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Molecular and Cellular Biology, June 2002, p. 4001-4010, Vol. 22, No. 12
0270-7306/02/$04.00+0 DOI: 10.1128/MCB.22.12.4001-4010.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
and Benoit Chabot*
Département de Microbiologie et d'Infectiologie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4
Received 4 January 2002/ Returned for modification 21 February 2002/ Accepted 11 March 2002
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Several studies have uncovered sequence elements within pre-mRNAs that positively or negatively influence splice site utilization (reviewed in references 1, 5, and 39). The interaction of cellular factors with these elements represents the most common way to modulate splice site utilization. Members of the family of SR proteins constitute one of the most important classes of cellular factors implicated in splice site selection (reviewed in reference 24). Recruitment of SR proteins on exonic splicing enhancers can increase U2AF65 or U2 snRNP binding to an upstream 3' splice site (32, 36, 55, 61). Positioning SR binding sites near a 5' splice site may also facilitate the stable recruitment of U1 snRNP (4, 14, 16, 17, 29, 34, 51).
A variety of elements repressing splice site utilization have also been reported. Some elements act by forming duplex structures that impair splice site recognition (2, 11, 13, 30). Other silencer elements require the contribution of trans-acting factors. The polypyrimidine tract binding protein (PTB) has often been associated with the activity of silencer elements (reviewed in references 58 and 60). PTB binding sites can overlap with those of U2AF65, leading to competition for binding to the 3' splice site region (37, 53). This type of repression is similar to the mechanism used by Drosophila melanogaster female-specific splicing factor SXL to prevent U2AF65 binding to the tra pre-mRNA, leading to the selection of a weaker downstream 3' splice site (22, 59). In other cases, PTB and SXL binding sites do not directly overlap with splice site regions (8-10, 20, 21, 27, 28, 46, 54). The mechanism of repression in these cases remains unclear.
Factors that have been initially identified as repressors or activators in one system can display the opposite behavior in different pre-mRNAs. This is the case for PTB, which has been reported as promoting splice site recognition in genes coding for calcitonin and the calcitonin gene-related peptide (40). Likewise, the binding of SR proteins to intron sequences can have a negative impact on splicing. For example, the binding of SR proteins to a purine-rich element located in the intron upstream of the IIIa exon of adenovirus L1 splicing unit inhibits splicing (33, 47). In this case, SR protein binding is thought to sterically interfere with the interaction of U2 snRNP with the 3' splice site region of exon IIIa. SR proteins have also been found in association with repressor elements in the Rous sarcoma virus (RSV) pre-mRNA (42) and the cystic fibrosis transmembrane regulator gene (45). In myotonic dystrophy, the overexpression of CUG-BP can promote aberrant inclusion of exon 5 in the cardiac troponin T pre-mRNA (48) or repress the inclusion of exon 11 in the insulin receptor pre-mRNA (49).
To study the control of alternative splicing, we used the hnRNP A1 gene as a model system. Exclusion and inclusion of exon 7B in the hnRNP A1 pre-mRNA generate two different mRNAs encoding the A1 and A1B proteins, respectively. We have previously identified elements capable of influencing the alternative splicing of exon 7B: CE6 base pairs with the 5' splice site region of exon 7B to decrease its use (2), CE4m represses the 3' splice site of exon 7B (3), and hnRNP A1 binding sites located on both sides of exon 7B promote exon skipping (3, 7). Our previous work on a 38-nucleotide (nt) intron element called CE9 uncovered an activity that can repress splicing to a downstream 3' splice site (52). In the present study, we identify a member of the family of SR proteins, SRp30c, as the factor that binds to CE9 and we provide evidence to support a direct role of SRp30c in the repressing activity of CE9.
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In vitro transcription.
Pre-mRNA substrates A, A3xPu, A2x, and A3x and derivatives were produced from plasmids linearized with HincII and transcribed with SP6 RNA polymerase (Amersham Pharmacia Biotech) in the presence of a cap analog and [
-32P]UTP (Amersham Pharmacia Biotech). The C3'-/2x transcript was obtained after linearization of the plasmid by ScaI and transcription with T3 RNA polymerase (Amersham Pharmacia Biotech). CE9, CE9.17, and CE9.12d RNAs and K+ RNA were produced from plasmids linearized with ClaI and transcribed with T3 RNA polymerase. CE9.8d RNA, CE9.7 RNA and mutated versions, and 3xPu RNA were obtained from plasmids linearized with EcoRI and transcribed with T3 RNA polymerase. Cold RNA was produced as described above except that the relative amount of [
-32P]UTP was reduced 2,000-fold. The purification of all RNA molecules was performed as described by Chabot (6).
In vitro splicing assays. HeLa nuclear extracts were prepared (15) and used in splicing reactions as previously described (7). Identification of lariat molecules and other splicing products was confirmed by performing debranching reactions in an S100 extract followed by migration relative to molecular weight standards. Competition with cold RNA was performed by preincubating the splicing mixture with the competitor RNA for 10 min at 30°C prior to the addition of the radiolabeled pre-mRNA substrate.
UV cross-linking. Radiolabeled RNA were incubated for 10 min under standard splicing conditions. One-half of the reaction mixture was irradiated for 10 min with UV and digested with RNase A as described by Côté et al. (12). The cross-linking assay in the presence of heparin was performed in the buffer used for mobility shift assays (see below). After incubation, one-half of the reaction mixture was irradiated for 10 min with UV and digested with RNase A. Cross-linking products were analyzed by electrophoresis on sodium dodecyl sulfate (SDS)-12.5% polyacrylamide gels.
Gel shift assays. RNA mobility shift assays were performed by incubating RNAs for 15 min on ice in splicing conditions prior to the addition of 1 mg of heparin/ml and incubation for 2 min on ice. The reactions were run on a 5% native acrylamide gel (29:1 acrylamide/bisacrylamide, 5% glycerol, 50 mM Tris [pH 8.8], 50 mM glycine) in Tris-glycine running buffer (50 mM Tris [pH 8.8], 50 mM glycine).
RNA affinity chromatography. Fifty nanomoles of synthetic RNA oligonucleotide corresponding to CE9 (CUGGAUUAUUCAACUG) or an adenovirus RNA (C RNA; AAUGUCUGCUACUGG; Dharmacon Research Inc.) was incubated 1 h on ice, protected from light, in a 100-µl reaction volume containing 100 mM Tris-HCl, pH 7.5, and 10 mM sodium periodate. The periodate-treated RNAs were coupled to 0.5 ml of agarose adipic acid hydrazide resin in accordance with the manufacturer's protocol (Amersham Pharmacia Biotech). The resin was washed twice with 10 ml of storage buffer (20 mM HEPES-KOH [pH 7.9], 100 mM KCl, 20% glycerol, 5.7 mM MgCl2, 1 mM dithiothreitol [DTT]) and kept as a 50% slurry at 4°C. The coupling efficiency, which was typically higher than 95%, was measured by comparing the absorbance at 260 nm of 1% of the input periodate-treated RNA to that of 10% of the unbound material. One hundred seventy-five microliters of HeLa nuclear extract containing 5.7 mM MgCl2, 0.90 mM ATP, 36 mM phosphocreatine, 3.58 mM DTT, and 1.25 U of RNAguard/ml was incubated with 50 µl of packed beads for 10 min at 30°C under agitation. The mixture was spun, and the supernatant was transferred to a second tube containing 50 µl of the same packed beads. The beads were washed four times with 1 ml of 70% buffer D (20 mM HEPES-KOH [pH 7.9], 100 mM KCl, 20% glycerol, 1 mM DTT) containing 5.7 mM MgCl2. The bound proteins were pooled and loaded on gel after the beads were boiled in 100 µl of loading dye (62.5 mM Tris-HCl [pH 6.8], 6 M urea, 10% glycerol, 2% SDS, 0.7 M mercaptoethanol, 0.003% bromophenol blue).
Recombinant protein purification. Recombinant glutathione S-transferase (GST)-ASF/SF2 and GST-SRp30c were purified with a glutathione-Sepharose column (Amersham Pharmacia Biotech) as described by the manufacturer in Rec buffer (20 mM piperazine-HCl [pH 9.5], 0.5 M NaCl, 1 mM DTT, 1 mM bacitracin, 20 µg of benzamidine/ml, 0.5 mM phenylmethylsulfonyl fluoride) in the presence of 3 mg of lysozyme/ml and 1% Triton X-100. The columns were washed with Rec buffer containing 0.1% Triton X-100 and eluted in elution buffer (200 mM piperazine-HCl [pH 9.5], 0.5 M NaCl, 1 mM DTT, 20 mM reduced glutathione). Trx (thioredoxin-His)-SRp30c was purified by standard Ni column chromatography as described by Lamontagne et al. (35). The purified proteins were extensively dialyzed against buffer D (20 mM HEPES [pH 7.9], 100 mM KCl, 20% glycerol, 0.5 mM DTT). The concentration of recombinant proteins was measured by the Bradford method using the protein assay from Bio-Rad and/or estimated from Coomassie blue-stained SDS-polyacrylamide gels, with serial dilutions of bovine serum albumin as the standard.
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FIG. 1. Multiple copies of CE9 inhibit the utilization of a downstream 3' splice site. (A) CE9 is a highly conserved 38-nt element in hnRNP A1. Shown is a schematic representation of the downstream portion of the hnRNP A1 alternative splicing unit with the distance of CE9 to the constitutive 3' splice site of exon 8 and the length of CE9 in nucleotides indicated. The alignment between the mouse and the human CE9 sequences shows a level of conservation that is greater than 90%. (B) Schematic representation of pre-mRNAs. The distance between the site of insertion and the 3' splice site in nucleotides is indicated. (C) Labeled pre-mRNAs were incubated in a HeLa nuclear extract for 2 h. C3'-/ and A3x are identical to C3'-/2x and A3x, respectively, except that they contain insertions of sequences complementary to CE9. Splicing products were run on an 8% acrylamide-8 M urea gel (C3'-/2x and C3'-/ ) or a 6.5% acrylamide-8 M urea gel (A3x and A3x ). A mixture (M) containing A3x and A3x was analyzed to rule out the presence of a nonspecific inhibitor in the A3x RNA preparation (lane 5). (D) CE9 activity is mediated by a trans-acting factor. Splicing was performed in a HeLa nuclear extract preincubated for 10 min with increasing amounts of an RNA containing multiple copies of CE9 (3x9f) as a competitor (lanes 2 to 4 and 6 to 8). Each set of competition was performed with 0.5 fmol of pre-mRNA and 62.5, 125, and 250 fmol of 3x9f RNA. The positions of the pre-mRNAs and of various lariat products are indicated.
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CE9 is bound by a nuclear factor of 25 kDa. To detect the interaction of factors with CE9 in a HeLa nuclear extract, we performed a gel shift assay. 32P-labeled RNA probes were incubated in the extract, and complexes were resolved on a nondenaturing gel. An RNA containing the complete 38 nt of CE9 formed a complex in a HeLa extract (Fig. 2B, lane 4). In contrast, a control RNA carrying only plasmid sequences did not form a complex (lane 2). Complex formation occurred in a nuclear extract depleted of ATP and kept at 0°C (data not shown). A smaller RNA carrying the first 17 nt (CE9.17) was also assembled into a low-mobility complex (lane 6). Although the last 12 nt of CE9 (CE9.12d) elicited complex formation, the majority of the RNA remained free (lane 8), suggesting that the interaction of cellular factors with the 3' end of CE9 is considerably weaker. These results indicate that the HeLa nuclear extract contains one or several factors that can specifically associate with CE9.
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FIG. 2. A 25-kDa protein interacts with CE9. (A) The sequence of CE9 RNA and portions of it used in gel shift assays are shown. Each RNA also contains at its 5' end 35 nt of unrelated sequence that is derived from pBluescript II KS(+) RNA. (B) Labeled transcripts were incubated for 15 min on ice in the presence (+) or absence (-) of a HeLa nuclear extract (NE). Complexes were separated on a nondenaturing 5% acrylamide gel. (C) Cross-linking assays were performed in the presence of labeled transcripts incubated for 10 min at 30°C (lanes 1 and 2) or on ice in the presence of 0.1 mg of heparin (lanes 3 and 4), followed by UV irradiation. Samples were fractionated on a 12.5% polyacrylamide-SDS gel. K+, control RNA containing 35 nt from pBluescript that are also present at the 5' end of the other transcripts.
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A CE9 RNA column retains a 25-kDa nuclear protein: SRp30c. To address the identity of the 25-kDa protein, we performed RNA affinity chromatography using the first 16 nt of CE9 covalently linked to an adipic acid hydrazide column (Fig. 3A). After the RNA column was loaded with a HeLa nuclear extract under splicing conditions, beads were washed. Bound proteins were fractionated onto an SDS-polyacrylamide gel and stained with AgNO3. A comparison of the profiles of proteins retained by a control RNA column and by the CE9 RNA column revealed a protein of approximately 25 kDa that was specifically retained by the CE9 RNA (Fig. 3B, compare lanes 1 and 2).
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FIG. 3. A 25-kDa protein binds to a CE9 RNA column. (A) Sequences of the RNAs that were covalently linked to the agarose adipic acid column. (B) Proteins from a HeLa nuclear extract bound to the different RNA columns were resolved on a 12.5% polyacrylamide-SDS gel and stained with silver nitrate. The positions of some of the molecular weight markers are shown (lane MW).
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Recombinant SRp30c binds specifically to CE9. To confirm that SRp30c can interact specifically with CE9, we used GST-tagged SRp30c (rSRp30c; kindly provided by Stefan Stamm, Erlangen, Germany) in a mobility shift assay. 32P-labeled CE9 RNA was incubated in the presence of increasing amounts of rSRp30c. Complex formation occurred with CE9 RNA but not with a control RNA (Fig. 4, compare lanes 2 to 5 with 11 to 14). The specificity of the interaction of SRp30c with CE9 was also assessed by testing the binding of ASF/SF2, a protein that displays 74% amino acid identity with SRp30c. The RS domain of SRp30c is the shortest of all those of SR proteins reported to date, an attribute that may explain why SRp30c is not as active as ASF/SF2 in its ability to confer splicing activity to a HeLa S100 extract (50). Recombinant ASF/SF2 did not form a complex with CE9 RNA (Fig. 4, lanes 6 to 9). Thus, despite the high degree of similarity between ASF/SF2 and SRp30c, only the latter can bind to CE9.
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FIG. 4. Recombinant SRp30c binds specifically to CE9 RNA. Labeled transcripts (see Fig. 2) were incubated for 15 min on ice in the presence of 0.1, 0.25, 0.5, and 1.0 µg of GST-SRp30c or GST-ASF/SF2. Complexes were separated on a nondenaturing 5% acrylamide gel. Samples in lanes 1 and 10 lack recombinant proteins.
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FIG. 5. SRp30c mediates the activity of CE9 in vitro. (A) Splicing reaction mixtures with a HeLa nuclear extract were preincubated for 10 min with 250 fmol of 3x9f RNA as the competitor (lanes 2 to 5 and 7 to 10). Increasing amounts of Trx-SRp30c (0.75 and 1 µg) and 1 µg of GST-ASF/SF2 were then added to the mixture, which was further incubated for 10 min (lanes 3 to 5 and 8 to 10) before the addition of the labeled A3x or A pre-mRNA, as indicated. (B) Mixtures for splicing reactions performed in a HeLa nuclear extract were preincubated for 10 min with increasing amounts of recombinant proteins (1 and 1.5 µg of Trx-SRp30c and GST-ASF/SF2) followed by the addition of labeled A2x or the A2x transcripts. Mock incubations were also performed as controls (lanes 1 and 6). A2x contains two copies of CE9, while A2x contains two copies of the complementary sequence of CE9. Note that two copies of CE9 are not sufficient to promote a consistent reduction in splicing efficiency in vitro in this system (compare lane 1 with lane 6). Likewise, a single copy of CE9 had no effect in the adenovirus system (not shown). Splicing products in both panels were resolved on a 6.5% acrylamide-8 M urea gel. The positions of the pre-mRNAs and various lariat products are indicated.
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The first 7 nt of CE9 are important for SRp30c binding. We used the first 16 nt of CE9 to recover SRp30c by affinity chromatography. To further narrow the sequence requirement for binding by SRp30c, we performed a gel shift assay with smaller RNA fragments. rSRp30c could bind specifically to the first 7 nt of CE9 but not to the immediately downstream 8-nt portion (Fig. 6 compare lanes 6 to 8 with 2 to 4, respectively). To probe the sequence requirement in this 7-nt portion, mutated versions of CE9.7 were tested in a mobility shift assay. Swapping the G at position 4 for the A at position 5 (CE9.7AG) or converting the G at position 3 into an A (CE9.7A3) abolished SRp30c binding (Fig. 6B, lanes 9 to 16).
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FIG. 6. The binding of SRp30c to CE9 correlates with repression activity. (A) Sequences of the various RNAs. CE9.16 was used in RNA affinity chromatography (see Fig. 3). Underlined nucleotides indicate mutations relative to the wild-type CE9.7 sequence. (B) Radiolabeled transcripts were incubated for 15 min on ice in the absence (lanes 1, 5, 9, and 13) or the presence of increasing amounts of GST-SRp30c (0.1, 0.25, 0.5, and 1 µg). Complexes were separated on a nondenaturing 5% acrylamide gel. (C) Splicing reactions were performed with a labeled adenovirus or C3' pre-mRNA derivatives (see Fig. 1B). The adenovirus substrates contained three copies of the shortest CE9.7 minimal sequence or mutant versions, while the C3' variants contained two copies of CE9.7 or mutant versions. The adenovirus and C3' splicing products were resolved on an 8% acrylamide-8 M urea gel and an 11% acrylamide-8 M urea gel (lanes 1 to 3 and 4 to 7, respectively). The positions of the pre-mRNAs and various lariat products are indicated.
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The repressing activity of SRp30c is not a general property of SR proteins bound to intron sequences. To address whether the binding of another SR protein in the intron can also promote repression, we replaced the three copies of CE9.7 with three copies of a purine-rich element that represents a high-affinity binding site for ASF/SF2 (38). A gel shift assay performed with the purine-rich element and the CE9.7 RNA reveals that recombinant ASF/SF2 binds to the purine-rich element (Fig. 7B, lanes 2 to 5). Notably, rSRp30c could bind to the purine-rich element at least as efficiently as to CE9.7 (Fig. 7B, lanes 6 to 9 and 11 to 14). The insertion of this purine-rich element at the same position as CE9 in the intron of an adenovirus pre-mRNA did not compromise splicing activity (Fig. 7C, compare lanes 2 and 3). Thus, the presence in an intron of high-affinity binding sites for ASF/SF2 did not elicit splicing inhibition. Although this element is also bound by SRp30c, its binding by the higher-abundance ASF/SF2 protein (25) may explain the lack of repression in a HeLa extract. Thus, targeting the binding of a different, yet closely related, SR protein in the intron does not promote splicing repression.
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FIG. 7. A purine-rich element bound by SR protein ASF/SF2 does not elicit repression when positioned in the intron. (A) Sequences of 3xPu and 3xCE9.7. (B) Radiolabeled transcripts were incubated for 15 min on ice in the absence (lanes 1 and 10) or presence of GST-SRp30c and GST-ASF/SF2 (0.1, 0.5, and 1 µg). Complexes were separated on a nondenaturing 5% acrylamide gel. (C) Splicing reactions were performed with labeled adenovirus containing either three copies of CE9.7 or three copies of the purine-rich element. Adenovirus splicing products were resolved on an 8% acrylamide-8 M urea gel. Note that the difference in the migration of pre-mRNAs and lariat products between A3xCE9.7 and A3xPu is due to extra nucleotides from pBluescript II KS(+) in the A3xCE9.7 substrate.
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The contribution of SR proteins to splicing has been associated principally with activation, either as generic splicing factors or as proteins that bind to exon enhancer elements to stimulate the use of an adjacent splice site (24). However, there are a few cases of splicing repression implicating the SR proteins. The binding of an SR protein to the RSV intron silencer element may be important to elicit repression but is not sufficient by itself (41, 42). As is the case with CE9, the RSV intron silencer does not affect U2 snRNP-dependent complex formation (19). It was proposed that the binding of SR proteins enhances U1 snRNP binding to another region of the RSV silencer, leading to the formation of a nonproductive spliceosome (26, 41, 44). Whether a similar mechanism is taking place with CE9 remains to be demonstrated.
The activity of CE9 is also reminiscent of the activity of intron silencer element 3RE, found in the adenovirus IIIa pre-mRNA (33, 47). Although recombinant SRp30c can also bind to 3RE to repress splicing, the natural contribution of SRp30c to splicing inhibition in this case remains unclear because the more abundant ASF/SF2 protein can also bind to 3RE and elicit repression (33, 47). In contrast, we have shown that ASF/SF2 does not strongly bind to CE9 and does not promote splicing repression of a CE9-containing pre-mRNA. CE9 does not appear to be functionally equivalent to 3RE. First, 3RE but not CE9 can function as an enhancer when placed downstream from a 3'splice site (33) (data not shown). Second, the proximity of 3RE to the branch site is apparently responsible for the interference with U2 snRNP binding to the branch site (33). In contrast, the assembly of U2-dependent complexes is not compromised by CE9 (52). CE9 is naturally located 100 nt upstream from the putative branch site of exon 8, and we have observed a similar level of inhibition when CE9 is positioned up to 300 nt from the adenovirus branch site (data not shown). Based on these considerations we conclude that the activity of 3RE and CE9 is likely mediated by different factors which use different mechanisms of splicing inhibition.
Finally, it is worth noting that the presence of binding sites for SR proteins in introns is not always associated with splicing inhibition. For tropomyosin, natural intron binding sites for SR proteins have been associated with enhancer activity (18). We have also shown previously that positioning the exonic splicing enhancer from the human fibronectin ED1 exon in the intron does not inhibit splicing (36). Finally, we have shown here that inserting several copies of a purine-rich element bound by ASF/SF2 at the same position as CE9 does not repress splicing.
Several observations suggest that CE9 may play a role in the control of hnRNP A1 alternative splicing. We have identified several elements that prevent efficient exon 7B inclusion in the hnRNP A1 pre-mRNA (3, 7). Among these, CE6 impairs the use of the 5' splice site of exon 7B by forming a stable duplex structure with this splice site (2). Duplex formation acting on the hnRNP A1 alternative splicing unit essentially creates a situation where the 3' splice sites of exon 7B and exon 8 are in competition for pairing with the 5' splice site of exon 7. By repressing the 3' splice site of exon 8, CE9 may therefore favor the production of the A1B mRNA.
At this point, however, the true contribution of the SRp30c-CE9 interaction to A1 splicing control in HeLa cells remains unclear. While a single copy of CE9 has an impact on exon skipping in vivo in a heterologous system, the expression of a minigene carrying a deletion of CE9 does not significantly affect the frequency of exon 7B inclusion (52). Moreover, the addition of several copies of CE9 in the A1 minigene is required to eliminate splicing to the 3' splice site of exon 8 in vivo (52). Likewise, whereas a single CE9 element is sufficient to shift splicing in vitro when a pre-mRNA containing the 3' splice sites of exon 7B and adenovirus major late exon 2 is used (52), two CE9 elements are required to affect exon 7B/exon 8 splicing in vitro when a simple one-intron pre-mRNA is used. We could not use a pre-mRNA containing the 3' splice sites of exon 7B and exon 8 to measure the effect of a single CE9 element on its natural downstream 3' splice site because splicing to exon 7B occurred exclusively even in the absence of CE9 (data not shown). The impact of a single CE9 element in its natural context may be difficult to observe in HeLa cells and extracts because the 3' splice site of exon 8 is weak and because CE9 may not be sufficiently robust to offset the activity of other elements (e.g., CE6, CE4, and CE1) that all favor exon 7B skipping. The contribution of CE9 to splicing control may become more important at higher concentrations of SRp30c. Notably, the abundance of SRp30c mRNA varies in a tissue-specific manner (50). It will be worth testing whether modifying the abundance of SRp30c can affect the inclusion frequency of exon 7B in HeLa cells and in other cell types.
M.J.S. was the recipient of a studentship from the FCAR/FRSQ. This work was supported by a grant from the Canadian Institutes of Health Research (CIHR) to B.C. B.C. is a Canada Research Chair in Functional Genomic and is a member of the Sherbrooke RNA/RNP group supported by the Université de Sherbrooke, the FCAR, and the CIHR (grant no. MGC-48372).
Present address: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605. ![]()
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