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Previous Article | Next Article 
Molecular and Cellular Biology, December 2003, p. 9327-9337, Vol. 23, No. 24
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.24.9327-9337.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Erika L. Lasda,1, Eric J. Wagner,1,3,
and Mariano A. Garcia-Blanco1,2*
Department of Molecular Genetics and Microbiology,1 Department of Medicine,2 Program in Molecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 277103
Received 20 May 2003/ Returned for modification 3 July 2003/ Accepted 10 September 2003
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ABSTRACT |
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 Top Abstract IntroductionMaterials and
METHODSResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and
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The mutually exclusive incorporation of exon IIIb or exon IIIc is regulated by the complex interplay of cis-acting elements in FGFR2 pre-mRNA and trans-acting factors, some of which appear to be cell type specific. To study the mechanism of regulation, we employed two cell lines derived from Dunning rat prostate tumors. The DT3 cell line is a well-differentiated carcinoma and expresses FGFR2(IIIb) exclusively, whereas the AT3 cell line is poorly differentiated and solely expresses FGFR2(IIIc) (51). Skipping of exon IIIb in AT3 cells is facilitated by the presence of weak splice sites flanking this exon, an exonic silencing sequence in exon IIIb, and two intronic silencing elements. The upstream intronic splicing silencer (UISS) and the downstream intronic splicing silencer (DISS), which resides within the intronic control element (ICE), flank exon IIIb (8, 13, 20, 50). The exonic silencing sequence functions to recruit hnRNP A1 to exon IIIb, thereby repressing its inclusion (16), while UISS and ICE antagonize exon IIIb definition by binding the polypyrimidine tract binding protein (PTB) and other factors yet to be characterized (8, 49, 50). The silencing of exon IIIb is countered in epithelial cells by the action of several cis-acting elements. Intronic activating sequence 1 (IAS1), which is located downstream of exon IIIb, serves as a binding site for the splicing factor TIA-1. The binding of TIA-1 to IAS1 has been demonstrated to activate the weak 5' splice site of exon IIIb as well as weak splice sites of other exons (15). IAS2 is located in the middle of the DISS within the ICE, while the intronic splicing activator and repressor (ISAR, also known as IAS3) is located over 700 nucleotides downstream of IAS2. Both of these cell-type-specific elements serve to activate the inclusion of exon IIIb (7, 13, 14). IAS2 and ISAR also function to repress exon IIIc inclusion in a cell-type-specific manner (7, 28, 50a). It is believed that IAS2 and a portion of ISAR, hereafter referred to as ISAR core, activate exon IIIb inclusion by creating a stem structure that disrupts the silencing activity of UISS and ICE (14, 28, 40, 49).
It has been shown that sequences within IAS2 and ISAR and their potential to form a stem have been highly conserved for over 600 million years (38), suggesting the importance of structure and perhaps sequence in the activation of exon IIIb inclusion and the repression of exon IIIc inclusion. Recently, it has been shown that similar stem structures are predicted to form in FGFR1 in several species (40). In addition, it has been shown that other sequences capable of stem formation can functionally substitute for IAS2 and ISAR (40).
In this study, we demonstrate that some, but not all, unrelated stem-forming sequences can functionally substitute for IAS2 and ISAR core. We also find that the 735 nucleotides separating IAS2 and ISAR core serve no function in the regulation of FGFR2 exon choice. Most importantly, we determine that the major function, if not the only function, of the IAS2-ISAR core stem in exon IIIb activation is to approximate intronic sequences upstream of IAS2 to sequences downstream of ISAR core. Splicing precursors where these sequences have been juxtaposed are capable of recapitulating epithelial cell-type-specific exon IIIb inclusion independent of IAS2 and ISAR core. Finally, we identified a GCAUG element downstream of ISAR core that is important for cell-type-specific exon IIIb inclusion and exon IIIc repression.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and
METHODSResultsDiscussionReferences |
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IAS2
(50a) with ClaI
and performing nondirectional annealed oligonucleotide cloning with the
following oligonucleotides: IAS2 Rep F, IAS2 Rep R, IAS2 Blue F, IAS2
Blue R, IAS2 PyPu F, and IAS2 PyPu R. To create pI12DE-Blue Blue,
pI12DE-Blue Blue(c), pI12DE-Blue(c) Blue, pI12DE-Blue(c) Blue(c),
pI12DE-PyPu, and pI12DE-PyPuBulge, a portion of the plasmids
containing substitutions in IAS2 were subcloned into pBluescript
(Stratagene) by using XbaI and XhoI. These plasmids
were then digested with NdeI and NsiI to remove ISAR,
and annealed oligonucleotide cloning was performed to replace ISAR core
within the wild-type ISAR sequence with the following
oligonucleotides: ISAR Blue F, ISAR Blue R, ISAR Blue(c) F, ISAR
Blue(c) R, ISAR PyPu F, ISAR PyPu R, ISAR PyPu Bulge F, and
ISAR PyPu Bulge R. The FGFR2 sequence cloned into pBluescript
was then subcloned back into pI12DE-WT digested with XbaI and
XhoI. The XbaI-XhoI fragment of pI12DE-WT
was subcloned into pBluescript (Stratagene) and then subjected to
QuikChange mutagenesis (Stratagene) to create the following minigenes:
pI12DE-U, pI12DE-G, pI12DE-+G,
pI12DE-Bulge, pI12DE-C5, pI12DE-C10, pI12DE-C15, pI12DE-C20,
pI12DE-C25, pI12DE-C30, pI12DE-C11, pI12DE-C13, pI12DE-C14, and
pI12DE-C16. To create pI12DE-LP, PCR amplification of
pI12DE-WT with the primer sets INT3BF2 with IAS2-cla-R and INT3CR
(7) with HinP1I-ISAR-F was
followed by digestion with SpeI/ClaI and
XhoI/HinP1I, respectively. The PCR products were
sequentially cloned into pI12 digested with XbaI and
ClaI and then with ClaI and XhoI.
pI12DE-STLP was cloned by PCR amplification of pI12DE-WT with
the primer sets INT3BF2 with del-stem-loop-R and del-stem-loop-F with
INT3CR (7), followed by
digestion with SpeI/ClaI and
BsmBI/XhoI, respectively. The PCR products were
sequentially cloned into pI12 digested with XbaI/ClaI
and then BsmBI/XhoI. pI12DE-STLP C15 was
cloned by PCR amplification of pI12DE-WT with INT3BF2
(7) and del-stem-loop-R
and PCR amplification of pI12DE-C15 with del-stem-loop-C15-F and INT3CR
(7), followed by digestion
with SpeI/ClaI and BsmBI/XhoI. The
PCR products were sequentially cloned into pI12 digested with
XbaI/ClaI and then with BsmBI/XhoI.
pI12DE-STLP C10-18 was cloned in the same manner as
pI12DE-STLP C15 except del-stem-loop-C10-18-F and INT3CR
(7) were used for PCR
amplification from pI12DE-C15. pI12DE-C10-18 was cloned by PCR
amplification of pI12DE-C15 with Intron2F and Core 10-18R, followed by
digestion with BsiWI and XbaI. The PCR product was
cloned into pI12DE-STLP C10-18 digested with XbaI and
BsiWI. All constructs utilizing PCR amplification were
sequenced for verification. All oligonucleotide sequences will be
provided upon request. Cell culture and transfection. AT3 and DT3 cells were maintained in Dulbecco's modified Eagle medium (low glucose) supplemented with 10% fetal bovine serum (HyClone). Stable transfections were performed as previously described (7), with one exception; after trypsinization, the cells were reseeded in 25-cm2 flasks containing 500 µg of Geneticin (Gibco)/ml for selection of stable cell populations.
RNA isolation and RT-PCR assay of transfected minigenes. Cellular RNA for reverse transcription (RT)-PCR and the Invader RNA assay was isolated by using the method of Chomczynski and Sacchi (10) or Trizol (Invitrogen). RT-PCRs using T7 and SP6 primers were performed as previously described (7). PCR products were either loaded directly onto 5% nondenaturing acrylamide gels or added to restriction digests with either AvaI or HincII (New England Biolabs). Analysis and quantification of PCR products from double-exon digests was performed as previously described (8). Analysis and quantification of PCR products from single-exon constructs (IIIb or IIIc) was performed in the following manner. Phosphorimager quantification of single-inclusion (U-IIIb-D or U-IIIc-D) and skipped bands (U-D) was performed with ImageQuant. A ratio of included to skipped products was calculated by dividing the moles of single inclusions by the sum of the moles of single inclusions and the moles of the skipped product.
Invader RNA assay. The Invader RNA assay (Third Wave Technologies, Madison, Wis.) was carried out essentially as described by Eis et al. (18) except that 1 µM probe was used for all probe sets and 0.25 µM Invader oligonucleotides were used for the IIIb-IIIc and U-IIIc probe sets as described in Wagner et al. (50a). To analyze all splice variants for double-exon minigenes, the Invader RNA assays were run in the biplex format by using the probe set combinations IIIb-D/U-D and IIIb-IIIc/U-IIIc as previously described (50a). To analyze splice variants for single-exon transfected minigenes, the Invader RNA assays were run in the biplex format by using the probe set combinations IIIb-D/U-D and IIIc-D/U-D. Different concentrations of each RNA splice variant were used to calculate a standard graph comparing attomoles of RNA to fluorescence. From the fluorescence readings, absolute levels of each splice variant were calculated (see the legends to Fig. 1 and 5).
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RESULTS |
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TopAbstractIntroductionMaterials and
METHODSResultsDiscussionReferences |
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The primary sequence of the proximal stem has been conserved from mammals to Strongylocentrotus purpuratus; of particular interest is the stretch of adenosines and uridines in IAS2 and ISAR, respectively (38). Also conserved is a predicted two-nucleotide bulge that was shown above to form in the rat stem structure. Although the presence of the bulge has been maintained throughout evolution, the nucleotide content of the bulge has varied, as has the primary sequence of the distal portion of the stem. These observations led us to question whether both the sequence of the proximal stem and the overall structure were critical for the proper regulation of FGFR2. In order to test the importance of sequence composition, we made several minigene constructs, as shown in Fig. 1B. Two of these minigenes [pI12DE-Blue Blue(c) and pI12DE-Blue(c) Blue] have the capacity to form a stem containing a two-nucleotide bulge, while the other two minigenes [pI12DE-Blue Blue and pI12DE-Blue(c) Blue(c)] cannot (Fig. 1B and C). These minigenes were transfected into DT3 and AT3 cells, and RNA from stably transfected cell populations was analyzed by RT-PCR or the Invader RNA assay (18, 50a). The transfections into AT3 cells were performed to control for any effect on the silencing of exon IIIb, which is mediated in part by silencers flanking IAS2. Alterations of IAS2 and ISAR were not expected to affect IIIc inclusion in AT3 cells, and indeed, we observed nearly exclusive use of exon IIIc with each minigene tested (data not shown). In DT3 cells transfected with pI12DE-Blue Blue(c) and pI12DE-Blue(c) Blue, we observed 80 and 76% exon IIIb inclusion, respectively (Fig. 1D). These heterologous stems allowed for even greater exon IIIb inclusion than did pI12DE-Rep (80 and 76% compared to 61%), which contains the authentic IAS2 and ISAR core sequences (Fig. 1C and D). The constructs that could not form a stem lost the ability to effectively include exon IIIb. To determine if the different stem structures were separately involved in activating exon IIIb and repressing exon IIIc, we made minigenes that contained only exon IIIb or exon IIIc as the internal exon (7). These single-exon constructs revealed that stem formation in epithelial cells activated exon IIIb inclusion and repressed exon IIIc inclusion (data not shown). These data and the recent report of Muh et al. (40) suggested that the replacement of IAS2 and ISAR core with presumably unrelated complementary sequences could elicit cell-type-specific regulation of exon IIIb inclusion.
We noticed, however, that all functional stems, including those reported in Muh et al. (40), contained stretches of purines in one strand and complementary pyrimidines on the other (Fig. 1C). Indeed, a stretch of five adenosines and two guanosines in IAS2 and a stretch of five uridines and two cytidines are highly conserved in vertebrates (38). To test whether a stretch of purines and a complementary track of pyrimidines were important, we generated pI12DE-PyPu, which contains alternating pyrimidines and purines in the stem structure (Fig. 1B and C). When this construct was transfected into AT3 cells, almost exclusive use of exon IIIc was observed (data not shown). When this construct was transfected into DT3 cells, pI12DE-PyPu included exon IIIb 40% of the time, which is lower than the 61% IIIb inclusion observed with pI12DE-Rep (Fig. 1D). This result suggested that the stretches of purines and pyrimidines, while not critical, may influence the proper regulation of FGFR2 alternative splicing (see below). Combining the results discussed above and the recent findings of Muh et al. (40), we concluded that many different stems can substitute for IAS2 and ISAR core; however, clearly not all stems regulate exon choice equally.
Deletion of the two-nucleotide bulge has no effect on FGFR2 splicing regulation. Bulges in RNA secondary structures tend to function by providing unique recognition sites for protein interactions, either by creating a specific RNA topography for protein binding in an otherwise helical structure or by kinking the RNA backbone, allowing access for protein recognition (24). In the case of the human immunodeficiency virus type 1 (HIV-1) trans-activation response element RNA stem structure, a tri-nucleotide bulge serves as a docking site for the binding of Tat. Mutating the bulge destroys the ability of the RNA to bind Tat (45, 46).
Given that
both the IAS2 and ISAR core in vitro structure probing and phylogenetic
data indicated the presence of a two-nucleotide bulge, we wanted to
determine if mutating the bulge would affect exon choice. To that end,
we created pI12DE-PyPuBulge, which contains the same stem as
pI12DE-PyPu with a deletion of the bulge (Fig.
1B and C). When this
minigene was transfected into AT3 cells, exon IIIc was included almost
exclusively (data not shown). Interestingly, when
pI12DE-PyPuBulge was transfected into DT3 cells, it gave
69% IIIb inclusion, which is higher than the 40 and 61%
IIIb inclusion observed with pI12 PyPu and pI12 Rep, respectively (Fig.
1D). This result
demonstrates that the presence of a two-nucleotide bulge is not
necessary for IIIb activation; indeed, the presence of this bulge
decreased exon IIIb inclusion in the context of the PyPu stem.
Moreover, given that pI12DE-PyPuBulge lacks any stretches of
pyrimidines and purines, these data argue against any role for the
conserved sequence composition in the proximal stem of the IAS2-ISAR
core structure in exon IIIb activation.
In order to analyze the
function of the GU bulge in the authentic stem formed between IAS2 and
ISAR core (Fig. 1A), we
created pI12DE-U, which contains a deletion of the U,
pI12DE-G, which contains a deletion of the G,
pI12DE-+G, which has an extra G in the bulge, and
pI12DE-Bulge, which has the bulge entirely deleted (Fig.
1E). These minigenes were
transfected into AT3 and DT3 cells, and RNA from stable cell
populations was pooled and analyzed by RT-PCR and the Invader RNA
assay. All of the minigenes that were transfected into AT3 cells
resulted in the nearly exclusive use of exon IIIc (data not shown).
None of the minigenes tested in DT3 cells resulted in a significant
decrease in exon IIIb inclusion (Fig.
1E). All the constructs
tested included exon IIIb in greater than 80% of all
single-inclusion splicing events. These results were confirmed with the
Invader RNA assay (data not shown), demonstrating that the bulge has no
apparent function in the proper regulation of exon
choice.
A six-nucleotide loop is
sufficient to mediate proper regulation of FGFR2 splicing.
The IAS2-ISAR core structure contains a
735-nucleotide loop. If loop sequences were to contribute to function,
deletion of the loop would be predicted to impact splicing regulation.
A previously reported loop deletion construct, which resulted in a
126-base pair loop, was shown to have no effect on FGFR2 exon choice
(7). Moreover, it was
recently shown that a deletion of the majority of the loop sequence
between IAS2 and ISAR core resulted in the proper epithelium-specific
inclusion of exon IIIb
(40). However, in this
study the minimal spacer region between IAS2 and ISAR was not
determined. To test the importance of these sequences, we created a
minigene with a six-nucleotide loop (Fig.
2A). The six-nucleotide loop minigene (pI12DE-LP) had a deletion of
the entire loop region between IAS2 and ISAR core with the exception of
two nucleotides downstream of IAS2, three nucleotides of unrelated
spacer sequence, and one nucleotide upstream of ISAR core. Transcripts
from this minigene included exon IIIc almost exclusively in AT3 cells
and included exon IIIb predominantly in DT3 cells (Fig.
2B). Remarkably,
pI12DE-LP was more tightly regulated than the wild-type
minigene, as exon IIIb was included in 92% of all single-exon
inclusion events in DT3 cells compared to 83% for pI12DE-WT, and
exon IIIc was included 98.5% of the time in AT3 cells compared
to 93% for pI12DE-WT (Fig.
2B). Analysis of all
possible splicing events in DT3 cells further suggested tight
regulation of pI12DE-LP, as double inclusion (transcripts that
include both IIIb and IIIc) decreased from 28% for pI12DE-WT to
18% for pI12DE-LP (data not shown). This suggests that
the decreased loop length allowed for greater repression of exon IIIc
in epithelial cells, perhaps because smaller loops may facilitate stem
formation. These results demonstrated that the loop was not essential
for proper splicing regulation in either cell
line.
|
STLP in
Fig.
3A). The deletion in STLP transcripts was designed to mimic the
approximation of sequences caused by the IAS2-ISAR core stem. RT-PCR
and the Invader RNA assay revealed that STLP transcripts
predominantly included exon IIIb in DT3 cells (70%) and, almost
exclusively, exon IIIc in AT3 cells (Fig.
3B and data not shown).
Cell-type-specific exon choice was achieved even though this construct
lacked IAS2, ISAR core, or any RNA stem. The fact that STLP
transcripts included IIIc predominantly in AT3 cells strongly suggests
that exon IIIb was not globally activated by intron shortening or by
unintended disruption of intronic silencer elements upstream of the
deletion. These results ruled out the first scenario, in which
double-stranded RNA binding trans-acting factors are required
for exon IIIb activation, and demonstrated that approximating sequences
upstream of IAS2 to sequences downstream of ISAR core was sufficient
for this activation.
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STLP (data not
shown; see Fig. 5C and
text below). These data suggest that although exon IIIb inclusion was
not significantly affected by the deletion of the stem-loop structure,
this structure was required for efficient exon IIIc repression in
epithelial cells. Sequences downstream of the ISAR core are important for exon IIIb inclusion in DT3 cells. Downstream of ISAR core we had observed high conservation of sequence from echinoderms to mammals (38). Understanding that strong sequence conservation within introns can be a good predictor of important cis elements, we performed a mutational analysis of the sequence downstream of ISAR core. Initially, we created six minigenes, which contained five-nucleotide substitutions sequentially positioned downstream of ISAR core (Fig. 4A). These minigenes were transfected into DT3 cells, and the isolated RNA from stable cell populations was analyzed by RT-PCR. The C5, C25, and C30 mutations (in nucleotides 1 to 5, 21 to 25, and 26 to 30 downstream of ISAR core) had no effect on exon IIIb inclusion compared to that for wild type, and the C10 and C20 mutations (in nucleotides 6 to 10 and 16 to 20) slightly decreased exon IIIb inclusion compared to that for wild type. When nucleotides 11 to 15 were mutated in the C15 construct, exon IIIb levels dropped from 85 to 51% (Fig. 4B), suggesting that these nucleotides play a key role in exon IIIb inclusion and IIIc repression in DT3 cells. To determine if these nucleotides were separately involved in activating exon IIIb and repressing exon IIIc, we created single-exon IIIb and single-exon IIIc minigenes and transfected them into the DT3 cell line. This analysis demonstrated that the primary effect of mutating these nucleotides is to lower IIIb inclusion from 60 to 36%; moreover, the C15 mutation also allowed a slight increase of from 21 to 32% in exon IIIc inclusion in DT3 cells (data not shown). These results suggest that an important cis element is located 11 to 15 nucleotides downstream of ISAR core and that its effect is more critical for exon IIIb inclusion than for exon IIIc repression. This region contains the sequence GCAUGCAUG, which has two overlapping GCAUG motifs, and the C15 mutation is the only one to destroy both GCAUG copies. These GCAUG motifs could be the regulatory elements downstream of ISAR core that are approximated by the IAS2-ISAR core RNA duplex.
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STLP C15 and pI12DE-STLP
C10-18, which contained the C15 and C10-18 mutations in minigenes
lacking the IAS2-ISAR stem-loop sequences (Fig.
5A).
The six
minigenes shown in Fig. 5A
were transfected into DT3 cells, and RNA isolated from stable
transfections was analyzed by RT-PCR (data not shown) and by the
Invader RNA assay (Fig. 5B
and C). As expected, the C15 and C10-18 mutations in the context of the
pI12DE-WT minigene led to a significant decrease in exon IIIb
inclusion. This decrease in exon IIIb inclusion was observed both by
measuring the percentage of single-inclusion transcripts that included
IIIb (100 x no. of U-IIIb-D transcripts/[no. of U-IIIb-D
transcripts + no. of U-IIIc-D transcripts]) (Fig.
5B) and by measuring the
percentage of all transcripts that included IIIb (100 x
[no. of U-IIIb-D transcripts + no. of U-IIIb-IIIc-D
transcripts]/[no. of U-D transcripts + no. of
U-IIIb-D transcripts + no. of U-IIIc-D transcripts +
no. of U-IIIb-IIIc-D transcripts]) (Fig.
5C). As noted above with
RT-PCR analysis, the mutations of the GCAUG element also led to
increased exon IIIc inclusion, suggesting that this element was
required for efficient repression of exon IIIc in DT3 cells. Analysis
of the deletion of IAS2, the loop region, and ISAR core (STLP)
by the Invader RNA assay (Fig.
5B and C) confirmed the
results of our RT-PCR analysis (Fig.
3). Specifically, the
overall inclusion of exon IIIb (the sum of the no. of U-IIIb-D and the
no. of U-IIIb-IIIc-D transcripts) was not affected in STLP
relative to that for WT (Fig.
5C), and all of the
decrease in IIIb single-inclusion transcripts observed in Fig.
5B could be accounted for
by an increase in double-inclusion transcripts. The data, however,
indicate that the STLP transcripts are not capable of
efficient exon IIIc repression. The C15 and C10-18 mutations in the
context of STLP transcripts led to precisely the expected
results: a decrease in exon IIIb inclusion due to the disruption of the
GCAUG element and a dramatic increase in overall exon IIIc inclusion
due to the combined losses of the GCAUG element and the IAS2-ISAR core
structure.
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DISCUSSION |
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TopAbstractIntroductionMaterials and
METHODSResultsDiscussionReferences |
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Bulge transcript (above), which
although fully functional does not have a tract of purines base paired
to a tract of pyrimidines, demonstrate that structure and not sequence
was important. Yet not all stem structures provided equal regulation.
Even though we cannot categorically explain these differences between
RNA structures, their relative stability, as calculated from the
predicted G (Fig.
1C), correlated well with
activation of exon IIIb inclusion (see discussion of the findings of
Brown et al., below). Thus, we conclude, in agreement with Muh et al.
(40), that base pairing
between IAS2 and ISAR core, rather than the unique sequences of these
elements, is critical for regulation of FGFR2 alternative
splicing.
Secondary structures have been proposed to act as
cis elements, and their action can be exerted in multiple
ways. RNA duplexes can be recognized, usually in a sequence-independent
fashion, by double-stranded RNA binding proteins
(19,
47). Specificity can be
provided by direct or indirect interactions with neighboring
cis-acting elements or single-stranded regions within the
structures (30). RNA
duplexes can also either occlude or favorably display other
cis-acting elements
(2,
3,
41). Finally, RNA
duplexes can approximate otherwise distant cis elements. In
this manuscript, we show that the major, if not the sole, function of
the IAS2-ISAR core structure in exon IIIb activation is to approximate
elements upstream of IAS2 to elements downstream of the ISAR core. To
properly mimic the propinquity mediated by the IAS2-ISAR core
structure, we deleted all the sequences from the 5' end of IAS2
to the 3' end of ISAR core, inclusively, in the plasmid of
STLP. STLP transcripts included exon IIIb
predominantly in DT3 cells and exon IIIc almost exclusively in AT3,
which indicated correct regulation in the absence of IAS2, ISAR core,
and the structure they form. Therefore, we conclude that the IAS2-ISAR
core structure works by bringing together other elements. Note that a
previous approximation that did not precisely mimic IAS2-ISAR core stem
formation did not promote exon IIIb inclusion in epithelial cells
(14). Approximation of
cis elements by RNA secondary structures has precedent in
pre-mRNA processing. The elaborate RexRE structure promotes
polyadenylation of human T-cell leukemia virus type 1transcripts by approximating two required elements, the canonical
AAUAAA hexanucleotide and the downstream G/U
element, which in the primary sequence lie hundreds of nucleotides away
(1). Brown et al.
(4) showed that many RNA
secondary structures can substitute for the RexRE to activate
polyadenylation in this context and suggested that the strength of the
base pairing interaction and the spatial distance between the
approximated elements were critical for activity. RNA duplexes have
also been shown to enhance splice site pairing in single intron
transcripts (9,
31,
42) and exon inclusion in
two intron transcripts in yeast
(25). In these cases, the
proximity of sequences presumably enhances the interactions between
splicing complexes assembled at the ends of introns. Here, we report a
case of duplex-mediated approximation of intronic control elements in
FGFR2 transcripts, a first for cell-type-specific regulatory
sequences.
Even though several questions remain about how approximation leads to exon IIIb activation in epithelial cells, parsimony suggests that the IAS2-ISAR core stem exists in both epithelium and mesenchyme, but only in the former are the approximated sequences recognized by epithelium-specific factors. The identity of the approximated sequences has not been unambiguously ascertained; however, data shown above and prior work suggest likely candidates. In this manuscript, we show that exon IIIb activation requires nucleotides 11 to 15 downstream of ISAR core. These nucleotides are found within the sequence (+10) GCAUGCAUG (+18), which contains two repeats of the previously characterized intronic activating sequence, GCAUG (5, 12, 23, 26, 29, 32, 39). This cis element has been shown to interact with several trans-acting factors and is believed to counteract the action of splicing repressors such as PTB (27, 33, 37). Thus, it is likely that the GCAUG-containing element is the relevant sequence downstream of ISAR core. Our previous work suggests that the pertinent sequence upstream of IAS2 is the intronic splicing silencer (ISS) immediately upstream of IAS2 (50). The juxtaposition of the GCAUG element to the ISS, which binds PTB, must be required to counteract the silencer and mediate inclusion of exon IIIb in epithelial cells.
Although our data indicate that
the cell-type-specific factor(s) that control(s) exon IIIb inclusion
need not interact with IAS2-ISAR core sequences, note that
approximation does not restore repression of exon IIIc in epithelial
cells. Careful quantification of exon IIIc (single-inclusion) and exons
IIIb and IIIc (double-inclusion) transcript levels by the Invader RNA
assay demonstrated reproducibly higher levels of exon IIIc inclusion in
STLP transcripts than in WT transcripts. While it is possible
that the topology of the STLP transcripts does not fully
reproduce the approximation created by the IAS2-ISAR core structure, it
is also conceivable that the IAS2-ISAR core structure plays a direct
role in repressing exon IIIc in epithelial cells. One could envision an
exon IIIc repressor that interacts with the IAS2-ISAR core and also
with adjacent single-stranded RNA cis elements. This model
might be similar to the interactions observed between the Sm protein
heterohexamer and multiple sites within U snRNAs
(35).
The tissue-specific use of exon IIIb appears to be regulated by the superimposition of three layers of control (Fig. 6). The first layer is determined by the weak splice sites bordering exon IIIb (e.g., the 5' splice site is GUAACA instead of the canonical GURAGU) and leads to poor exon definition. The second layer is provided by dual ISSs, upstream and downstream of exon IIIb, which mediate their action via PTB and other unidentified repressors. Whereas exon IIIb silencing is dominant in fibroblasts, all the required factors for silencing are also present in epithelial cells. In these cells, however, a cell-type-specific third layer that requires the IAS2-ISAR core structure activates exon IIIb. We propose that exon IIIb activation is carried out by approximation of the GCAUG element to the neighborhood of the ISS. Only in epithelial cells is this element active, very likely because of the differential expression of factors that recognize the GCAUG element. The coordinated repression of exon IIIc requires an overlapping but not identical set of elements and factors.
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ACKNOWLEDGMENTS |
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This research was supported by PHS grants to M.A.G.-B. (GM63090). E.J.W. and A.P.B. acknowledge the support of a Department of Defense predoctoral fellowship, and A.P.B. also acknowledges the support of a PHS training grant.
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FOOTNOTES |
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A.P.B. and E.L.L. contributed equally to this paper.
Present address: Program in Molecular Biology and Biotechnology, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599.
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