From the Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom
Received for publication, October 3, 2002, and in revised form, February 7, 2003
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ABSTRACT |
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Polypyrimidine tract-binding protein (PTB) is an
abundant widespread RNA-binding protein with roles in regulation of
pre-mRNA alternative splicing and 3'-end processing,
internal ribosomal entry site-driven translation, and mRNA
localization. Tissue-restricted paralogs of PTB have previously been
reported in neuronal and hematopoietic cells. These proteins are
thought to replace many general functions of PTB, but to have some
distinct activities, e.g. in the tissue-specific regulation
of some alternative splicing events. We report the identification and
characterization of a fourth rodent PTB paralog (smPTB) that is
expressed at high levels in a number of smooth muscle tissues.
Recombinant smPTB localized to the nucleus, bound to RNA, and was able
to regulate alternative splicing. We suggest that replacement of PTB by
smPTB might be important in controlling some pre-mRNA alternative
splicing events.
The importance of post-transcriptional mechanisms of gene
regulation has been emphasized by the relatively modest number of genes
in the human genome (1, 2). Alternative splicing, RNA editing, and
alternative translational initiation all allow for more than one
protein isoform to be produced by individual genes. Alternative
splicing is the most prevalent of the post-transcriptional mechanisms
for producing protein isoforms. Conservative estimates predict that
one- to two-thirds of human genes are alternatively spliced, and some
of these genes have the potential to produce thousands of isoforms
(reviewed in Refs. 3-7).
Regulation of alternative splicing involves the interaction of cellular
trans-acting factors with specific cis-acting
regulatory elements within a target pre-mRNA (8, 9). These
regulatory interactions influence the recognition of splice sites by
the splicing machinery. Such regulation can be positive, involving activator factors and enhancer sequences. Conversely, repressor proteins can mediate their influence via silencer elements. Although some model systems of regulated splicing involve the presence or
absence of a single regulatory protein, the majority of examples appear
to be more complex, with regulatory decisions being achieved by
particular combinations of regulatory factors, each of which is
expressed more widely than the splicing event that is being regulated (7-9).
The heterogeneous nuclear ribonucleoproteins
(hnRNPs)1 are a group of
abundant and widespread nuclear proteins with diverse roles in
pre-mRNA and mRNA function, including the regulation of
alternative splicing (10, 11). Polypyrimidine tract-binding protein
(PTB)1 (reviewed in Refs. 12 and 13), also known as
hnRNP-I, is a prominent member of this family. PTB was originally
identified as a potential splicing factor due to its ability to bind to
polypyrimidine tracts at 3'-splice sites (14-16). However, it was
subsequently recognized to act as a splicing repressor at particular
splice sites (17-31). PTB also plays roles in nuclear pre-mRNA
3'-end processing (32, 33), cytoplasmic internal ribosomal entry site-driven translation (34), mRNA localization (35), and regulation of mRNA stability (36). Consistent with these varied roles, PTB can shuttle between the nucleus and cytoplasm, but is
predominantly localized in the nucleus (37). The optimal RNA binding
sequence for PTB (UCUU in a pyrimidine-rich context) is found within
silencer elements that act by binding PTB (26). These elements are
often found within the 3'-splice site polypyrimidine tract; and in some
cases, PTB acts by directly competing for binding to the polypyrimidine
tract with the splicing factor U2AF65 (23, 27). However,
PTB-binding sites are also found in other locations in the region of
PTB-regulated exons, so it may also be able to inhibit splicing in
other ways (reviewed in Ref. 13).
Consistent with its expression pattern, most PTB-mediated repression of
specific exons is widespread. Regulated selection of the exons occurs
in a small subset of tissues where the repressive action of PTB is
either absent or in some way modulated. PTB exists in two major
alternatively spliced isoforms, termed PTB1 and PTB4, which arise from
skipping or inclusion, respectively, of exon 9, which encodes a
26-amino acid insert. A minor isoform, PTB2, is produced by inclusion
of exon 9 using an internal 3'-splice site, giving a 19-amino acid
insert. In at least one case, these isoforms have differential
activity, with PTB4 being more repressive upon We have been investigating two alternative splicing events that are
regulated in smooth muscle (SM) cells. In In an attempt to understand how the tropomyosin and actinin splicing
events are regulated, we investigated the expression of PTB isoforms in
dedifferentiating rat aorta smooth muscle (RASM) cells. We found no
change in the ratio of PTB1 and PTB4. However, in a number of SM
tissues, we detected expression of a novel PTB paralog that is distinct
from PTB, nPTB/brPTB, and ROD1. We refer to the new paralog as smPTB
due to its initial identification and high levels of expression in SM
tissues. smPTB is ~70% identical to PTB and has additional 36- and
22-amino acid inserts in two of the linker regions separating RNA
recognition motif (RRM) domains. Recombinant smPTB binds RNA in
vitro and has splicing inhibitory activity. We propose that
expression of smPTB may play a role in switching subsets of alternative
splicing events in SM and other cells.
Analysis of Rat Aorta SM and Tissue RNAs--
Primary RASM cells
were isolated by enzymatic dispersion and cultured as described (43,
44). Total RNA from SM cells and tissues was made using TRI
reagent (29). Splicing patterns of the different genes were analyzed by
reverse transcription (RT)-PCR. Oligo(dT) was used for reverse
transcription by avian myeloblastosis virus reverse transcriptase (45).
PCR was carried out with a 32P-end-labeled primer using a
hot start of 92 °C for 3 min, followed by enzyme addition at
80 °C. Thirty cycles were carried out at 94 °C for 30 s,
annealing temperature (variable) for 30 s, and 72 °C for
60 s, with a final extension at 72 °C for 2 min. A 55 °C
annealing temperature was used for primer sets 5'-vin/3'-vin, TM1/TM4,
P30/31 (smPTB + PTB) (see Fig. 4), P37/Pd3'2 (smPTB + PTB) (see Fig.
2), P37/P38 (PTB-specific), and P12/P38 (human PTB plasmid markers). A
58 °C annealing temperature was used for primer set P32/P33
(smPTB-specific). A 60 °C annealing temperature was used for primer
set EF1a5'/3'-act. In each case, the 5'-primer is stated first.
For the PTB plasmid marker PCR, 10% Me2SO was included.
Products were analyzed on 4% denaturing polyacrylamide gels. The
sequences of the oligonucleotides used for PCR are as follows: EF1a5',
5'-ATCAGCCAGGAACAGATG-3'; 3'-act, 5'-ACATGAAGTCGATGAAGGCCTG-3'; 5'-vin, 5'-GGTGATTAACCAGCCAATGATGAT-3'; 3'-vin,
5'-CTTCACAGACTGCATGAGGTT-3'; TM1, 5'-CGAGCAGAGCAGGCGGAG-3'; TM4,
5'-CAGAGATGCTACGTCAGCTTCAGC-3'; P12, 5'-AAGAGCCGTGACTACACACGC-3'; P30,
5'-GACCTGCCCTC(A/T)G(A/G)(T/A)GACAG-3'; P31,
5'-GCGTTCTCCTTC(C/T)TGTTGAACA-3'; P33, 5'-TCGGTGCACATCGCCATAGGCA-3'; P37, 5'-AAGAGCCGAGACTACACACGC-3'; P38, 5'-GAGGCTTTGGGGTGTGACTCT-3'; and
Pd3'2, 5'-TTGCCGTCCGCCATCTGCACTA-3'.
Cloning--
Constructs were prepared by standard cloning
techniques (45). Full-length smPTB and PTB were cloned into vectors for
in vivo transfection (pCMVSPORT), localization (pEGFPN1 or
pEGFPC1), and overexpression in Escherichia coli (pET21).
Rapid amplification of cDNA ends (RACE) was used to amplify smPTB
in two halves using gene-specific primers from day 0 rat aorta RNA.
3'-RACE (45) was carried out as a first round with oligo(dT) and P32
(see below) as the forward gene-specific primer (30 cycles of 94 °C
of 30 s, 55 °C for 30 s, and 72 °C for 60 s). This
was followed by nested PCR with a forward gene-specific primer (P10)
under the same cycle conditions. For 5'-cDNA, SMART RACE
(Clontech) was used. Reverse transcription was
carried out using Moloney murine leukemia virus reverse transcriptase
with oligo(dT) plus a SMART oligonucleotide (P77). The first round of
PCR was carried out (30 cycles of 94 °C for 30 s, 58 °C for
30 s, and 68 °C for 60 s) with a 5'-primer mixture at a
ratio of 5:1 P79/P78 plus the gene-specific 3'-reverse primer P33 (see
above). Advantage GC2 polymerase (Clontech)
was used. This was followed by 25 cycles (94 °C for 30 s,
60 °C for 30 s, and 68 °C for 2 min) of nested PCR with a
5'-primer (P80) and a gene-specific primer (P11 or P41). The
oligonucleotides used for RACE were as follows: P10,
5'-TTCCTCAAGCTGCAGGCTTGGCCA-3'; P11,
5'-GGGAGCCTGAGGTGACCATTGAGC-3'; P32, 5'-ATGACAGTCAGCCCTCTCCGGTC-3'; P41, 5'-GAGCTGAGGCCATGTTCTGGACCTGGACCGGA-3'; P77,
5'-AAGCAGTGGTAACAACGCAGAGTACGCGGG-3'; and P80,
5'-AAGCAGTGGTAACAACGCAGAGT. The sequence of the complete rat
smPTB open reading frame has been deposited in the
GenBankTM/EBI Data Bank (accession number AY223520).
In Situ Hybridization--
35S-Labeled sense and
antisense RNA probes corresponding to mouse smPTB amino acids 100-182
were transcribed in vitro and hybridized to day 10, 14, and
15 mouse embryo sections as described (46).
Localization of smPTB--
PAC-1 or HeLa cells grown on
coverslips were transiently transfected, using LipofectAMINE
(Invitrogen), with PTB or smPTB tagged with green fluorescent protein
(GFP) at the C or N terminus. After 24 or 48 h, the coverslips
were inverted on a microscope slide, and GFP was visualized using a
Zeiss fluorescent microscope with an MC100 camera attached for photographs.
Expression and Purification of Recombinant
Proteins--
C-terminally His6-tagged recombinant
proteins were expressed and purified as previously described (46) with
one modification for smPTB. To solubilize smPTB, it was necessary to
re-extract the pellet after lysis using the non-detergent sulfobetaine
(1 M; NDSB-201) in lysis buffer (47).
UV Cross-linking--
High specific activity
[ Cell Culture, Transfection, and Analysis of Cellular
RNA--
PAC-1 cells were grown in Dulbecco's modified Eagle's
medium containing 10% fetal calf serum. Transient transfection was
carried out using LipofectAMINE; total RNA was isolated using TRI
reagent; and RT-PCR for Alternative Splicing Changes in RASM Cells--
In view of the
evidence for the involvement of PTB in controlling alternative splicing
of Identification and Expression of smPTB--
Having observed the
switch in alternative splicing of
Further analysis of PTB expression using primers P37 and Pd3'2, which
prime within exons 8 and 12, respectively, produced a strikingly
different result. A novel band (labeled smPTB in Fig.
2, C and D) larger than PTB4 was the major PCR
product in day 0 samples, but not in day 4 or PAC-1 samples. Cloning
and sequencing of this PCR product showed that it was derived from a
PTB-related gene that was distinct from PTB and the known paralogs ROD1 (40) and nPTB (38, 39). We refer to this new PTB
paralog as smPTB due to its high expression in a number of SM
tissues (see below). RT-PCR using smPTB-specific primers P32 and P33
confirmed that it was expressed in day 0 RASM cells, but not in day 4 or cultured PAC-1 cells (Fig. 2D). smPTB was also expressed
in other SM tissues such as the uterus and vas deferens.
smPTB Is Expressed from an Intronless Gene--
At the time that
we identified smPTB, no corresponding sequences could be identified by
BLAST searches of available expressed sequence tag or genomic
data bases. However, using 3'- and 5'-RACE, full-length smPTB cDNA
was isolated from day 0 RASM cell RNA. The open reading frame encodes a
588-amino acid protein with a predicted molecular mass of 63.7 kDa and
with 53-74% amino acid identity to PTB, nPTB, and ROD1 (Fig.
3). Pairwise BLAST analyses showed that
smPTB is more closely related to PTB than to either of the other genes.
Subsequent to cloning the full-length smPTB cDNA, the corresponding
rat gene sequence was identified using the ENSEMBL Trace Database,
whereas the mouse gene was identified by BLAST analysis of annotated
mouse genomic data bases and was located in contig 132920, corresponding to chromosome X A1.1. Both the rat and mouse genes are
intronless. The mouse gene contains three possible polyadenylation
addition signals giving a message size of 4.16, 5.09, or 6.53 kb. As
determined by Northern blot analysis, the size of the rat smPTB
mRNA is close to 6 kb (data not shown).
smPTB has the same overall structural organization as PTB (Fig. 3),
with four RRM domains and the same unusual fifth Expression of smPTB--
Expression of smPTB relative to PTB was
monitored across a range of rat tissues using PCR primer pair
P37/Pd3'2, corresponding to PTB exons 8 and 12, respectively. These
primers also allow detection of alternatively spliced PTB isoforms in
both exons 9 and 11, respectively. We have found exon 11 to be skipped
in a small proportion of PTB mRNA, giving rise to the PTB1tr (where "tr" is truncated) and PTB4tr
isoforms (Fig. 4).2 smPTB was
expressed most prominently in two SM tissues, the aorta (day 0) and
uterus, where it was more abundant than PTB. It was also readily
detected in the testis, thymus, skin, and lung (Fig. 4), although in
these tissues, PTB was more abundant. It was not highly expressed in
the stomach or small or large intestine, all of which contain SM cells.
Longer exposures showed that smPTB expression could be detected in most
tissues.
We next investigated smPTB expression by in situ
hybridization, which allows direct analysis of expression without PCR
amplification. Expression of smPTB was analyzed by in situ
hybridization to day 10, 14, and 15 mouse embryo sections. This allowed
us to conduct an unbiased survey of smPTB expression across multiple
tissues at a stage of embryonic development when many SM cells express late markers of differentiation (e.g. SM myosin heavy
chain). Whereas the sense smPTB control probe produced no signal, the antisense probes hybridized in a number of places, indicated by the
light areas in the dark-field images (Fig.
5, lower panels). The most
prominent signal was seen in the terminal bronchioles of the lung. The
signal was readily detected at embryonic day 14.5 and was higher by
embryonic day 15.5. Hybridization was also seen in the skin,
intercostal muscles, and the venous plexus of the liver. In day 10 embryo sections, hybridization to the maternal uterus could be observed
(Fig. 5, left panels). The lung, skin, and uterus are all
tissues that showed relatively high levels of smPTB expression by
RT-PCR (Fig. 4). The embryo sections did not include the aorta, which
was one of the tissues with the highest smPTB signal upon RT-PCR (Fig.
4). Nevertheless, the in situ hybridization data were
generally in agreement with the RT-PCR data and indicated that smPTB is
differentially expressed in mouse embryos.
smPTB Is a Nuclear Protein That Binds RNA--
To start to address
the possible functions of smPTB, PAC-1 cells were transiently
transfected with expression vectors for smPTB fused to GFP at either
the C or N terminus (Fig. 6). Like PTB-GFP (12), GFP-smPTB localized almost completely to the nucleus, despite lacking sequences equivalent to the N-terminal half of the PTB
bipartite nuclear localization signal (see above). Identical results
were obtained with both N- and C-terminal GFP fusions and in HeLa
cells. In a small number of PAC-1 cells, fluorescence was observed in
the cytoplasm as well as the nucleus. These results demonstrate that
smPTB is predominantly localized to the nucleus, consistent with a role
in regulation of splicing, but that, like PTB, it may also be able to
play additional cytoplasmic roles.
To examine the activities of smPTB in vitro, we
overexpressed C-terminally His-tagged smPTB in E. coli.
Recombinant smPTB migrated upon SDS-PAGE with an anomalously high
mobility of 83 kDa compared with the expected size of 65 kDa. A
similarly sized product was obtained by in vitro translation
of smPTB in reticulocyte lysate (data not shown). Recombinant smPTB was
analyzed for RNA binding by both UV cross-linking and electrophoretic
mobility shift assays. RNA probes containing various regulatory
elements from smPTB Is a Splicing Repressor--
We tested the activity of
recombinant smPTB in a number of in vitro splicing assays,
but were able to observe only nonspecific inhibitory activity (data not
shown). At this stage, we do not know whether this is due to the
recombinant protein lacking full activity or to the lack of an
essential cofactor. We also tested the activity of smPTB as a splicing
regulator by cotransfection with tropomyosin and actinin splicing
reporter constructs. It had relatively modest effects on splicing of
The data reported here confirm the existence and tissue-specific
expression of mRNA for a fourth PTB paralog, smPTB, in rat and
mouse. We have also observed additional cross-reactive bands in Western
blots using anti-PTB antiserum and protein samples from day 0 RASM
cells (data not shown). In the future, we aim to further analyze and
verify smPTB expression using specific antisera raised against the
recombinant protein. We have demonstrated that recombinant smPTB has
various properties in common with PTB, including predominant nuclear
localization (Fig. 6), RNA binding (Fig. 7), and splicing repression
(Fig. 8). At present, it is not clear which alternative splicing events
might be specific targets of endogenous smPTB. The correlation between
smPTB mRNA expression and regulation of
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tropomyosin exon 3 than PTB2 or PTB1 (29). In addition to the PTB isoforms, at least two
paralog genes, nPTB/brPTB and ROD1, with ~70% amino acid
identity to PTB have been identified. nPTB/brPTB (38, 39) is expressed
predominantly in neuronal cells, whereas ROD1 is expressed mainly in
hematopoietic cells (40). Cells expressing nPTB or ROD1 tend to express
less PTB; and in the case of the alternative N1 exon of
c-src, nPTB is less repressive than PTB, contributing to the
neuronal selection of the N1 exon (39). The effect of nPTB on other
splicing events is similar to that of PTB (29). Although no functional
data have been reported on ROD1, a reasonable proposal is that, in neuronal or hematopoietic cells expressing nPTB or ROD1, many PTB-repressed splicing events will be unaffected, whereas a specific subset will be altered. Alterations in the expression of the
alternatively spliced isoforms of PTB or in the expression of paralog
genes therefore provide one way in which PTB activity can be modulated.
-actinin, a SM-specific
exon is repressed by PTB in non-SM cells, leading to inclusion of the
mutually exclusive alternative non-muscle (NM) exon (see Fig.
1A) (28, 41). In
-tropomyosin (
-TM), exon 2 is
included only as a result of repression of the mutually exclusive exon
3 in SM cells (see Fig. 1C) (42). This repression is
mediated in part by high affinity PTB-binding sites on either side of
exon 3 (21, 26). In vitro splicing experiments have shown
that PTB mediates a low level of exon 3 repression in non-muscle extracts (29). However, in vivo, full repression is observed only in SM cells. In this respect, PTB-mediated repression of
-TM
exon 3 differs from all other characterized splicing events regulated
by PTB.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]UTP-labeled RNA probes (20 fmol) were incubated
with recombinant protein in 12 mM Hepes, pH 7.9, 100 mM KCl, 3% glycerol, 0.1 mM EDTA, 0.3 mM dithiothreitol, and 50 µg/ml E. coli
rRNA for 20 min at 30 °C. For competitive binding experiments, PTB4
and smPTB were premixed before addition of the RNA. Heparin was added
to 0.25 mg/ml, and the reaction was left for 5 min at room temperature.
Samples were irradiated on ice at 254 nm in a Spectrolinker
cross-linker with a controlled energy dose of 1.92 mJ. RNA was digested
with RNases T1 (1 unit/µl) and A (0.4 mg/ml), and the samples were
run on SDS-polyacrylamide gels.
-actinin was carried out as previously
described (29).
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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-TM and
-actinin and of the differential activity of the
alternatively spliced PTB isoforms upon
-TM splicing (29), we
decided to analyze expression of PTB in a well characterized model cell
system in which these events are regulated. Freshly isolated RASM cells
are initially highly differentiated, but, over the course of 4-6 days
in culture, become dedifferentiated and cease to express a number of
genes associated with the differentiated contractile state (43). We
analyzed alternative splicing of
-TM,
-actinin, and
vinculin/meta-vinculin in RNA from RASM cells at day 0 or 4 in culture and from the PAC-1 pulmonary artery SM cell line (Fig.
1). RT-PCR analysis of
-actinin
splicing indicated that, at day 0, the major product included the SM
exon, with a small amount containing the larger NM exon (Fig.
1A). A small quantity of product corresponding to skipping
of both the NM and SM exons was also observed. Like the SM isoform,
this "double-skipped" product, which has not been reported before,
would result in a nonfunctional EF-hand domain. By day 4, there were
roughly equal amounts of the SM and NM isoforms, with a decrease in the
amount of the skipped product. The cultured PAC-1 cells were similar to
the day 4 cells, but with more NM than SM inclusion and no double-skipped product. RT-PCR analysis of vinculin (Fig.
1B) showed that the meta-vinculin isoform was
expressed as a minor isoform only in day 0 RASM cells and was
undetectable in day 4 and PAC-1 cells. Mutually exclusive splicing of
-TM exons 2 and 3 produced products of identical size. To
differentiate them, the radiolabeled PCR products were digested with
XhoI, which cuts within exon 2 to produce a 145-nucleotide
product, or with PvuII, which cuts exon 3 products to
produce a 150-nucleotide band (Fig. 1C). Double digests
showed that the PCR product could be fully digested by both enzymes.
The almost complete XhoI digestion and PvuII
resistance of the day 0 PCR product showed that fully differentiated RASM cells predominantly expressed the exon 2-containing
-TM isoform. By day 4, PvuII digested a greater proportion of
the PCR product compared with XhoI, indicating a substantial
switch toward inclusion of exon 3 instead of exon 2. In comparison, in cultured PAC-1 cells, the majority of
-TM RNA contained exon 3. PAC-1 cells commonly show a greater degree of regulated splicing than
observed here (21, 42, 48), but they served as a useful undifferentiated control sample. Taken together, the data indicate that
the three alternative splicing events analyzed in RASM cells showed a
substantial switch toward the non-SM pattern after 4 days in
culture.
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Fig. 1.
Alternative splicing of
-actinin (A), vinculin
(B), and
-tropomyosin
(C) in SM cells. RNA harvested from day 0 (0) and day 4 (4) primary RASM cells and from the
PAC-1 SM cell line was analyzed by RT-PCR using a one end-labeled PCR
primer. The n lanes are PCR-negative controls
with no template added. The sizes of amplified bands (bp) are
shown to the left of each panel, and bands marked by an
asterisk are SM-specific splicing products. For
-TM, the
two spliced products, which are the same size, were differentiated by
digestion with XhoI (X lanes) or PvuII
(P lanes), which are specific for exon 2- and exon
3-containing products, respectively. The U lanes
are undigested PCR products, whereas the XP lanes
are double digests.
-TM and
-actinin, both of which
are regulated by PTB, we next analyzed expression of the PTB isoforms.
RT-PCR was carried out using primers P37 and P38, which correspond to
exons 8 and 11, respectively. This analysis allows the detection of
alternative splicing of exon 9, which gives rise to the PTB1 and PTB4
isoforms. Unlike
-TM,
-actinin, and vinculin, alternative
splicing of the PTB1 and PTB4 isoforms showed no significant changes
between the day 0 and 4 RASM and PAC-1 samples (Fig.
2B). Therefore, despite the fact that PTB4 has been shown to be a more active repressor of
-TM
exon 3 compared with PTB1 (29), changes in the ratio of the PTB
isoforms do not cause the switch in
-TM and
-actinin splicing in
dedifferentiating RASM cells. RT-PCR was not carried out under
conditions that would allow quantitative analysis of absolute levels of
expression. Nevertheless, we consistently observed that the levels of
PTB products appeared to be lower in day 0 cells than in day 4 cells.
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Fig. 2.
Novel PTB in differentiated SM cells.
A, alternative splicing of PTB exons 9 and 11. Inclusion of
exon 9 produces PTB4, whereas skipping leads to PTB1. Skipping of exon
11 produces mRNAs that encode truncated isoforms PTB1tr and PTB4tr.
The positions of primers P37, P38, and Pd3'2 used for PCR in
B and C are indicated. B, RT-PCR of
RNA harvested from RASM cells at day 0 (0) or day 4 (4) or from PAC-1 cells. PCR primers P37 and P38 correspond
to PTB exons 8 and 11, respectively. C, same as
B, but using primers P37 and Pd3'2, corresponding to PTB
exons 8 and 12, respectively, which also detect the novel smPTB
paralog. D, same as B and C, but using
primers specific for the smPTB paralog. Tissues from the uterus and vas
deferens were also tested with smPTB-specific primers. Size
markers are either HaeIII ØX174 (M lanes;
sizes are indicated in bp) or PCR products from a mixture of PTB1,
PTB2, and PTB4 plasmids.
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Fig. 3.
Amino acid alignment of rat PTB, smPTB, ROD1,
and nPTB. The amino acid sequences of the four rat paralogs
were aligned using Clustal, followed by some manual adjustment.
Positions identical between the four genes are shown by
asterisks, whereas conservative and semiconservative
alterations in the RNP2 and RNP1 boxes are marked by colons
and periods, respectively. The bipartite N-terminal nuclear
localization signal of PTB is underlined. RRM domains are
shown in blue, with the RNP2 and RNP1 boxes in
red. RRMs are as defined by PROSITE, with the exception that
RRM3 is extended to include the fifth -strand (underlined LTKD(Y/F))
determined by NMR (49).
-strand within RRM3
(49). The degree of identity to PTB within RRM1-4 is 78, 89, 71, and
75%, respectively. Within the characteristic RNP1 and RNP2 motifs,
most amino acid changes are conservative (Fig. 3). PTB and nPTB have
bipartite nuclear localization signals (37), the N-terminal half of
which appears to be lacking in both smPTB and ROD1. smPTB contains
sequences equivalent to PTB exon 9, which defines the PTB4 isoform. The
larger size of smPTB is accounted for by additional inserts of 36 amino
acids between RRM1 and RRM2 and 22 amino acids between RRM2 and RRM3.
These two linkers are the most divergent regions both between smPTB and
PTB, and also between rat and mouse smPTBs. Strikingly, the linker
region between RRM3 and RRM4 is identical in smPTB and the other
paralogs. This region was not observed in the NMR structure of PTB RRM3
and RRM4, suggesting that it is extremely flexible (49). The absolute
conservation suggests that the linker serves an important role and
perhaps takes up a defined structure upon RNA binding. PTB has three
sites that can be cleaved by caspase-3 (50). The most efficiently
cleaved site (LKTD138S) is conserved in nPTB and ROD1, but
not in smPTB. However, the next major site (AAVD170A in
PTB) is reasonably conserved in smPTB (SAVD178T),
suggesting that it may also be a target of caspase-3 during apoptosis.
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Fig. 4.
Comparison of PTB and smPTB expression in rat
tissues. RNA from rat tissues was analyzed by RT-PCR using primers
P37 and Pd3'2 (Fig. 2, A and C), which detect
both PTB and smPTB. The no DNA lane is a PCR-negative
control, whereas the PTB lane is a positive
control containing PTB plasmid markers. The identities of the bands are
shown to the right.
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Fig. 5.
In situ localization of the
smPTB transcript in mouse embryos. Sections of mouse
embryos at embryonic days 10 (E10) and day 14 (E14) were hybridized to sense and antisense riboprobes as
described under "Experimental Procedures." After developing, the
sections were stained with hematoxylin and photographed under
bright-field (upper panels) and dark-field (lower
panels) illumination. Hybridization to the sense probes gave
little or no signal (not shown). GT, giant trophoblasts;
C, costal cartilage; VP, hepatic venous plexus;
TB, terminal bronchioles.
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Fig. 6.
Nuclear localization of PTB-GFP, smPTB-GFP,
and GFP-smPTB in PAC-1 cells. Fusion proteins were transfected
into PAC-1 cells. Cells were visualized by green fluorescence
(left panels), phase contrast (middle panels),
and Hoechst staining for nuclei (right panels).
-tropomyosin were used in UV cross-linking assays with
recombinant smPTB and PTB4 (Fig. 7,
A and B). Both PTB and smPTB cross-linked to RNA
probes that contained the PTB-binding elements P3 and DY (probes 1-4),
but not to probe 5, which contains a UGC motif regulatory element.
Whereas PTB cross-linked with roughly equal efficiency to probes 1-4,
smPTB cross-linked more efficiently to probes 1 and 4, which contain
the DY regulatory sequence. These data confirm that smPTB is an
RNA-binding protein. To compare the affinity with which smPTB and PTB
bind to RNA, we carried out a competitive UV cross-linking assay. PTB4
and smPTB were premixed before incubation together with
-TM RNA
probe 1. While one protein was held at a constant concentration of 0.5 µM, the concentration of the other protein was varied.
smPTB was readily able to displace PTB (Fig. 7C, lanes
1-6). In contrast, titration of PTB4 led to a more gradual increase in its own cross-linking signal, and there was little evidence
of smPTB displacement (lanes 7-13). Similar results were seen with RNA probes 3 and 4 (data not shown). These data suggest that
smPTB binds to the RNA probes tested with higher affinity compared with
PTB.
View larger version (37K):
[in a new window]
Fig. 7.
smPTB binds RNA. A, schematic
representation of RNA probes from -TM regulatory sequences used for
UV cross-linking. Exon 3 is shown by the box. PTB-binding
regulatory elements P3 and DY are shown by rectangles, with
the vertical lines denoting the optimal PTB-binding sites.
Upstream regulatory element (URE) and DUGC
(diamonds) contain UGC (or CUG) motifs. B, UV
cross-linking of probes 1-5 to recombinant smPTB or PTB4.
C, competitive UV cross-linking of smPTB and PTB4 to probe
1. Lanes 1-6, PTB4 at a constant concentration of 0.5 µM, with smPTB at 0, 0.1, 0.25, 0.5, 1, and 1.5 µM; lanes 7-13, smPTB at a constant
concentration of 0.5 µM, with PTB4 at 0, 0.1, 0.25, 0.5, 1, 1.5, and 2 µM, respectively.
-TM constructs in PAC-1 SM and other cell types (data not shown).
This could be because there is already abundant PTB in these cells, and
smPTB does not have a significantly different activity on this
substrate. We also tested the effects of smPTB cotransfection with the
pA
-actinin splicing reporter into HeLa cells. This reporter
predominantly spliced to include the NM exon (Fig.
8). In control experiments, the pA
reporter has been shown to be unresponsive to other overexpressed proteins, including
-galactosidase, hnRNP-C, hnRNP-L, and
PTBtr1.2 Consistent with previous results (29),
overexpression of PTB led to skipping of both mutually exclusive exons
(Fig. 8, lanes 5 and 6). Overexpression of smPTB
had a similar effect, leading to enhanced skipping of both the NM and
SM exons (lanes 3 and 4), although it was not as
potent compared with PTB. This result establishes that smPTB has the
ability to act as a splicing repressor, with activity similar (but not
identical) to that of PTB.
View larger version (27K):
[in a new window]
Fig. 8.
smPTB is a splicing repressor. The
-actinin splicing reporter pA (200 ng) was cotransfected into HeLa
cells with two increasing concentrations (80 and 800 ng) of smPTB
(lanes 3 and 4) and PTB4 (lanes 5 and
6) expression plasmids. RNA was analyzed by RT-PCR using a
labeled PCR primer. Lane 1, no template (n);
lane 2, pA with no cotransfection; lane 7,
size markers, with sizes (bp) shown to the right.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-TM and
-actinin
alternative splicing in primary RASM cells and its high expression in
some SM tissues (Figs. 1, 2, and 4) initially suggested that it may
play a key role in regulating these two splicing events. However, some
SM tissues such as the intestine did not express smPTB, and
cotransfection of smPTB with either
-TM or
-actinin reporters did
not cause a significant switch toward the characteristic SM-specific
splicing pattern of each gene. One possibility is that smPTB plays no
role in these regulated splicing decisions. Other possibilities are that smPTB may simply replace the activity of PTB in these systems, that it may require a specific cofactor for activity in these systems,
or that it may not be active in the presence of PTB. Currently, we
cannot distinguish between these possibilities, although the fact that
it binds RNA in vitro with higher affinity than PTB argues
against the third possibility. In the future, it will be of interest to
test the activity of smPTB using in vitro splicing and
translation assays and also in cells after knockdown of endogenous PTB
by RNA interference (31).
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ACKNOWLEDGEMENT |
---|
We thank Christine Witchell for providing the rat aorta cells.
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FOOTNOTES |
---|
* This work was supported in part by Wellcome Program Grant 059879 (to C. W. J. S.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY223520.
Basic Science Lecturer supported by the British Heart Foundation.
§ To whom correspondence should be addressed. Tel.: 44-1223-333655; Fax: 44-1223-766002; E-mail: cwjs1@mole.bio.cam.ac.uk.
Published, JBC Papers in Press, February 10, 2003, DOI 10.1074/jbc.M210131200
2 M. C. Wollerton and C. W. J. Smith, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
hnRNPs, heterogeneous nuclear ribonucleoproteins;
PTB, polypyrimidine
tract-binding protein;
nPTB, neurally enriched PTB;
brPTB, brain
enriched PTB;
smPTB, smooth muscle PTB;
SM, smooth muscle;
NM, non-muscle;
RASM, rat aorta smooth muscle;
-TM,
-tropomyosin;
RRM, RNA recognition motif;
RT, reverse transcription;
RACE, rapid
amplification of cDNA ends;
GFP, green fluorescent protein;
contig, group of overlapping clones.
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