Dimethyl Sulfoxide Affects the Selection of Splice Sites*

Lucie BolducDagger, Benoit Labrecque, Mélanie Cordeau, Marco Blanchette§, and Benoit Chabot

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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' -/-, 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 [alpha -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.

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).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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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.

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' -/- 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.

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.


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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.

To address whether Me2SO has a similar activity on 3'-splice site selection we tested a pre-mRNA (C3' -/-; 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 beta -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.

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).


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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.

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.


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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.

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).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger 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.

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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