From the International Centre for Genetic Engineering and
Biotechnology, Padriciano 99, Trieste 34012, Italy and the
Laboratorio de Fisiología y Biología
Molecular, Departamento de Fisiología, Biología
Molecular y Celular, Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, Ciudad Universitaria, Pabellón
II (C1428EHA) Buenos Aires, Argentina
Received for publication, September 20, 2002, and in revised form, November 4, 2002
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ABSTRACT |
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Using hybrid minigene experiments, we have
investigated the role of the promoter architecture on the regulation of
two alternative spliced exons, cystic fibrosis transmembrane regulator
(CFTR) exon 9 and fibronectin extra domain-A (EDB). A specific
alternative splicing pattern corresponded to each analyzed promoter.
Promoter-dependent sensitivity to cotransfected regulatory
splicing factor SF2/ASF was observed only for the CFTR exon 9, whereas
that of the EDB was refractory to promoter-mediated regulation.
Deletion in the CFTR minigene of the downstream intronic
splicing silencer element binding SF2/ASF abolished the specific
promoter-mediated response to this splicing factor. A systematic
analysis of the regulatory cis-acting elements showed that
in the presence of suboptimal splice sites or by deletion of exonic
enhancer elements the promoter-dependent sensitivity to
splicing factor-mediated inhibition was lost. However, the basal
regulatory effect of each promoter was preserved. The complex
relationships between the promoter-dependent sensitivity to
SF2 modulated by the exon 9 definition suggest a kinetic model of
promoter-dependent alternative splicing regulation that
possibly involves differential RNA polymerase II elongation.
In most eukaryotic cells, transcription and pre-mRNA
processing (capping, splicing, and cleavage/polyadenylation) are
coordinately regulated within the nucleus both in a temporal and
spatial fashion (for review see Refs. 1-3). The phosphorylated
C-terminal domain (CTD)1 of
RNA polymerase II (pol II) provides key molecular contacts with these
mRNA processing reactions throughout transcriptional elongation and
termination. For example, transcripts originating from polymerases
without a CTD (T7 polymerase and pol III) cannot be spliced or
polyadenylated and, indeed, expression of a form of pol II lacking the
CTD does not abrogate transcriptional activity but actually depresses
pre-mRNA processing (4). The CTD can physically interact with
several pre-mRNA processing factors (4-8), including SR proteins
(9), and with transcriptional factors that may have a dual role in
transcription and splicing regulation.
The connection between transcription and splicing might have important
functional implications in vivo, because the
promoter-dependent recruitment of regulatory splicing
factor and/or changes in RNA pol II elongation and/or its
phosphorylation state may influence the subsequent splice site
selection (10). EDA transcript can be modified by changes in the
promoter region of the gene, using a transient expression system
combined with a promoter swapping processing of the alternative
splicing fibronectin (11). It has been clearly shown that this effect
is not because of the different mRNA levels produced by each
promoter but is related to qualitative properties of the promoters. In
addition, the sensitivity to overexpressed SR proteins, which induce
EDA exon inclusion, depends on the promoter, thus suggesting that the
promoter can modulate regulatory splicing factor action through the
participation of the CTD (12). An alternative but not exclusive
mechanism suggests that promoters may control alternative splicing via
the regulation of pol II elongation or processivity (13, 14). In this
case the splice site selection would be critically related to the
timing of presentation of the splicing regulatory elements contained in
the nascent transcripts. The differential behavior of the Alternative splicing of the cystic fibrosis transmembrane regulator
(CFTR) exon 9, in contrast with fibronectin EDA, is inhibited by SR
proteins, thus representing an interesting model for the study of the
contribution of the promoter architecture on alternative splicing. The
exon 9, at an early stage of synthesis of the CFTR transcript, emerges
first from the elongating polymerase followed by the downstream
intronic splicing silencer (ISS) element in intron 9. CFTR exon 9 recognition is modulated by exonic regulatory elements and splicing
signals at the intron-exon boundaries. At the 3'-end of intron 8 the
presence of a variable number of dinucleotide TG (from 9 to 13)
followed by a T repeat (T5, -7, or -9) generates polymorphic variants
in the population. A high number of TG repeats and a low number
of T tracts induces exon skipping (15-17). Serine arginine-rich proteins (SR proteins) and, in particular the splicing factor 2 (SF2/ASF), interact with the ISS and negatively regulate CFTR exon 9 splicing (18).
In this study we have used the CFTR exon 9 and the FN EDB alternative
splicing as models to further analyze the interplay between the
promoter architecture and alternative splicing. FN EDB was considered
because it shares similar but not identical structure with the
previously studied FN EDA exon (11). We have analyzed the sensitivity
of promoters to overexpressed SF2/ASF, the role of splice site
strength, and the splicing regulatory elements in the mediation of the
promoter effect on the alternative splicing. Our results indicate that
the promoter has no effect on the FN EDB but can affect CFTR exon 9 alternative splicing. This CFTR exon 9-specific effect is mediated by
the presence of the ISS silencer elements binding SR proteins. The exon
recognition mediated by the splice sites and by the ESE modulates the
promoter-dependent sensitivity to SF2/ASF, suggesting a
kinetic role of transcription elongation in splicing.
Construction of Hybrid Minigenes--
The 1508-bp
PstI-PstI fragment of the
The CF, U2, and H4 promoters were then digested with
SacI/MluI, XbaI/BssHII, and
XbaI/AflIII, respectively, and cloned in the
BssHII site in the first exon of the Analysis of the Hybrid Minigene Expression--
We transfected
Hep3B cells with the DOTAP reagent with 3 µg of each reported
minigene and with the control empty vector pCG (0.5 µg) (20) or with
different amounts of the splicing factors SF2/ASF codifying plasmid.
RNA extraction was performed after 48 h, and the RNA was digested
with Dnase-Rnase free. RT-PCR was done as described (18) with one of
the two direct primers, Exon-specific Alternative Splicing Regulation by the Promoter
Architecture--
Differences in pol II promoter structure have been
found to modify the processing of the fibronectin EDA alternative
spliced transcript (11, 13). To evaluate the specificity of this
phenomenon we have analyzed the effect of several promoters on two
alternative spliced exons, the FN EDB and the CFTR exon 9. The FN EDB
exon is homologous to the EDA, is positively regulated by several SR proteins (21, 22), and contains enhancer elements both in the exon
(ESE) and in the intron (ISE) (21-23). On the other hand, CFTR exon 9 inclusion is modulated by multiple and well characterized exonic and
intronic cis-acting elements (18, 24) and is inhibited by SR
proteins (18, 25). EDB and CFTR exon 9 hybrid minigene constructs were
prepared with the transcription driven by
These amplification experiments with the primers Role of Promoters from Intronless Genes in Pre-mRNA Splicing
Processing--
Previous studies have shown that some promoters are
not competent regarding pre-mRNA processing. This occurs not only
for bacterial T7 RNA polymerase but also for eukaryotic RNA pol IIII promoters. In addition, the expression of a form of pol II with truncation at the CTD strongly affects pre-mRNA processing (4). We
have addressed the question of whether the promoters transcribing intronless genes can be considered functionally competent regarding splicing. If the promoter-mediated regulation of splicing is related to
differential loading of splicing factors to the polymerase, we can
suppose that there is no reason for intronless promoters to load such
regulatory factors. Two pol II promoters that form intronless genes U2
and H4 were cloned in the CFTR exon 9 minigenes. Their effect on
alternative splicing was analyzed with the primers located in exon 1 and at the FN/exon 3 junction of the The Intronic Splicing Silencer Element Modulates the
Promoter-mediated Response to SF2/ASF in CFTR Exon 9 Alternative Splicing--
SR proteins can bind directly to specific
RNA sequences and to other splicing factors. In addition they can
associate, via the CTD, with the transcription machinery. These
interactions could mediate promoter regulation of alternative splicing.
In the case of the EDA exon, alternative splicing was shown to be critically dependent on the interaction of SF2/ASF with an ESE that
induces exon inclusion (12). With regard to the CFTR exon 9, SF2/ASF
requires intact splicing silencer elements (ISS and ESS) to induce exon
exclusion, the ISS being the main target of SR-protein interaction
(18). We have analyzed the promoter-mediated response to SF2/ASF in
relation to changes at these splicing silencer elements. For this
purpose three representative promoters ( CFTR Exon 9 Sensitivity to SF2/ASF Depends on Promoter
Architecture--
We have analyzed the kinetics of the response to
SF2/ASF in the CFTR exon 9 and in the EDB exon in order to investigate
the role of SR proteins in relation to the promoter-mediated regulation of splicing. Constructs containing three representative promoters, Promoter-dependent Sensitivity to SF2/ASF Is
Modulated by 5'- and 3'-Splice Site Recognition Efficiency and by
Exonic Splicing Enhancer--
The promoter architecture may
differentially modulate the pol II processivity, which may as a result
affect the interactions between the ISS and the SR proteins. The first
element found by the elongating polymerase is the exon 9, which is
followed by the ISS in intron 9. To modulate the recognition of CFTR
exon 9 we have prepared minigene constructs with nucleotide
substitutions at the splice sites or with the ESE deleted. We
have previously shown that variations at or near the splice sites
increase the proportion of CFTR transcripts without exon 9. This occurs
by increasing the number of TG tracts or by reducing the T repeats at
the 3'-end of intron 8 or with an G to A mutation at the 5'-splice site
that introduces the use of a weak cryptic splice site in the exon (26).
Recognition of exon 9 was reduced in the TG13-T3 variant, in the G
In the absence of SF2/ASF, the percentage of inclusion of exon 9 in
mature mRNA was higher for the FNMut promoter, intermediate for the
CF, and poor for the In this study we have analyzed the role of the promoter
architecture on the regulation of alternative splicing of two exons, the fibronectin EDB and the CFTR exon 9. We show that the promoter structure can modify the splicing pattern of the CFTR exon 9 but not
that of the EDB exon. The "structural unit" of the CFTR responsible for this selective promoter effect includes not only the exon 9 itself,
together with its regulatory elements, but also the flanking introns.
The CFTR exon 9 represents a particular case of non-evolutionary
conserved alternative splicing where the recognition of the exon is
weakened by the SF2/ASF interaction at the downstream intronic ISS
element (18). In the first instance, we have analyzed the effect of
disrupting the ISS element under several promoter contexts with or
without overexpression of SF2/ASF. Mutation of the ISS abolished the
splicing inhibition induced by SF2/ASF regardless of the kind of
promoter (Fig. 4). This indicates that an intact ISS is necessary for
the promoter modulation.
We subsequently analyzed the sensitivity to SF2/ASF in the presence of
an intact ISS element in relation to the promoter and to the
composition of the exonic regulatory elements. Our results show that
sensitivity to overexpressed SF2/ASF on the splicing pattern varies
with the promoter structure. The highest splicing inhibition was
observed for the Our results provide additional evidence to clarify the mechanisms that
could be involved in the coupling of transcription and alternative
splicing. Previous studies originally suggested that the promoter can
modulate alternative splicing by differentially recruiting splicing
factors to an exonic enhancer element (11, 12). In fact, in the EDA
exon sensitivity to the cotransfected SR proteins was critically
dependent on the promoter driving transcription; the Our results reinforce an alternative mechanism of splice site selection
mediated by the promoter that takes into account the different timing
of presentation of the critical regulatory elements to the elongating
polymerase. Experiments in which RNA pol II pause sites affect
alternative splicing have suggested a kinetic link between
transcription and splicing. This has been shown by delaying the
transcription of an essential splicing inhibitory element required for
regulation of tropomyosin exon 3 (28). At the early stages of the
synthesis of the CFTR transcript, the exon 9 emerges first from the
elongating pol II followed by the downstream ISS. Therefore the
alternative splicing decision will involve an initial definition of the
CFTR exon 9 by splice sites and ESE recognition followed by subsequent
silencing mediated by the intronic ISS. Its role could be imagined as
that of a "decoy exon" presenting strong binding sites for the SR
protein SF2/ASF. A highly processive polymerase would favor the
simultaneous presentation of the exon 9 and the ISS, resulting in exon
exclusion. If the pol II elongation is slower, the assembly of exon 9 spliceosomal complex will be favored because it does not have to
compete with the ISS complex that will be later transcribed. In
addition, the promoter influences the sensitivity to cotransfected
SF2/ASF only when the emerging exon is better defined (Fig.
7D). All this is consistent with the fact reported earlier
that the Mutations reducing the exon definition of CFTR exon 9 (such as the T5
allele at the end of intron 8) are frequently found in patients with
monosymptomatic forms of cystic fibrosis (16). The resulting
pathological aberrant exon skipping presents tissue-specific and
individual phenotypic variability. It has been previously suggested
that this variability results from the differential abundance of
cell-specific trans-acting factors (18, 24). The discovery
of the promoter modulation of alternative splicing suggests that
factors regulating splicing could be acting through promoters and
influence not only physiological alternative splicing but also
pathological splicing, thus explaining the phenotypic variability
frequently found in splicing pathology. It is expected that the
differential use of transcriptional factors at the promoter affecting
the pol II processivity might result in variable expression of the
pathological splicing processes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-globin
promoter compared with the FN promoter on EDA alternative splicing has
recently been found to correlate with different transcriptional
processivity (13). The higher processive transcription elongation
mediated by the
-globin promoter resulted in excess of the EDA
exclusion, whereas the FN promoter gave the reverse effect.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-globin minigene in
pBS KS containing the promoter region of the
1-globin along with the
entire three exons of the gene was used for the preparation of
the hybrid minigenes. The
1-globin minigene contains a unique BstEII site in the third exon that was used to clone the
previously reported BstEII-BstEII cassette
containing different versions of the CFTR exon 9 embedded in the
fibronectin EDB genomic sequences (18). The sequences of the
fibronectin and CMV promoters used have been previously described; the
mutant version of the fibronectin promoter contains disruptive point
mutations at the
170 cAMP-response (CRE) and
150 CCAAT regulatory
elements (11, 19). The 900-bp SspI and
HindIII fragments containing the FNWT, FNMut, and CMV promoters until the second exon of the
1-globin were
obtained from the original pSVEDA plasmids (11) and subcloned in the corresponding sites of the
1-globin minigene in pBS KS, generating the FNWT
, FNMut
, and CMV
plasmids. The promoter sequences of CF (1450 bp), of snRNA U2 (603 bp), and of H4 (865 bp) were amplified from genomic DNA, cloned in pBS KS, and entirely sequenced. The oligonucleotides used were: H4Dir,
5'-gatctagagtacacgggaggatgtgca-3'; H4Rev,
5'-ctcacgcgtctctgccggacatgaccgctg-3'; U2 Dir,
5'-gatctagaccttcggcttccctgactgg-3'; U2 Rev,
5'-gtgatgcgcgcagatactacacttgatcttagc-3'; CfproDir,
5'-gagagctcgccttagatgtgtcggcaa-3'; Cfprorev,
5'-ctctacgcgtagctcggttggccacct-3'.
1-globin minigene,
generating the CF
, U2
, and H4
plasmids. Different
variants of the CFTR exon 9 with different numbers of TGmTn repeats,
with or without the ISS elements, with the G to A mutation at the
5'-splice site or with the ESE deletion were obtained as a
BstEII cassette from the previously described constructs
(18). The BstEII cassette was cloned in the unique
BstEII site located in the third exon of the
1-globin of
the FNWT
, FNMut
, CMV
, CF
, U2
, and H4
plasmids to
generate the hybrid minigenes shown in Fig. 1.
1, 5'-cgcacgctggcgagtatggtg-3' or
2,
5'-caacttcaagctcctaagccactgc-3', and with the reverse primer B2,
5'-taggatccggtcaccaggaagttggttaaatca-3'. Amplified products were
routinely loaded on 1.7% agarose-EtBr gels. For quantitation of the
PCR reactions, [
-32]dCTP was included in the PCR
reaction mixture, and the products were loaded on 6% native
polyacrilamide gel, dried, and exposed to a Cyclone
(PerkinElmer). The counts of each splicing band were corrected
by the number of C/G present in the PCR product sequence. In
dose-response experiments splicing inhibitions were calculated as
percentages on the basis of the difference between the basal value
corresponding to transfection with the control pCG plasmid and those
obtained in the presence of different amounts of SF2/ASF.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-globin, FNWT, FNMut,
CMV, and CF promoters (Fig. 1). These
minigenes were transfected in Hep3B cells, and the pattern of splicing
was analyzed by RT-PCR with the primers
2 and B2, which can detect
the inclusion and exclusion of the corresponding alternative spliced
exons. Transfection experiments with the CFTR exon 9 showed that the percentage of exon 9 inclusion was dependent on the promoter that drives transcription (Fig.
2A). Inclusion of exon 9 in
mature mRNA was predominant with the FNMut promoter (about 90% of
exon 9 included) and poor with the
1-globin promoter (about 65% of exon 9 included), whereas intermediate splicing patterns were detected
for the other constructs. On the contrary, transfection experiments
with the FN EDB minigenes showed a percentage of EDB exon inclusion of
about 10% that was not affected by the promoter driving the
transcription (Fig. 2B).
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Fig. 1.
Schematic representation of the CFTR exon 9 (A) and EDB (B) hybrid minigenes used
in transfection experiments. -globin, fibronectin EDB, and
human CFTR exons are indicated in black, shaded,
and white boxes, respectively. The transcription
of the hybrid minigenes is driven by the indicated promoters. The
location of the polymorphic TGmTn variants at the 3'-end of CFTR intron
8 and the exonic (ESS and ESE) and
intronic (ISS) regulatory elements is indicated. The 5'-SS G
A substitution disrupts the conserved GT 5'-splice site, creating a
nearby weak cryptic splice site in the exon 9 ag/gcaagt (indicated by
an asterisk) (26). The boxed region is the
intronic splicing enhancer (ISE) in the EDB intron. Primers
1,
2, and B2 used in RT-PCR analysis are shown as
superimposed arrows.
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Fig. 2.
Effect of the promoters on CFTR exon 9 alternative splicing. Hep3B cells were transfected with 3 µg of
the indicated minigenes, and RNA splicing variants were detected by
RT-PCR and analyzed on 1.7% agarose gel. The amplified transcripts
with and without exon 9 or the EDB exon resulting from amplification
with different primers are indicated as black,
shaded and white boxes that correspond
to -globin, fibronectin EDB, and CFTR exon 9, respectively. The
numbers below each lane show the quantification of the
percentage of exon 9 inclusion; data represent the mean of four
independent experiments done in duplicate. The RNA splicing variants,
detected by radioactive PCR, were resolved on 6% native polyacrilamide
gels and quantitated using a Cyclone. M is the molecular mass
marker 1 Kb. A, RT-PCR amplification of the indicated
CFTR exon 9 minigenes with primers
2 and B2. B,
transfection experiments with the indicated EDB minigenes. The RNA
splicing variants were detected by RT-PCR with primers
1 and
B2. C, transfection experiments of CFTR exon 9 minigenes analyzed with primers
2 and B2.
2 and B2 were
specifically aimed only at detection of the splicing of the alternative
spliced exon. To determine whether the effect of the promoters
is specific for the alternative spliced exon we analyzed full-length
transcripts originating from the pre-mRNA splicing of the entire
hybrid minigene. These transcripts, including the two constitutively
spliced introns from the
1-globin gene, were detected with primers
located in exon 1 and at the FN/exon 3 junction of the
1-globin
gene. Fig. 2C shows that the two full-length transcripts
with or without the exons were correctly processed. Once again the
percentage of exon 9 inclusion was differentially affected by the
promoters, whereas no effect was evident for the EDB exon (data not
shown). The different behaviors of the FN EDB and CFTR exon 9 indicate
that specific exonic elements, and possibly context determinants, are
important in mediating the effect of the promoter in the recognition of
alternative spliced exons. Peculiar features of the alternatively
spliced CFTR exon 9 may be involved in the
promoter-dependent regulation.
1-globin gene, which detect the
entire length of the transcript. RT-PCR analysis showed that the U2 and
H4 promoters have a normal capacity to splice introns from pre-mRNA
transcripts (Fig. 3). The amount of
transcript with the inclusion of the exon 9 was similar to the CF
promoter. This data indicates that the promoters driving intronless
genes contain all the elements required for intron processing.
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Fig. 3.
Effect of promoters from intronless genes on
splicing processivity. The indicated minigene variants were
transfected in Hep3B cells and analyzed by RT-PCR with 1 and B2
primers. The numbers below each lane represent the
quantification of the percentage of exon 9 inclusion and are the mean
of three independent experiments done in duplicate.
-globin, CF, and FNMut) were
selected and cloned upstream of two CFTR silencer variants, one
consisting of deletion of the ISS element in intron 9 and the other
represented by a single exonic nucleotide substitution (see Fig.
1A). This latter adenine to cytosine substitution in position 146 of the exon (A146
C) occurs in the previously reported ESS element (Ref. 18 and data not shown). The CFTR minigene constructs
were cotransfected with the SF2/ASF plasmid and the resulting splicing
pattern analyzed by RT-PCR amplification. Fig. 4, lanes 3 and 5 shows that the deletion of both ISS and ESS together and of the ISS
alone resulted in a practically complete exon 9 inclusion from mature
mRNA, regardless of the promoter driving transcription. In the same
CFTR silencer variants, no significant changes in the percentage of
exon inclusion were observed after overexpression of SF2/ASF (Fig. 4,
lanes 4 and 6). These results indicate that the
promoter effect on CFTR exon 9 alternative splicing requires the
interaction of SF2/ASF at the ISS element.
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Fig. 4.
Effect of the deletion of splicing silencer
elements on the splicing inhibition mediated by SF2/ASF. Hybrid
minigene variants with different promoters contained deletion of the
intronic splicing silencer elements (ISS) and/or mutation
146A C in the exon. This mutation affects the exonic splicing
silencer. Minigenes were cotransfected in Hep3B cells with 500 ng of
the empty vector (pCG, odd bars) or with the same amount of
SF2/ASF plasmid (even bars). RT-PCR analysis was done with
the primers
2 and B2 and the resulting products analyzed on 1.7%
agarose gel (upper panel). In the lower panel the
histograms display the percent of exon 9 inclusion detected by
radioactive PCR and quantified using a Cyclone representing the
mean ± S.D. of at least two independent experiments done in
duplicate. WT plasmid, lanes 1 and 2; minigenes
with deletion of the ISS and with A146
C mutation,
lanes 3 and 4; minigenes with the deletion of the
ISS, lanes 5 and 6.
-globin, CF, and FNMut, driving transcription of either the CFTR exon 9 or EDB, were cotransfected with increasing amounts of the SF2/ASF plasmid and the resulting splicing pattern analyzed by RT-PCR
amplification. In the CFTR exon 9, SF2/ASF induces exon skipping (18).
However, the dose-response curves obtained with an increased amount of
SF2/ASF showed that the splicing inhibition was strictly dependent on
the promoter driving transcription (Fig. 5A). Each promoter showed a
characteristic dose-response curve. The strongest response was observed
for the
-globin promoter, whereas the FNMut showed a small
inhibitory effect mediated by the SR protein. The CF promoter showed an
intermediate splicing inhibition. In addition, we have also analyzed
the response of the two intronless promoters, U2 and H4. These two
promoters responded equally well to the SF2/ASF-mediated inhibition,
further indicating the fully competent capacity of these promoters in
splicing processivity (data not shown). On the other hand,
cotransfection of the EDB minigene constructs with SF2/ASF showed an
increase in the EDB+ variant as previously reported (21, 22) (Fig.
5B). However, and in contrast with the CFTR exon 9 system,
quantitative analysis of the percentage of the EDB inclusion induced by
the splicing factors indicates that the magnitude of the response to
SF2/ASF was independent from the promoter driving transcription (Fig. 5B). To exclude the possibility that the ISE element in the
EDB intron +1 (which is absent in the CFTR exon 9 minigenes) could be
responsible for the promoter-dependent regulation of CFTR
exon 9, we have analyzed EDB minigenes without this
cis-acting element. As shown in Fig. 5C, the
deletion of the ISE element did not affect sensitivity to the splicing
factor, thus indicating that the ISE element is not responsible for the
different promoter-mediated processing of the CFTR exon 9 splicing.
Altogether, these results point to an important role exerted by
multiple cis-acting elements located in the CFTR
exon 9 and in the nearby flanking introns in mediating the
promoter-specific effect on alternative splicing.
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Fig. 5.
Dose-response curves of exon 9 and EDB
splicing induced by SF2/ASF as a function of the promoter. Hep3B
cells were transfected with 3 µg of the indicated minigenes and with
500 ng of the empty vector (pCG) or with increasing
amounts of SF2/ASF plasmid (50, 100, 250, and 500 ng). RNA splicing
variants were detected by RT-PCR with primers 2 and B2 and analyzed
on 1.7% agarose gel. The percentage of exon inclusion is indicated at
the bottom of each lane and represents the mean of at least
three independent experiments done in duplicate. The graph
shows the splicing inhibition induced by SF2/ASF for each promoter.
Splicing inhibitions were calculated as percentages on the basis of the
difference between the basal value corresponding to transfection with
the control pCG plasmid and those obtained in the presence of different
amounts of SF2/ASF.
A mutation at the 5'-SS, and in the ESE deletion and was increased in
the T7 variant. Minigene constructs with three promoters (
-globin,
CF, and FNMut) were cotransfected alone or with increasing amounts of
the SF2/ASF coding plasmid to analyze the basal effect of the promoter
and its specific sensitivity to the splicing factor, respectively.
1-globin promoter (Fig.
6). This pattern was independent from the
variations introduced at the splice sites or at the ESE, indicating
that the basal differences of the promoter are independent from the
exon definition. On the contrary, the dose-response study to
cotransfected SF2/ASF showed a different sensitivity according not only
to the promoter but also to the changes in exon recognition. In
contrast to the TG11T5 minigenes analyzed in Fig. 5, the sensitivity to
SF2/ASF for the two splice sites variants and for the ESE deletion was
not differentially affected by the promoters (Fig.
7, A-C). In these cases the
minigenes responded with similar dose-response curves to the increasing amount of the splicing factors, indicating that in the presence of weak
splice sites or when the ESE is deleted the promoter-mediated sensitivity to the splicing factor SF2/ASF is lost. On the contrary, a
change in the length on the polymorphic polypyrimidinic tract from T5
to T7 gave a different sensitivity to SF2/ASF, according to the
promoters (Fig. 7D). These results indicate that exon
definition does not affect the basal promoter-specific alternative
splicing pattern but the specific sensitivity of the promoter to
SF2/ASF.
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Fig. 6.
Effect of the promoters on CFTR exon 9 minigenes with variations at the splice sites and ESE. The
indicated hybrid minigenes with different promoters ( -globin, FNMut,
and CF) and with weak 5'-splice site (5'-SS G
A) or changes at the 3'-splice site (GT13T3 and
GT11T7) or deletion at the ESE (
ESE) were transfected in
Hep3B cells and analyzed with
2 and B2 primers. The numbers
below each lane are the percentage of exon 9 inclusion and
represent the mean of at least two independent experiments done in
duplicate.
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Fig. 7.
Dose-response curves of exon 9 splicing
induced by SF2/ASF as a function of the promoter and splice site
variations and ESS deletion. Hybrid minigenes transcribed with
different promoters containing mutation at the 5'-splice site
(5'-SS G A, panel
A) or variations at the TGmTn repeats (GT13T3 and
GT11T7, panel B and D, respectively)
or deletion of the ESE (
ESE, panel C)
were transfected in Hep3B cells with the control plasmid pCG (500 ng)
or with increasing amounts of SF2/ASF coding plasmid (50, 100, 250, and
500 ng, respectively). The splicing pattern was then analyzed by RT-PCR
with
2 and B2 primers and resolved on 1.7% agarose gels. The
percentage of exon 9 inclusion, shown at the bottom of each
lane, is the mean of three independent experiments and was calculated
as indicated in the legend of Fig 5. The dose-response curves at the
right of each panel show the corresponding splicing
inhibition induced by SF2/ASF.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-globin promoter and the lowest for the fibronectin
promoter. This promoter-dependent sensitivity to SF2/ASF
depends, however, on the strength of the splice sites and on the
presence of an intact ESE, two critical cis-acting elements
involved in exon definition. According to the exon definition model,
pairing between the splice sites across an exon, facilitated by the
presence of exonic enhancer elements, forms the basic unit of initial
recognition by the splicing machinery (27). This model may be
particularly important in those genes with large introns. In the
presence of weak splice sites or in the case of the deletion of the
ESE, the promoters did not differentially affect sensitivity to the
splicing factor (Fig. 7, A-C). This strongly suggests that
the effect of the promoter on the CFTR exon 9 splicing depends both on
the definition of the exon and on the presence of an intact ISS
element. In addition, the promoters maintain a different splicing
pattern on the basal level of CFTR inclusion in all the different
variants affecting the exon definition (Fig. 6).
-globin
promoter did not respond to SF2/ASF and the FN promoter responded,
strongly inducing exon inclusion. However, in the CFTR exon 9 the
-globin promoter showed a higher sensitivity to SF2/ASF than the FN
promoter. This indicates that the promoter itself is not responsible
for recruiting splicing factors to the site of transcription and cannot
mediate a different loading capacity of SR proteins to critical
cis-acting elements (Fig. 5). This is also consistent with
the efficient splicing processivity of promoters from intronless U2 and
H4 genes (Fig. 3).
-globin promoter has a higher processivity than the FN
promoter and that this promoter causes EDA exon exclusion (13, 29).
However, in contrast to the EDA, the sensitivity to SF2/ASF in the CFTR
exon 9 is higher than that of the promoter with a higher pol II
processivity, i.e. the
-globin. This difference can be
explained by the different targeting of the splicing factor. In fact,
in the EDA SF2/ASF directly participates in the exon definition
targeting in the exon itself. On the contrary, in CFTR exon 9 this
splicing factor, interacting with the ISS element located in the
downstream intron, causes exon skipping. In this dynamic and
cotranscriptionally splicing decision a higher processivity of the pol
II determined by the promoter is associated with a higher sensitivity
to SF2/SF2 in the CFTR exon 9 because of the downstream location of the
ISS.
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ACKNOWLEDGEMENTS |
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We thank Davide Marconi for help in preparation of the promoters, Hector Torres for suggestions regarding intronless genes, and Ann Crum for proofreading the manuscript.
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FOOTNOTES |
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* This work was supported by Telethon-Italy Grant GGP02453 and by a grant from the Associasione Italiana Ricerca Cancro (AIRC).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.
§ A Howard Hughes Medical Institute International Research Scholar and a career investigator of the Consejo Nacional de Investigaciones Científicas y Técnicas of Argentina.
¶ To whom correspondence should be addressed. Tel: 39-040-3757337; Fax: 39-040-3757361; E-mail: baralle@icgeb.org.
Published, JBC Papers in Press, November 5, 2002, DOI 10.1074/jbc.M209676200
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ABBREVIATIONS |
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The abbreviations used are: CTD, C-terminal domain; CFTR, cystic fibrosis transmembrane regulator; FN EDB, fibronectin extra domain-B; FN EDA, FN extra domain-A; ISS, intronic splicing silencer; ESS, exonic splicing silencer; ESE, exonic splicing enhancer; ISE, intronic splicing enhancer; pol II, polymerase II; SR proteins, serine arginine-rich proteins; SF2/ASF, splicing factor 2; WT, wild type; RT, reverse transcriptase; CMV, cytomegalovirus.
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