From the Département de Microbiologie et d'Infectiologie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4
Received for publication, December 28, 2000, and in revised form, February 19, 2001
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ABSTRACT |
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Depending on the cell lines and cell types,
dimethyl sulfoxide (Me2SO) can induce or block
cell differentiation and apoptosis. Although Me2SO
treatment alters many levels of gene expression, the molecular
processes that are directly affected by Me2SO have not been
clearly identified. Here, we report that Me2SO affects splice site selection on model pre-mRNAs incubated in a nuclear extract prepared from HeLa cells. A shift toward the proximal pair of
splice sites was observed on pre-mRNAs carrying competing 5'-splice
sites or competing 3'-splice sites. Because the activity of recombinant
hnRNP A1 protein was similar when added to extracts containing or
lacking Me2SO, the activity of endogenous A1 proteins is
probably not affected by Me2SO. Notably, in a manner
reminiscent of SR proteins, Me2SO activated splicing in a
HeLa S100 extract. Moreover, the activity of recombinant SR proteins in
splice site selection in vitro was improved by
Me2SO. Polar solvents like DMF and formamide similarly
modulated splice site selection in vitro but formamide did
not activate a HeLa S100 extract. We propose that Me2SO
improves ionic interactions between splicing factors that contain
RS-domains. The direct impact of Me2SO on
alternative splicing may explain, at least in part, the different and
sometimes opposite effects of Me2SO on cell differentiation
and apoptosis.
Me2SO1 is a
polar solvent used to promote cell differentiation of tumor cell lines.
For example, the treatment of mouse erythroleukemic and neuroblastoma
cells with 2% Me2SO induces morphological changes and
differentiation in red blood cells and neurons, respectively (e.g. see Refs. 1, 2). Me2SO also induces
differentiation of the human U937 monoblast leukemia cell line into
monocyte/macrophage (3) and stimulates the differentiation of a human
ovarian adenocarcinoma cell line (4). Paradoxically, Me2SO
prevents the terminal differentiation of myoblasts (5, 6), inhibits the
differentiation of adipocytes (7), blocks the differentiation of
antibody-producing plasma cells (8), and interferes with the
differentiation of chick embryo chondrocytes (9). Whereas
Me2SO has been used to induce apoptosis in some cell lines
(10, 11), it inhibits cell density-dependent apoptosis of
CHO cells (6). Thus, depending on the cell line, Me2SO can
have completely different effects on differentiation and apoptosis.
The cellular mechanisms that are affected by Me2SO remain
unclear. Because Me2SO facilitates DNA uptake during
transfection procedures (e.g. see Ref. 12),
Me2SO has been proposed to affect the integrity of cell
membranes. Because Me2SO alters protein kinase C activity
and the expression of integrin complexes (6, 13), Me2SO may
alter intracellular signaling processes, which in turn may have a
broad impact on many aspects of gene expression. Me2SO
treatment promotes changes in the abundance of certain mRNAs and in
the ratio of spliced isoforms (14-17). Among the genes reported to be
affected in their alternative splicing is the NCAM
pre-mRNA. A 2% Me2SO treatment of N2a cells promotes
an increase in the inclusion of neuro-specific NCAM
exon 18 (18, 19). Me2SO alters the alternative splicing of
other genes including the amyloid precursor protein (20), the serotonin
5-HT3 receptor-A mRNA (21), and p53 (22, 23). Me2SO has
also been associated with an effect on c-Myc mRNA elongation,
maturation, and stability (23-25), and on the translation of some
mRNAs (1). Whether any of the above changes result from a direct
effect of Me2SO on RNA synthesis, maturation, and/or
stability is currently unknown.
Because treating cells with Me2SO can have a strong effect
on the alternative splicing of many pre-mRNAs and because the
mechanism of action of Me2SO remains unclear, we performed
a series of experiments in nuclear extracts to assess whether
Me2SO directly affects the activity of the splicing
machinery. We find that Me2SO can have drastic effects both
on 5'-splice site and on 3'-splice site selection in vitro.
Notably, other solvents of the same category (e.g. DMF and
formamide) also perturb splice site selection.
Treatment of Cells with Me2SO and in Vivo Alternative
Splicing Assays--
Me2SO was purchased from various
suppliers including EM Science and Fisher Scientific Inc. DMF and
formamide were from Calbiochem. N2a and HeLa cells were cultured at
37 °C in Dulbecco's modified Eagle's medium supplemented with 10%
bovine calf serum. For treatment with Me2SO, medium
containing 2% bovine calf serum was used. Following treatment, total
RNA was isolated using the guanidinium-HCl protocol as described in
Chabot (26). RNase T1 protection assay was performed according to
Melton et al. (27) using a uniformly labeled 530-nt NCAM
antisense RNA probe. Exon 17/exon 19 splicing yields a 303-nt protected
fragment while the inclusion of exon 18 produces a 452-nt fragment.
Products were resolved on a 5% denaturing acrylamide gel. The reverse
transcriptase-PCR assay used to amplify products corresponding to exon
7B inclusion and exclusion has been described in Chabot et
al. (28).
Substrate Pre-mRNAs and in Vitro Splicing Assays--
pC5'
Recombinant A1 and SR Proteins--
Recombinant GST·A1,
GST·SRp30c were purified using a glutathione-Sepharose column
(Amersham Pharmacia Biotech). Bacterial lysis was in buffer A (50 mM piperazine-HCl, pH 9.8, 0.5 M NaCl, 1 mM EDTA, 1 mM dithiothreitol, 20 µg/ml
bacitracine, 1 mM benzamidine, 0.5 mM
phenylmethylsulfonyl fluoride in the presence of 3 mg/ml lysozyme and
1% Triton X-100. Elution from the columns was performed with buffer B
(200 mM piperazine-HCl, pH 9.8, 0.5 M NaCl, 1 mM EDTA, 1 mM dithiothreitol, 20 mM
reduced glutathione). The purified proteins were dialysed against
buffer D (20 mM Hepes pH 7.9, 100 mM KCl, 20%
glycerol, 1 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride).
Me2SO Affects Alternative Pre-mRNA Splicing in
Vivo--
Me2SO can promote cell differentiation, a
process that is often associated with a change in the alternative
splicing profile of specific genes. One example of this effect is found
in the mouse N2a neuroblastoma cell line. The treatment of N2a cells with 2% Me2SO induces neuronal cell differentiation and
improves the frequency of inclusion of the neurospecific exon 18 in the NCAM pre-mRNA (Refs. 2, 31; Fig.
1A). A similar effect was observed on the hnRNP A1 pre-mRNA. In this case, we
monitored the inclusion frequency of alternative exon 7B following the
treatment of HeLa cells for 5 h with 5% Me2SO (Fig.
1B). Although the effect was less dramatic than for the
NCAM pre-mRNA, Me2SO treatment significantly
improved the inclusion of exon 7B.
Me2SO Affects Splice Site Selection in Vitro--
To
determine whether Me2SO can modulate splice site selection
directly, we tested the effect of adding Me2SO to splicing
reactions incubated in nuclear extracts prepared from HeLa cells. We
used model pre-mRNAs derived from the hnRNP A1 alternative splicing unit (29). C5'
The effect of Me2SO on 5'-splice site selection was as
strong on a pre-mRNA that was synthesized in the absence of cap
analogue (data not shown). Thus, the reduction in distal 5'-splice site usage was independent of the cap structure at the 5'-end of the pre-mRNA. Me2SO also affected 5'-splice site selection
in a model pre-mRNA carrying two copies of the 5'-splice site of
exon 7 (data not shown). Identical effects were seen with
Me2SO solutions obtained from different suppliers, and the
deionization of Me2SO did not change its activity on
5'-splice site selection (data not shown). Transient exposure of
nuclear extracts to Me2SO (i.e. incubation in
the presence of Me2SO followed by dialysis) did not affect 5'-splice site usage (data not shown). Thus, Me2SO needs to
be present in splicing mixtures to affect splice site selection.
Because Me2SO had a strong effect on the alternative
splicing of a pre-mRNA carrying A1 binding elements (C5' 4/4), we
asked whether Me2SO compromised the activity of the hnRNP
A1 protein. We have shown previously that hnRNP A1 promotes distal
5'-splice site utilization on this pre-mRNA (29). In nuclear
extracts containing Me2SO, the addition of hnRNP A1
efficiently shifted selection toward the distal 5'-splice site (Fig.
3A, lanes 6-10). The effect
was specific because the addition of similar amounts of GST or gene 32 protein had no effect (data not shown). Notably, the profile of
stimulation obtained with increasing amounts of recombinant A1 was
similar to the profile obtained in a nuclear extract lacking
Me2SO (Fig. 3A, lanes 1-5; compare
the slopes in Fig. 3B). Because the activity of recombinant
hnRNP A1 is not compromised by the presence of Me2SO, it is
unlikely that Me2SO affects the activity of the endogenous
A1 proteins.
To address whether Me2SO has a similar activity on
3'-splice site selection we tested a pre-mRNA (C3' Me2SO Activates SR Proteins--
The effect of
Me2SO on splice site selection is reminiscent of the
activity of SR proteins, which tend to activate splicing of the
proximal pair of splice sites (32, 33). Although Me2SO did
not stimulate overall splicing activity in nuclear extracts (Fig.
5A, lanes 1 and
2), we asked whether Me2SO could mimic the generic splicing activity of SR proteins. This activity was initially defined by the capacity of SR proteins to activate splicing in a HeLa
S100 extract, either as a mixture of SR proteins or individually (33,
34). U2AF65 also activates splicing when added to a HeLa
S100 extract (35). Notably, the addition of Me2SO to a HeLa
S100 extract stimulated splicing as efficiently as the addition of the
recombinant SR protein ASF/SF2 (Fig. 5A, lanes
3-5). The addition of Me2SO to a S100 extract also
stimulated the formation of complexes, as judged by native gel analysis
(Fig 5B, lanes 7-9). These results suggest that
Me2SO increases the activity of residual amounts of SR or
U2AF proteins in the S100 extract. The level of splicing stimulation
varied considerably in different preparations of S100 extract. Although
Me2SO and recombinant ASF/SF2 restored splicing activity in
a similar manner, splicing to the distal 5'-splice site was not
detected, as is the case in a nuclear extract (lanes 1 and
2). We have shown previously that distal 5'-splice site selection on this pre-mRNA requires hnRNP A1 (29). The failure to
activate distal 5'-splice site use is probably because of the fact that
S100 extracts contain small amounts of hnRNP A/B proteins as compared
with nuclear extracts (not shown).
The above result suggests that Me2SO may affect the
activity of SR proteins. To further examine this possibility, we tested the effect of adding Me2SO to splicing reactions
preincubated with a recombinant SR protein. At the concentrations used
and in the absence of Me2SO, the recombinant SR protein
GST·SRp30c had little effect on 5'- splice site selection when using
the C5' 4/4 pre-mRNA (Fig.
6A, lanes 1-3).
However, in the presence of Me2SO, the same amount of
GST·SRp30c stimulated proximal 5'-splice site utilization
(lanes 4-12). Thus, the simultaneous addition of
Me2SO and SR produced a shift toward proximal use that was greater than the sum of their individual contribution. Because recombinant SR proteins display more activity in the presence of
Me2SO, a similar effect on the endogenous SR proteins may
be responsible for the activity of Me2SO in nuclear
extracts.
DMF and Formamide Also Modulate Splice Site Selection--
To
understand the chemical basis for the activity of Me2SO in
alternative splicing, we tested other solvents. At equivalent percentages, both DMF and formamide were at least as active as Me2SO at modulating 5'-splice site selection (Fig.
7A). Surprisingly, although DMF and formamide shared with
Me2SO the ability to modulate 5'-splice site selection,
formamide was unable to activate splicing in a HeLa S100 extract (Fig.
7B).
We have observed that the addition of Me2SO to nuclear
extracts can have strong effects on splice site selection while having minimal effects on the efficiency of splicing. In contrast, the addition of Me2SO to a splicing-deficient HeLa S100 extract
stimulated splicing in a manner reminiscent of the activity of SR
proteins. The effect of Me2SO on splice site selection was
also similar to the activity of SR proteins because Me2SO
shifted selection toward the proximal pair of splice sites. Consistent
with the notion Me2SO stimulates the activity of SR
proteins, we found that the combination of Me2SO and SRp30c
produces a shift that is greater than the sum of their individual
contribution. Thus, a general stimulation in the activity of all
endogenous SR proteins most probably explains why Me2SO
influences splice site choice in vitro. Likewise, the
addition of Me2SO to a S100 extract may stimulate the
residual amounts of SR proteins present in this extract.
An additional mechanism by which Me2SO may affect splice
site selection is through the inactivation of the hnRNP A/B proteins, which are known to antagonize the activity of SR proteins in splice site selection (36, 37). However, we have
observed that the activity of recombinant hnRNP A1 proteins in splice
site selection is not affected by the presence of Me2SO.
This result suggests that the activity of endogenous A1 proteins is
probably not affected by Me2SO.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
, pC5' 4/4 and pC3'
/
have been described in Blanchette and
Chabot (29). pNCAM3' was constructed by substituting the 3'-splice site
of exon 7B and exon 7B sequences in pC3'
/
for the equivalent
regions of alternative exon 18 of the mouse NCAM gene (403 bp of exon 18 and 111 bp of upstream intron sequences). Splicing
substrates were produced from plasmids linearized with ScaI,
and transcribed with T3 RNA polymerase in the presence of cap analogue
and [
-32P]UTP (Amersham Pharmacia Biotech). RNA
purification was performed as described in Chabot (26). HeLa nuclear
extracts and S100 extracts were prepared (30) and used in splicing
reactions as described previously (28). Although Me2SO,
DMF, and formamide were always added last, the order of addition did
not affect the outcome.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Me2SO promotes exon inclusion
in vivo. A, mouse N2a cells were
treated 48 h with 2% Me2SO. Total RNA was analyzed
for changes in the alternative splicing of NCAM exon 18. A
RNase T1 protection assay was used to monitor the ratio of exon 18 inclusion (E18+) or exclusion (E18 ).
B, HeLa cells were treated with 5% Me2SO for
5 h. Three dishes of cells were tested for each treatment and
total RNA was analyzed for changes in the alternative splicing of exon
7B in the hnRNP A1 pre-mRNA. The percentage of exon 7B inclusion on
endogenous A1 transcripts was determined by using a reverse
transcriptase-PCR assay as described in Chabot et al. (28).
Control PCR reactions were performed with plasmids containing the
cDNA from A1 (lacking exon 7B, lane 1), or
A1B (containing exon 7B, lane 2). The values
were plotted as percentage of inclusion on a histogram that shows
standard deviations.
/
contains two competing 5'-splice sites and a
unique 3'-splice site (Fig.
2A). C5'
/
is spliced
almost exclusively to the proximal 5'-splice site (Fig. 2B,
lane 1). In contrast, the presence of A1 binding elements in
C5' 4/4 promotes efficient splicing to the distal 5'-splice site
(lane 5). The addition of Me2SO at a final
concentration of 0.8, 1.6, and 2.4% did not affect the splicing
efficiency of C5'
/
RNA, and 5'-splice site selection remained
exclusively proximal (Fig. 2B, lanes 2-4). In contrast, Me2SO promoted a strong reduction in the use of distal
5'-splice site in C5' 4/4 pre-mRNA (lanes 6-8). The
highest concentration of Me2SO (lane 8) produced
a 5-fold decrease in the use of the distal 5'-splice site. In some
experiments, the reduction in distal 5'-splice site use was accompanied
by an increase in the production of lariat products derived from the
proximal 5'-splice site (e.g. see Fig. 5A,
lane 2).
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Fig. 2.
Me2SO affects 5'-splice site
selection in vitro. A, structure of
the pre-mRNAs used to assay modulation of 5'-splice site selection.
C5' /
and C5' 4/4 have been described previously (29). The C5' 4/4
pre-mRNA contains two CE4 elements, which are binding sites for
hnRNP A1. B, incubation of the pre-mRNAs in HeLa
extracts was for 2 h in the presence of different percentages of
Me2SO (0, 0.8, 1.6, 2.4%). Labeled RNA products were
fractionated on a denaturing 11% polyacrylamide gel. The position and
structure of the proximal and distal lariat products are shown.
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Fig. 3.
Me2SO does not affect the
activity of recombinant hnRNP A1. A, HeLa extracts
lacking or containing 2.4% Me2SO were supplemented with
recombinant hnRNP A1 proteins (0.125, 0.25, 0.5, and 1 µg) and
splicing of the C5' 4/4 pre-mRNA was monitored. The position of the
distal and proximal lariat products are indicated. B,
diagram depicting the A1-mediated stimulation of distal 5'-splice site
usage in extracts lacking or containing Me2SO. The almost
identical slopes suggest that the activity of recombinant A1 is not
affected by Me2SO.
/
; Fig.
4A), which is spliced
predominantly to the distal 3'-splice site (Ref. 29; Fig. 4B,
lane 1). C3'
/
splicing was sensitive to increasing amounts of
Me2SO (Fig. 4B, lanes 2-4). At the
highest concentration of Me2SO (lane 4), more
than 50% of splicing occurred at the proximal 3'-splice site. We also
tested a derivative of C3'
/
in which the central portion was
substituted for the 3'-splice site region and a portion of
NCAM alternative exon 18 (NCAM3' RNA). Although splicing of NCAM3' RNA was less sensitive to
Me2SO than C3'
/
, Me2SO promoted a stronger
reduction in the use of the distal 3'-splice site than of the proximal
3'-splice site (Fig. 4B, lanes 5-9). Alternative
splicing of a
-globin pre-mRNA carrying duplicated 3'-splice
sites was also affected by Me2SO (data not shown).
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Fig. 4.
Me2SO affects 3'-splice site
selection in vitro. A, structure of
the pre-mRNAs used to assay modulation of 3'-splice site selection.
C3' /
is derived from the hnRNP A1 gene (29). The NCAM 3'
pre-mRNA is an hybrid pre-mRNA containing the 5'-splice site of
exon 7, the 3'-splice site of NCAM alternative exon 18 and the
3'-splice site of adenovirus L2 exon. B, in vitro
splicing assays of model pre-mRNAs. Incubation was for 2 h in
HeLa extracts containing increasing percentages of Me2SO
(0, 0.8, 1.6, 2.4% in lanes 1-4, and 0, 0.8, 1.6, 2.4, 3.2% in lanes 5-9). Labeled RNA products were fractionated
on denaturing 11% (for C3'
/
) or 6.5% (for NCAM3') polyacrylamide
gels. The position of the pre-mRNA and proximal and distal lariat
products is shown.
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Fig. 5.
Me2SO rescues splicing in a HeLa
S100 extract. A, splicing reactions were performed in a
HeLa nuclear extract (NE) and in a HeLa S100 extract. The
extracts were incubated in the absence or in the presence of 3.2%
Me2SO. The S100 extract was also supplemented with 0.5 µg
of recombinant ASF/SF2 protein (lane 5). The pre-mRNA
substrate used was C5' 4/4. B, a HeLa nuclear extract
(NE), a HeLa S100 extract or S100 extract supplement with
3.2% Me2SO(S100 + DMSO) were
incubated with a model pre-mRNA derived from the adenovirus major late
transcription unit. Following incubation for the time indicated (in
minutes), samples were fractionated in a native 5% polyacrylamide gel.
The position of the nonspecific complex H and of splicing complex A is
indicated.
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Fig. 6.
Me2SO activates SR proteins.
A, using the C5' 4/4 pre-mRNA, the activity of the
recombinant SR proteins GST·SRp30c was tested in the absence and in
the presence of increasing concentrations of Me2SO. The
GST·SRp30c protein (0.5 and 1 µg) was preincubated in nuclear
extract 15 min at 30 °C before adding the pre-mRNA and
Me2SO. B, diagram depicting the SRp30c-mediated
reduction in distal 5'-splice site usage in extracts lacking or
containing different concentrations of Me2SO.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
Modulating and stimulating activities
of DMF and formamide. A, in vitro splicing assays with
the model pre-mRNA C5' 4/4 were carried out in HeLa extracts in the
presence of different percentages of DMF or formamide (0, 1.6, 2.4, 3.2, and 4%). For comparison, splicing of the same pre-mRNA in a
HeLa nuclear extract containing 4% Me2SO is shown
(lanes 6 and 12). The position of the
pre-mRNA and proximal and distal lariat products is shown.
B, splicing reactions with the C5' 4/4 pre-mRNA were set
up in HeLa S100 extracts in the presence of increasing amounts of
Me2SO, DMF, or formamide (0, 2.4, 3.2, and 4%). For
comparison, a splicing reaction performed in a HeLa nuclear
extract is shown (NE, lane 13).
Because Me2SO, DMF, and formamide decrease the melting temperature of DNA and RNA duplexes, it is possible that changes in 5'-splice site selection may be caused by the weakening of the base pairing interactions between U1 snRNA and 5'-splice sites. However, this explanation is unlikely for the following reasons. First, a reduction in U1 snRNP binding should lead to more distal 5'-splice site utilization, as is the case when U1 activity is reduced through oligonucleotide-targeted RNase H digestion (data not shown). Moreover, increased selection of a proximal 5'-splice site is associated with improved U1 snRNP binding (38). Second, splice site selection on a transcript that is normally spliced to a distal 5'-splice site because of a duplex structure in the pre-mRNA was insensitive to Me2SO.2 Thus, the effect of Me2SO, formamide and DMF on 5'-splice site selection cannot be explained by a reduction in the stability of base pairing interactions. However, it is possible that this denaturing activity contributes to the reduction in overall splicing activity observed at higher concentrations of solvents.
Although our results suggest that Me2SO affects the activity of SR proteins, the mechanism by which SR proteins become activated remains unclear. Western analysis using an antibody that recognizes phosphorylated epitopes on SR proteins revealed no change in the overall and relative abundance of phosphorylated SR proteins upon incubation with Me2SO (data not shown). Moreover, Me2SO did not affect the binding of SR proteins to a purine-rich RNA splicing enhancer element (data not shown). Me2SO also did not modify the solubility of SR proteins when extracts were incubated with increasing concentrations of MgCl2 (data not shown). Although Me2SO is regarded as a relatively inert solvent for pharmacological applications, it improves the solvation of cations and stimulates nucleophilic reactions. DMF and formamide share with Me2SO this chemical property. Thus, one possibility is that Me2SO improves the solvation of positive charges on proteins. This may influence the structure at the surface of proteins and facilitate ionic contacts between charged domains of interacting proteins. Consistent with this view, modulation of 5'-splice site selection in vitro is known to be sensitive to the ionic conditions of the reaction (39). Splicing proteins that carry charged domains include SR and U2AF proteins, which have RS-domains rich in positively and negatively charged amino acids (arginines and phosphorylated serines, respectively). Interactions between the RS-domain containing proteins ASF/SF2, U1 snRNP-70 kDa, and U2AF35 have been proposed to occur early during spliceosome assembly (40). Moreover, these interactions are sensitive to the phosphorylation state of ASF/SF2 (41). Thus, Me2SO may activate splicing in a S100 extract by improving the quality of the ionic interactions between residual amounts of SR and U2AF proteins. Because the amount and activity of these proteins are in excess in a nuclear extract, this would explain why Me2SO stimulates generic splicing in a S100 but not in a nuclear extract.
Even though nuclear extracts contain sufficient amounts of SR proteins for generic splicing, their activity in splice site selection is not maximal because adding more SR proteins can have a strong effect on the selection of splice sites (32, 33). Because a similar effect can be obtained by adding kinases that phosphorylate the RS domains of SR proteins (42), the profile of charged residues at the surface of SR proteins is crucial for their activity as splicing regulators. Moreover, the requirement for charged residues at the surface of SR proteins appears different for generic and alternative splicing because dephosphorylation of ASF/SF2 is essential for constitutive splicing, but is not required for the protein to function as a splicing regulator (41). Thus, Me2SO may affect the presentation of charged residues that are important for the activity of SR proteins in splice site selection. Because Me2SO and DMF activate splicing in a HeLa S100 extract, Me2SO and DMF may also affect the presentation of different residues that are important for generic splicing. In contrast, because formamide affects splice site selection but cannot activate a S100 extract, formamide may only affect the presentation of residues that play a role in splice site selection.
Our results raise the possibility that the documented effect of
Me2SO on cell differentiation may be caused, at least in
part, by changes in the activity of SR proteins, which in turn affect splice site selection. This conclusion is supported by the observation that DMF can mimic the effect of Me2SO both in
differentiation assays (5, 43-45), and in splicing assays in
vitro. Depending on the cell types, Me2SO can either
promote or block differentiation or apoptosis. These opposite outcomes
may be explained if different subsets of pre-mRNAs are expressed in
different cell types. For example, alternative splicing is often used
to control the production of proteins involved in programmed cell death
such as Fas, Bcl-2, Bax, and Ced-4 (46). Hence, Me2SO may
alter the alternative splicing of a pre-mRNA to favor the
production of a repressor protein in one cell type, while an inducer
may be produced from another gene in a different cell type.
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ACKNOWLEDGEMENTS |
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We thank Jude Beaudoin for performing the RNase T1 protection assay on NCAM, and Johanne Toutant for help with cell culture. We thank Stephan Stamm for providing the GST·SRp30c plasmid, and Martin Simard for the SR protein preparations.
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FOOTNOTES |
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* This work was supported in part by a grant from the National Cancer Institute of Canada with funds from the Canadian Cancer Society (to B. C.).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.
Recipient of a studentship from the Fonds pour la Formation de
Chercheurs et l'Aide à la Recherche (FCAR).
§ Recipient of a studentship from the Medical Research Council of Canada. Present Address: Dept. of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3204.
¶ Research Scholar from the Fond de la Recherche en Santé du Québec and member of the Sherbrooke RNA/RNP group supported by the FCAR. To whom correspondence should be addressed. Tel.: 819-564-5295; Fax: 819-564-5392; E-mail: b.chabot@courrier.usherb.ca.
Published, JBC Papers in Press, March 1, 2001, DOI 10.1074/jbc.M011769200
2 M. Cordeau and B. Chabot, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: Me2SO, dimethyl sulfoxide; DMF, N,N-dimethylformamide; nt, nucleotide(s); bp, base pairs; hnRNP, heterogeneous nuclear ribonucleoprotein; NCAM, neural cell adhesion molecule; GST, glutathione S-transferase; PCR, polymerase chain reaction.
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REFERENCES |
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