From the Department of Pediatrics and Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110
Received for publication, September 29, 2000, and in revised form, December 14, 2000
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ABSTRACT |
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We have been using the caspase-2 pre-mRNA as
a model system to study the importance of alternative splicing in the
regulation of programmed cell death. Inclusion or skipping of a
cassette-type exon in the 3' portion of this pre-mRNA leads to the
production of isoforms with antagonistic activity in apoptosis. We
previously identified a negative regulatory element (In100) located in
the intron downstream of alternative exon 9. The upstream portion of
this element harbors a decoy 3' acceptor site that engages in
nonproductive commitment complex interactions with the 5' splice site
of exon 9. This in turn confers a competitive advantage to the
exon-skipping splicing pattern. Further characterization of the In100
element reveals a second, functionally distinct, domain located
downstream from the decoy 3' acceptor site. This downstream domain
harbors several polypyrimidine track-binding protein (PTB)-binding sites. We show that PTB binding to these sites correlates with the
negative effect on exon 9 inclusion. Finally, we show that both domains
of the In100 element can function independently to repress exon 9 inclusion, although PTB binding in the vicinity of the decoy 3' splice
site can modulate its activity. Our results thus reveal a complex
composite element that regulates caspase-2 exon 9 alternative splicing
through a novel mechanism.
Pre-mRNA alternative splicing is an important mechanism for
higher eucaryotes to regulate cell type- and developmental
stage-specific gene expression. It provides a potential for an
extraordinarily high level of diversity in generating multiple, often
functionally distinct, protein isoforms from a single gene. In addition
to the basic splicing signals (5' splice site, branch point sequence and pyrimidine tract-AG), numerous sequence elements have been identified in exons or introns that can influence in various ways the
function of the splicing machinery (reviewed in Refs. 1-3). These
regulatory elements can, in some cases, mediate their effects in
cis, for example, through the formation of stem-loop
structures (e.g. Ref. 4), although they will usually
interact with trans-acting factors. Such factors often form
multicomponent complexes that can contain combinations of known
constitutive splicing factors, including
hnRNPs,1 snRNPs, and
serine-arginine (SR) proteins as well as novel specific alternative
splicing factors (e.g. Refs. 5-7). However, little is known
about the precise mechanisms by which these specific complexes interact
with and influence the function of the splicing machinery. Similarly,
the process of splice site selection in complex pre-mRNAs is still
a poorly understood phenomenon (8-10).
One mechanism for intronic elements to function in repressing splicing
was first described in Drosophila. The sex-specific splicing
factor Sex-lethal (SXL) regulates the splicing of transformer (tra) pre-mRNA by competing with U2AF65 for
binding to the polypyrimidine tract of the regulated 3' splice site.
This permits the use of an alternative 3' splice site that is normally
not selected, thus resulting in the production of a functional TRA
protein only in female flies (11-16). Direct competition with
constitutive splicing factors has been shown to be a conserved strategy
for vertebrate alternative 3' splice site choice and often involves the
polypyrimidine tract binding protein (PTB/hnRNP I) (15, 17-19). PTB
was first identified ~10 years ago (20, 21) and then found to have
features of an hnRNP protein (22). It was only recently that PTB was
recognized as an important player in alternative splicing regulation
(reviewed in Ref. 23). Nevertheless, the precise mechanism by which PTB
influences splicing is still unclear, even though it has been
implicated in the alternative splicing of a number of genes.
We have been using the caspase-2 (also known as Ich-1 (interleukin-1 Plasmid Constructions and Splicing Substrates--
C2 and C3
have been described previously (24). Various portions of the In100
element were amplified using PCR with specific oligonucleotides. Each
fragment was either inserted in pcDNA3 (Invitrogen) in
front of the T7 RNA polymerase promoter or at a BglII site
in intron 9 of C3 to generate the respective C3 derivative (e.g. C3In50up, C3In50dn). In50up Transfections and RT-PCR--
HeLa cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum and were seeded at In Vitro Splicing Assays--
Splicing substrates were
synthesized using T7 RNA polymerase (Promega) except for i9
derivatives, which was with T3 RNA polymerase. Final concentrations of
reagents were as follows in a 10-µl reaction: 500 ng of linearized
DNA template, 0.4 mM ATP and CTP, 0.1 mM GTP
and UTP, 0.84 mM GpppG cap analogue (Amersham Pharmacia
Biotech), 10 mM dithiothreitol, 0.5 unit/µl RNasin
(Promega), 1× Transcription Buffer (Promega), 20 µCi of
[ UV Cross-linking Assays--
Splicing reactions were set up as
above except that higher specific activity radiolabeled substrates were
used. Trans-competition assays were carried out as above, except that
350-500 fmol of cold RNA competitors were used and the pre-incubation
was for 10 min on ice. 6-µl aliquots were transferred onto a 96-well
microtiter plate previously cooled at Immunoprecipitation Assays--
Cross-linking was carried out as
described above except that, following the RNase treatment, samples
were incubated with 20% fetal calf serum (as a normal serum control)
or with various antibodies. Pre-blocked Protein A/G-agarose beads were
then added with further incubation and gentle rocking. The RNA-labeled
proteins retained on the beads after several washings were eluted and
resolved on SDS-PAGE.
Electrophoretic Separation of Splicing Complexes--
This
procedure was adapted from Refs. 39 and 40. 4-µl aliquots were
removed from standard splicing reactions at indicated time points and
mixed with 1 µl of heparin at 1 mg/ml. 0.5 µl of loading buffer
(1× TBE, 20% glycerol, 1% bromephenol blue, and 1% xylene cyanol)
was then added and the samples loaded on nondenaturing 4%
polyacrylamide gels (acrylamide:bisacrylamide = 80:1), which had
been pre-electrophoresed at 200 V for 30 min in 50 mM
Tris-glycine. Electrophoresis was then continued under the same
conditions for 4-5 h at room temperature. ATP depletion of the nuclear
extracts was achieved as in Ref. 41 by pre-incubating the extracts at
room temperature for 20-30 min. without addition of hexokinase and
glucose. ATP, MgCl2, and creatine phosphate were omitted
from these splicing reaction mixtures.
The In100 Element Can Be Separated into Two Functionally Distinct
Regions--
Deletion mutagenesis experiments revealed a negative
regulatory element (In100) located in the intron downstream of
alternative exon 9 (Ref. 32 and Fig. 1B, compare
lanes 1 and 2). To assess the
potential involvement of trans-acting factors and dissect the
functional domains of the In100 element, a series of small RNAs
corresponding to various regions of the element (Fig.
1A) were produced in
vitro and used in splicing trans-competition assays. Specifically,
cold RNA competitors (0.5-1 pmol) were mixed with HeLa splicing
reactions and pre-incubated for 10 min at 30 °C.
32P-Labeled pre-mRNA substrates were then added and the
incubation continued for 2 h. When added in trans to a splicing
reaction containing C2 substrates, the In100 RNA promoted a complete
shift toward the production of the casp-2S exon 9-inclusion
mRNA (Fig. 1B, lane 3). This was
detected by monitoring the relative ratio of casp-2S to
casp-2L mRNAs and corresponding lariat molecules (see
Fig. 1B; a black arrow represents
casp-2L, and a white arrow marks
casp-2S products/intermediate). This observation suggested that In100 mediated its negative effect on exon 9 inclusion at least in
part through the involvement of trans-acting factors. These factors are
titrated away by the excess amount of In100 competitor RNA and the
inhibition is relieved. The same de-repression could also be observed
with RNA competitors spanning the entire 3' portion of the element
(In75dn and In50dn; Fig. 1B (lanes 7 and 8) and see Fig. 3). In contrast, competitor RNAs
spanning the upstream (In50up, In75up, In30up, and In20up) portion of
the element were unable to induce any shift in the splicing profile when added in trans (Fig. 1, B (lanes
4-6) and A, respectively). This is consistent
with the presence of a sequence resembling a 3' acceptor site in this
region, which may act as a decoy 3' splice site for interacting with
the 5' splice site of exon 9 (32). According to this model, the
exon-skipping splicing pattern would be competitively favored over the
exon inclusion through a cis-mediated mechanism and this fragment would
not be expected to have any effect when added in trans.
Finally, a centrally located fragment as well as a minimal RNA
transcript spanning the last 25 nucleotides of the In100 element were
also unable to release the inhibition of exon 9 inclusion (Fig.
1B, lanes 9 and 10 and lanes 11 and 12, respectively). These
results thus define a minimal region (In50dn; denoted in
black in Fig. 1A) downstream of the decoy 3'
splice site in In100 that requires interaction with nuclear factors for
its activity.
PTB Specifically Interacts with the In100 Regulatory
Element--
We used a UV cross-linking assay as a first step toward
the identification of nuclear factors interacting with In100. HeLa nuclear extracts were first pre-incubated under splicing conditions with the respective RNA competitors before C2 substrates were added and
the samples UV-irradiated for 10 min. Following RNase treatment, the
covalently cross-linked, RNA-labeled proteins were resolved on
SDS-PAGE. Using this method, we identified two bands with apparent
molecular masses of 60 and 35 kDa, respectively, that were specifically
competed away by the addition of cold In100 RNA in trans
(Fig. 2B, compare
lanes 2 and 3 with lane
1). The 60-kDa protein could be titrated away specifically
by the In50dn fragment, whereas the 35-kDa protein was competed away by
the In50up fragment (Fig. 2B, lanes 6 and 7 and lanes 4 and 5,
respectively). These results show that functionally distinct domains of
the In100 regulatory element are recognized by different protein
factors. Several observations led us to consider PTB as a likely
candidate for the 60-kDa band observed on SDS-PAGE in our cross-linking experiments. First, putative PTB-binding sites are present in the
In50dn portion of the element (see Fig. 2A): specifically, two copies of the "core" PTB consensus motif UCUU(C) within a pyrimidine-rich context (38) and two copies of the UUCUCU(CU), which
was identified previously as a candidate PTB splicing repressor motif
(19, 42). Second, PTB was one of the known splicing factors in this
range of molecular weight. Third, longer gel migrations resolved the
60-kDa species as a doublet, typical of PTB. To test whether the 60-kDa
cross-linking band was PTB, we used a cross-linking assay followed by
immunoprecipitation with specific antibodies. Specifically, a labeled
RNA substrate consisting of the complete In100 element was used in UV
cross-linking as before (Fig. 2C, lane
1) except that aliquots were taken, incubated with
antibodies, and the immunoselected proteins recuperated using Protein
A/G-agarose beads. Using this strategy, we show that a monoclonal
antibody directed against PTB was able to immunoprecipitate
specifically the 60-kDa doublet cross-linked on In100 (Fig.
2C, lane 3). In contrast, no proteins
were selected if a control serum or an unrelated antibody was used
(Fig. 2C (lane 2) and data not shown,
respectively). These results clearly identify the 60-kDa band as
PTB.
We next wanted to map precisely where PTB was binding on the In100
element. To do this, 32P-labeled short RNA substrates (see
Fig. 1A) were used to carry out the UV cross-linking assay
as described above. As expected, PTB in the nuclear extract
cross-linked exclusively on the In50dn region of the element (Fig.
2D, compare lane 4 to lane
3). Furthermore, consistent with the distribution of the
PTB-binding sites, the entire In50dn fragment was required for strong
binding of PTB, and an RNA containing only two (In25) or one (In40) out
of four sites (see Figs. 1A and 2A) permitted
only a weak interaction or no detectable interaction (Fig.
2D, lanes 6 and 5,
respectively). This suggested that PTB may bind cooperatively on the
PTB-binding site array, although we have not yet studied in detail the
precise stoichiometry of this interaction. The other protein species
cross-linked to In100 (including the 35-kDa species) all seem to
interact specifically with the In50up fragment, with the exception of
U2AF65, which cross-linked with equivalent efficiency to
both In50up and In50dn substrates (Fig. 2D, compare
lanes 3 and 4). The identity of
U2AF65 was assessed on the basis of its comigration with
purified U2AF65 cross-linked on In100 as well as by
immunoprecipitation with a specific antibody (Fig. 2D,
compare lanes 2-4 with lane
1 and Ref. 32).
Finally, we used site-specific mutagenesis to confirm that the
consensus PTB-binding sites were indeed required to maintain strong PTB
cross-linking to In50dn. Multiple purine insertions were made in each
of the four sites present in In50dn, and the mutated fragment was used
in a UV cross-linking assay using purified histidine-tagged PTB
(In50dn PTB Can Repress Exon 9 Inclusion--
To address the functional
significance of PTB binding to the In100 element, we used the in
vitro splicing competition assay. Splicing reactions were carried
out using the C2 substrate (which contains In100) with increasing
amounts of cold In50dn RNA added in trans as described (see
Fig. 1). This promoted a concentration-dependent derepression of exon 9 inclusion (Fig. 3,
lanes 2-4). Addition of purified His-PTB protein
to this de-repressed reaction shifted the splicing profile back toward
the exon 9 skipping splicing profile (Fig. 3, lanes
5 and 6). In contrast, addition of similar amounts of purified histidine-tagged hnRNP A1 or other RNA-binding proteins to the de-repressed reaction had no effect on the splicing profile (Fig. 3, lane 7 and data not shown).
Finally, In50dn Both Domains of the In100 Element Can Function Independently to
Repress Exon 9 Inclusion--
The upstream portion of the In100
element, which acts as a decoy 3' acceptor site can facilitate the
skipping of exon 9 on its own, i.e. when the downstream
PTB-binding region is deleted from the substrate (32). In this isolated
context, mutating all of the 3' splice site signals (branch point,
Py-tract, and AG dinucleotide) abolished completely the effect of
In50up (see In50up PTB Can Affect 5' Splice Site Selection--
The complete In100
element was shown to be able to negatively affect 5' splice site
selection of exon 9 in the context of a substrate where the 3' splice
site of the alternative exon 9 is mutated (32). According to the decoy
3' acceptor site model we proposed for In50up, it is expected that this
domain should be functionally active in the context of such a 553 substrate. On the other hand, we were interested in testing if PTB
binding to In50dn could also affect 5' splice site selection. To
address these questions, the In50up and In50dn fragments (wild-type and mutated) were individually inserted in C3-553 to replace the In100 sequence. The splicing profile of these substrates was assessed in vitro following incubation in HeLa nuclear extracts under
splicing conditions. As expected, in the presence of the In50up
fragment, splicing proceeded principally to the distal 5' splice site
of exon 8 (Fig. 5, compare
lane 2 to lane 1). Mutating
the branch site, Py-tract, and the AG dinucleotide in In50up abolished
this inhibitory effect on the proximal 5' splice site and shifted the relative splicing profile from 90% distal 5' splice site usage to
~50% usage of both splice sites (Fig. 5, lane
3). We observed that these mutations also reduced overall
splicing efficiency. Interestingly, the In50dn fragment also affected
5' splice site selection and led to predominant usage of the distal 5'
splice site (Fig. 5, In50dn-553, lane
4). Mutating the CU-rich sequences in In50dn promoted a
small, but reproducible, shift in the relative splicing ratio, from
90% distal/10% proximal to ~70% distal/30% proximal (Fig. 5,
In50dn PTB Binding Sites Downstream of the Decoy 3' Acceptor Site Can
Modulate Its Recognition--
Although we had observed that each of
the two domains of In100 element could function independently, we were
intrigued by their juxtaposed arrangement in the pre-mRNA and asked
if they could influence each other in any way. If placed in a substrate containing only exon 9 with its 5' splice site and no downstream 3'
splice site, In100 could be used as a bona fide
3' acceptor site in this i9In100 substrate, although with greatly
reduced efficiency (Fig. 6A
(lanes 1 and 2) and Ref. 32). In
contrast, In50dn could not be used as a 3' acceptor site when placed
downstream of exon 9 in an isolated i9In50dn substrate (Fig.
6A, lanes 5 and 6).
However, utilization of the decoy 3' acceptor site increased 5-fold if
the In50dn domain was deleted from i9In100 to yield i9In50up (Fig.
6A, lanes 3 and 4). This
suggested that PTB binding to In50dn may affect the recognition of the
decoy 3' splice site and the formation of spliceosome complexes on this
site. We then compared splicesome assembly on minimal substrates
containing or lacking PTB binding sites. When incubated in Hela nuclear
extracts under splicing conditions, the i9In100 substrate, which
contains the intact In100 element, supported the formation of
apparently normal spliceosomal complexes A, B, and C. However, the
splicing complex formation was much less efficient (or less stable)
with i9In100 than with C3i9, a substrate containing the natural 3' splice site of exon 10 (see Ref. 32 and Fig. 6B, compare
lanes 9-12 with lanes
1-4, respectively). When tested in this assay, i9In50up,
which does not contain the PTB-binding sites, promoted the formation of
a slightly faster migrating complex (Fig. 6B, lanes 5-8). In addition, this complex formed
very efficiently on i9In50up, as can be assessed by the level of
substrate already converted into the retarded species at the 15-min
time point (Fig. 6B, compare lane 8 with lane 12). Interestingly, in contrast to normal U2-dependent splicesomal complexes that are
ATP-dependent, this complex was partially resistant to ATP
depletion (Fig. 6B, lanes 13 and
14). Finally, consistent with the results obtain in splicing
assays, the presence of In50dn by itself did not promote formation of
any complexes detectable by native gel electrophoresis (Fig.
6B, lanes 15 and 16). Taken
together, these results show that, under certain conditions, PTB is
capable of modulating the activity of the adjacent In50up element,
possibly by promoting the formation of a distinct, aberrant complex on
the decoy 3' acceptor site.
In this study, we have characterized a repressor element (In100)
located in the intron downstream of caspase-2 alternative exon 9. We
have showed previously that the upstream portion of this element
consists of a decoy 3' acceptor site that interacts nonproductively
with the 5' splice site of the alternative exon, which in turn
competitively favors skipping of exon 9. We now uncover in this element
a second functionally distinct domain (In50dn) juxtaposed downstream of
the decoy 3' acceptor site. PTB binding to CU-rich motifs in In50dn
correlated well with the repressor activity of this domain.
Interestingly, this negative effect of PTB binding site-containing
In50dn can also be observed in the context of competing 5' splice
sites. Finally, we provide evidence supporting that PTB can modulate
recognition of the adjacent decoy 3' acceptor site and discuss the
possible implications for regulation.
In100 Contains Two Domains--
The In100 element is composed of
two juxtaposed subdomains that can repress exon 9 inclusion using
distinct mechanisms. This bipartite architecture is also found in other
exonic or intronic elements, although in those cases it usually
involves the overlapping of positive and negative elements (19, 42,
46-50). In a few instances, two different types of splicing enhancers
(e.g. Pu-rich and CA-rich) were found in the same exon
(51-54). Another example of an intronic repressor that can be
separated into two mechanistically distinct subelements was identified
in the hnRNP A1 pre-mRNA downstream of the alternative exon 7B;
however, the mechanism of action is different (55). The upstream
portion of hnRNP A1 intronic element can specifically repress the 3'
splice site of the alternative exon, and the downstream portion binds
hnRNP A1 and may interact with a second hnRNP A1 molecule bound in the
upstream intron, thus favoring exon skipping through a looping-out of
the alternative exon (55). We have observed that the complete In100
element can also negatively affect the 3' splice site selection of exon 9 although we have not yet characterized which domain of the element was responsible for this effect (Côté and Wu, unpublished results). A recent study suggests that intrinsically defective splice sites and
negative elements play important roles in distinguishing the authentic
constitutive splice sites from the vast number of pseudo-splicing signals (56). Interestingly, the In100 element in the casp-2 pre-mRNA presents just this architecture: a pseudo-splice site juxtaposed with a repressor element. Our results suggest that the
adjacent repressor element may help in preventing the decoy 3' splice
site from being used in splicing. A data base search suggests that
In100 sequence motif, a decoy 3' splice site juxtaposed to a
pyrimidine-rich element, may be present in other genes undergoing alternative splicing and may represent a general intronic splicing regulatory element (data not shown). The involvement of such a pseudo-splice site in alternative splicing regulation might explain the
phylogenic conservation of pseudo-splicing signals in mammalian introns. Further investigation is necessary to prove the generality of
such splicing regulatory motifs.
PTB Can Repress Exon 9 Inclusion--
PTB has been implicated in
the alternative splicing of several pre-mRNAs (15, 17, 19, 38, 42,
57-62). Nevertheless, how PTB mediates its negative effect on splicing
is still unclear. In one scenario, PTB was found to bind in the 3'
splice site region of some alternative exons, preventing constitutive
splicing factors to recognize these sites. This is the case in the
GABAA receptor
To gain some more insights into the mechanism by which PTB acts, we
have used 553 splicing substrates harboring the wild type (binds PTB)
or mutated (no PTB binding) In50dn fragment (see Fig. 5,
In50dn-553 and In50dn Involvement of Other Trans-acting Factors--
We have focused our
discussion exclusively on PTB in the preceding section, but it is clear
that additional sequence elements and trans-acting factors are also
involved in the alternative splicing of these various pre-mRNAs.
Similarly, in the caspase-2 pre-mRNA, PTB binding to In50dn is most
likely part of a coordinated mechanism (including the decoy 3' acceptor
site) for maintaining the low level of inclusion of exon 9 in most cell
types. There may be additional trans-acting factors involved in this
event. In this sense, we have previously reported that certain SR
proteins as well as hnRNP A1 were able to modulate exon 9 alternative
splicing (24), although this effect could be observe independently of the presence of In100 in the
pre-mRNA.2
Nevertheless, this does not necessarily rule out the possibility of a
cross-talk between two distinct regulatory pathways in specific cellular contexts. We have not yet been able to identify the 35-kDa protein that specifically interacts with the In50up portion of the
element (see Fig. 2B). In addition, we have noticed the
appearance of a new cross-linked species upon addition of increasing
amount of cold In100 RNA competitor in trans (see Fig.
2B, lanes 2 and 3). It is
possible that sequestering of PTB or the 35-kDa protein with large
amounts of In100 RNA permits the binding of a regulatory protein to the
casp-2 pre-mRNA. This could reflect certain physiological situations in which the repression of exon 9 inclusion needs to be
relieved, for instance, in neuronal cells where significantly more exon
9 inclusion has been observed. Finally, a unique 50-kDa band was also
detected when the minimal In40 RNA was used in UV cross-linking
experiments (see Fig. 2D, lane 5). One
potential candidate for this 50-kDa protein is CUG-BP/hNab50, a
conserved 50-kDa hnRNP protein that has been associated with certain
splicing phenotype in myotonic dystrophy (66, 67). Interestingly, it was recently proposed that CUG-BP could compete for PTB binding on a
repressor element upstream of a neurospecific exon in the clathrin
light chain B pre-mRNA (68). Further experiments will be required
to determine the identity of these proteins as well as their role in
caspase-2 alternative splicing regulation.
Caspase-2 Alternative Splicing Regulation--
The results
presented in Figs. 4 and 6 might seem somewhat difficult to reconcile,
namely each domain of the element is able to work independently of the
other in inhibiting exon 9 inclusion, suggesting they are functionally
redundant, although they mediate their effect via distinct mechanisms.
On the other hand, the presence of PTB-binding sites immediately
downstream of the 3' acceptor site in In50up can suppress the usage of
this 3' splice site. This effect of PTB-binding sites was also examined
by monitoring the formation of spliceosomal complexes. When PTB-binding
sites are present, seemingly normal complexes are formed, but with
greatly reduced efficiency (see Fig. 6B, lanes
9-12). In contrast, the decoy 3' acceptor site by itself
without the downstream PTB-binding sites promoted the more efficient
formation of a faster migrating complex that is now partially resistant
to ATP depletion. One possibility consistent with the results shown in
Fig. 6 is that PTB binding in the vicinity of the decoy 3' acceptor
site may contribute to the suppression of the usage of the decoy splice site as a normal 3' splice site. It is conceivable that the weaker PTB
binding at other sites in the pre-mRNA may be sufficient to silence
the decoy 3' acceptor site in In50up when the In50dn domain is not
present. In support of this hypothesis, two additional UCUU motifs can
be found just upstream of In100 and cross-linking experiments are
indicative of a residual binding of PTB in the absence of In100 (32).
Alternatively, this bipartite arrangement might be required only in
tissues where inclusion of exon 9 needs to be de-repressed and where
additional factors may be involved (e.g. neurons).
Interestingly, identification of a brain-enriched form of PTB (nPTB)
has been reported (19, 42, 69). Recently, it was reported that this
nPTB and the previously known PTB show significant differences in their
properties (65). Specifically, nPTB binds more strongly than PTB to the
downstream conserved sequence regulatory element in the
c-src pre-mRNA, while being a weaker repressor of
splicing in vitro (65). It is an attractive possibility that
this new PTB protein might also interact with In50dn in neuronal tissues.
It is our hope that further investigation in these avenues will reveal
new information on the communication among different components of the
splicing machinery as well as between spliceosome and signal
transduction pathways.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
converting enzyme homologue 1) or Nedd2) pre-mRNA as a model system
to study the importance of alternative splicing regulation in the
process of programmed cell death (24). Inclusion or skipping of 61-base
pair exon 9 in the 3' end of this pre-mRNA leads to the formation
of two mRNAs encoding protein isoforms with antagonistic activities
in apoptosis (25). CASP-2L is derived from the skipping of
alternative exon 9 and can induce cell death in a variety of cells
(25-29). CASP-2S is a truncated version of the protein
produced because of a premature termination codon created by the
inclusion of exon 9. Overexpression of CASP-2S has been
shown to prevent apoptotic death (25, 30). Recently, the generation of
caspase-2-deficient mice has provided evidence that suggests an
important role for caspase-2 as both a positive and negative cell death
effector (31). We have previously implicated SF2/ASF and hnRNP A1 as
modulators of caspase-2 exon 9 alternative splicing (24). Using a
systematic mutagenesis approach, we have identified an intronic
regulatory element (In100) located 140 nucleotides downstream of exon
9, that can repress inclusion of the alternative exon. The upstream
portion of that element harbors sequences highly resembling a typical
3' acceptor site. We have shown that this upstream region can behave as
a normal 3' splice site in certain conditions and can promote the
assembly of stable U1 snRNP-dependent complexes on the 5'
splice site of nearby exon 9. Based on our results, we proposed a model
in which this region would act as a "decoy" 3' acceptor site,
engaging in nonproductive splicing complexes with the 5' splice site of
the alternative exons, thus competitively favoring the pairing of exons
8 and 10 (32). We now show that PTB interacts with a region downstream of the decoy 3' acceptor site in In100 and represses inclusion of the
alternative exon. Furthermore, we find that each domain of the In100
element can function independently to repress exon 9 inclusion using
distinct mechanisms.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and In50dn
were
generated using overlapping mutagenic oligos. Oligos were hybridized,
blunt-ended using Klenow fragment polymerase and inserted either in
pcDNA3 or at a BglII site in intron 9 of C3
to generate C3In50up
and C3In50dn
. Specific mutations were
introduced in the upstream (In100up
) or downstream (In100dn
)
portion of the In100 fragment using "PCR overlap-extension
mutagenesis" (33). Mutagenic fragments were digested and substituted
back into the C2 plasmid DNA. PCR was again used to mutate exon 9 3'
splice site AG to a CU. This fragment was then substituted into
C3In50up, C3In50up
, C3In50dn, or C3In50dn
) at HindIII
and BamHI sites to generate the respective 553 derivative.
C3i9 consists of the Ex9-10 splicing unit with the In100 sequences
deleted. i9 and i9In100 were generated by linearizing the corresponding
C3i9 or C2i9 (containing In100) plasmid at the BglII site
described above. i9In50up and i9In50dn were generated in a similar
manner from the corresponding C3 constructs. Plasmids containing small
fragments of the element were linearized at a BamHI site
(present in 3' oligos), and all C2 and C3 derivatives were linearized
at an XhoI site in exon 10.
2 × 105 cells/well in a
six-well dish, 24 h prior to transfection. Transfection was done
using a standard calcium phosphate precipitation procedure with 3 µg
of DNA. Transfection efficiency was routinely
60%, as evaluated by
cotransfection with a green fluorescent protein marker. Cells were
harvested 48 h after transfection, and the RNA extracted using
Trizol reagent (Life Technologies, Inc.). Spliced products derived from
the expressed minigenes were detected using RT-PCR as described
previously (24) and resolved on 6% polyacrylamide 1× TBE gels.
-32P]UTP, and 1 unit/µl RNA polymerase. Samples
were then treated with 0.1 unit/µl DNase I (Promega) for 15 min and
ethanol-precipitated, and the full-length transcripts were gel-purified
as described (34). Synthesis of cold competitor RNAs was in a scaled-up
100-µl reaction with the following modifications: 0.5 mM
UTP and 0.42 mM cap analogue were used, and
[
-32P]UTP was omitted. HeLa cell nuclear extracts were
prepared according to previously established protocols and contained
20 mg/ml total proteins (35). Splicing reactions were set up and
processed as previously described (36) except that some batches of
nuclear extracts were supplemented with 1 unit of creatine kinase
(Sigma). 2 fmol of RNA substrates was added and the samples incubated
at 30 °C for 2 h unless otherwise mentioned. For
trans-competition experiments, 0.5-1 pmol of cold RNAs were
pre-incubated with nuclear extracts at 30 °C for 10 min prior to
addition of the substrate and incubation under splicing conditions.
Splicing products were resolved on 8% polyacrylamide, 8 M
urea gels. The identity of lariat molecules was determined by
performing a debranching reaction in a S100 extract (37) followed by
gel migration alongside molecular weight markers. The other products
were identified according to size and comigration with pertinent
partial splicing substrates.
20 °C and irradiated with
1 Joule in a UV Stratalinker 1800 (Stratagene). Samples were then
treated for 30 min. at 37 °C with one volume of RNase A (5 mg/ml).
Radiolabeled cross-linked proteins were boiled for 5 min in 1× SDS
loading buffer and separated on 12.5% SDS-PAGE. Histidine-tagged PTB
expression vector was a gift from Dr. J. G. Patton and was
expressed and purified from Escherichia coli as described
(38). Purified U2AF65 was a generous gift from Dr. Rui-Ming
Xu. Histidine-tagged hnRNP A1 was expressed and purified from E. coli using standard procedure.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (36K):
[in a new window]
Fig. 1.
The In100 element can be dissected into two
functionally distinct regions. A, structure of the C2
substrate and RNA competitors used. C3 is identical to C2 except that
In100 is deleted. B, in vitro splicing assays in
the presence of cold competitor RNAs. HeLa nuclear extracts were
pre-incubated for 10 min at 30 °C in the presence of 0.5-1 pmol of
cold competitor RNAs (as indicated above each
lane). 32P-Labeled C2 substrate was then added
with the incubation continued under splicing conditions for 2 h.
The positions of casp-2S and casp-2L mRNAs
and splicing intermediates are indicated by open and
black triangles, respectively. Addition of cold
In100 or In75dn RNAs led to a shift toward more inclusion of exon 9, whereas In50up, In25dn, or In40 did not have the effect.
View larger version (48K):
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Fig. 2.
PTB specifically interacts with the In100
regulatory element. A, putative PTB binding sites are
indicated above the sequence. The core PTB consensus motifs UCUU(C) are
in lowercase, and the UUCUCU(CU) motifs identified
previously as candidate PTB splicing repressor sites (Refs. 19 and 42)
are indicated by a line. Also marked are the splicing
signals in the decoy 3' acceptor site (BS; consensus:
YNYYRÅY, where Y represents pyrimidines; R,
purine, and N, any nucleotide) followed by a long stretch of
pyrimidines ((pY)n) and (NY)AG (putative 3' cleavage site is
indicated by an arrow). B, two specific bands are
revealed by UV cross-linking/competition experiments. Splicing
reactions were set up as before using the C2 substrate with In100,
In50up, or In50dn as cold RNA competitor (0.25-0.5 pmol). Aliquots
were UV-irradiated and treated with RNase A, and the RNA-labeled
proteins resolved on 12.5% SDS-PAGE. Arrows indicate
positions and sizes of cross-linked proteins specifically interacting
with distinct portion of the In100 element. C, UV
cross-linking/immunoprecipitation experiment. Samples were processed as
before for cross-linking except that antibodies were added following
the RNase A treatment. After incubation on ice, protein A/G-agarose
beads were mixed in and the incubation continued at 4 °C. The beads
were then thoroughly washed, and the retained proteins were resolved on
12.5% SDS-PAGE. FCS, fetal calf serum. D, a UV
cross-linking experiment was carried out as before with various
portions of In100 as labeled RNA transcripts in the presence of HeLa
nuclear extracts or purified U2AF65. Position of the PTB
doublet is indicated on the right. E, UV
cross-linking experiment was carried out using purified His-tagged PTB
with different labeled RNA transcripts as shown above the
autoradiograph.
; Fig. 2A). As was observed in the nuclear
extract, His-PTB cross-linked specifically with the In50dn RNA
substrate, and the signal of this cross-linking is as strong as that
detected with the total In100 element (Fig. 2E,
lanes 1-3). As expected, no detectable
cross-linking was observed when the In50dn
was used (Fig.
2E, lane 4). Taken together, these results revealed a specific interaction of PTB with consensus CU-rich
sequences located in the downstream portion of In100.
, in which PTB binding sites were mutated (see Fig.
2E), was unable to promote derepression of exon 9 inclusion
when used as a trans-competitor (Fig. 3, lane 8).
These results strongly suggest a role for PTB binding to In50dn in the
repression of alternative exon 9 inclusion.
View larger version (49K):
[in a new window]
Fig. 3.
PTB can repress exon 9 inclusion.
Splicing reactions were carried out as before with In50dn added in
trans to de-repress exon 9 inclusion (lanes
2-4). Following pre-incubation with cold In50dn RNA,
purified His-tagged PTB (lanes 5 and
6) or hnRNP A1 (lane 7) was added to
the reaction and the incubation continued for 2 h. De-repression
was reversed by addition of purified PTB (lanes 5 and 6) but not by hnRNP A1 (lane 7).
No de-repression was detected when In50dn RNA was used as a
competitor in trans (lane 8). The
positions of casp-2S and casp-2L mRNAs, and
splicing intermediates are marked by open and
black triangles, respectively.
in Fig.
4A). In contrast, when tested
in the context of the C2 substrate, i.e. in the presence of
both portions of the element, these mutations had no effect on the
splicing profile (Fig. 4, A (In100up
) and
B (lane 2)). One possible explanation for these observations is that in this particular situation, PTB binding to the downstream portion of In100 was responsible for the
repression observed. To test this hypothesis, we mutated the PTB-binding sites either in the context of the complete In100 element
or in the isolated In50dn (see Fig. 4A,
In100dn
and In50dn
, respectively). The
mutant DNA constructs were transfected in HeLa cells and the resulting
splicing products were analyzed using RT-PCR with oligonucleotides
located in the flanking exons 8 and 10. First, mutating PTB-binding
sites downstream of the "functional" decoy acceptor site while
keeping In50up intact had no effect on the level of exon 9 inclusion
(Fig. 4B, lane 3). Similarly, complete
removal of the upstream portion of the In100 element (In50up) while
retaining In50dn did not promote any change in the splicing profile
(Fig. 4B, lane 5), suggesting In50dn
could indeed function on its own to inhibit exon 9 inclusion.
Supporting this idea, mutating the CU-rich PTB-binding sequences in
this context, in the absence of In50up, promoted a strong derepression of exon 9 inclusion, indicating the involvement of PTB binding in the
negative effect (Fig. 4B, lane 6).
Interestingly, retaining only two out of four PTB-binding sites in
In50dn yielded an intermediate effect on the splicing profile (Fig.
4B, lane 4), suggesting again the
requirement of multiple PTB molecules interacting with the element to
maintain sufficient repression. These results demonstrate that each
domain of the In100 element, the upstream "decoy 3' splice site"
sequence and the downstream PTB binding region, can function
independently of each other to repress alternative exon 9 inclusion.
View larger version (30K):
[in a new window]
Fig. 4.
Both domains of In100 can function
independently. A, structure of C3 substrates with the
various fragments reinserted in the downstream intron 9. The level of
repression of exon 9 inclusion mediated by each fragment is indicated
by + or signs. B, C3 derivatives were transfected
into HeLa cells. Cells were harvested 48 h after transfection, and
RNA splicing products were detected using RT-PCR. The positions of
products derived from casp-2S and casp-2L
mRNAs are as indicated.
-553, lane 5).
This result suggests that, in this In50dn-containing substrate, PTB
binding may be required for its effect on 5' splice site selection.
Since in this context, the splicing machinery has to choose between two
competing 5' splice sites for pairing with a common downstream 3'
splice site, one attractive hypothesis is that PTB might act at the
level of U1 snRNP binding. We have performed a specific RNase H
protection assay to monitor U1 snRNP occupancy on each respective 5'
splice site (32, 43-45). No significant difference in the profile of U1 snRNP-dependent protection was observed using this assay
whether the In50dn element was present in the transcript or not (data not shown). This suggests that the repression effect of PTB on the
alternative exon 9 inclusion is likely mediated by influencing events
other than U1 snRNP binding (see "Discussion").
View larger version (39K):
[in a new window]
Fig. 5.
In50dn can affect 5' splice site
selection. Substrates were incubated in HeLa nuclear extracts for
2 h under splicing conditions. Position of the splicing products
generated from the use of the proximal (Prox.) or distal
(Dist.) splice site is indicated by a white or
black rectangle, respectively. The
asterisk on the left denotes aberrant splicing
products not reproducibly observed in our splicing reactions.
View larger version (42K):
[in a new window]
Fig. 6.
PTB binding sites downstream of the decoy 3'
acceptor site can modulate its recognition. A, standard
splicing reactions (2 h) were set up using substrates containing exon 9 and its 5' donor site with downstream intron sequences containing
In100, In50up, or In50dn. Position of lariat intermediates and 5'-exon
generated by the first step of the splicing reaction is indicated.
B, gel electrophoresis analysis of splicing complexes formed
on In100 or other labeled RNAs as depicted above the autoradiograph.
Aliquots from standard splicing reactions were taken at times indicated
above each lane, and splicing complexes were
resolved on a native Tris-glycine gel. The small
gray rectangle depicts In50up. The
small black rectangle represents
In50dn. Heterogeneous (H) and spliceosomal complexes
(A, B, and C) are indicated on the
left.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-2 pre-mRNA, where multiple PTB
binding sites are clustered around the branch site of a neurospecific
exon and prevent efficient binding of U2 snRNP (19). Direct competition
for binding with U2AF65 was also reported in the repression
of mutually exclusive exons in the
- and
-tropomyosin
pre-mRNAs (15, 17). In other cases, there may be complex mechanisms
involved. In the GABAA receptor
-2 pre-mRNA, the PTB
binding sites upstream cooperate with another PTB site in the
alternative exon itself to down-regulate splicing of the downstream
intron, thus suggesting an effect of PTB on the use of the downstream
5' splice site (19). Interestingly, similar to what we observed with
In100, this effect was highly dependent on the presence of a competing
splicing event (19, 32). A somewhat similar mechanism was also
described for the regulation of the Src N1 alternative exon, where PTB
binding to the upstream polypyrimidine tract represses splicing of the
downstream intron (42). In this case, however, the inhibition was not
dependent on the presence of a competing splice site (63). It was
recently shown that PTB also assembled onto a repressor element
downstream of the N1 exon (64). A model was proposed in which
cooperative assembly of PTB on each side of the alternative exon could
promote bridging of the N1 exon, similar to what was suggested for the hnRNP A1 pre-mRNA (55, 64). This model could also be envisioned for
the
-tropomyosin pre-mRNA since PTB interacts with specific elements in the introns flanking mutually exclusive exon 3 and plays a
role in repressing both its 3' splice site as well as its 5' splice
site (38, 59). In contrast, several observations argue against such a
model for the role of PTB in regulating alternative splicing of the
caspase-2 pre-mRNA. First, the In100 element requires the context
of competing splicing events to mediate its effect. Second, we could
not detect any PTB binding to the upstream intron and this intron could
be substituted for a different intronic sequence (from the human
-globin gene) without affecting In100 activity (32). Finally,
PTB-binding sites in the casp-2 In100 regulatory element are adjacent
to a decoy 3' splice site, and our results suggest that the binding of
PTB downstream of this decoy splice site prevents effective usage of
that site by the splicing machinery. Therefore, the regulatory role of
PTB in casp-2 alternative splicing and its mechanism of action appear
to be distinct from previously described systems.
-553,
respectively). We observed that abolishing PTB binding to In50dn
increased the ratio of proximal to distal 5' splice site utilization,
which suggested that PTB was mediating its negative effect by somehow
modulating recognition of the alternative exon 5' splice site.
Interestingly, this effect on 5' splice site selection did not appear
to be mediated through a direct reduction of U1
snRNP-dependent complexes, although we cannot exclude the
possibility that a transient interaction with the U1 particle might not
be detectable in the RNase H protection assay used. Alternatively,
later steps in 5' splice site scanning could be affected
(e.g. U6 snRNA interaction with the 5' splice site).
Finally, the effect observed upon mutation of the In50dn element in the
context of these 553 substrates is partial as compared with the
respective substrates containing intact acceptor sites. This could
suggest that an intact 3' splice site is required upstream of the
alternative exon, possibly because of favorable exon-bridging interactions that would permit its optimal inclusion. However, upon
complete deletion of the intronic element, even in the context of 553 substrates, a full switch to proximal 5' splice site utilization is
observed (32). Therefore, we think that either residual binding of PTB
and/or binding of other factors to the mutated element is responsible
for the partial switch.
![]() |
ACKNOWLEDGEMENTS |
---|
We acknowledge Smitha Rajasekhar and Michael Nolan for their excellent technical assistance as well as Dr. Zhi-Hong Jiang, who has initiated this project. We thank Drs. Marc T. McNally and Woan-Yuh Tarn for their generous gifts of 2'-O-methyl oligos, and Dr. Rui-Ming Xu for providing purified U2AF65. Drs. Gideon Dreyfuss, Maria Carmo-Fonseca and Adrian Krainer have generously provided antibodies against hnRNP C and A1, U2AF65, and SF2/ASF, respectively. We are grateful to Drs. Benoit Chabot, Douglas Black, James G. Patton, and Marco Blanchette for providing various reagents, helpful discussions, and critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health grants (to J. Y. W.).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 by a postdoctoral fellowship from the Natural Sciences
and Engineering Research Council of Canada.
§ Present address: Lady Davis Inst. for Medical Research, Montreal, Quebec H3T 1E2, Canada.
¶ Supported by a scholarship from the Leukemia and Lymphoma Society. To whom correspondence should be addressed: Dept. of Pediatrics and Dept. of Molecular Biology and Pharmacology, Washington University School of Medicine, 4938 Parkview Pl., MPRB Rm. 3107, St. Louis, MO 63110. Tel.: 314-286-2798; Fax: 314-286-2892; E-mail: jwu@molecool.wustl.edu.
Published, JBC Papers in Press, December 14, 2000, DOI 10.1074/jbc.M008924200
2 Z. Jiang and J. Y. Wu, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: hnRNP, heteronuclear ribonucleoprotein; PTB, polypyrimidine track-binding protein; snRNP, small nuclear ribonucleoprotein; oligo, oligonucleotide; RT, reverse transcription; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; nPTB, brain-enriched form of polypyrimidine track-binding protein.
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