From the Departments of Medicine (Cardiology) and
¶ Physiology and Biophysics, Case Western Reserve University
School of Medicine, Cleveland, Ohio 44106-4958
Received for publication, August 5, 2002, and in revised form, December 16, 2002
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ABSTRACT |
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Isoforms of the smooth muscle myosin phosphatase
targeting subunit 1 (MYPT1) are generated by cassette-type alternative
splicing of exons. Tissue-specific expression of these isoforms is
thought to determine smooth muscle-relaxant properties and unique
responses to signaling pathways. We used mini-gene deletion/mutation
constructs to identify cis regulators of splicing of the
chicken MYPT1 central alternative exon. Comparisons of alternative exon
splicing were made between smooth muscle cells of the fast-phasic
contractile phenotype (gizzard), in which the central alternative exon
is skipped, and slow tonic contractile phenotype (aorta), in which the
alternative exon is included. We demonstrate that splicing of the
alternative exon requires a cis-enhancer complex in the vicinity of the alternative exon 5'-splice site. This complex consists
of two UCUU motifs in an intronic U-rich sequence (putative PTB
(polypyrimidine tract binding) or T cell inhibitor of
apoptosis-1 binding sites), an intronic 67-nucleotide enhancer
that has similarities with the cardiac Troponin T MSE3 enhancer, and a
potentially novel exonic splicing enhancer. The exonic enhancer
contains the palindromic sequence UCCUACAUCCU present in many other
transcripts where alternative splicing of exons occurs, suggesting that
it may be more broadly active. The exonic enhancer is adjacent to a
potentially novel exonic silencer element that contains a 13-nucleotide
imperfect palindromic sequence. This silencer, in conjunction with a
distal intronic silencer, is proposed to mediate the silencing of
splicing of the MYPT1 central alternative exon in the fast phasic
smooth muscle phenotype.
Smooth muscle tissues show considerable phenotypic and functional
diversity. Much of the phenotypic diversity in smooth muscle tissues is
generated by the alternative splicing of exons from single gene
transcripts. As many as 50% of vertebrate genes are estimated to have
alternative splicing of exons as a mechanism by which multiple protein
isoforms with different or modified functions are generated from a
single gene (1-3). The process of splice site selection and exon
definition for exons that are constitutively spliced has been well
described (4-7). Binding of U1 small nuclear
ribonucleoprotein particle
(snRNP)1 to the consensus
5'-splice site sequence and binding of splicing factor 1 (SF1) and U2
snRNP auxiliary factor (U2AF) to the consensus 3'-splice site sequences
(branchpoint and polypyrimidine tract, respectively) commits the exon
to the splicing pathway (7, 8). A number of factors are known to
contribute to the "weakening" of the splicing reaction so that
splicing of an exon may be regulated. These factors include exon and
intron size, RNA secondary structure, and the extent to which the
5'-splice site, branchpoint, and polypyrimidine tract sequences match
the consensus sequences for U1 snRNP, SF1, and U2AF binding,
respectively (9-11).
A number of cis-elements that regulate the splicing of
alternative exons have been identified (reviewed in Refs. 10, 12-14). We are interested in defining the cis-regulatory elements
that account for the great variety in exon splicing that generates diversity in smooth muscle. This aspect of smooth muscle phenotypic diversity has not been examined. We have selected the MYPT1 subunit of
smooth muscle myosin phosphatase as a model gene to address this
question for several reasons: 1) MYPT1 isoforms are thought to
determine smooth muscle relaxant properties and tissue-specific relaxant responses to signaling pathways (15, 16). 2) Isoforms of MYPT1
are generated by cassette-type alternative splicing of single exons in
the central and 3'-end of the pre-mRNA. Splicing of each exon is
tissue-specific and developmentally regulated (15, 17). Around the time
of hatching (ED21), the MYPT1 isoforms in the chicken gizzard (phasic
fast contracting smooth muscle) completely switch from central exon-in
and 3'-exon-out to central exon-out and 3'-exon-in. In contrast, the
slow tonic contracting aorta smooth muscle throughout development
expresses MYPT1 isoforms in which the central alternative exon is
included, and the 3' alternative exon is skipped (15, 17).
We hypothesized that cis-acting silencer(s) may mediate the
silencing of splicing of the MYPT1 central alternative exon that occurs
in the phasic gizzard tissue in vivo around the time of hatching. We have previously shown that smooth muscle phenotypic diversity is rapidly lost under routine cell culture conditions, in
which phasic smooth muscle cells (SMCs) revert to the more embryonic
tonic phenotype and display tonic/slow contractile properties in
vitro (17, 23). We therefore developed a novel in vivo (whole animal) plasmid delivery method to identify
cis-elements that regulate splicing of the MYPT1 central
alternative exon and may be important in the generation of smooth
muscle phenotypic diversity.
Data Base Search to Identify Candidate Regulatory
Sequences--
Several strategies were used to identify candidate
splicing regulatory sequences. First, the intron sequences
(GenBankTM accession numbers AF110177 and AF110176) were
compared with sequences in the NCBI data base (BLAST search) to
identify sequences that are also present in introns from other genes.
Second, the intron sequences were scanned with sequences known to
regulate the splicing of other pre-mRNAs using the GeneQuest
program (DNASTAR, Madison, WI). Third, the alternative exon sequence
was searched for matches to previously characterized ESE sequences,
including optimal binding sites for the SR proteins SC35, SF2/ASF,
SRp40, and SRp55, as determined by functional systematic
evolution of ligands by exponential
enrichment (SELEX) experiments (18, 19). Finally, the corresponding
chicken and rat MYPT1 sequences were compared with each other to
identify sequence identities and dissimilarities between them within
the alternative exon and flanking introns. This approach identified a
number of regions/sequences in the alternative exon and downstream
intron that were potentially significant (described under
"Results") and facilitated targeted deletion and mutation analyses
to determine their functional significance.
Construction of Wild-type and Mutant MYPT1 Mini-genes--
A
5.4-kb genomic fragment containing the chicken MYPT1 central
alternative exon and flanking introns and exons (Fig. 2A)
was generated by PCR using the Expand Long Template PCR system (Roche Diagnostics). Genomic DNA from chicken liver was amplified using a sense oligonucleotide at +1576 (5'-AAAGGATCCAGACTTGCCTATGTTGCACC-3') and an antisense oligonucleotide at +1925
(5'-CACGAGCTCAAGTTGAACCCGTTGTACTC-3') of the chicken MYPT1 M133
cDNA sequence (20). BamHI and SacI restriction enzyme recognition sequences were placed at the 5'-ends of
the oligonucleotides. The genomic fragment was cloned into the pCR2.1
vector using the Original TA cloning kit (Invitrogen). The exon and
splice site sequences in the cloned genomic fragment were confirmed by
DNA sequencing to match the sequence as previously determined (17). The
entire MYPT1 genomic fragment was removed from the pCR2.1 vector by
restriction digestion at BstXI sites in the pCR2.1 vector
and cloned into the BstXI site of pRc/RSV, a eukaryotic
expression vector (Invitrogen), upstream of the bovine growth hormone
polyadenylation signal (Fig. 2B).
Large deletions in the introns were engineered in several steps. First,
the alternative exon was deleted along with 2522 and 2169 nucleotides
of the upstream and downstream introns, respectively (termed MCS
vector). This vector was generated using the Expand Long
Template PCR system (Roche Diagnostics) and sense
(5'-GGTCACCATATGCCGCGGGCCCCAGAAAGATTCAG-3') and antisense
(5'-CGGGTCCGGATCGATGGCCGGCCACAAAACAATGTG-3')
oligonucleotides that created a linker region with the following
unique restriction sites at the site of the deletion: FseI,
ClaI, BspEI, PshAI, BstEII, NdeI, SacII, EcoO109I, and
ApaI. Next, a Tsp45I fragment containing the
alternative exon surrounded by 267 and 602 nucleotides of the upstream
and downstream introns, respectively, was cloned into the
BstEII site of the MCS vector, generating the
A chimeric vector was engineered by an exact replacement of chicken
MYPT1 alternative exon with the corresponding exon from the rat MYPT1
gene. This vector was generated using the Expand Long Template
PCR system. Briefly, two oligonucleotides
(5'-CTGTAAATAAGATTTTGTTTTAGC-3' and 5'-GTGTGGGATTTTCTCTTGTCTTTT-3'),
which hybridize immediately upstream and downstream of the chicken
alternative exon, were used to PCR-amplify the entire chicken MYPT1
expression vector, minus the chicken alternative exon. The rat MYPT1
exon was PCR-amplified using two phosphorylated oligonucleotides that
begin at the first nucleotide of the corresponding rat MYPT1 exon
(5'P-AGAGTCTTCAAATTTGCGAACA-3') and at the last nucleotide of the rat
exon (5'P-CTTCTATGGTAAGTAGATCCTTCA-3'). These two PCR-amplified
fragments were ligated as blunt-ended fragments to generate a chimeric
vector containing a precise replacement of the chicken alternative exon
sequence with the rat exon sequence. The identity of this chimeric
vector was confirmed by DNA sequencing.
All of the remaining deletions and mutations in the MYPT1 mini-gene
(shown in Figs. 4-7) were generated using the Expand Long Template PCR
system with specific oligonucleotides to set the boundaries of the
deletion. All of the deletions in the An in Vivo System of Gene Delivery--
An in vivo
plasmid DNA delivery system to smooth muscle was implemented by
adapting methods previously described in striated muscles (22). We
optimized expression conditions from an injected plasmid DNA by
performing DNA dose-response curves and time course experiments with a
vector containing luciferase as a reporter gene. 1-50 µg of plasmid
DNA containing either the SV40 early promoter driving transcription of
the Sea Panzy (Renilla reniformis) luciferase gene or the
RSV promoter driving transcription of the Firefly (Photinus)
luciferase gene were injected. White leghorn chickens (Gallus
gallus, 1-2 days old, hatched from eggs obtained from Squire
Valleevue Farm, Cleveland, OH) were anesthetized with inhaled
Halothane (Halocarbon Laboratories) and, after depilation, a
longitudinal midline abdominal incision was made to expose the gizzard.
Each plasmid in a volume of 20-30 µl of 1× phosphate-buffered saline, 0.015% Evan's Blue dye was injected into each of the two lobes of the gizzard smooth muscle using a 50-µl Hamilton syringe. The skin was then sutured and the bird awakened. The birds were sacrificed by decapitation 1-7 days later, and the gizzard smooth muscle was isolated by dissection and homogenized in 0.75-1.75 ml of
1× passive lysis buffer from the Dual-Luciferase Reporter Assay system
(Promega). After the lysates were centrifuged, the supernatants were
assayed for either Renilla or Firefly luciferase activity as
described by the manufacturer, using either a single sample luminometer
or a TriLux scintillation counter.
In separate experiments, chicken gizzards were injected with 10 µg of
the plasmid pDsRed1-N1 (Clontech), which expresses
the red fluorescent protein under the control of the cytomegalovirus promoter. Additional chicken gizzards were injected with pUC plasmid as
controls. One day later, the gizzards were harvested, frozen in
OCT, and cryosectioned. Sections were examined by fluorescence microscopy on the rhodamine channel. Control and experimental samples
were photographed with identical exposures using a Spot RT digital
camera. Sections were then formalin-fixed, permeabilized, and stained
with phalloidin-FITC (Sigma) as per the manufacturer's recommendations. Phalloidin specifically binds to filamentous actin and
thus identifies muscle cells in the section. The same approximate
region that was photographed on the red channel was rephotographed on
the fluorescein channel to identify the phalloidin labeled smooth
muscle cells.
The MYPT1 mini-gene expression vectors were injected into gizzard
tissue as described above, with the exception that chicks were 1-2
weeks old and 25 µg of plasmid DNA was injected in 50 µl of 1×
phosphate-buffered saline, 0.15-0.3% Evan's Blue dye. One day later
the gizzards were removed and the blue-stained portions of the smooth
muscle tissue were dissected. The dissected tissues were immediately
frozen in liquid nitrogen and processed for total RNA isolation using
either Ultraspec II (Biotecx Laboratories, Inc.) or the Absolutely RNA
Miniprep kit (Stratagene).
Tissue Culture and Transfections--
Smooth muscle cells were
isolated from gizzards and aorta of embryonic chicks as previously
described (17). Briefly, gizzard or aorta tissue was removed from
ED14-15 embryos, minced into fine fragments, and digested with
repeated rounds of collagenase treatment. Pooled individual cells were
cultured on tissue culture plastic with a 50:50 mixture of Dulbecco's
modified Eagle's medium and F-12 media in the presence of 10% serum
(heat-inactivated fetal bovine serum, HyClone Laboratories). The cells
were passaged when sub-confluent with a 1:4 split. Smooth muscle cells
were used for transfection after 1-3 passages. These cultures have been previously characterized and shown to be greater than 90% smooth
muscle cells as determined by staining for RT-PCR of RNA--
The injected and transfected RNA samples were
analyzed by RT-PCR for the presence of exon-included and exon-skipped
MYPT1 mini-gene transcripts. The sense oligonucleotide
(5'-GGCCAGTACCTCTGACATTGATGA-3') was complementary to chicken MYPT1
cDNA at +1620 (20) and the antisense oligonucleotide
(5'-GCTGGCAACTAGAAGGCACAGTCG-3') was complementary to the bovine
growth hormone polyadenylation signal present in the pRc/RSV expression
vector at position +745 (see Fig. 2B). Thus, this PCR primer
pair did not detect the endogenous MYPT1 mRNA (data not
shown). Visualization of the RT-PCR products was accomplished by
5'-end-labeling the sense PCR oligonucleotide with a Cy5 fluorescent
label (IDT and Research Genetics). RT reactions were performed with 0.5 and 5-10 µg of input total RNA from transfected and injected RNA
samples, respectively. RNA was reverse-transcribed using the antisense
primer and AMV Reverse Transcriptase (AMV RT) enzyme (Roche
Diagnostics) in a volume of 50 µl. The PCR reactions were performed
with Taq DNA Polymerase (Roche Molecular Biochemicals) or
Taq Bead Hot Start Polymerase (Promega) in a volume of 50 µl. The reactions consisted of 23-27 (transfected) and 30-32
(injected) cycles of 95 °C for 45 s, 60 °C for 1 min, and
72 °C for 1 min, with a final extension step at 72 °C for 7 min.
RT-PCR products were separated on 6% native polyacrylamide gels. The
fluorescence from the Cy5 label was detected using a Storm 860 Imager
(Amersham Biosciences). The exon-included and exon-skipped MYPT1 RT-PCR products were quantified using Amersham Biosciences ImageQuaNT software.
Several controls were performed to verify the specificity and accuracy
of the RT-PCR reactions. RT-PCR was performed on RNA from cells that
were injected or transfected with a pRc/RSV vector containing no
insert. Also, to verify that the RT-PCR products were
RNA-dependent, RT-PCR reactions were performed omitting the AMV RT. To verify that the RT-PCR reactions were quantitative, PCR
reactions were performed using two different amounts, 0.5 and 2 µl,
of input cDNA from each RT reaction. Each pair of PCR reactions was
judged by the following two criteria: 1) that there would be
approximately a 4-fold increase in total signal resulting from the
4-fold difference in input cDNA to verify that the PCR reaction was
not saturated and 2) that the ratio of exon inclusion to exon skipping
would not change significantly between the two PCR reactions. This
control was performed with all of the RNA samples analyzed in this
study. Finally, to verify that the injection procedure did not
adversely affect the splicing machinery, RT-PCR was performed on a
number of injected RNA samples to detect the endogenous MYPT1
transcript. These controls revealed that splicing of endogenous
MYPT1 was not affected by the injection procedure (n = 6).
Statistics--
Quantification of exon-included and exon-skipped
MYPT1 RT-PCR products was obtained as described above. Because the
hypothesis to be tested was that cis-elements mediate
tissue-specific silencing of the splicing of the alternative exon in
phasic smooth muscle, all data are presented as the percentage of
exon-skipped/total MYPT1 mRNA. The values shown in this study
represent the mean ± S.D. from at least three independent
injections or transfections of each mini-gene construct. Data groups
were compared with Student's t test or one-way analysis of
variance followed by a Bonferroni t test for comparison of
pairs of data points as appropriate using Prism version 3.02 (GraphPad). p < 0.05 was considered significant.
An in Vivo System to Identify cis Regulators of Alternative
Splicing--
We developed an in vivo (whole animal)
mini-gene delivery system modeled after those used in transient gene
delivery to skeletal and cardiac muscle (22), taking advantage of the
unique property of the gizzard as a solid smooth muscle organ, which
could be directly injected with recombinant plasmid DNA. To optimize
the experimental system, a plasmid containing the highly sensitive luciferase reporter driven by the viral RSV promoter was used. The
naked plasmid DNA was directly injected into the gizzard of 1- to
7-day-old chicks as described under "Experimental Procedures." Dose-response and time course data were generated (data not shown). The
highest level of luciferase activity was observed 1 day after injection
of 10 µg of plasmid DNA, and the activity decreased steadily between
days 2 and 7. Injection of a plasmid with a red fluorescent protein
(RFP) reporter demonstrated abundant expression of RFP in smooth muscle
fibers in the region of injection (Fig. 1A). The smooth muscle fibers
are identified by the presence of filamentous actin bound by
phalloidin-FITC (Fig. 1B). No red fluorescence was observed
in regions remote from the site of injection (not shown), nor in
control gizzards injected with pUC plasmid (Fig. 1C).
Dose-response experiments demonstrated a linear relationship between
amount of injected plasmid DNA (from 1 to 50 µg) and luciferase activity (data not shown). Thus, transcripts arising from 25 µg of
plasmid DNA injected into the gizzard were analyzed 1 day after injection in all subsequent experiments.
Pre-mRNA Splicing from an MYPT1 Mini-gene Construct Is
Regulated--
We tested an MYPT1 mini-gene expression cassette that
contained the chicken central alternative exon and flanking introns and
exons (Fig. 2, A and
B) in phasic and tonic SMCs and in a non-muscle (liver
carcinoma) cell line. In the phasic SMCs (injected gizzard), 27 ± 4% of the spliced products generated from the MYPT1 mini-gene
construct lacked the alternative exon (Fig. 2C, lane 1). In contrast, in tonic SMCs (cultured gizzard SMCs), as well as
in non-muscle cells (LMH cells), 11 ± 2% of transcripts
generated from the MYPT1 mini-gene construct lacked the alternative
exon (lanes 2 and 3). Cultured gizzard and aortic
SMCs gave identical results for all mutant constructs that were tested
in both cell types (data not shown), thus only the results with
cultured gizzard SMCs are reported here. This 2.5-fold greater
exclusion of the alternative exon in the phasic compared with tonic
SMCs is statistically significant (p < 0.001) and
suggests that some of the genetic information necessary for the
silencing of the MYPT1 central alternative exon is present within the
mini-gene construct.
Role of the Introns in the Regulated Splicing of the Central
Alternative Exon--
To test the role of the introns in the regulated
splicing of the MYPT1 central alternative exon, ~2.25 and 1.6 kb of
the upstream and downstream introns were deleted, respectively (Fig.
3A). The deletions were
designed so as not to alter the splice sites and immediately adjacent
sequences. Removal of these large regions from the adjacent introns
resulted in virtually complete loss of exon skipping of the MYPT1
mini-gene mRNA in gizzard cells in vivo (phasic), in
cultured gizzard cells (tonic), and in cultured non-muscle LMH cells
(construct Identification of a Distal Intronic Silencer of Splicing--
A
series of overlapping deletions of the downstream intron were generated
to locate potential silencer sequences (Fig.
4). Deletion of the proximal 660 nucleotides ( Identification of a Proximal Intronic Enhancer of
Splicing--
Deletion of the proximal 660 nucleotides ( A Second Potent Enhancer of Alternative Exon Inclusion Is Present
in the Intron--
Immediately adjacent to the 5'-splice site of the
MYPT1 alternative exon is a 19-nucleotide pyrimidine-rich sequence
containing two UCUU motifs (Fig.
6A), which is a putative TIA-1
(28, 29) or PTB (30) binding site. This sequence was not included in the original large intron deletions due to our desire to avoid changing
sequences that were near the splice sites. Deletion of the
19-nucleotide pyrimidine-rich sequence caused a dramatic shift toward
exon skipping in phasic, tonic, and non-muscle cells (86, 81, and 84%
exon skipping, respectively; Fig. 6B, lane 2).
Mutation of both UCUU motifs to CCCC, which would be predicted to
abolish binding of both PTB and TIA-1, caused an equivalent shift
toward exon skipping in phasic, tonic, and non-muscle cells (92, 84, and 83%; Fig. 6B, lane 3), suggesting that the
UCUU sequences were required for inclusion of the alternative exon in
all cell types. Interestingly, if only one of the two UCUU motifs sites was mutated (Cmut2, lane 4), the shift to exon skipping was
close to that of the mutation of both UCUU motifs in the phasic SMCs (77% versus 92%; Fig. 6A, compare lane
4 with 3) but not in tonic SMCs and non-muscle cells
where endogenous exon inclusion normally predominates (34-39%
versus 83-84%; Fig. 6B, compare lane
4 with 3). To distinguish between the possibilities
that the specific UCUU motifs were required versus a
requirement for U-rich sequence, all three Cs in this
polypyrimidine stretch were mutated to Us (Umut1 + 2, lane
5). This mutation had no significant effect on exon splicing in
the phasic SMCs and caused a small shift toward exon skipping in the
tonic/non-muscle cells (Fig. 6B). This suggests that it is
the U-rich nature of the sequence and not specifically the UCUU motif
that is necessary for enhancer function.
Role of the Exon in Its Alternative Splicing--
To test the role
of the exon sequence itself in alternative splicing, we took advantage
of the fact that the corresponding exon in the rat MYPT1 gene is
similar in size and sequence, but is constitutively spliced (17), and
thus may lack regulatory sequences. A chimeric MYPT1 mini-gene was
engineered in which the chicken alternative exon sequence was precisely
replaced with the corresponding rat exon sequence, preserving the
chicken introns and splice site sequences. This replacement results in
the mutation and deletion of 36 and 18 nucleotides, respectively, of
the 123 nucleotides contained within chicken alternative exon (Fig.
7C). This deletion and
mutation of the chicken MYPT1 alternative exon (rat-chicken chimera)
resulted in nearly complete exon skipping in phasic, tonic, and
non-muscle cells (Fig. 7, A and B, lane 2) and suggested that one or more sequences that enhance splicing is present in the chicken alternative exon. To rule out the possibility that undesired mutations were introduced elsewhere in the mini-gene during the creation of the chimera that crippled its ability to be
properly spliced, re-introduction of the chicken alternative exon
sequence into this vector restored splicing of the alternative exon
(data not shown), verifying the integrity of the chimera construct.
Identification of Specific Regulatory Sequences within the
Chicken MYPT1 Central Alternative Exon--
Comparison of the chicken
and rat exon sequences identified an 11-nucleotide palindromic
sequence, UCCUACAUCCU, that is present in the chicken alternative exon
but absent from the rat exon sequence (Fig. 7C,
sequence B). This palindromic sequence is located within a
stretch of sequence where only 6 of 31 nucleotides (19%) are identical
(flanking sequences are designated A and C in
Fig. 7C), whereas 63 of 92 nucleotides (68%) are identical
outside of this region. Deletion of this 31-nucleotide sequence from
the chicken MYPT1 mini-gene construct resulted in a significant
increase in exon skipping (data not shown), suggesting that this region
contained an enhancer of splicing.
Initially, we focused on a potential role for the palindrome sequence
in regulating splicing of the MYPT1 alternative exon. Our strategy for
mutating the palindrome involved searching the entire MYPT1 alternative
exonic sequence, including the 11-nucleotide palindromic sequence, for
matches to previously characterized ESEs. The data from this search
(see "Experimental Procedures") revealed that the palindrome
contains a good match (UACAUC) to the optimal binding site for the SR
protein SRp55 (18) that overlapped with less ideal matches for SRp40
and SC35 binding. Mutations were made to each half of the palindrome to
improve or destroy the putative SRp55 binding site while also partially or completely disrupting the palindrome. A mutation that disrupts both
half-sites of the palindrome and the putative SRp55 binding site
(lane 5, Mutpal1 + 2) significantly increased exon skipping in phasic, tonic, and non-muscle cell types (75, 45, and 47%, respectively; Fig. 7, A and B, lane
5), identifying this 11-nucleotide sequence as a critical ESE of
exon inclusion. To determine if the putative SRp55 binding site played
a role, this site was mutated to improve the predicted binding of SRp55
while at the same time disrupting the 3'-half-site of the palindrome
(lane 4, Mutpal2). This mutation increased exon skipping in
phasic, tonic, and non-muscle cell types (39, 23, and 21%,
respectively; Fig. 7, A and B, lane 4), suggesting that the ESE is not functioning through SRp55. This
is supported by the effect of mutation of only the 5'-half-site of the
palindrome (lane 3, Mutpal1), which destroys the putative SRp55 binding site but has a minimal effect on the splicing of the
alternative exon in all cell types (lane 3). The fact that this palindrome does not resemble any of the other known splicing enhancer sequences raises the possibility that it represents a novel
ESE.
The mutation of the palindromic sequence significantly reduced
the splicing of the alternative exon but not to the level observed with
the rat/chick chimera, suggesting that additional enhancer sequences
may be present in the exon. We thus turned our attention to the 10- and
9-nucleotide sequences flanking the palindrome and contained within the
31-nucleotide sequence (Fig. 7C). Our strategy for mutating
these two sequences was to replace them with the corresponding
sequences in the rat exon (Fig. 7A), because the rat exon
sequence did not splice in the context of the rat/chick chimera and
thus presumably lacked enhancer sequences. Mutation of the upstream
10-nucleotide sequence to the corresponding rat sequence (lane
6) significantly increased exon skipping in phasic, tonic, and
non-muscle cell types (83, 33, and 50%; Fig. 7, A and B, lane 6) to a degree similar to the mutation of
the palindromic sequence. Simultaneous mutation of both the
10-nucleotide sequence and the adjacent palindrome (lane 7)
produced a significant additive effect toward exon skipping in the
tonic/non-muscle cells where the alternative exon is normally included
but a minimal additive effect in the phasic SMCs (lane 7).
Of note, the level of exon skipping in the tonic SMCs after mutation of
the two putative enhancer sequences is not equivalent to that of the
rat/chick chimera (compare Fig. 7B, lane 7 with
2) suggesting that an additional enhancer element may be present.
The 9-nucleotide sequence immediately downstream of the
11-nucleotide perfect palindrome is part of a 13-nucleotide imperfect palindrome (UUAAACACAAGUU, Fig. 7C), in which the first U is
also common to the perfect palindrome described above. Mutation of six
nucleotides of this sequence to the corresponding rat sequence (Fig.
7A, lane 8) nearly abolished exon skipping in
phasic SMCs (2%; Fig. 7, A and B, lane
8) and abolished the difference in levels of exon skipping between
phasic, tonic, and non-muscle cells (Fig. 7B;
p = NS), suggesting that this sequence mediates the
greater silencing of alternative exon splicing in the phasic SMCs
compared with tonic SMCs. Thus, the chicken MYPT1 central alternative
exon contains a bipartite regulatory region consisting of a
22-nucleotide ESE and a 9-nucleotide exonic splicing silencer (ESS).
In this study, we have used deletion and mutation analysis
of a MYPT1 mini-gene to identify cis-regulators of splicing
of the central alternative exon. This study was performed using
cultured SMCs and non-muscle cells as well as a novel in
vivo injection system. Our data support a model in which multiple
cis-enhancers clustered near the alternative exon 5'-splice
site are required for its inclusion in MYPT1 mRNA in slow tonic
smooth muscle and non-muscle cells (Fig.
8A). The splicing enhancers
appear to be countered by two silencer elements, one in the exon and
one distal in the downstream intron. We propose that these function to
silence the splicing of the alternative exon in the fast-phasic smooth muscle phenotype.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ui+di (upstream intron and downstream intron deleted) vector in Fig. 3
(lane 2).
ui (lane 3) was generated by cloning
a HindIII-EcoO109I fragment from the
ui+di
vector containing the upstream intron deletion into the
HindIII and EcoO109I sites of the wild-type MYPT1
mini-gene vector.
di (lane 4) was generated by cloning a
EcoNI-SmaI fragment from the
ui+di vector
containing the downstream intron deletion into the EcoNI and
SmaI sites of the wild-type MYPT1 mini-gene vector. Finally,
di+1
and
di+2
(lanes 5 and 6) were
generated by insertion of one and two copies, respectively, of the 1-kb
"stuffer" DNA sequence derived from phage lambda DNA (21). This was
accomplished by removing one or two copies of the 1-kb stuffer sequence
from the vector Bluescript (Stratagene, gift from G. Screaton) by
digestion with SacII and ApaI, which were then
inserted into the unique SacII and ApaI sites of
the vector
di.
1,
2, and
3 series
(Figs. 4 and 5) contained a unique ApaI restriction enzyme
recognition sequence at the site of the deletions so that the deletions
could be confirmed by ApaI digestion. Long PCR was also
used to make all of the desired point mutations by including the
mutation(s) in the oligonucleotides used for PCR. PCR cycle number was
limited to minimize the occurrence of second-site mutations, and
multiple independent clones of each construct were tested in
transfection assays. All clones were sequenced to verify that they had
the desired mutations and that the alternative exon and splice site
sequences were intact.
-actin (23). LMH, a
chicken hepatocellular carcinoma cell line (ATCC CRL-2117), was
cultured on 0.1% gelatin with Waymouth's MB 752/1 medium in the
presence of 10% serum. Transfections were performed on 24-well dishes.
Cells were plated 1-2 days before each transfection at a density of
1.2 × 105 cells/well (LMH) and 1.5 × 105 cells/well (SMCs), such that on the day of the
transfection they would be 50-70% confluent. Each mammalian
expression vector containing the wild-type or a mutant MYPT1 mini-gene
(0.15 µg of plasmid per well) was transfected into these cells using
LipofectAMINE (Invitrogen) at a LipofectAMINE to DNA ratio (wt:wt) of
12-18:1 (LMH) and 14:1 (gizzard). Serum-containing media was added to the cells 5 h after transfection. RNA was isolated 24 h after transfection using TRIzol reagent (Invitrogen).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
RFP expression in gizzard SMCs in
vivo. A, RFP expression is evident at
the membrane and as aggregates in the cytosol of smooth muscle cells of
the gizzard 1 day after plasmid DNA injection. B,
co-staining with phalloidin FITC shows the actin filaments and
preserved muscle architecture in the region where RFP expression is
abundant. C, no significant red fluorescence is detected in
control gizzard tissues injected with pUC plasmid DNA. Magnification
was 100× in all panels.
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Fig. 2.
Splicing of pre-mRNA derived from a MYPT1
mini-gene is regulated. A, schematic diagram of the
genomic fragment cloned in this study. This fragment contains the
chicken MYPT1 central alternative exon and flanking introns and exons.
The solid box represents the alternative exon, whereas the
open boxes represent the flanking exons. Horizontal
lines connecting these boxes represent the introns.
Sizes of exons and introns are not drawn to scale but are as shown in
numbers below each exon and intron. The numbers
and letters above the diagram refer to exon and intron
labels used in Ref. 17. V-shaped lines above (inclusion of
alternative exon) and below (skipping of alternative exon) the diagram
represent the endogenous MYPT1 splice variants observed in adult aorta
and gizzard. B, schematic diagram of the MYPT1 mini-gene
expression vector used in this study. The genomic fragment described in
A was cloned into the pRc/RSV mammalian expression vector in
which the viral RSV promoter drives transcription of the MYPT1
mini-gene. The bovine growth hormone (bGH) polyadenylation
signal (striped box) is located immediately downstream of
the MYPT1 mini-gene to allow for 3'-end formation of the mini-gene
mRNA. The arrows indicate the approximate location of
the oligonucleotides that were used in all of the RT-PCR reactions in
this study. C, representative RT-PCR reactions performed on
RNA samples from gizzards that were injected with (Gizz Inj)
or smooth muscle and non-muscle cells that were transfected with the
wild-type MYPT1 mini-gene (Gizz Trans and LMH).
The RT-PCR products were separated, visualized, and quantified as
described under "Experimental Procedures." The percentages of exon
skipping observed in the injected and transfected RNA samples are shown
underneath the appropriate lanes. Labels to the
left of the gel refer to exon included (407 nucleotides) and
exon-skipped (284 nucleotides) RT-PCR products; n.s.,
nonspecific RT-PCR products.
ui+di; Fig. 3A, lane 2, and Fig. 3B). In addition, removal of either the upstream or
downstream regions individually also resulted in significant loss of
exon skipping (
ui and
di; Fig. 3A, lanes 3 and 4). This suggested either that intron size was crucial
to exon skipping and/or that critical regulatory sequences were
deleted. To distinguish between these two possibilities, the downstream
intron was re-lengthened by the addition of "stuffer"
(non-regulatory) DNA sequence derived from phage lambda DNA (21).
We focused on the downstream intron, because our sequence analysis had
suggested that a number of regulatory elements might be located there
(described below). Insertion of one copy of the lambda sequence
(
di+1
) resulted in little or no change in the level of exon
skipping (compare lanes 4 and 5). Insertion of
two copies of the lambda sequence (
di+2
), which increased the
size of the intron to 0.4 kb larger than its wild-type length,
increased exon skipping in both phasic (lane 6) and tonic SMCs (Fig. 3B) to the level observed with the wild-type
mini-gene in tonic SMCs (10-11%). These results suggest that the
relatively large introns surrounding the alternative exon weaken the
splicing reaction, allowing for the process to be regulated. The
re-lengthening of the introns resulted in significantly less exon
skipping as compared with the wild-type construct in phasic SMCs
(10.4% versus 27%, p < 0.001), but not in
tonic SMCs (10.3% versus 10.9%, p = NS;
Fig. 3B), suggesting that sequences that repress splicing of
the alternative exon in the phasic SMCs may be present within the
downstream intron.
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Fig. 3.
Role of the introns in the regulated splicing
of MYPT1. A, schematic diagrams of the wild-type and
mutant MYPT1 mini-genes used in this figure are shown at the
top and are patterned after Fig. 2A. The
elongated rectangles in 5 and 6 represent the 1-kb stuffer DNA sequence described under "Results."
In this and subsequent figures only pictures of gels from
representative RT-PCR reactions from gizzards that were injected with
MYPT1 mini-gene constructs are shown below the schematic diagrams to
conserve space. Lanes are arranged in the same order as the diagrams of
the deletion constructs (numbers 1-6). RT-PCR products were
separated, visualized, and quantified as described under
"Experimental Procedures" and labeled as described in Fig.
2C. B, bar graph showing quantification of exon
skipping for each wild-type and mutant mini-gene in gizzard tissue,
cultured SMCs, and LMH cells. ui, upstream intron;
di, downstream intron; , 1 kb lambda phage DNA.
1) identified this region as a splicing enhancer and is
described below. Deletion of either the mid or distal 600-700
nucleotide portions of the intron (
2 and
3) reduced exon skipping
in phasic SMCs (11 and 7% exon skipping, respectively) to a level that
was close to the level observed in tonic SMCs (7 and 4%, respectively)
and non-muscle cells (8 and 3%, respectively; Fig. 4, A and
B, lanes 3 and 4). Smaller deletions
identified a 237-nucleotide sub-region of
2 that had a significant
effect on exon skipping (
2.2, lane 6), whereas deletion
of the remaining 363 nucleotides had no effect (
2.1, lane
5).
3 was subdivided into three smaller deletions of 363, 128, and 258 nucleotides (
3.1,
3.2, and
3.3, respectively).
3.2
had no effect on splicing of the MYPT1 alternative exon (data not
shown), indicating that a 100-nucleotide region that contained several
overlapping matches to a number of genes in the NCBI data base (BLAST
search) were not functionally significant.
3.3 also had no
significant effect on exon skipping (lane 8), whereas
3.1 caused the same shift toward exon inclusion as the entire
3 (7.8% versus 7.4% in phasic SMCs, and 4.1% for each in tonic
SMCs; compare lane 7 with lane 4).
3.1 was
further subdivided into three smaller deletions of 148, 132, and 105 nucleotides (
3.1A,
3.1B, and
3.1C, respectively).
3.1A and
3.1B both caused partial shifts toward exon inclusion compared with
3.1, whereas
3.1C had no effect on splicing (data not shown).
These results suggest that the silencer sequence is contained within a
continuous region covered by
2.2,
3.1A, and
3.1B. Therefore, a
526-nucleotide deletion covering this region (
2.2/
3.1B) was
constructed that also caused a large shift toward exon inclusion (8%
exon skipping in phasic SMCs, lane 9). This deletion
eliminated the difference in exon skipping between phasic and tonic
SMCs (8% versus 6%, p = NS), suggesting
that a silencer of alternative exon splicing that is more active in
phasic than tonic SMCs is contained within this region.
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Fig. 4.
Serial deletions identify a distal intronic
silencer of exon inclusion. A, schematic diagrams of
the wild-type and mutant MYPT1 mini-genes used in this figure. The
bars below the diagram of the wild-type mini-gene
(diagram number 1) represent deletions made in each of the
mutant mini-genes. Numbers underneath these bars
represent the size of each deletion. Candidate regulatory sequences
referred to in the text are identified by various symbols in the
diagram and defined in the box on the left.
Representative RT-PCR reactions are shown, labeled and analyzed as in
Fig. 3A. B, bar graphs showing
quantification of exon skipping as in Fig. 3B.
1) resulted
in an increase in the amount of exon-skipped MYPT1 mRNA (Figs.
4A and 5B, lane 2), suggesting that
this sequence contains one or more enhancers of exon inclusion. This
sequence contains two UGCAUG motifs, first identified as regulators of
splicing of fibronectin alternative exons (24, 25), as well as matches
to intron L of the chicken embryonic MHC (ceMHC) gene (26) and intron 7 of the chicken interferon alpha/beta receptor 1 (IFNAR1) gene (27)
(Figs. 4A and 5A). Alignment of these MYPT1,
ceMHC, and IFNAR1 sequences revealed five blocks of sequence that were
highly identical between the three genes, ranging from 83 to 93%
identity within these blocks (Fig.
5A). Serial deletions were
generated to test the role of each sequence in enhancing the splicing
of the alternative exon. Deletion of 216 nucleotides that contained blocks numbers 1a, 1b, and 2 (
1-1 + 2, lane 4) caused a
shift toward exon skipping in all cell types (53-71%) that was even more dramatic than the original
1 deletion (38-48% exon skipping, Fig. 5, B and C, compare lane 4 with
lane 2). A 67-nucleotide deletion that removed only block
number 1a (
1-1a, lane 5) caused essentially the same
shift toward exon skipping in all cell types, as did the entire
1-1 + 2 deletion (compare lanes 4 and 5), suggesting that only block number 1a is required for splicing. Consistent with
this hypothesis, a 157-nucleotide and a 168-nucleotide deletion that
removed blocks 1b and 2 (
1-1b+2) and blocks 4a and b (
1-4a+b), respectively, had little or no affect on splicing of the alternative MYPT1 exon (lanes 6 and 7). Finally, a
167-nucleotide deletion that deletes the two UGCAUG motifs (
1U) had
no significant effect on MYPT1 splicing (lane 3), suggesting
that these two motifs are not essential for the function of the
enhancer contained within
1. Taken together, these results suggest
that the
1 region contains a 67-nucleotide enhancer of exon
inclusion that is active in phasic, tonic, and non-muscle cells.
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Fig. 5.
Serial deletions identify a proximal
67-nucleotide intronic enhancer of exon inclusion. A,
three schematic diagrams of the MYPT1 alternative exon and downstream
intron are shown at the top of this figure. Superimposed on
each of these diagrams are ovals that represent regions of
high sequence identity (>83%) between MYPT1 versus ceMHC
(first diagram), MYPT1 versus IFNAR1
(second diagram), and MYPT1 versus ceMHC
versus IFNAR1 (third diagram). The percent
identities are shown above each oval, and the number of
nucleotides in each match is shown below the ovals. The
numbers above the lines represent distances
between the exon and the various matches. The MYPT1, ceMHC, and IFNAR1
sequences within consensus match number 1a are shown below the
schematic diagrams. The ceMHC and IFNAR1 nucleotides that differ from
the MYPT1 sequence are underlined. B, schematic
diagrams of the wild-type and mutant MYPT1 mini-genes used in this
figure. These diagrams are represented in identical manner to the
diagrams in Fig. 4A. Representative RT-PCR reactions are
shown, labeled and analyzed as in Fig. 3A. C,
bar graphs showing quantification of exon skipping as in
Fig. 3B.
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Fig. 6.
Deletion/mutation analysis identifies an
intronic pyrimidine-rich sequence required for exon inclusion.
A, sequences of the wild-type and mutant MYPT1 mini-genes.
The sequences surrounded by the open-ended boxes represent
the last eight nucleotides of the alternative exon. The remaining
sequences represent the nucleotides that are at the 5'-end of the
downstream intron (please note that the wild-type splice site sequence
shown in this figure represents a correction of this sequence as
reported in Ref. 17). The underlined nucleotides highlight
the UCUU motifs that are described under "Results." Deleted and
mutated nucleotides are indicated by a gap and closed
boxes, respectively. Representative RT-PCR reactions are shown,
labeled and analyzed as in Fig. 3A. B, bar
graphs showing quantification of exon skipping as in Fig.
3B.
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Fig. 7.
The alternative exon contains a novel
bipartite enhancer/silencer element. A, sequences of
the wild-type and mutant MYPT1 mini-genes used in this figure are shown
at the top. The sequences surrounded by the open-ended
boxes represent the last 48 and 36 nucleotides of the chicken
MYPT1 alternative exon and the corresponding exon in rat, respectively.
The remaining sequences represent the six nucleotides that are at the
5'-end of the downstream intron. The small capital letters
(construct number 2) represent the nucleotides in the rat
exon that differ from the chicken exon. The underlined
nucleotides represent the 11-nucleotide palindrome discussed in the
text. The nucleotides surrounded by small boxes represent
the nucleotides that were changed in each of the mutant MYPT1
mini-genes. Representative RT-PCR reactions are shown, labeled and
analyzed as in Fig. 3A. B, bar graphs
showing quantification of exon skipping as in Fig. 3B.
C, alignment of the chicken alternative exon and the
corresponding rat exon. Dots, identical nucleotides in
chicken and rat sequences; dashes, gaps required for optimal
alignment. The nucleotides that are marked by vertical lines
represent the three regions that were mutated in A. B indicates the 12-nucleotide sequence that is missing in
the rat exon. This sequence contains the 11-nucleotide perfect
palindrome referred to in the text. A and C refer
to the 10-nucleotide and 9-nucleotide sequences that are upstream and
downstream, respectively, of the palindrome. The six
underlined nucleotides represent the optimal SRp55 binding
site described in the text.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 8.
Proposed model for MYPT1 alternative
splicing. A, inclusion of the alternative exon appears
to require at least three distinct splicing enhancers. The putative
factors that recognize these enhancers are represented by the
open ovals. Because removal of any one of these enhancers
significantly reduces exon inclusion in all cell types tested, we
propose that the splicing factors that recognize these sequences form
an enhancer complex that is required for basal splicing of the exon.
The location of this enhancer complex suggests that it may be involved
in stabilizing binding of U1 snRNP to the 5'-splice site. The two
solid ovals represent silencers that appear to
preferentially inactivate the enhancer complex in the phasic SMCs.
B, putative stem-loop RNA structure showing potential
base-pairing between the 11-nucleotide perfect palindrome (ESE) and the
5' donor sequence, whereas the 13-nucleotide imperfect palindrome (ESS)
forms part of a 20-nucleotide loop. The two palindromes overlap each
other by one nucleotide suggesting a steric interference model for the
ESE and ESS. The arrow indicates the 5'-splice site
(exon-intron junction).
Intronic Enhancer Elements-- We identified an enhancer complex that is necessary for splicing of the alternative exon in all cell types tested. The requirement of cis-enhancer sequences for the alternative exon to be efficiently spliced is consistent with the general rule that the sequence and/or structure of alternative exons are unfavorable for recognition and splicing by the basal splicing machinery (9-11). Indeed, the MYPT1 central alternative exon 5'-splice site deviates from consensus (AAG:GUGUGG versus CAG:GURAGU), the alternative exon 3'-splice site contains a poor polypyrimidine tract, and the relatively large flanking introns (2.3-2.7 kb in chicken) have a negative effect on exon splicing in the mini-gene context (Figs. 2 and 6) (17). One of the enhancers present in the intron is a 19-nucleotide U-rich sequence containing two UCUU motifs located immediately downstream of the alternative exon 5'-splice site. Deletion of the U-rich sequence or mutation of six residues from U to C led to a dramatic shift toward exon skipping (Fig. 6A), suggesting that these two sites function as splicing enhancers. Based on their presence in other transcripts that undergo alternative splicing, this regulatory motif may be considered a putative TIA-1 or a PTB binding site (28-30). TIA-1 binds to U-rich sequences immediately downstream of the 5'-splice site and enhances splicing, perhaps by stabilizing binding of U1 snRNP to a weak 5'-splice site (29). On the other hand, PTB binds to UCUU motifs and functions in most instances as a negative regulator of the splicing of alternative exons (14). The identification of the trans-acting factors that bind to this site in the MYPT1 transcript and how they might influence splicing of the exon will require further study.
A 67-nucleotide sequence required for inclusion of the alternative exon was identified in the intron ~190 nucleotides downstream of the alternative exon. The specific nucleotides required for the function of this second putative enhancer remain to be determined. However, within the 32-nucleotide region that is highly identical between MYPT1, ceMHC, and IFNAR1 (Fig. 5A) is the sequence UUUCCCUG, which is also present in the central portion of MSE3, a muscle-specific enhancer of exon inclusion of chicken cardiac troponin T (31, 32). This suggests the possibility that this is an MSE3-like enhancer, perhaps functioning through a CUG-binding protein. Deletion of this enhancer sequence, like the deletion or mutation of the other two enhancer sequences identified in this study, resulted in significant reduction of a exon splicing in tonic and phasic SMCs and non-muscle cells. This suggests that these enhancers function to enhance the splicing of a weakly spliced (alternative) exon but are not involved in the tissue-specific regulation of the splicing of this exon.
Exonic Enhancer Element-- The third identified enhancer sequence resides within the alternative exon itself. It consists of an 11-nucleotide perfect palindrome (UCCUACAUCCU) that is absent from the corresponding constitutively spliced rat exon, along with the 10 nucleotides immediately upstream of the palindrome. As was observed with the U-rich intronic enhancer, mutation of both sequences produces additive effects in cells where the alternative exon is normally spliced (i.e. tonic SMCs and non-muscle cells). In the cell type where the endogenous exon is excluded (i.e. phasic SMCs) a single mutation results in nearly complete exon exclusion, and there is little additive effect of the double mutations. This suggests a model in which the enhancers of alternative exon splicing are more susceptible to silencing in the phasic SMCs than in the tonic SMCs, as would be expected based on the splicing of the endogenous exon.
Interestingly, this exact UCCUACAUCCU palindromic sequence is present
in or near the alternatively spliced exons of many other transcripts,
including the human DSCAM, mouse and rat CamK1-, and chicken
c-ski (NCBI data base search, not shown). This suggests that
this specific 11-nucleotide sequence may function as a novel enhancer
of alternative exon splicing in a number of cell types. Alternatively,
it was possible that it would function through the well-described ESEs
that bind SR proteins. We scanned the exon sequence against a
SELEX-generated data base of SR binding sites and identified several
sequences where SR proteins might bind (data not shown). A particularly
good match to the consensus binding sequence for the SR protein SRp55
(18) was contained within the palindromic sequence (UACAUC) that
overlapped with less ideal matches for SRp40 and SC35 binding. However,
mutations of the palindromic sequence to sequences that would be
predicted to abrogate (CAAAUC) or improve (UACGUA) binding of the SR
proteins did not alter splicing of the exon in the former case and
actually reduced exon splicing in the latter case. It is therefore
proposed that the 11-nucleotide palindrome represents a novel ESE.
Silencer Elements-- Two silencers of exon inclusion were identified in the MYPT1 mini-gene construct. One of these silencer sequences is located within the distal portion of the downstream intron, whereas the other silencer is found within the alternative exon (ESS). An interesting feature of these silencers is that, unlike the enhancers, mutation of either one of them completely abolishes the difference in splicing observed between phasic and tonic SMCs, suggesting that the silencers play an important role in determining smooth muscle phenotypic diversity. The ESS is found adjacent to the ESE and just upstream of the alternative exon 5'-splice site. Mutation analysis identified six nucleotides in this sequence that are critical for exon skipping, but the exact boundaries of the ESS remain to be defined. The mutated sequence within the ESS is AC-rich and thus resembles A/C-rich exon enhancers (ACE elements) (33, 34). However, in this case the sequence appears to function as a cis-silencer. This is similar to several other alternatively spliced exons where bipartite elements containing ESEs and ESSs have been described (35-40), but the MYPT1 ESE/ESS sequences appear to be different from these elements.
An interesting feature of the MYPT1 ESS is that it is part of an
imperfect 13-nucleotide palindrome sequence, where only a G residue in
the 11th position interrupts the palindrome
(UUAAACACAAGUU). This imperfect palindrome overlaps by one
nucleotide the upstream palindrome enhancer sequence. In the modeling
of the RNA secondary structure (41), this 13-nucleotide sequence forms
part of a 20-nucleotide loop in the stem-loop where the 5'-splice site
and the 11-nucleotide perfect palindrome are base-paired to one another (Fig. 8B). This potential structure suggests a model in
which the stem-loop structure prevents U1 snRNP from binding to the 5'-splice site and directing splicing unless a trans-acting
factor bound to the 11-nucleotide palindrome disrupts the stem-loop. The ESS would function in this model by sterically interfering with the
binding of a protein to the ESE. This stem-loop model is similar to
that proposed for the regulated splicing of the human tau
alternative exon 10 associated with inherited dementia (42) and the
chicken -tropomyosin alternative exons (43, 44). In each instance,
both formation of a stem-loop structure that masks the 5'-splice site
as well as specific regulatory sequences are proposed to regulate
splicing. The fact that deletion of the 11-nucleotide perfect
palindrome causes an effect that is the opposite of mutation of the
palindrome, i.e. a shift toward exon inclusion (data not
shown) is consistent with a potential role for secondary structure in
MYPT1 pre-mRNA splicing. However, proof of the importance of the
proposed secondary structure will require an examination of the effects
of compensatory mutations that alter the sequence but maintain the
proposed base-pairing scheme.
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ACKNOWLEDGEMENTS |
---|
We thank Katherine M. Joyce, Anjum Jafri, and Yaling Qiu for technical assistance with transfections and RT-PCR, and Tasso Konstantakos and Qi-Quan Huang for technical assistance with the in vivo injections. We also thank Drs. Adrian R. Krainer and Luca Cartegni for providing the high score functional motif analysis of the MYPT1 SR protein binding sites, and Dr. Hua Lou for critically reading the manuscript.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health (NIH) Grants K08HL-03275 and R01 HL-66171 (to S. A. F.).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.
§ Supported in part by NIH-National Research Service Award Training Grant T32-HL07887.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF110177 and AF110176.
To whom correspondence should be addressed: 422 Biomedical Research Building, 2109 Adelbert Rd., Cleveland, OH
44106-4958. Tel.: 216-368-0488; Fax: 216-368-0507; E-mail:
saf9@po.cwru.edu.
Published, JBC Papers in Press, December 30, 2002, DOI 10.1074/jbc.M207969200
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ABBREVIATIONS |
---|
The abbreviations used are: snRNP, small nuclear ribonucleoprotein particle; ceMHC, chicken embryonic myosin heavy chain; ED, embryonic day; ESE, exonic splicing enhancer; ESS, exonic splicing silencer; RFP, red fluorescent protein; IFNAR1, interferon alpha/beta receptor 1; MYPT1, myosin phosphatase targeting subunit 1; PTB, polypyrimidine tract-binding; SELEX, systematic evolution of ligands by exponential enrichment; SF1, splicing factor 1; SMCs, smooth muscle cells; U2AF, U2 snRNP auxiliary factor; RSV, Rous sarcoma virus; FITC, fluorescein isothiocyanate; RT, reverse transcriptase; MSE, muscle-specific enhancer; AMV, avian myeloblastosis virus; SR, arginine-serine rich.
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