of KCl in the renaturation buffer
(lanes 1-4). In addition to the expected bands
corresponding to each form II RNA, a new band appeared that migrated at
a position intermediate between exon 3 form II RNA and 3'Ex3 form II
RNA. These data were consistent with an intermolecular association
between the 139-nucleotide exon 3 RNA and the 88-nucleotide 3'Ex3 RNA.
In this reaction mixture, 3'Ex3 RNA was present at 2-fold excess over
exon 3 RNA. Therefore, the exon 3 RNA II band was fainter than the 3'Ex
3 RNA II band, since most of the exon 3 form II RNA was in the
"heterodimer-like" form.
The ability of the exon 3 RNAs to form a slower migrating dimer-like
RNA species that was bound by ERev was perplexing, since computer
analysis failed to predict stable inter- or intramolecular structures
based on Watson-Crick base pairing. In order to define nucleotides
essential for the RNA-RNA interaction, we generated mutated transcripts
with base substitutions within 3'Ex3 RNA (Fig. 4B). 32P-Labeled
RNAs were incubated in the presence or in the absence of recombinant
ERev protein, and the reaction complexes were examined by EMSA (Fig.
4A). In L21 and L22 RNAs, two adenine to uridine substitutions within a 20-nucleotide-long purine tract prevented interactions necessary to generate form II RNAs (Fig. 4A,
lanes 5-8). Substitution of the AAAAU sequence
with UUUUA near the 3' end of S12 RNA also abolished formation of RNA
II (Fig. 4A, lanes 11 and
12). In contrast, the substitutions in B1 and S11 RNAs had
no effect on RNA structure. Mutations that abolished RNA II formation
also prevented Rev-RNA complex formation. Rev binding to B1 and S11
RNAs appeared to be weaker compared with wild type RNA (Fig.
4A, lanes 3 and 4 and
lanes 9 and 10). pKS RNA also migrated
as two bands, but ERev protein did not affect migration of either
(lanes 13 and 14). Taken together, the
data indicated that ERev specifically recognized a unique structure in
exon 3 RNA, which appeared to be mediated by purine-purine
interactions.

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Fig. 4.
Nucleotide substitutions in exon 3 RNA affect
RNA structure and ERev binding. A, EMSA analysis was
performed with 3'Ex3 RNA probes containing nucleotide substitutions and
recombinant ERev protein. 32P- Labeled RNAs (88 bases;
1 × 104 cpm) were incubated in the absence
(lanes 1, 3, 5,
7, 9, 11, and 13) or in the
presence of 1.4 nM (lanes 2,
4, 6, 8, 10, 12,
and 14) of ERev. Complexes were resolved by non-denaturing
polyacrylamide gel electrophoresis on a 4% polyacrylamide gel. The
wild type 3'Ex3 RNA probe migrated as two bands designated RNA I and
RNA II. ERev-RNA complexes are indicated as Rev/RNA. pKS is a
nonspecific RNA control derived from Bluescript KS-II plasmid.
B, base substitutions in 3'Ex3 RNA (pEX322) are
highlighted, and the slash mark
denotes the 5' splice site.
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Binding of Cellular Proteins to Sequences in the 3' Half of Exon 3 RNA--
The presence of ESE elements within exon 3 and their
proximity to the 5' splice site (6) suggested that cellular proteins may bind to RNA sequences juxtaposed with the Rev binding site. Three
tandem purine-rich repeats, denoted as A repeats and composed of the
sequence AAAGAAGAA, were identified in the 3' half of exon 3 (Fig.
5A). Binding of purified HeLa
SR proteins to RNAs containing individual or composite repeat elements
was examined in electrophoretic mobility shift assays. The 3'Ex3 RNA,
which contains three copies of the A repeat (Fig. 5A), was
shifted into a very slow migrating complex by purified HeLa SR proteins
(Fig. 5B, lane 2). Factors in the SR
protein preparation bound to both the fast (RNA I) and slow (RNA II)
migrating forms of the 3'Ex3 RNA, since both forms were shifted into a
protein-RNA complex (Fig. 5B). Furthermore, SR proteins
shifted heat-denatured form I RNA (Fig. 3B). This contrasts
with the binding of ERev, which bound exclusively to the slower
migrating form of 3'Ex3 RNA. The oligoribonucleotide probe L2 is
shorter by 9 bases from the 5' end and 10 bases from the 3' end
compared with 3'Ex3 and contains two A repeats (Fig. 5A).
Unlike the 3'Ex3 RNA, which migrated as two bands on non-denaturing gels, the L2 RNA migrated as a single species, implying that the terminal bases in 3'Ex3 RNA contribute to the formation of higher order
structures (Fig. 5B, lanes 1-3).
Proteins in the purified HeLa SR protein sample shifted the L2 RNA
probe, yielding a protein-RNA complex, which migrated faster than the
3'Ex3 RNA-protein complex (Fig. 5B, lanes
3 and 4). The oligoribonucleotide probe A (Fig. 5A), which contains a single copy of the A repeat, formed
complexes that migrated faster than those formed on the longer RNA
probes (Fig. 5B, lanes 5 and
6). To delineate the specific sequence elements in the 3'Ex3
region required for SR protein interactions, we examined the relative
binding activities of SR proteins to the L2 RNA probe by UV
cross-linking in the presence of various cold competitor RNAs (Fig.
5A). Protein and RNA mixtures were irradiated with UV light,
digested with RNase A, resolved by SDS-gel electrophoresis, and
analyzed by phosphorimage analysis. L2 RNA was cross-linked to two
proteins in the SR protein preparation of ~35 and 50 kDa (Fig.
5C, lane 2). The 50-kDa product is a nonspecific
RNA-binding protein that cross-links to a wide variety of RNAs (39).
Addition of cold homologous L2 RNA competitor reduced formation of the 35-kDa cross-linked product by 68% when added at a 50-fold excess relative to the L2 RNA probe (Fig. 5C, lane
4). The unlabeled A RNA, which contains a consensus site for
ASF/SF2, reduced complex formation on the L2 probe by 60% (Fig.
5C, lane 7). The other competitors,
which contain consensus binding sites for SC35 and SRp40 proteins or
the KS negative control sequence failed to compete with L2 for binding
to the 35-kDa polypeptide. Thus, the A repeat containing the ASF/SF2
consensus binding element appeared to be the minimal sequence in exon 3 recognized by the 35-kDa protein.

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Fig. 5.
Cellular SR proteins bind to sequence
elements in the 3' half of exon 3 RNA. A, sequences of
RNA probes and competitors. The purine-rich repeat elements designated
as A are indicated. B, EMSA analysis was
performed with 32P-labeled 3'Ex3 RNA and
33P-end-labeled synthetic oligoribonucleotides designated
L2 and A in the absence (lanes 1, 3,
and 5) or presence (lanes 2,
4, 6) of 3.8 µg of purified HeLa SR proteins.
The 33P-end-labeled oligoribonucleotide probes migrated as
one band identified as RNA oligo at the bottom of
the gel. The RNA-SR protein and oligoRNA-SR protein complexes are
indicated as RNA/SR or RNA oligo/SR.
C, UV cross-linking competition binding assay of SR proteins
to the [32P]GTP-labeled L2 oligoribonucleotide with
unlabeled oligoribonucleotides. Binding reactions were performed as in
panel B except that the indicated unlabeled
oligoribonucleotides were present at 5-fold (lanes
3, 6, 9, 12, and
15) or 50-fold (lanes 4, 7,
10, 13, and 16) molar excess compared
with labeled L2 oligoribonucleotide. Reactions were UV-irradiated and
protein resolved by SDS-PAGE and analyzed on phosphorimager.
D, UV cross-linking of HeLa SR proteins to 5' end-labeled A
or KS RNA probes followed by immunoprecipitation with anti-ASF/SF2
monoclonal antibody and SDS-polyacrylamide gel electrophoresis. KS is
the nonspecific RNA control. The positions of the molecular size
markers are shown at the left.
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In order to determine whether the 35-kDa polypeptide was ASF/SF2, as
previous data suggested (6), purified HeLa SR proteins were mixed with
5' end-labeled A- or KS RNAs and subjected to UV cross-linking,
followed by immunoprecipitation with an anti-ASF/SF2 monoclonal
antibody and SDS-gel electrophoresis (Fig. 5D). A UV cross-linked protein product of 35 kDa was detected with the A RNA
probe (lane 1) but not with the KS RNA probe
(lane 2). In summary, EMSA, UV cross-linking competition,
and immunoprecipitation experiments indicated that the SR protein,
ASF/SF2, binds to repeated RNA elements proximal to the 5 ' splice site
of exon 3. Although ERev and SR protein binding sites appeared to
overlap, we believe that they are not identical. Therefore, an
important question is whether both viral and cellular proteins can bind
simultaneously to this 55-nucleotide purine-rich region.
Rev and SR Protein Interactions on the 3' Exon 3 RNA--
Having
shown that ERev and SR proteins bind independently to
cis-acting RNA elements located in the same region of exon
3, we next asked whether the viral and cellular proteins bind RNA competitively or simultaneously. Purified SR proteins and
32P-labeled 3'Ex3 RNA were cross-linked by UV irradiation
in the presence of increasing amounts of ERev protein. After digestion with RNase A, the complexes were fractionated by SDS-PAGE and visualized by autoradiography. ERev did not interfere with ASF/SF2 cross-linking to 3'Ex3 RNA in this assay, but rather, caused a modest
but reproducible increase in ASF/SF2 binding (Fig.
6A, lanes
3-5). ASF/SF2 was not cross-linked to the control pKS RNA (Fig. 6A, lane 1).

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Fig. 6.
ERev and HeLa SR proteins bind simultaneously
to a composite element in exon 3 in vitro.
A, UV cross-linking of 100 ng of HeLa SR proteins to the
32P-labeled 3'Ex3 RNA in the absence (lane
2) and in the presence of 100, 200, and 500 ng of ERev
protein (lanes 3, 4, and 5,
respectively). The complexes were separated by SDS-PAGE and visualized
by autoradiography. pKS RNA is the nonspecific control (lane
1). B, wild type ERev or mutant ERev-M27 were combined
with HeLa SR proteins and complex formation with
32P-labeled 3'Ex3 RNA (88 bases; 1 × 104
cpm) was examined by EMSA. The ERev-M27 mutant was previously predicted
to be defective for RNA binding (20). RNA was incubated in the absence
of protein (lane 1), or with individual proteins:
1 µg of ERev (lane 2), 1 µg of ERev-M27
(lane 3), or 100 ng of HeLa SR proteins
(lane 4). Complex formation was examined with
combinations of ERev (1 µg) and HeLa SR proteins (100 ng)
(lane 5) or mutant ERev-M27 plus HeLa SR proteins
(lane 6). The novel RNA-ERev-SR protein complexes
in lane 5 is identified as Complex.
C, pull-down assay was performed with 1 µg of
Ni2+-bead-immobilized His-tagged ERev (lane
1) and 8 µg of purified SR proteins, and then
SDS-polyacrylamide gel electrophoresis was carried out for
fractionation of the bound proteins. The protein retained by
interaction with ERev were visualized by Western blotting
(WB) using anti-ASF/SF2 antibody (lanes
2 and 3) or anti-monoclonal antibody 104 (lanes 4 and 5).
|
|
We next performed EMSA experiments with labeled 3'Ex3 RNA and SR
proteins in combination with either wild type or mutated ERev proteins.
The ERev mutant, M27, has a 4-amino acid alanine substitution
(ERLE93-96AAAA) and was shown to be defective for viral RNA transport
and alternative splicing in vivo (20). The mutation in
ERev-M27 disrupts a predicted
-helical region and was previously
suggested to be defective for RNA binding. This prediction was
confirmed by the observation that recombinant ERev-M27 protein, in
contrast to wild type ERev, failed to bind 3'Ex3 RNA in
vitro (Fig. 6B, compare lanes 2 and 3). Addition of 100 ng of purified HeLa SR proteins to
the binding reaction yielded diffuse complexes that migrated near the
position of RNA II and higher (Fig. 6B, lane
4). Addition of 100 ng of purified HeLa SR proteins in
combination with 1 µg of ERev resulted in the formation of a new
complex, which migrated more slowly than the ERev-RNA complex (Fig.
6B, lane 5; designated as
Complex) and which was absent in reactions containing SR
proteins and ERev-M27 (lane 6). These data
indicated that ERev bound to RNA in association with factors present in
the SR protein fraction and that complex formation was dependent on the
ability of ERev to bind RNA. Although EMSA and UV cross-linking studies
indicated that ASF/SF2 and ERev bind to 3'Ex3 RNA, we cannot rule
out the possible involvement of additional proteins in formation of
the novel complex. We are currently in the process of identifying the
cellular proteins that bind exon 3 RNA in association with ERev.
We next asked whether there was an interaction between recombinant ERev
and SR proteins in the absence of exon 3 RNA with an affinity pull-down
assay. Purified HeLa SR proteins were incubated with
Ni2+-chelate Sepharose beads in the absence or presence of
His-tagged ERev protein. After incubation, the beads were washed
extensively and proteins that were bound to the affinity matrix were
fractionated by SDS-PAGE electrophoresis and visualized by Western
blotting using anti-ASF/SF2 antibody or monoclonal antibody 104, which recognizes phosphorylated SR proteins (Fig. 6C). ASF/SF2 was
retained on the Ni2+-chelate matrix through its association
with His-ERev (Fig. 6C, lane 2), since
there was no binding to beads alone (lane 1).
Besides ASF/SF2, two other phosphoproteins in the SR protein
preparation (~55 and 25 kDa) interacted with His-ERev (Fig.
6C, lane 3). It remains to be
determined whether ERev directly associates with various SR proteins or
interacts with a complex of factors.
 |
DISCUSSION |
In this report we have identified the binding site for EIAV Rev
and show that it is located in a 55-nucleotide region proximal to the
5' splice site of exon 3 RNA. We also show that elements within this
relatively short purine-rich region were bound by cellular SR proteins.
Indeed, ASF/SF2 was shown to bind a 34-nucleotide sequence (L2)
contained within the longer purine-rich tract. Exon 3 RNAs that
contained the 55-nucleotide purine-rich tract formed a higher order
intermolecular structure and it was this form of RNA, which was
specifically bound by ERev in vitro. Heat denaturation of
the RNA prior to EMSA reactions, or base substitutions that prevented
formation of the dimer-like structure, abolished ERev binding. In
contrast, SR protein binding was independent of structural features in
exon 3 RNA. These differences in recognition of the RNA by the
individual proteins suggested that Rev and ASF/SF2 binding sites may
overlap but are nevertheless distinct entities. In combination, ERev
and SR proteins formed a novel complex on exon 3 RNA and,
concomitantly, complexes that were seen with the individual proteins
were diminished. The new complex was not formed when SR proteins were
combined with an ERev mutant, which was defective for RNA binding
activity. UV cross-linking experiments confirmed that binding of ERev
and SR proteins to the RNA was not mutually exclusive. Furthermore,
ERev was shown to interact with SR proteins in the absence of RNA
in vitro. Thus, EIAV presents a subtle variation on the
HIV-1 Rev system, where ASF/SF2 does not bind to the RRE alone but is
recruited to the RRE subsequent to Rev binding (46).
Other RREs are large, stem-loop structures, which contain a small,
discrete element that binds Rev with high affinity; adjacent lower
affinity Rev binding sites cooperate with the primary binding site to
support optimal Rev/RRE function. HIV-1 Rev binds to a purine-rich
structure, which is stabilized by non-Watson-Crick G-A and G-G base
pairs (40, 41). Although computer-assisted folding of the high affinity
binding site for EIAV Rev did not predict a stable secondary structure,
native gel electrophoresis of this region indicated the presence of two
discrete forms, only one of which was recognized by ERev in
vitro. It is likely that unusual base pairing in this purine-rich
region forms a unique structural feature recognized by ERev. It seems
unlikely that this particular dimer-like structure is required for EIAV
Rev/RRE function in vivo. On the other hand, purine-rich RNA
elements elsewhere in EIAV RNA (for example, in exon 4) could interact with the RRE in exon 3 to provide a similar structured element in
vivo. Since ERev does not interact with RNA in the absence of
cellular proteins in vivo, it is possible that association of ERev with cellular factors modifies its binding affinity and specificity. The analogy of ERev-ASF/SF2-RRE associations to Tat-cyclin T-TAR RNA interactions is noteworthy. In the latter case, various lentivirus Tat proteins bind to RNA in association with cellular cyclin
T proteins; the latter cooperate with Tat to stably bind RNA and
subsequently act on the transcription complex (42).
Although the distantly related, genetically complex retroviruses employ
conserved mechanisms and similar cellular pathways to control virus
gene expression, they display variation in the ways that the components
are recognized and assembled. For example, the Rev proteins encoded by
EIAV and HIV-1 have no sequence similarity, yet both bind to their
cognate RNA elements, associate with CRM-1 (exportin 1), and couple
viral pre-mRNA to a protein export pathway (3, 43-45).
Furthermore, the RNA elements with which Rev proteins interact are
quite diverse with respect to their sequences, structures, and genomic
locations. The HIV-1 RRE forms a large secondary structure with
multiple stem-loop elements and is located in the middle of the
env gene. The HTLV-I RxRE also forms a large stem-loop structure but is located at the 3' end of the viral RNA. In contrast, the EIAV Rev binding site lies in a relatively short purine-rich region
located in exon 3 adjacent to a 5' splice site and is not predicted to
form a typical base-paired structure. The various locations of the Rev
binding sites among the viruses may be related to differences in their
genetic organization and gene expression programs. For example, the
location of the ERev binding site next to a splice site in EIAV exon 3 could facilitate alternative splicing and negative autoregulation of
ERev synthesis. The varied structures of the RREs would also determine
the manner in which both Rev and cellular proteins interact with these
RNAs. The RNA binding domains of HIV-1 Rev and HTLV-I Rex proteins are
arginine-rich, whereas that of EIAV Rev has few and interspersed
arginine residues. The short, purine-rich EIAV RRE and the distinct RNA
binding domain of ERev suggest that this ERev-RRE interaction would be
different compared with other retroviruses and might require cellular
cofactors for optimal stability.
In a previous study, we suggested that the juxtaposition of ERev and SR
protein binding sites in EIAV RNA could influence two alternative
mechanisms for Rev-induced alternative splicing (6) EIAV Rev-induced
exon 3 skipping was proposed to result either from mutually exclusive
binding of ERev and SR proteins to RNA or from simultaneous binding and
subsequent disruption of spliceosomal complex assembly or activity. The
data presented here are consistent with the latter mechanism. It is of
particular interest that Rev proteins from distantly related viruses
such as EIAV and HIV-1 bind to RNA in association with particular SR proteins and have been reported to inhibit splicing in vivo
and in vitro (46). Although SR proteins, especially ASF/SF2,
have been studied primarily in the context of constitutive and
alternative splicing in the presence of an exonic splicing enhancer
element (47), it has been reported that ASF/SF2 can act as a negative regulator of splicing in some contexts (48, 49). It is likely that the
inhibitory effects of SR proteins in these situations may be analogous
to Rev-SR protein effects at the RRE. We are currently testing the
possibility that ERev-ASF/SF2 interactions interfere with the assembly
or catalytic activity of the spliceosomal complex.
 |
ACKNOWLEDGEMENTS |
We thank Gisela Heidecker, Barry
Morse, and Huey-Jane Liao for helpful discussions.
 |
FOOTNOTES |
To whom correspondence should be addressed. Tel.:
301-846-5611; Fax: 301-846-6863; E-mail: derse@ncifcrf.gov.
Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M008996200
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
 |
ABBREVIATIONS |
The abbreviations used are:
HIV, human
immunodeficiency virus;
HTLV, human T-cell lymphotrophic virus;
PCR, polymerase chain reaction;
ERev, equine infectious anemia virus Rev
protein;
RRE, Rev-responsive element;
RxRE, Rex-responsive element;
PAGE, polyacrylamide gel electrophoresis;
EIAV, equine infectious
anemia virus;
EMSA, electrophoretic mobility shift assay;
ESE, exonic
splicing enhancer;
SR, serine- and arginine-rich.
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