From the Institute for Cancer Research, Fox Chase
Cancer Center, Philadelphia, Pennsylvania 19111, the § Cell
and Molecular Biology Graduate Group, University of Pennsylvania,
Pennsylvania 19104, and the ¶ Division of Cellular and
Molecular Medicine, University of California at San Diego,
La Jolla, California 92093-0651
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ABSTRACT |
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Retroviruses display a unique form of alternative splicing in which both spliced and unspliced RNAs accumulate in the cytoplasm. Simple retroviruses, such as avian sarcoma virus, do not encode regulatory proteins that affect splicing; this process is controlled solely through interactions between the viral RNA and the host cell splicing machinery. Previously, we described the selection and characterization of novel avian sarcoma virus mutants. These viruses were separated into two classes based upon analysis of splicing intermediates produced in infected cells and in a cell-free system. One class, which included mutants with altered polypyrimidine tract or branch point sequences, showed significant accumulation of intermediates, suggesting that splicing was regulated in step 2. The other class, which included mutants with deletions of exonic enhancer sequences, did not accumulate splicing intermediates, suggesting that splicing was regulated before step 1 of the splicing reaction. In this report, we show that a mutant blocked at step 1 fails to form a stable spliceosomal complex, whereas one blocked at step 2 shows a defect in its ability to transit through the last spliceosomal complex. Using UV cross-linking methods, we show that regulation at each step is associated with specific changes in the binding of cellular splicing factors. Regulation at step 1 is correlated with decreased cross-linking of the factor U2AF65, whereas regulation at step 2 is correlated with enhanced cross-linking of the factor SAP49. Because these mutations were isolated by selection for replication-competent viruses, we conclude that retroviral splicing may be regulated in vivo through altered binding of constitutive splicing factors.
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INTRODUCTION |
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Pre-mRNA splicing, the process by which introns are removed, takes place in the nucleus within a large structure termed the spliceosome and can be viewed as a two-step process (for recent reviews, see Refs. 1 and 2) (see Fig. 1B). In step 1, the pre-mRNA is cleaved at the 5'-splice site (5'-ss),1 and a branched lariat-exon 2 intermediate is formed. In step 2, the two exons are ligated together, and the branched intron is released. In vertebrates, several loosely conserved sequence elements within the pre-mRNA are important for recognition by the splicing machinery, including sequences at the 5'-ss, the 3'-ss, the polypyrimidine tract (PPyT), and the branch point (BPS) (3). Additional RNA elements have been described within the intron and the exon that can promote or inhibit splicing (reviewed in Refs. 4 and 5).
The RNA splicing elements are recognized by trans-acting cellular components that include both small nuclear ribonucleoproteins (snRNPs) and proteins known as splicing factors (6-8). The snRNPs are composed of a highly structured RNA to which a number of shared as well as unique proteins are bound. The RNA component of some snRNPs recognizes elements in the splicing signals in pre-mRNAs through base pairing interactions. The U2 snRNP recognizes the BPS, and the U1 snRNP recognizes the 5'-ss. Two other snRNPs required for splicing, U4 and U6, participate in formation of the mature spliceosome (6, 9). It is believed that the RNA components of the snRNPs and pre-mRNA cooperate to catalyze both steps in the splicing reaction (10).
The non-snRNP protein components of the spliceosome appear to assist in splice site selection, organization of snRNPs, and regulation of alternative splicing events. A subset of splicing factors contains motifs of repeating serine-arginine peptides, and these factors are called SR or SR-related proteins (11, 12). SR proteins have distinct as well as overlapping functions; one role of SR proteins appears to be in the activation of splicing by exonic splicing enhancers (ESEs). The ESE elements in the downstream exon promote splicing at the upstream 3'-ss usually through interaction with specific SR proteins (13-15).
The limited base pairing potential between small nuclear RNAs and the pre-mRNA splicing signals, as well as the degenerate nature of these signals, suggests that the binding of snRNPs must be assisted by proteins. Several proteins have been implicated in tethering the U2 snRNP to its target element, the BPS. They include the splicing factor U2AF65 (U2-associated factor of 65 kDa), which binds to the PPyT and has RNA annealing activity (16, 17). Some spliceosome-associated proteins (SAPs) can bind to pre-mRNA as well as to small nuclear RNA (8, 18). For example, the U2 snRNP-associated SAP49 and other SAPs have been shown to bind to pre-mRNA upstream of the BPS (19). It appears that the combined binding of U2AF65 and several SAPs in the vicinity of the BPS may stabilize the binding of the U2 snRNP to the pre-mRNA.
The molecular basis for the control of alternative splicing in vertebrate cells has been the focus of several recent in vitro studies (20, 21). In some cases, it appears that a protein factor can inhibit splicing by binding to a nearby RNA sequence (20). In other cases, a protein factor can activate splicing by binding near a particular splice site (21, 22). Retroviruses display a unique form of regulated splicing that allows simultaneous expression of spliced and unspliced RNAs within the same cell. The integrated viral DNA produces a single primary transcript that may either be spliced, to allow for expression of the viral envelope glycoproteins, or may remain unspliced, to allow for expression of viral structural proteins and enzymes. In addition to serving as an mRNA, the unspliced RNA also comprises the genome for progeny virions. In the case of retroviruses with the simplest genomes, such as the avian sarcoma virus (ASV), approximately two-thirds of the primary transcripts remain unspliced, and the limited splicing that takes place uses a common 5'-ss and one of two 3'-ss (see Fig. 1).
ASV requires an appropriate balance between spliced and unspliced RNAs for efficient replication. We have exploited this feature as a genetic selection tool to study the control of splicing at the env splice site. We demonstrated that a specific insertion into the BPS, upstream of env, increased the efficiency of splicing and created a replication defect (23). Upon prolonged passage, replication-competent viruses that contained spontaneous second-site suppressor mutations were isolated. The suppressor mutations were of two types: one type comprised base substitutions in the BPS, and the other deletions of the ESE within the env exon. Both types of suppressors down-regulated splicing. Up-regulation of splicing and a concomitant replication defect were also created by lengthening of the PPyT (24). In this case, selection for replication-competent mutants revealed a third type of suppressor comprising U to C transitions within the PPyT, which also down-regulated splicing. When the different types of suppressor mutants were examined in more detail, we found evidence for regulation at two distinct steps in the splicing reaction. The first class (mutants that include ESE deletions) showed a partial block in splicing prior to step 1, and the second class (mutants with alterations in the BPS or PPyT) showed a partial block subsequent to step 1, but prior to step 2 (24, 25). In this report, we describe a more detailed in vitro characterization of RNA splicing control and of the protein factors that form complexes with ASV RNAs that include these two classes of splicing suppressor mutations.
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EXPERIMENTAL PROCEDURES |
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Plasmids-- The plasmids used to prepare in vitro RNA splicing substrates have been described elsewhere (24).
In Vitro Transcription and Splicing-- In vitro transcription and splicing assays were carried out as described (24). Radioactive 32P-labeled run-off transcripts were synthesized in vitro using T7 polymerase and were then gel-purified for UV cross-linking experiments. For in vitro splicing reactions, RNA (105 cpm) was incubated with 3-10 µl of nuclear extract (~30 µg, prepared as described previously (24)), ATP (0.5 mM), creatine phosphate (20 mM), MgCl2 (3.2 mM), RNase inhibitor (5 Prime-3 Prime, Inc.) (0.25 units/reaction), dithiothreitol (2 mM), KCl (100 mM) and 20 mM HEPES, pH 7.6 in a 25-µl volume. Reactions were incubated at 30 °C for the indicated times and stopped by addition of an equal volume of proteinase buffer. One-hundred µg of proteinase K was then added, and the reaction was incubated for 15 min at 37 °C. The RNA products were then purified by phenol/chloroform extraction and ethanol precipitation. Samples were resuspended in formamide loading buffer and heated to 70 °C for 5 min prior to loading on 6% polyacrylamide gel containing 7 M urea. The gels were dried and subjected to autoradiography.
Splicing Complex Formation Assays--
Conditions for gel shift
assays have been described (26). In vitro splicing reactions
were incubated at 30 °C for the indicated times, after which they
were brought to a final concentration of 0.5 mg/ml heparin and
incubated for 5 min at 30 °C. Samples were then loaded directly onto
a 4% polyacrylamide gel (running and gel buffer was 50 mM
Tris, pH 8.0, and 50 mM glycine) and subjected to
electrophoresis at 200 V for 5 h. The gels were then dried and
subjected to autoradiography. Because it is unusual to detect
significant amounts of complex C in splicing reactions, we analyzed our
most efficiently spliced substrate, BG-uuu, in a similar manner (data
not shown). As we detected amounts of complex C similar to that
observed with I(17), we attribute the increased detection of this
complex to some general feature of our system.
UV Cross-linking-- For UV cross-linking, the splicing reactions were incubated for the indicated times and then placed on ice in an inverted Eppendorf tube top. UV exposure was for 5 min at 10 cm from the source (Sylvania G64T6 lamp). After UV irradiation, the samples were treated for 15 min with an RNase mixture (Ambion Inc.) that contained RNases A and T1. An equal volume of 2× Laemmli loading buffer was added to the samples, which were heated to 95 °C for 5 min and then loaded onto 10% SDS protein gels. The gels were then fixed, dried, and subjected to autoradiography.
Two-dimensional Gels-- Non-equilibrium pH gradient gel electrophoresis (NEPHGE) was carried out as described (27). Cross-linking reactions were treated with RNase, incubated with an equal volume of sample buffer (9.5 M urea, 2% Nonidet P-40, 5% 2-mercaptoethanol, 1.6% ampholyte, pH 5-8, and 0.4% ampholyte, pH 3-10) at 65 °C for 10 min, and then layered on top of a tube gel (9.2 M urea, 4% acrylamide (10:1 acrylamide/bisacrylamide ratio), 20% Nonidet P-40, 1.6% ampholyte, pH 5-8, 0.4% ampholyte, pH 3-10, 0.01% ammonium persulfate, and 0.1% TEMED). A solution of 100 mM NaOH was placed in the lower chamber, and 10 mM H3PO4 was placed in the upper chamber. Samples were subjected to electrophoresis at 500 V for 1 h and then at 750 V for 2 h. Each tube gel was then transferred to the top of a 10% SDS protein gel without a stacking gel. The protein gels were run until the dye reached the bottom, at which time they were transferred to nitrocellulose for blotting. To determine the location of cross-linked species and the location of specific proteins in the nuclear extract on the same gel, the membrane was subjected to chemiluminescence Western analysis (Pierce). The membrane was then washed overnight in a large volume of phosphate-buffered saline to remove chemiluminescence reagents and exposed to x-ray film to detect the location of 32P-labeled cross-linked species.
Production of Anti-SAP49 Antibodies and Immunoblotting-- An anti-SAP49 rat serum was prepared using a fragment of SAP49 comprising the RNA-binding domains (amino acids 1-180) (the SAP49 cDNA was kindly provided by Robin Reed (28)). The pET28 system (Novagen) was used for Escherichia coli expression and purification through histidine tags at both termini. The bacteria were lysed with a French press, and the recombinant protein was purified by TALON metal chelate chromatography (CLONTECH). Polyclonal antibodies were raised in rats by the Laboratory Animal Facility at the Fox Chase Cancer Center. Immunoblotting and chemiluminescence detection were performed according to the recommendations of the manufacturer (Pierce).
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RESULTS |
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Substrates and Experimental Design--
Fig.
1A shows the organization of
the ASV genome and the location of RNA elements involved in splicing
and the minigene RNA substrates used in this study. The nucleotide
sequences of the regions spanning the BPS to the env 3'-ss
for RNAs analyzed in this study are shown in Fig. 1C. Two
mutations, I(17) and IS1cu (24), were constructed by site-directed
mutagenesis. Both increase splicing efficiency and cause a replication
defect. The suppressor mutations listed were obtained by selecting for
replication-competent viruses in cultures transfected with viral DNAs
that contain the I(
17) or IS1cu mutations (Fig. 1C). These
suppressor mutations all down-regulate splicing. Several of the
suppressor mutations cause a partial block in step 2 of the splicing
reaction detected by the accumulation of lariat-exon 2 intermediates
both in cell-free splicing extracts and in cells infected with the
mutant viruses (Fig. 1C) (24). An additional substrate used
in our studies is not described in Fig. 1, but has been described
elsewhere (24). This substrate replaces the weak wild type BPS with the
strong
-globin BPS and adds a three-uridine extension onto the PPyT and it is thus denoted BG-uuu. The splicing of BG-uuu is very efficient
compared with the wild type, and it was therefore used as a positive
control when appropriate.
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Suppressor Mutations Affect the Formation of Splicing
Complexes--
We were interested in determining if suppressor mutants
with partial blocks at either step in splicing could be distinguished from each other, or from an efficiently splicing RNA substrate, in
their ability to form splicing-specific complexes. For these analyses,
we chose the parental, oversplicing mutant I(17) and the I
51 and
IS1 suppressor derivatives that cause partial blocks in splicing before
step 1 and at step 2, respectively (24, 25). As illustrated in the
in vitro splicing assay shown in Fig.
2A, the lariat-exon 2 intermediates accumulated with IS1, but not with I
51, and spliced
product was detectable only with the I(
17) parent. The association of
a substrate RNA with the splicing machinery is known to occur through a
stepwise process; several stages in this process can be visualized on a
native gel. In the initial stage, the RNA is found in a heterogeneous,
but fast migrating complex, designated H (26, 29), which contains
mainly heterogeneous nuclear ribonucleoproteins (30). After the H
complex, RNAs can be found in splicing complexes, designated A, B, and
C in order of increasing size and time of appearance. Step 2 splicing
takes place in complex C, after which the spliced RNA is released, and the spliceosome disassembles (31). This complex is usually short-lived and often difficult to detect.
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Substrate RNAs That Are Blocked before Step 1 of Splicing Show
Reduced Cross-linking of U2AF65--
We next wanted to
determine if the partial blocks in splicing could be correlated with
changes in the binding of cellular proteins.
32P-Radiolabeled RNA substrates were incubated with HeLa
cell nuclear extracts, and the bound proteins were cross-linked by
exposure to UV light (32). Following digestion with RNase, proteins
that contained cross-linked [32P]RNA adducts were
analyzed by polyacrylamide gel electrophoresis followed by
autoradiography. We first examined the cross-linked products from RNA
substrates containing the ESE deletion suppressor mutations (Fig.
3B). Typical patterns from
in vitro splicing reactions with these RNA substrates are
shown in Fig. 3A. The block in splicing was less severe with
the I36 suppressor compared with the I
51 suppressor. When
normalized for total radioactive content, only an ~2-fold reduction
in splicing was typically observed between I
36 and the I(
17)
parent. This is consistent with our previous observation that I
36
only partially suppresses I(
17) oversplicing in vivo
(23).
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Substrates That Are Blocked at Step 2 in Splicing Show Enhanced
Cross-linking of a 50-kDa Protein--
We next asked if a partial
block at step 2 is also correlated with differential binding of
cellular proteins. In vitro splicing patterns for the seven
RNA substrates used in these analyses are shown in Fig. 4A.
As expected, the BG-uuu substrate was spliced most efficiently, and no
splicing was detected with the wild type substrate (24). Results with
I(17) and IS1 were the same as those shown in Fig. 3A. The
substrate IS1cu, with an extended PPyT, was spliced more efficiently
than IS1, from which it was derived (Fig. 4A, compare
lanes 5 and 6). Splicing patterns of RNA
substrates derived from replication-competent viruses that contain
additional mutations that suppress IS1cu (IS1cuU(
15)C and
IS1cuU(
9,
15)C) are also shown (Fig. 4A, lanes
7 and 8). As can be seen by comparing the patterns,
the two suppressors of IS1cu restored a step 2 block in
splicing similar to that seen in IS1 (Fig. 4A, compare
lanes 5, 7, and 8). These results are consistent
with previous results from in vivo analyses (24).
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Identification of the 50-kDa Protein as SAP49-- Based on its predicted molecular mass and the observed migration of the 50-kDa cross-linked species on two-dimensional gels, we suspected that this protein might correspond to the splicing factor SAP49 (36). We employed NEPHGE to determine if the 50-kDa cross-linked species comigrated with SAP49. We raised polyclonal antibodies to the bacterially expressed N-terminal domain of SAP49. Serum from one rat contained highly specific antibodies that recognized a single species in one- and two-dimensional Western analyses (two-dimensional analysis shown in Fig. 7, upper panels). An in vitro cross-linking reaction was performed with a novel PPyT mutant RNA substrate that displayed a step 2 block and relatively strong cross-linking to the 50-kDa protein (data not shown) and with a wild type RNA substrate as a control. Following digestion with RNase, proteins were fractionated on a two-dimensional NEPHGE gel. The proteins were subsequently transferred to nitrocellulose, and the membrane was probed with our anti-SAP49 antibody and detected by chemiluminescence (upper panels). The same membrane was subjected to autoradiography to identify the cross-linked species (lower panels). The upper panels show that the anti-SAP49 antibody recognized a single protein on the NEPHGE gels. Two major cross-linked species were detectable on the gels shown in the lower panels, as expected from the one-dimensional gels (Fig. 6). Alignment of the two films revealed that the 50-kDa cross-linked species comigrated with SAP49 (Fig. 7, lower panels, closed arrowheads). We conclude that the 50-kDa protein corresponds to SAP49. In a similar NEPHGE experiment, Staknis and Reed (39) found that cross-linked SAP49 demonstrated a slightly slower and more acidic migration than silver-stained SAP49 from nuclear extracts, an alteration attributed to the RNA adducts. In our experiments, RNA nucleotide adducts did not significantly affect the protein migration on the NEPHGE gels. We attribute this to technical differences in our protocol.
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DISCUSSION |
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In this report, we have examined virus-host interactions through in vitro studies of RNA splicing substrates derived from retroviral mutants selected for their ability to restore viral growth. We have shown previously that the relative splicing efficiencies observed in vivo can be recapitulated in vitro. Here we have demonstrated that one mechanism of regulation, which involves accumulation of splicing intermediates in vivo, is characterized by the accumulation of spliceosomal complexes in vitro. We further characterized the substrates by examining the proteins that are in contact with the RNA. Differential UV cross-linking of two proteins, U2AF65 and SAP49, is described. We also demonstrated that the suppressor mutations comprising ESE deletions result in diminished cross-linking of U2AF65. This supports the findings of others that exonic enhancer elements act indirectly to promote U2AF65 binding to the PPyT (13, 37-39). We also observed a decrease in U2AF65 cross-linking in the case of a natural suppressor mutation that converts a five-uridine stretch in the PPyT to four (Fig. 4B, lanes 6 and 7). This is consistent with in vitro mutagenesis studies (34, 35). With the substrates tested, enhanced cross-linking of SAP49 correlated with accumulation of spliceosomal complex C, indicative of a block at step 2. Furthermore, this increased cross-linking of SAP49 occurred early in the splicing reaction, suggesting that this protein is involved in the early establishment of the block, but that its effect is manifested late in the splicing reaction. Because the suppressor mutations arose naturally during viral replication, the differences in factor binding observed in vitro are likely to contribute to the regulation that was selected for in vivo.
All of the substrates tested that were blocked in step 2 demonstrated increased cross-linking of SAP49. One potential explanation is that this factor is a component of C complexes, and the increased cross-linking merely reflects the accumulation of this complex. We addressed this possibility by examining cross-linking profiles at early time points in the splicing reaction (Fig. 6). Under the conditions used, we found increased cross-linking of SAP49 at the earliest time tested. This time point was prior to detection of any splicing complexes, which indicates that the enhanced association of SAP49 with the substrate occurred before accumulation of C complexes. This suggests that the increased binding of this protein is likely to be involved in the establishment of the block at step 2, as opposed to being a consequence of the block.
The SAP49 protein is a component of the essential splicing factor SF3b
and is associated with the U2 small nuclear RNA (8, 40). SAP49 has been
shown to cross-link efficiently to RNA substrates in complexes A and B
and also to bind both U2 snRNP and the pre-mRNA (36, 41). It has
been proposed that SAP49 is involved in tethering the U2 snRNP to the
BPS (28). How can it be that a factor involved in constitutive splicing
is also involved in the establishment of a block within the splicing
pathway? We speculate that SAP49 may bind to the mutant substrates in
an inappropriate manner and that this causes the spliceosome to stall
after step 1. This interpretation is supported by the following
observations. First, UV cross-linking experiments reported in the
literature indicate that SAP49 normally binds to RNA substrates only in
the presence of ATP. However, one of our step 2-blocked RNA substrates,
(IS1cuU(9,
15)C) can be cross-linked to SAP49 in the absence of
exogenous ATP (data not shown). Second, suppressor mutations that
affected SAP49 binding in our studies comprised U to C substitutions in
the PPyT (IS1cuU(
15)C and IS1cuU(
9,
15)C). Previous studies
indicate that SAP49 normally binds to RNA substrates upstream of the
BPS (28). Although we have not yet mapped SAP49 cross-linking sites on
the suppressor RNA substrates, we had not expected that changes in the
PPyT would affect its cross-linking. The fact that they do suggests
that SAP49 may bind inappropriately to these substrates and that such binding may contribute to the block in step 2.
Another protein that demonstrated variable cross-linking was identified
as U2AF65. This essential splicing factor has been shown to
bind to the PPyT of pre-mRNAs early in the splicing reaction (33).
U2AF65 also contains RNA annealing activity (17) and has
been shown to contact the BPS of the pre-mRNA (16). These results
suggest that U2AF65 binds to pre-mRNA early and
promotes the association of U2 snRNP with the BPS. Thus, both
U2AF65 and SAP49, along with other proteins, act to tether
the U2 snRNP to the BPS (19). Generally, our data are consistent with
this model for U2AF65 and SAP49 functions. However, we did
not find an absolute correlation between increased U2AF65
cross-linking and efficiency at step 1. Splicing of one of the substrates tested, IS1cuU(9,
15)C (Fig. 4), was fairly efficient at
step 1, but did not cross-link U2AF65 well. In our system,
the substrates that demonstrated low U2AF65 cross-linking
and that were efficient for step 1 of splicing also showed significant
cross-linking to SAP49. This suggests that SAP49 and U2AF65
may have some complementary functions in step 1 of splicing. We noted
that cross-linking of U2AF65 was less efficient than SAP49
under reduced ATP conditions, indicating that SAP49 may bind prior to
U2AF65 in our system. Experiments are underway to address
this issue directly.
Retroviruses display a unique form of alternative splicing in which both spliced and unspliced RNAs accumulate in the cytoplasm. Here we have performed in vitro analyses on several sets of mutant and suppressor viruses in which the ratio of spliced to unspliced RNA was altered. One set demonstrated decreased splicing efficiency that correlated with decreased association with U2AF65. A second set demonstrated decreased splicing efficiency that correlated with increased association with SAP49. This result suggests that splicing efficiency can be decreased through increased binding of constitutive splicing factors.
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ACKNOWLEDGEMENTS |
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We thank Drs. Robin Reed and James Patton for providing useful reagents and Sharon Jamison and the Laboratory Animal Facility of the Fox Chase Cancer Center for technical support. HeLa cell pellets were provided by the Cell Culture Center and National Center for Research Resources. We also thank Drs. John Taylor and Robert Perry for helpful comments on this manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant CA-47486 and Institutional Grant CA-06927, by a grant from the Pew Charitable Trust, and by an appropriation from the Commonwealth of Pennsylvania.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.
Searle Scholar. Supported by grants from the Cancer Research
Coordination Committee of the University of California and from the
National Institutes of Health.
** To whom correspondence should be addressed: Inst. for Cancer Research, Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111. Tel.: 215-728-3668; Fax: 215-728-2778; E-mail: r_katz{at}retro2.fccc.edu.
1 The abbreviations used are: ss, splice site(s); PPyT, polypyrimidine tract; BPS, branch point sequence; snRNP, small nuclear ribonucleoprotein; SR, serine-arginine; ESE, exonic splicing enhancer; SAP, spliceosome-associated protein; ASV, avian sarcoma virus; NEPHGE, non-equilibrium pH gradient electrophoresis; TEMED, N,N, N',N'-tetramethyl-ethylenediamine.
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REFERENCES |
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