From the Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/Université Louis Pasteur, 1 Rue Laurent Fries, BP 163, 67404 Illkirch Cedex, C.U. de Strasbourg, France
Received for publication, October 18, 2000
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ABSTRACT |
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9G8 protein belongs to the conserved
serine/arginine-rich (SR) protein family, whose members exhibit
multiple functions in constitutive and alternative splicing. We have
previously shown that 9G8 primary transcripts are subjected to
alternative splicing by excision/retention of intron 3 and to a tissue
specific modulation. Because both 5'- and 3'-splice sites of intron 3 appear to be suboptimal in vertebrates, we tested the 9G8 intron 3 as a
novel model system of alternative splicing. By using an in
vitro approach and a mutational analysis, we have identified two
purine-rich exonic splicing enhancers (ESE) located in exon 4 and a
(GAA)3 enhancer located in exon 3. These elements act in
concert to promote efficient splicing activation both in
vitro and in vivo. Titration experiments with an
excess of exonic enhancers or SR-specific RNA targets strongly suggest
that SR proteins are specifically involved in the activation process.
Although ASF/SF2 was expected to interact the most efficiently with ESE
according to the enhancer sequences, UV cross-linking coupled or not to
immunopurification demonstrates that 9G8 is highly recruited by the
three ESE, followed by SC35. In contrast, ASF/SF2 only binds
significantly to the (GAA)3 motif. S100 complementation
experiments with individual SR proteins demonstrate that only 9G8 is
able to fully restore splicing of intron 3. These results, and the fact
that the exon 3 and 4 ESE sequences are conserved in vertebrates,
strongly suggest that the alternative splicing of intron 3 represents
an important step in the regulation of the expression of 9G8.
Alternative splicing of nuclear pre-mRNA is a widespread
mechanism for controlling gene expression in higher eukaryotes, which allows the formation of various RNA isoforms from a single primary RNA
transcript (1, 2). The differential recognition and alternative choice
of 5'- and 3'-splice sites, which also represent the earliest events of
the spliceosome formation, may be under developmental, tissue, or
sex-specific controls. These regulations are predicted to require both
cis- and trans-acting factors, which modulate
either positively or negatively the alternative splicing process (3,
4). The study of many alternative splicing systems has allowed the
identification and characterization of cis-acting elements,
which activate or repress competing splicing reactions. Among them, the
best characterized are the exonic splicing enhancers (ESE),1 which stimulate the
use of nearby weak 3'-splice sites (for review, see Ref. 5). Some of
these ESE are purine-rich, and they are present in various model
systems. However, much less is known about the trans-acting
factors and the precise mechanisms responsible for alternative splicing
regulation. Important progress has been made in Drosophila
models, especially for the genes involved in the sex determination
pathway for which the role of several specific trans-acting
factors, namely, Sex-lethal (Sxl), Transformer (Tra) and Transformer-2
(Tra-2) have also been well documented (for review, see Refs. 2, 6).
However, in vertebrate systems, the advances on the identification of
the cellular factors involved in splicing regulation are far more limited.
A family of essential splicing factors, the serine/arginine (SR)-rich
proteins, represents one of the attractive candidates for the
activation and regulation of the splice site choice in the alternative
splicing process (7-9). SR proteins contain one or two N-terminal
ribonucleoprotein-type RNA binding domains, which are required for RNA
binding, and a C-terminal arginine/serine (RS) domain, which may
promote protein-protein interactions with other components of the
splicing machinery. The SR proteins are required for the constitutive
splicing reaction, mainly for the formation of the early
prespliceosomal complex by stabilizing U1 small nuclear
ribonucleoprotein at the 5'-splice site and in spliceosome formation,
most likely by mediating interactions between U1 small nuclear
ribonucleoprotein and U2AF, bound to the 5'- and 3'-splice sites,
respectively (7, 8, 10). SR proteins have been shown to intervene in
alternative splicing both in vitro and in vivo in
a concentration-dependent manner, by modulating 5'-splice
site choice in pre-mRNA containing competing 5'-splice sites
(11-14). They also participate in splicing activation of introns
containing weak 3'-splice sites by binding to purine-rich ESE (7, 9) as
well as in splicing repression (15).
To better understand the basis of their specific functions in
alternative splicing regulation, analyses of the SR protein abilities
to recognize specific RNA targets have been performed. High affinity
RNA targets have been defined by a conventional SELEX approach for
ASF/SF2, SC35, 9G8, SRp20, and SRp40 (16-18). Specific targets for SR
species can contain purine-rich sequences, for ASF/SF2 for instance
(16) or pyrimidine-rich sequences, for SC35 or SRp20 (17). Importantly,
most of these high affinity targets act as splicing enhancers under the
control of the SR species, which specifically recognizes them (16, 17).
By using a complementary approach based on functional SELEX, exonic
enhancers have also been identified, with more degenerate sequences
that are specific of the appropriate SR protein, validating both
conventional and functional SELEX approaches (19-21). Most of these
prototypical motifs represent strong enhancers, which are subject to
regulation according to their location in the pre-mRNA and/or their
involvement in a more complex activation region. Such elements,
for which interacting SR species have been unambiguously defined,
include the purine-rich element of the doublesex
(dsx) exon 4, recognized by ASF/SF2 (22); the Pu1 and Pu2
motifs of the bidirectional splicing enhancer of the adenoviral E1A
gene, which interact with 9G8; and ASF/SF2 or SC35, respectively (23),
and a constitutive splicing enhancer identified in the During a previous study (26) we identified and characterized several
9G8 mRNA isoforms in human fetal tissues. Among them, the 2.4-kb
isoform, containing the entire intron 3, was predominant in kidney,
whereas the classic 1.4-kb mRNA isoform was predominant in liver,
suggesting that a tissue-specific modulation of intron 3 splicing takes
place. Further studies of intron 3 alternative splicing as a model
system appeared to be interesting for several aspects. First, intron 3 of the 9G8 gene exhibits the hallmarks of an alternative intron (see
also "Results"). There are only a few examples of regulated
excision of introns, and the best characterized example is that of the
intron 3 of the P element in Drosophila whose regulation
does not involve SR species (27). Second, among the SR protein family,
the transcripts of ASF/SF2 (28), SRp55, SRp40, and SRp30c (29), or
SRp20 (30) have also been shown to undergo alternative splicing. Only
the alternative splicing of SRp20, which occurs by inclusion/skipping
of exon 4, has been analyzed in details, but the precise identification of the cis-acting elements has not yet been carried out
(31). Finally, as in the case of the SRp20 gene for which the inclusion of exon 4 is expected to lead to the synthesis of a SRp20 truncated for
an important part of the RS domain, the retention of the intron 3 in
the 9G8 mRNA should allow the synthesis of a putative 9G8 isoform,
which preserves only a reduced part of the RS domain (26). Thus, in
addition to the modulation of absolute levels of the whole SR species,
the alternative splicing of the 9G8 intron 3 could result in the
synthesis of shorter isoforms with particular properties in splicing regulation.
Because the 5'- and 3'-splice sites of 9G8 intron 3 are intrinsically
weak, we have focused our study on the cis-acting elements that control the activation of these splice sites, as well as the
trans-acting factors that mediate the activation effect. We have identified strong exonic elements in exons 3 and 4, which are
absolutely required for the splicing of intron 3. Moreover, we show
that 9G8 is primarily involved in the splicing activation mechanism.
In Vitro Constructions
The mutated exon 4 E3-E4 constructions are made by the replacement of
one or both halves of the exon 4 with oligonucleotides generating the
mPuA, mPuB,
The cons. 5'-E3-E4 construct contains a consensus 5'-splice site,
whereas cons. 5'3'-E3-E4 contains consensus 5'- and 3'-splice sites.
The first construct was obtained from the E3
To synthesize the RNA competitors corresponding to sequences of
wild-type or mutated exon 4 halves, paired oligonucleotides were
introduced between the KpnI/XbaI sites of the
Bluescript SK vector (Stratagene). The (GAA)3 RNA
competitor, which contains the last 21 nt of exon 3 and the first G of
intron 3, was obtained in the same manner. Constructs for expressing
the ASF/SF2 (sequence A10: GCACAGGACGAAGCUGCACC), SC35 (sequence 7:
AGGCGCAGUAGGGUAUGCUG), and 9G8 (sequence 102:
GACAACGACGACGACUAGAA) competitor RNA were previously described
(17).
Northern Blot--
Human and mouse multiple tissue Northern
blots (CLONTECH) were probed with human or mouse
full-length cDNA of 9G8, respectively, as well as with human or
mouse intron 3 probes as previously described (26). The approximate
size of the various species was determined according to the migration
of RNA markers.
UV Cross-linking and Immunopurification Assays--
For UV
cross-linking, 3 µl of HeLa cell nuclear extract, S100 extract, or SR
protein preparation were preincubated for 10 min at room temperature in
a medium containing 50 mM KCl, 1 mM MgCl2, 25 mM creatine phosphate, 0.76 mM ATP, 8-10% glycerol, and 100 ng of Escherichia
coli tRNA, in a 10-µl volume. Interactions between proteins and
RNA (400,000 cpm of [ In Vitro Splicing Assays--
In vitro splicing
assays with 7 fmol of E3-E4 and mutated E3-E4 transcripts were
performed as previously described (23, 33) with 10 µl of HeLa cell
nuclear extract supplemented with 2 µl of cytoplasmic S100 extract in
the presence of 48 mM KCl and 2.6 mM
MgCl2 and the basic components of the splicing reaction. Complementation assays were done with 9 µl of S100 fraction
supplemented with 1.5 µl of nuclear extract in the presence of 60 mM KCl and 2.6 mM MgCl2. Different
amounts of baculovirus-purified recombinant proteins (17) were added to
the transcript. Titrations assays were done in the presence of excess
of cold RNA competitor sequences.
Recombinant Proteins--
Recombinant baculoviruses expressing
9G8, ASF/SF2, SC35, or SRp20 and the purification of the individual SR
proteins were as described previously (17). At the end of the
purification step, all proteins were dialyzed against buffer D
(34).
Transient Transfections and RT-PCR Analysis--
HeLa and NIH3T3
cells were grown at 37 °C in 5% CO2 in Dulbecco's
modified Eagle's medium supplemented containing 2.5% fetal calf serum
or 4.5 g of glucose per liter and 10% calf serum, respectively. Cells (~1 × 106/60-mm plate) were transfected using
the CaCl2 procedure according to a previous study (35),
with 0.5 µg of reporter construct and 9.5 µg of carrier
pBluescript-SK plasmid DNA. After 14 h at 37 °C in 2% of
CO2, the medium was replaced with fresh medium and the
cells were placed at 37 °C in 5% of CO2. Approximately 10-15% of cells were transfected, as confirmed by cotransfection of a
lacZ-expressing vector. At 48 h after transfection, cells were
harvested and total RNA was extracted with TRIzol reagent (Life
Technologies, Inc.) and DNase-treated. Reverse transcription was
carried out on 1.5 µg of total RNA, using Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.), for 1 h at
42 °C. Two specific primers were used for the analysis of the splicing reaction by PCR. Primers sequences were:
5'-AACCATTATAAGCTGCAAT-3' for the RTase reaction and PCR and
5'-GTACCCTCGAGAAGCTTGCATG-3', specific for the expression vector, for
the PCR. The cycle number was kept to 30, and the products were
resolved on 1.5% agarose gel.
The 9G8 Intron 3 Carries Sequences Indicative of an Alternative
Intron and Its Splicing Is Modulated in Human and Mouse
Tissues--
Previous studies have shown that the splicing of intron 3 of the human 9G8 gene was regulated by alternative splicing in
embryonic tissues (Fig. 1A and
see Ref. 26). To determine whether regulation of intron 3 has been
maintained during evolution, we first cloned and sequenced DNA around
the exon 3/intron 3 and intron 3/exon 4 junctions in various
vertebrates (Fig. 1B). As previously noted for the human 9G8
gene, the 3'-end of intron 3 of all species analyzed is particularly
unusual. A purine-rich sequence of at least 10 residues is located
immediately upstream to the CAG 3', which pushes the standard U-rich
sequence 15 residues upstream of the intron 3/exon 4 junction. In
contrast, the 5'-splice sites appear to be less altered, although they
never contain more than 5 successive consensus residues and the
important G residue at position +5 is absent (in human and mouse) or
flanked by two nonconsensus residues at positions +4 and +6 (Fig.
1B). Thus, this sequence comparison indicates that the 3'-
and 5'-splice sites are suboptimal, in four distant species, strongly
suggesting that the alternative splicing of intron 3 might occur in
many vertebrates.
To test this hypothesis, we have performed Northern blot analyses with
9G8 mRNA or intron 3 probes on total RNA isolated from adult human
and mouse tissues (Fig. 1, C and D). Previous
analysis had only been performed on embryonic human tissues (26). As shown in Fig. 1C and as previously observed with embryonic
tissues (26), four mRNA isoforms, from the classic 1.4-kb mRNA
to the largest 3.6-kb mRNA, are identified with a 9G8 cDNA
probe that results from alternative splicing (excision/retention) of
intron 3 and use of two polyadenylation sites (see Fig. 1A
for their structure). Interestingly, the alternative splicing of 9G8
mRNA is also modulated in the adult tissues. Indeed, the retention of complete intron 3, as estimated by the accumulation of the 2.4-kb
isoform relative to that of the 1.4-kb mRNA, is weak in liver,
lung, heart, and skeletal muscle, whereas it is highest in kidney and
in pancreas (compare the left and the right
panels in Fig. 1C). The same kind of splicing
pattern is obtained from mouse tissues (Fig. 1D), the
2.4-/2.6-kb species doublet being poorly separated. We still observe a
splicing modulation of intron 3, because the weakest retention of
intron 3 was detected in skeletal muscle, whereas the relative
retention of intron 3 was highest in testis, kidney, and brain. RT-PCR
analysis of total RNA from various mouse tissues also revealed that,
relative to the minus intron 3 mRNA isoform, the intron
3-containing isoform is predominant in kidney and brain (data not
shown). Thus, data in Fig. 1 clearly show that intron 3 alternative
splicing is subjected to modulation according to the tissues,
i.e. modulation that could be more dramatic if considering
individual cell types within tissues.
Identification of Exonic Splicing Enhancers--
Because the 5'-
and 3'-splice sites of intron 3 are suboptimal, we hypothesized that
the splicing of intron 3 must require additional cis-acting
sequences. To identify these elements, we used an in vitro
splicing assay. The standard E3-E4 pre-mRNA includes the major part
of exon 3, the intron 3 whose size has been reduced from 875 to 469 nt
to obtain more efficient splicing (see "Experimental Procedures")
and the whole exon 4 followed by 23 nt from the intron 4. The splicing
of the E3-E4 pre-mRNA was analyzed in Fig.
2B (lane 2), in
which only the lariat intron 3 is shown, or in Fig. 7 (lane
2) in which both final products and free exon 3 are shown. Despite
the weakness of the 5'- and 3'-splice sites of intron 3, we observed
that the splicing was relatively efficient, as judged by the
accumulation of the lariat intron (Figs. 2B and 3B are examples). This
suggests that splicing of intron 3 may require some cis
enhancers to compensate for the weakness of the wild-type splice
sites.
As shown in Fig. 2A, purine-rich elements of 12 or 17 residues are present in each half of exon 4, labeled A and
B. To determine whether exon 4 contains enhancer sequences
that are required for the splicing of intron 3, each half of exon 4 was
replaced in the pre-mRNA E3-E4 by exonic sequences of the
constitutive exon 2, generating E3-E4
Immediately upstream the 5'-splice site of intron 3 (between positions
Involvement of Trans-acting Factors to Activate Splicing of Intron
3--
To test whether trans-acting factors are involved in
the splicing activation of intron 3, titration experiments using short RNA containing each half of exon 4 or high affinity RNA specific for
individual SR proteins as competitors were performed. A control RNA in
which the upstream (GAA)3 motif of E3-E4 was removed and the 5'-splice site improved in a consensus site was also used (cons.
5'-E3-E4). With this RNA, the effects of competitors were expected to
be linked only to the exon 4 enhancers. In a second control, the exon 4 enhancers were further deleted and the 3'-splice site was improved in a
consensus site (cons. 5'3'-E3-E4). It should be noted that both control
pre-mRNA (Fig. 4 lanes
12 and 20) are spliced more efficiently than the
standard E3-E4 pre-mRNA (lane 2) as expected.
The results of titration experiments of the E3-E4 pre-mRNA are
shown in Fig. 4. As observed with the control mPuA RNA sequence (lane 5), the half A sequence used in 750 and 1500 times
excess relative to the substrate is a poor splicing competitor
(lanes 3 and 4). This result was
unexpected, because mutants in the half A sequence lead to
inhibition of splicing (Fig. 2) and will be discussed later. In
contrast, the half B sequence (lanes 6 and 7), used at twice less excess, almost completely inhibited
the splicing reaction, whereas the control RNA mPuB was without effect (lane 8). Interestingly, the same behavior of the
competitors was observed with the cons. 5'-E3-E4 RNA (lanes
12-16), indicating that the factors that are titrated by
the half B enhancer are involved in the activation of the weak
3'-splice site. In contrast to the splicing competition revealed on
these two substrates, the splicing of the cons. 5'3'-E3-E4
pre-mRNA, which lacks the identified exonic enhancers, is not
significantly competed by any RNA (lanes 20-24).
This demonstrates that, in our experimental conditions, the titration
of the factors involved in the activation of the weak 3'-splice site
doesn't alter the multiple events of the constitutive splicing
reaction analyzed with this control pre-mRNA. That one or more SR
species could be involved in the activation was suggested by a specific
titration assay of individual SR species. Indeed, we observed that a
9G8-specific RNA competitor identified previously (17) is as efficient
as the B competitor and highly inhibits splicing of the E3-E4 and cons.
5'-E3-E4 pre-mRNA even at a moderate excess (375-fold;
lanes 11 and 19). In contrast, the
ASF/SF2- and SC35-specific competitors (17) were less efficient than
the half B competitor (compare lanes 9 and
10 with 7 and lanes 17 and
18 with 15). As control assays, we show that the three SR-specific competitors tested individually were inefficient (lanes 25 and 26) or weak (lane
27) at inhibiting constitutive splicing of the cons. 5'3'-E3-E4
pre-mRNA. Thus, the data demonstrate that some
trans-acting factors are specifically involved in splicing activation of intron 3 and suggest that the 9G8 protein itself might be
one of these factors.
SR Proteins Bind to the Enhancer Sequences--
To identify the
putative factors that interact with the cis enhancers of
exon 3 and 4, we performed UV cross-linking experiments using nuclear
extracts, S100, and total SR preparations (Fig. 5A). Comparison of the
cross-linking patterns with probe A reveals a specific broad band
corresponding to ~35-kDa proteins, which is obtained with the nuclear
extract and the SR preparation (lanes 1 and
3) but not with the S100 extract (lane 2).
Significantly, the mPuA probe does not reveal such
interactions (Fig. 5A, lanes 4-6).
Similar results were obtained in comparing cross-linking with the B and
mPuB probes (lanes 7-12), strongly suggesting
that both purine-rich elements of exon 4 are effective targets for the
SRp30 species of the nucleus. Finally, a comparable analysis with the
(GAA)3-containing probe from exon 3 shows that two specific bands correspond to proteins of ~35 and 20 kDa, indicating that some
SRp30 and possibly SRp20 species interact with the upstream (GAA)3 enhancer (Fig. 5A, lanes
13-15). Finally, the results presented in Fig.
5A did not reveal any significant interaction of SR species of higher molecular weight with the exon 3 or exon 4 enhancers.
Because the SRp30 proteins includes at least four individual species,
we identified what species primarily bind(s) to the exon 4 enhancers.
For that, an UV cross-linking with nuclear extract was followed by
immunopurification analysis using specific antibodies for 9G8, SC35, or
ASF/SF2 (Fig. 5B). We observed that the half A RNA interacts
preferentially with 9G8 (lanes 2-4), whereas the half B RNA interacts more uniformly with the three SRp30 species but
still with a preference for 9G8 (lanes 6-8). As
expected, no significant interaction was revealed with the mPuA RNA
(lanes 10-12). Because immunopurification
experiments give qualitative rather than quantitative data, mainly due
to possible differences in the affinity of the individual antibodies
for their respective antigen, we developed a complementation assay in
which individual SR proteins are added to an S100 extract to
reconstitute splicing assays before UV cross-linking was performed
(Fig. 6). The results show that 9G8 and
SC35 interact efficiently with the A or B probes (Fig. 6A
and B, lanes 3 and 4), the
strongest interaction being between the 9G8 and B RNA probe (Fig.
6B, lane 4). In contrast, ASF/SF2 interacts
moderately with B and only weakly with the A probes (Fig. 6B
and A, lane 2). We have confirmed that the three SRp30 proteins interact efficiently with an RNA probe specific for each
of them (Fig. 6B, lanes 10,
12, and 14) with slightly less probe cross-linked
to ASF/SF2 (lane 10).
Finally, we show that the exon 3 (GAA)3 enhancer interacts
more uniformly with ASF/SF2, SC35, and 9G8 (Fig. 6A,
lanes 10-12), its cross-linking pattern with the
three SRp30 being similar to that of the SR-specific targets in Fig.
6B (lanes 10, 12, and 14). As expected, interactions of control mPuA or mPuB RNA
are very weak with most SR species (Fig. 6A lanes
6-8, Fig. 6B lanes 6 and
7) and are only moderate for 9G8 (Fig. 6B,
lane 8). Taken together, data of Fig. 6 indicate that the
exon 4 enhancers interact preferentially first with 9G8 and second with
SC35, whereas the exon 3 enhancer is recognized more similarly by the 3 SRp30 species. Significantly, the strongest interactions between the B
half of exon 4 and 9G8 fully complement results of titration
experiments (Fig. 4), showing that both the B half and 9G8-specific
probes are the strongest competitors of splicing. In contrast, the
weakest interaction between the A half and 9G8 might explain why the A half is a poor competitor of the intron 3 splicing.
Activation of Intron 3 Splicing with Individual SR Species--
To
analyze the capacities of individual SR species to promote the splicing
of 9G8 intron 3, we carried out complementation assays containing S100
extract and the standard E3-E4 pre-mRNA or the cons. 5'3'-E3-E4 RNA
as a suitable pre-mRNA control lacking exonic enhancers (Fig.
7). Complementation of the S100 extract with the 9G8 protein results in an efficient splicing of the E3-E4 pre-mRNA (lane 6) and of the control substrate
(lane 15) as shown by the accumulations of intron 3 and
mRNA relative to the unspliced pre-mRNA. In contrast to 9G8,
ASF/SF2 or SC35 only poorly activates the splicing of the E3-E4
pre-mRNA (lanes 4 and 5).
Unexpectedly, however, relative to the assay without exogenous SR in
which a residual splicing is observed (lane 12), these two
SR species do not efficiently activate the splicing of the control RNA,
either individually (lanes 13 and 14)
or mixed together (lane 19). Because these two SR species
are active in promoting splicing of other well characterized
pre-mRNA (data not shown), the reasons why they are inappropriate
in promoting splicing of the control cons. 5'3'-E3-E4 remain obscure.
We have confirmed that a mix of 9G8 and ASF/SF2 activates the splicing
of the E3-E4 or control pre-mRNA as the 9G8 alone (compare
lanes 8 with 6 and lanes 17 with 15, respectively), suggesting that ASF/SF2 and SC35
(see also lane 18) do not activate unidentified splicing
silencers. However, the unexpected behavior of ASF/SF2 or SC35 species
was not general. Although SRp20 does not strongly activate the splicing
of the E3-E4 pre-mRNA (lanes 7 and
9), in agreement with the fact that the downstream exonic
enhancers have been shown to not react with SRp20 (see Fig. 4), this SR
protein is able to activate efficiently splicing of the control RNA
(lanes 16 and 18). In conclusion, among the SRp30 species that interact significantly with the upstream or downstream enhancers of exons 3 and 4 of 9G8 gene, we demonstrate that only 9G8 activates efficiently the splicing of intron 3 when the
SR species are tested individually. In contrast, SRp20, ASF/SF2, and
SC35 appear to be unable to perform, alone or as a mix, an efficient
E3-E4 splicing through an activation of its enhancers.
The Upstream and Downstream Enhancers Are Required in Vivo for
Splicing Intron 3--
Finally, it was important to determine whether
the enhancer motifs identified in vitro are also required
in vivo. For a better comparison with the in
vitro experiments, we have used the same E3-E4 constructs as those
used in Figs. 2 and 3, with the region of interest being inserted in a
mammalian expression vector. The constructs were transfected in HeLa or
NIH3T3 cells, and the splicing of the various RNA was analyzed by
RT-PCR (Fig. 8). Transfection of the
standard E3-E4 construct in both cells leads to the formation of the
intron 3 (
We have also assessed the exon 3 (GAA)3 enhancer by testing
As observed for several pre-mRNA models, relative
concentrations of SR proteins at the sites of which splicing occurs are crucial for the choice between alternative splicing reactions. As the
local concentrations of SR proteins at the multiple splicing sites in
the nucleus depend on their absolute levels in the cell, it is
important to understand how the cell regulates the general level of
each SR protein in the nucleus. Several features have prompted us to
perform in this first study an identification of the
cis-acting elements and trans-acting factors
involved in the splicing activation of intron 3 of 9G8. First, we
observed that the intron 3 splicing is modulated according to the
tissues (Fig. 1), which may result in a regulation of 9G8 protein
expression. Second, according to the sequences of the 5'- and 3'-splice
sites of intron 3 for various vertebrates (Fig. 1), it is expected that alternative splicing of intron 3 occurs ubiquitously in vertebrates, strongly suggesting that this event is of primary importance. Third,
the 9G8 SR protein itself appears to be involved in very different
splicing events. It has been shown to be specifically involved in a
transcriptionally coupled alternative splicing of the fibronectin EDI
exon (38), in the activation of a bidirectional splicing enhancer
present in adenoviral E1A pre-mRNA (23), and in the activation of
Drosophila doublesex splicing enhancer in conjunction with
Drosophila Tra and Tra-2 proteins in a heterologous system
(22).
Among the multiple features associated with the alternative splicing of
the 9G8 intron 3 model, some appear to be unusual and other more
classic. First, an alternative splicing based on an excision/retention
of an intron is rather infrequent, most probably because intron
retention may create a conflict between the splicing and the transport
machineries. Several observations document this aspect; (i) an
alternative mRNA isoform with an intron has a tendency to
transiently accumulate in the nucleus before it is exported toward the
cytoplasm, as shown for the tumor necrosis factor Given the weakness of the 5'-splice site and mainly of the 3'-splice
site of the intron 3, the identification of multiple exonic enhancers
was not unexpected. In vitro and in vivo analyses show that both the exon 4 purine motifs and the exon 3 (GAA)3 motif bear an enhancer activity for the intron 3 splicing. In in vitro assays, the presence of the three
motifs is required to obtain a maximal splicing efficiency
(Figs. 2 and 3). However, in culture cells (HeLa and 3T3), which
perform efficient splicing of endogenous 9G8 intron 3 (data not shown),
we observed that the presence of the (GAA)3 motif may be
dispensable if both exon 4 motifs are preserved, whereas the exon 4 motif B may be also dispensable in the presence of the
(GAA)3 motif and the exon 4 motif A (Fig. 8). In cells in
which the splicing machinery is less optimal for the 9G8 pre-mRNA
splicing, it is likely that the involvement of the three enhancers is
important in promoting a significant but incomplete splicing of intron 3.
Identification of exonic enhancers similar to the (GAA)3
motif of exon 3 that lead to an activation of a downstream 5'-splice site has been demonstrated very recently (23, 42, 43). The novel motif
we identified is separated from the 5'-splice site by 4 nt (Fig. 3). In
the bidirectional splicing enhancer that we previously characterized in
the E1A pre-mRNA (23), only the Pu1 motif, distant by 10 nt from
the 12 S 5'-splice site, exhibits an enhancer activity for this splice
site. In contrast, the Pu2 motif, separated from this 5'-splice site by
only 1 nt, exhibits a slight repressor activity. Therefore, from both
model systems we have studied, we suggest that an upstream
cis enhancer element should not be in close contact with the
5'-splice site to be fully active but has to be separated by at least 4 nt from the exon/intron limit.
In contrast, the organization of the 9G8 exon 4, with purine motifs in
each half is more classic. It is reminiscent of what exists in the
cassette exon 5 of troponin C, which contains in the 5'-half a first
purine-rich motif important for the exon 5 recognition (37) and in the
3'-half a second purine motif, which has the capacity to dictate splice
site selectivity when two 5'-splice sites are in competition (44).
Several shorter purine motifs with enhancer activity are also
distributed in exon 2 of The presence of repressor elements or splicing silencer elements has
been reported in several pre-mRNA undergoing alternative splicing
(15, 47-51), with some of them recruiting SR proteins (15, 51). These
elements, which may be located in the vicinity of the enhancer
elements, may act in a coordinated network for the on/off splicing
regulation. In our case, several features reveal that the occurrence of
cis repressors represents only a very remote hypothesis.
First, the complete change of exon 4 by sequences of 9G8 exon 2 leads
to a complete absence of splicing (Fig. 2), which is better explained
by a removal of enhancer elements of the exon 4 rather than by a
removal of a combination of enhancers and repressors. Second, in
in vivo analysis (Fig. 8), the precise mutation of only one
enhancer element, A or B in exon 4 or the (GAA)3 motif in
exon 3, does not result in a significant inhibition of splicing, which
could be expected if active repressors are still present within the
pre-mRNA. Third, the analysis of the control pre-mRNA, in which
almost all the exon 4 and the (GAA)3 motif have been
removed but the 5'- and 3'-splice sites were made consensus (the cons.
5'-3'-E3-E4), shows a very efficient splicing (Figs. 4 and 7),
suggesting that no strong repressor cis activity remains in
exon 3 and intron 3 sequences.
Given their richness in G/A residues, the enhancers A and B of exon 4 were anticipated to be recognized primarily by ASF/SF2. Indeed, parts
of enhancer A have a good match (6 of 8 nt) with the octamer
AGAAGAAC consensus of Tacke and Manley (16) and with the
"functional" consensus (6 of 7 nt) determined by Liu et
al. (19). In the same way, parts of enhancer B are a good match
with the decamer consensus (16) (7 of 10 nt) or with the "functional" consensus (7 of 7 nt). The same feature was also anticipated for the (GAA)3 motif located in exon 3. Unexpectedly, we show that ASF/SF2 interacts only moderately with the
exon 4 enhancers (Figs. 5 and 6), although it recognized more
efficiently the (GAA)3 enhancer.
In contrast to ASF/SF2, 9G8 interacts efficiently with the three
enhancers (Figs. 5 and 6). Interaction of 9G8 with motif A could
be explained at least partly by the recognition of its 3'-part
(GAAGGCGAC), which is reminiscent of a repetition of GAC triplets as
well as interaction with motif B by the presence of a GAC triplet.
However, interaction of 9G8 with the (GAA)3 motif (Fig. 6),
although also resembling a (GAC)3 motif, is not fully explained on the basis of its known target consensus (17).
Interestingly, it has been shown also that 9G8 and ASF/SF2 both
activate splicing of the fibronectin EDI exon, through action of an
ESE, of sequence GAAGAAGAC (38). It is possible that 9G8 protein bears
a more extended capacity to interact with RNA targets containing
G/A-rich sequences or that 9G8 is helped by coactivators that could
allow its RNA binding capacities to be extended. Other lines of
evidence indicate that 9G8, through interactions with exon 3 and 4 enhancers, is the primary SR species involved in the intron 3 activation. First, titration experiments by RNA targets specific for
each SRp30 species demonstrate that the 9G8-specific target is the most
efficient in inhibiting intron 3 splicing (Fig. 4). Second, complementation experiments with individual SR proteins (Fig. 7) show
that only individual 9G8 protein promotes an effective splicing of
intron 3. Although SC35 interacts significantly with the exon 3/exon 4 enhancers, this SR protein is unable to activate intron 3 splicing if
added in complementation assays (Fig. 7), supporting the notion that
intron 3 splicing absolutely requires 9G8 to be activated in human
cells. Examination of exon 3 and 4 sequences in other vertebrates
species shows that the putative enhancers have very similar sequences
(data not shown). In particular, the exon 3 sequences upstream of the
5'-splice site, although encompassing a repetition of arginine residues
encoded by CGX (X is any residue) or AGPu
triplets may be arranged as repetitions of GAA or GAG triplets, arguing
that the activation mechanism for intron 3 splicing may be preserved in
all vertebrates.
A possible mechanism for controlling the expression of splicing factors
is a feedback autoregulation that may take place at the level of
alternative splicing. In the case of SRp20 in mammals, the
autoregulation might be negative, because its overexpression leads to
the inclusion of the cassette exon 4, which contains a premature stop
codon and precludes the synthesis of the classic SRp20 protein (31). In
the case of the 9G8 mRNA, the retention of intron 3 should also
preclude the synthesis of the entire protein (26). However, it appears
that the identified exonic enhancers, which respond positively only to
the 9G8 protein itself are not sufficient to promote an entire
autoregulation mechanism, because the presence of the highest amounts
of 9G8 protein should theoretically accentuate the splicing of intron 3 and cannot lead to a decrease of the 9G8 synthesis. In the conditions
of our in vitro analysis, we were also unable to detect any
antagonistic effects between 9G8 and other SR proteins (Fig. 7) such as
those demonstrated previously with ASF/SF2 and SC35 for the chicken
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
globin exon
2, specifically recognized by SC35 (24). However, as suggested recently
(25), it is likely that SR proteins may also be involved in alternative splicing regulation by interacting with multiple cis-acting
elements, which present only moderate affinity for one specific SR
species, exhibit cross-reactivity with several SR species, or require
some accessory factors to be active. Because a limited number of
alternative splicing regulation examples are well established, it is
important to analyze other models to get additional information.
EXPERIMENTAL PROCEDURES
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DISCUSSION
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--
Plasmids encoding the intron 3 pre-mRNA substrates are derived from an initial construct in which
a 1118-bp SphI/XmnI fragment of the human 9G8
gene containing the last 144 bp of exon 3, 875 bp of intron 3, exon 4, and the first 23 bp of intron 4 was cloned between the
SphI and HincII sites of pGEM 3Zf+ plasmid
(Promega). The exon 3-exon 4 (E3-E4) construct is derived from
the previous wild-type minigene by generating two deletions, internal
to the intron 3, which reduce its size to 469 bp. The first is a
SpeI/EcoRV deletion to remove 162 bp; the second
is created by an opening at the NcoI site, followed by a
digestion with the exonuclease Bal31, filling-in and
rejoining of the ends. In addition, an NheI site was created
in the middle of exon 4 to allow us to modify easily the exon 4 sequences.
A, or
B sequences (see Fig. 2 for the exact
sequences). The constructs mutated at the exon 3 (GAA)3 enhancer were generated by two strategies. In the first, we created short deletions by an S1 nuclease digestion at the level of a BspMI site occurring 8 bp upstream of the exon 3/intron 3 junction. A 5-bp deletion (construct
I) or a 6-bp deletion was
obtained (see Fig. 3A). In a second strategy, the
(GAA)3 sequence was substituted for a SpeI site
by PCR-based mutagenesis, creating the
II construct (see Fig.
3A). Short oligonucleotides were inserted in the
SpeI site to recreate one (GAA)3 motif (in the
II+(GAA)3 construct) or two (GAA)3 motifs in
the
II+(GAA)6 construct. Primer sequences and further
details are available on request.
II-E4 construct in which
we changed the 5'-splice site sequence AG|GTATTT to
AG|GTAAGT using a PCR approach with the appropriate
oligonucleotides. By the same method, we changed the 3'-splice site
sequence AACTTGAAAAATAG| to AACTTTTTTTATAG| for the cons.
5'3'-E3-E4 construct, and the exon 4 length was decreased by a
XbaI digestion before in vitro transcription to
obtain an enhancerless exon of 14 nt long. All in vivo
constructs were obtained by insertion of the
HindIII/EcoRI fragments from the above constructs
between the HindIII and EcoRI sites of the pXJ42
plasmid (32). All clones were verified by sequencing.
-32P]ATP-labeled transcript) were
performed in the same medium as above, with addition of 1 mM dithiothreitol, 0.1% Nonidet P-40, 30 ng/µl bovine
serum albumin, and 10 units of RNasin for 15 min at room temperature in
a 15-µl volume. The reaction mixtures were then exposed to UV light
(254 nm) for 10 min and subsequently treated with RNases (250 ng of
RNase A, 100 units of RNase T1), for 1 h at 37 °C. Aliquots of
the assays were then analyzed directly or immunopurified with
monoclonal antibodies directed against ASF/SF2 or 9G8 or polyclonal
antibody against SC35 as previously described (17). Each antibody
recognizes its corresponding SR protein specifically. After dilution of
the assays to a volume of 100 µl with IP buffer (50 mM
Tris-HCl, pH 7.5, 150 mM NaCl), the antibodies prebound to
protein G-Sepharose were added and incubated overnight at 4 °C.
After extensive washing, the proteins bound to the Sepharose were
eluted with the 2× SDS loading buffer, heated, and then resolved by
SDS-polyacrylamide gel electrophoresis on a 12% gel.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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Fig. 1.
Splicing pattern of 9G8 mRNA isoforms in
human and mouse tissues. A, schematic diagram of the
major isoforms of the 9G8 mRNA. Exons (from E1 to
E8) are designated by boxes and introns by
lines. Retained intron I3 is schematized by a
crosshatched box. The open reading frame of 9G8 (Met codon
in E1) is marked gray. B, alignment of
genomic sequences at the 5'- and 3'-ends of intron 3 for various
vertebrate species. The residues matching with the 5'-splice site
consensus are underlined, and the purine-rich sequences
lying at the position of the standard polypyrimidine tract at the
3'-end are underlined by a dotted bar.
C, Northern blot analysis of the expression of the 9G8 gene
in human tissues using a cDNA 9G8 probe (on the left
panel) or an intron 3 probe (on the right panel). The
approximate sizes of the various isoforms are indicated between both
panels. Note that the amount of brain RNA blotted on membrane is
presumably lower than that of other tissues and that the minor 2.0-kb
mRNA is detected with the 3'-half but not the 5'-half of intron 3, but has not been characterized further (26). D, Northern
blot analysis of the 9G8 expression in mouse tissues with the same
probes as defined in C, but specific to mouse.
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Fig. 2.
Purine-rich elements in exon 4 activate the
weak 3'-splice site of intron 3. A, sequences of the
natural and mutated exon 4 present in various constructs are given. The
A and B halves correspond to the wild-type sequence except a 2-nt
change to create an NheI site (UCUCGC to GCUAGC). A and
B sequences originate from the 9G8 exon 2 and in mPuA and mPuB
sequences, the purine-rich sequences have been specifically altered.
B, in vitro splicing of various transcripts
containing the indicated mutations. Only the lariat intron, which
accumulates as a doublet band after a 2-h incubation and gives a good
indication of the splicing efficiency, is shown. The bands marked by an
asterisk result from internal cleavages by nucleases
unrelated to splicing.
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Fig. 3.
The (GAA)3 motif in exon 3 is
required for efficient splicing of downstream intron 3. A, sequence of the natural and mutated transcripts in the
exon 3 region immediately upstream of the 5'-splice site. The purine
residues of the (GAA)3 motif are in boldface.
B, in vitro splicing of various transcripts
containing the indicated mutations, shown as in Fig. 2. The
asterisk indicates a cleavage product unrelated to
splicing.
A and
B pre-mRNA,
respectively. Compared with the initial E3-E4 RNA, we observed that
replacement of the A or B half by the
A or
B sequences (Fig.
2B, lanes 3 and 4, respectively)
results in a significant impairment of splicing. In addition,
concomitant replacement of both halves in the E3-E4 RNA by the
AB
sequences leads to a dramatic splicing inhibition (lane 5).
To determine whether the purine motifs in each half of exon 4 were
involved in the activation process, these motifs were replaced by
neutral sequences giving rise to the mPuA and mPuB constructs (see Fig.
2A). As shown above, the replacement of each purine-motif
induced a decrease of the splicing reaction (Fig. 2B,
lanes 6 and 7). An almost complete
splicing inhibition was observed after the replacement of both purine
sequences (in the mPuAB transcript, lane 8). Thus, our
results suggest that the purine sequences A and B of exon 4 are
directly involved and cooperate in promoting the intron 3 splicing,
most likely through an activation of the weak 3'-splice site.
13 and
5, see Fig. 3A), we noticed a (GAA)3
motif. In other alternative splicing models such motifs have been
demonstrated to activate weak 3'-splice sites upstream (36, 37). In the 9G8 pre-mRNA, this motif might serve as a cis enhancer
to activate the 3'-splice site of intron 2, when the intron 3 is not
recognized by the splicing machinery. However, because the 5'-splice
site of intron 3 is not optimal (Fig. 1B), it is possible
that the (GAA)3 motif is also involved in the activation of
the downstream 5'-splice site. To analyze this possibility, progressive
deletions and/or replacements of this motif were performed as well as
reinsertion of various sequences (see Fig. 3A). Typical
results are shown in Fig. 3B. The construction
I, in
which the first GAAGA residues of the (GAA)3 motif are
deleted and only a single mutation is present in the upstream GCCGGC
sequence (Fig. 3A), exhibits a strong inhibition of splicing
(Fig. 3B, lanes 3 and 2,
compare the accumulation of the lariat intron with the remaining
pre-mRNA). A similar construction (G10), which deletes one
additional C nt upstream of the (GAA)3 triplet but creates
a GAGAA sequence instead of a CAGAA in the previous construct, is
slightly less deleterious than
I (data not shown). Thus, the
(GAA)3 motif rather than immediately upstream sequence is
primarily involved in the intron 3 activation. The substrate
II in
which a ACUAGU replaced the (GAA)3 motif (Fig.
3A) showed poor splicing as expected (Fig. 3B,
lane 4). An insertion of an exogenous (GAA)3
sequence resulted in a significant but incomplete reactivation
(lane 5). In contrast, the insertion of two
(GAA)3 motifs allowed a strong splicing reactivation,
because the splicing efficiency was close to that of the initial E3-E4 RNA (compare lanes 1 and 6). Together
these results demonstrate that the (GAA) repeats are involved in the
activation of the 5'-splice site of intron 3.
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Fig. 4.
Competitor RNA containing one purine element
of exon 4 or SR protein-specific targets specifically inhibit splicing
of the E3-E4 pre-mRNA. 10 fmol of the pre-mRNA substrates
(E3-E4, lanes 1-11; cons. 5'-E3-E4,
lanes 12-19; and cons. 5'3'-E3-E4, used as a
control constitutive transcript, lanes 20-27)
were spliced for 2 h in the presence of the indicated RNA
competitors, at the -fold molar excess indicated above the
lanes. Lane 1, nonspliced pre-mRNA
(NS); lanes 2, 12, and 20,
splicing without RNA competitor. The A and B competitors correspond to
the exon 4 halves A and B, respectively, as shown in Fig. 2. Note that
the B competitor was used at half the molar excess as that of the A
competitor, and the 9G8-specific RNA competitor is used at half the
molar excess as those for ASF/SF2 and SC35. The asterisk
indicates a cleavage product common to the 3 pre-mRNA, unrelated to
splicing.
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Fig. 5.
The purine-rich elements of exons 3 and 4 specifically bind SR proteins. A, UV cross-linking
experiments. The uniformly labeled RNA probes indicated above
the lanes were incubated in the presence of nuclear
extract (NE), S100 cytoplasmic extract (S100), or
total SR preparation (SR), respectively, under splicing
conditions, then subjected to UV cross-linking and RNase treatment
before gel electrophoresis analysis. Cross-linked proteins
corresponding to SRp30 and SRp20 proteins are indicated. B,
UV cross-linking/immunopurification experiments. RNA probes indicated
above the panels were incubated in the presence of nuclear
extract and subjected to UV and RNase treatment as above. One aliquot
of each assay was analyzed directly as in A (lanes
1, 5, and 9). Other aliquots were
analyzed after immunopurification with specific antibodies against
ASF/SF2, SC35, or 9G8. The aliquots loaded in lanes
1, 5, and 9 were one-fourth the amount
used for the other lanes.
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Fig. 6.
Individual SR proteins cross-link to
purine-rich elements in reconstituted splicing assays. The A, B,
and mutant sequences from exon 4 and the 22-nt
(GAA)3-containing probe from exon 3 were uniformly labeled
and used as indicated above the lanes. They were incubated
in the presence of cytoplasmic S100 extract, supplemented or not by 100 ng of individual SR proteins, under splicing conditions. After UV
irradiation and RNase digestion, the samples were analyzed as in Fig.
5. A, the probes A, mPuA, and (GAA)3 are used as
indicated above the panels. B, the probes B,
mPuB, as well as specific targets for the individual SR proteins
ASF/SF2, SC35, and 9G8 (17) are used.
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Fig. 7.
9G8 specifically promotes splicing of 9G8
intron 3 in S100 complementation assays. The E3-E4 pre-mRNA
and the control pre-mRNA cons. 5'-3'-E3-E4 were subjected to
splicing in the presence of S100 extract, complemented or not by
individual SR proteins. 200 ng of each individual SR protein were
added. Lanes 1 and 10, nonspliced RNA
(NS); lanes 2 and 11,
splicing in the presence of nuclear extract (NE);
lanes 3 and 12, splicing in the
presence of S100 only; lanes 4-9 and
13-19, complementation by individual SR as indicated
above the lanes. At the bottom of the gel, only
regions surrounding the exon 3 and the mRNA products are
shown.
) mRNA isoform (lanes 1 and
10) that is expected for culture cells that efficiently
splice the intron 3 of the endogenous 9G8 gene. Mutations within the
half A result in a modest (mPuA, lane 2) or high level
(
A, lane 5) of splicing inhibition. In contrast to what
has been observed in vitro, mutation within the half B does
not result in a detectable effect (lanes 3 and 6). However, the concomitant mutations of both halves result
in a strong (mPuAB, lane 4) or in a complete inhibition
(
AB, lane 7) of intron 3 splicing, similarly to what was
observed in vitro (Fig. 2), indicating that the purine
motifs in both halves of exon 4 have an enhancer activity in
vivo and act together for the intron 3 splicing.
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Fig. 8.
The identified enhancers of exon 3 and 4 function in vivo to activate intron 3 splicing.
The E3-E4 and mutant templates used for in vitro studies
were transiently transfected in HeLa or NIH3T3 cells. RT-PCR assays
using specific primer for the transfected constructs were performed on
total RNA from transfected cells and analyzed by agarose gel (see
"Experimental Procedures" for details). Lanes 1 and
10, E3-E4 construct; lanes 2-9 and
11-15, analysis of the various mutants.
I corresponds to the
(GAA)3-altered construct as described in Fig. 3.
ImPuA or
ImPuB are in
addition mutated in the A or B enhancer of exon 4, respectively. The
amplified DNA products are 790 bp (+intron 3) and 320 bp
(
intron 3) in size, respectively.
I (see the sequence in Fig. 3) and
I mPuA constructs. In HeLa cells, the (GAA)3 mutation only weakly alters the splicing
of the transcripts (compare lanes 8 and
9 with lanes 1 and 2, respectively). As 3T3 cells are less efficient than HeLa cells in splicing transfected 9G8 constructs (Fig. 8; compare lanes 12 with
2 for the splicing of the mPuA construct), we have done the
same analysis with 3T3 cells (right panel, lanes
10-15). Interestingly, the mutation of the
(GAA)3 enhancer alone (lane 11) or in
conjunction with mPuA or mPuB (in lanes 13 and
15) significantly impairs the splicing of the initial
constructs (lanes 10, 12, and
14, respectively), indicating that the upstream enhancer has
a significant role in the activation of the 5'-splice site of intron 3 and in the alternative splicing of intron 3. Thus, the data of Fig. 8
demonstrate that the identified enhancers of exon 3 and 4 also exhibit
splicing activities in vivo and that they cooperate to lead
to an efficient splicing of intron 3.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
mRNA with
intron 3 (39); (ii) the efficient transport of nonspliced or
incompletely spliced RNA of human immunodeficiency virus requires the
involvement of the viral protein Rev, to relieve their nuclear
sequestration (Ref. 40 and references therein). In this respect, it is
not known whether the 9G8 mRNA isoform, including intron 3, which
accumulates significantly in certain tissues (Fig. 1), is
preferentially located in the cytoplasm or nucleus. In addition, the
exact status of this specific 9G8 mRNA isoform, which contains a
premature stop codon in the retained intron 3, relative to the mRNA
surveillance mechanism (41), is not clearly defined.
tropomyosin (43) or in exons 6 and 8 of
rat
tropomyosin (45). We have shown that the A enhancer, although
interacting less strongly than the B enhancer with SR proteins (Figs. 5
and 6) and being a weaker splicing competitor than B (Fig. 4), appears
to be more efficient than B within the E3-E4 pre-mRNA at promoting
splicing activation in vitro and in vivo (Figs. 2
and 8, respectively). This may be explained by the proximity of the A
enhancer relative to the weak 3'-splice site of intron 3, because it
has been demonstrated that the activity of splicing enhancers increases
when they approach the 3'-splice site (46). Regarding the ESE we have
identified in exon 3 and 4 for splicing of intron 3, it should be
stressed that they could also be involved in the splicing of intron 2 and intron 4 in vivo because the retention of 875 nt of
intron 3 creates an internal composite exon of 1125 nt with the
flanking exons 3 and 4. Thus, it is possible that the ESE are also
involved in the definition of this very large composite exon, by
reinforcing its flanking 3'- and 5'-splice sites and facilitating their
cross-talk.
tropomyosin exon 6A splicing (52). This raised the question of the
biological significance of the identified exonic enhancers, which could
be involved only in a general activation mechanism for the weak
5'- and 3'-splice sites of intron 3. However, as the specific features of intron 3 are conserved in all vertebrates, it is likely that the
alternative splicing of intron 3 constitutes only a part of a more
complex mechanism, which might regulate finely the expression of the
9G8 protein. Consistent with this hypothesis, we have revealed by
RT-PCR analysis the occurrence of an extended network of alternative reactions occurring inside the whole intron 3 in
vivo.2 By Northern blot
analysis, a minor internal splicing has also been suspected previously
(26). Such internal splicing reactions were not significantly detected
in our initial in vitro splicing assays of whole intron 3- containing pre-mRNA (data not shown) and could not be revealed
during the present study because intron 3 was partially deleted of
internal sequences. Finally, we cannot exclude that the splicing of
intron 3 represents also a target for another regulated cascades, as it
has been shown for the MKK3/6-p38 signaling pathway, which alters the
balance between splicing factors in the nucleus and induces alternative
splicing modulation in vivo (53). Our study, which has
identified the exonic splicing enhancers of 9G8 intron 3, suggests that
more detailed in vivo studies, using whole intron 3 constructs will be required for a broader understanding of the
alternative events and regulations occurring on the intron 3 region.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. R. Gattoni, J. Marie, C. F. Bourgeois, and P. Blader for fruitful discussions and critical reading of the manuscript. We are grateful to L. Kister and G. Hildwein for excellent technical assistance and the Institut de Génétique et de Biologie Moléculaire et Cellulaire core services for providing materials and technical help. We thank also Drs. P. Criqui-Filipe and S. M. Maira for cell culture advice.
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FOOTNOTES |
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* This work was supported by funds from INSERM, CNRS, the Hôpital Universitaire de Strasbourg, the Association pour la Recherche contre le Cancer, and a studentship from the Ministère de l'Enseignement Supérieur et de la Recherche (to F. L.).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.
Present address: UPR 41 CNRS, Faculté de Medecine, Avenue du
Pr L. Bernard, 35043 Rennes Cedex, France.
§ To whom correspondence should be addressed. Tel.: 33-3-88-65-33-61; Fax: 33-3-88-65-32-01; E-mail: stevenin@igbmc.u-strasbg.fr.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M009510200
2 F. Lejeune and J. Stevenin, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: ESE, exonic splicing enhancers; SR, serine/arginine-rich proteins; E1-E8, exons 1 through 8; Pu, purine; Py, pyrimidine; nt, nucleotide(s); kb, kilobase(s); bp, base pair(s); RT-PCR, reverse transcription-polymerase chain reaction; SELEX, systematic evolution of ligands by exponential enrichment; cons., consensus.
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