Overexpression of SRm160 stimulates CD44 v5 inclusion dependent on Ras signaling.
Because of the biological importance of CD44 splice variants in tumorigenesis, we explored the regulation of CD44 alternative splicing. The CD44 variable exon sequences were compared between species, and potential ESEs in the v5 exon were computationally identified (9
). Among the species examined (human, rat, and mouse), conserved GAA repeats were observed in the v5 exon. Both experimental and computational analyses indicate that GAA repeats are a hallmark of ESEs that activate splicing (9
). Interestingly, SRm160, an SR-containing splicing coactivator, has been shown to be important in the splicing of pre-mRNAs that contain GAA repeats (8
). These observations suggested that SRm160 might be involved in the regulation of v5 inclusion.
To determine whether SRm160 has an important role in regulating CD44 alternative splicing, we used a CD44 v5 reporter minigene construct (23
). This minigene contains a luciferase-based splicing reporter, in which the CD44 v5 exon and its flanking introns were inserted upstream of an intact Photinus
luciferase gene (Fig. ). Inclusion of the v5 exon produces a fusion protein containing the coding sequence of the Photinus
luciferase, whereas exclusion of the v5 exon positions a stop codon upstream of the luciferase coding region. Thus, the luciferase activity directly indicates the levels of CD44 v5 inclusion. Previous work has demonstrated that the ratios of the v5 inclusion and exclusion are consistent whether they are measured by RT-PCR of spliced mRNA or luciferase assays (45
FIG. 1. SRm160 stimulates CD44 v5 inclusion. (A) The luciferase-based v5 minigene schematic. Exons are shown as boxes and introns are denoted as straight lines. Lines above and below each intron illustrate different joining of exons by alternative splicing. Positions (more ...)
The CD44 v5 minigene construct was cotransfected in 293T cells with either a control vector or a plasmid ectopically expressing SRm160, and a Renilla luciferase plasmid served as an internal transfection control. One transfection (Fig. , vector) included the CD44 v5 minigene and an empty plasmid vector. The second transfection (Fig. , SRm160) included the v5 minigene and the plasmid encoding SRm160. This series was repeated with the addition of treatment with PMA, which activates the Ras pathway. The level of luciferase was normalized to 1.0 following transfection with the control plasmid, which corrects for any stimulation by PMA alone of either transcription or inclusion of v5. Under these conditions, SRm160 overexpression increased the level of luciferase, i.e., CD44 v5 inclusion, by 2.4-fold in comparison with control vector transfection (Fig. ). This increase was entirely dependent upon PMA stimulation, as very little change (1.1-fold) was produced by SRm160 overexpression in non-PMA-treated cells. These observations suggested that stimulation of CD44 alternative splicing by SRm160 is regulated by Ras signaling. To confirm this hypothesis, a constitutively activated form of Ras, H-Ras V12, was ectopically expressed in 293T cells along with either control or SRm160 expression plasmids using the same experimental design (Fig. ). As expected, SRm160 specifically stimulated luciferase expression by ~3.7-fold in cells that overexpressed H-Ras V12. These results are consistent with the idea that the stimulation of v5 inclusion by SRm160 is dependent on Ras signaling.
The stimulation of luciferase expression from the CD44 v5 minigene is an indirect measure of alternative splicing. To directly assay the v5 inclusion in mRNA, we analyzed the ratios of variants by RT-PCR with primers flanking the v5 exon (Fig. ; the location of primers relative to spliced mRNAs is indicated by arrows). Both the included and excluded variants can be detected in one PCR, with the former yielding a longer product. In the absence of Ras activation, the levels of v5 inclusion were similar in cells ectopically overexpressing SRm160 or control vector (0.20 and 0.13, respectively). However, in the presence of cotransfected activated Ras, overexpression of SRm160 stimulated the level of v5 inclusion to 0.75 (Fig. ). These results show that SRm160 activity can regulate CD44 alternative splicing through processes influenced by Ras signaling.
A potential mechanism explaining the activity of SRm160 in stimulation of the v5 exon is the presence of GAA-containing ESEs. This was addressed by testing the activity of a mutant form of v5 exon where the 10-nt GAA-containing ESE segment (ATGAAGAGGA) was replaced with a random sequence (19
). In agreement with previously described results (19
), we observed, in the presence of activated Ras, an ~50% reduction in inclusion of the v5 exon when this GAA mutant was compared to the wild-type v5 construct. More importantly, the SRm160 stimulatory effect on v5 inclusion was almost entirely abolished by this mutation (0.10 versus 0.15, without and with SRm160, respectively) (Fig. ). A control mutation with a 10-nt substitution immediately downstream of this GAA segment displayed stimulatory effects very similar to the wild-type v5 construct upon SRm160 overexpression (ratios of inclusion to exclusion: 0.23 versus 1.09, without and with SRm160, respectively) (data not shown). To test whether the 10-nt GAA-containing ESE segment is sufficient to allow Ras-dependent alternative splicing, we used a reporter construct, pSXN (6
), that contains an alternatively spliced 33-nt exon 2. Random-sequence insertion in this exon 2 predominantly results in exon skipping. Interestingly, insertion of the 10-nt GAA segment into the pSXN exon 2 promoted inclusion of this exon. This inclusion was moderately stimulated (1.6-fold ± 0.1-fold) in response to SRm160 in a Ras signaling-dependent manner (data not shown). These observations indicate that the v5 GAA-containing ESEs and possibly other elements are important for SRm160's stimulation of v5 inclusion.
Knockdown of SRm160 inhibits inclusion of the CD44 v5 exon.
To further investigate the role of SRm160, we determined the effects of SRm160 reduction on v5 inclusion. SRm160 siRNA transfection was used to reduce the level of this protein. Transient transfection of the SRm160 siRNA produced an ~10-fold knockdown of SRm160 mRNA as measured by RT-PCR analysis relative to cells transfected with a control siRNA (Fig. ). Protein analysis using immunoblotting with an SRm160-specific antibody showed that the levels of a series of bands of the SRm160 protein were undetectable in cells treated with SRm160 siRNA (Fig. ). These clustered polypeptide bands for SRm160 have been described previously and probably represent different degrees of phosphorylation of this highly repetitive SR protein. In total, these results indicate that RNAi knockdown was successful.
FIG. 2. SRm160 knockdown inhibits v5 inclusion. (A) Quantitative RT-PCR analysis of the SRm160 mRNA harvested from HeLa cells transiently transfected with either control siRNA or SRm160 siRNA. Primers specific to SRm160 were used. Amplification of DHFR mRNA was (more ...)
The v5-Photinus luciferase reporter construct was cotransfected in HeLa cells with the H-Ras V12 plasmid, the internal control Renilla luciferase plasmid, and either SRm160 siRNA or control siRNA. Again, the level of expression of the v5-Photinus luciferase reporter was normalized to 1.0 for the control siRNA. There was about a fivefold reduction of v5 inclusion in cells treated with the SRm160 siRNA in comparison to those treated with the control siRNA (Fig. ). These results were verified by measuring mRNA levels of v5 inclusion versus exclusion by RT-PCR. As anticipated, the inclusion form of v5 was reduced in SRm160 siRNA-treated cells. The ratio of included to excluded forms in SRm160 siRNA-treated cells was reduced to 0.29 from 0.74, the level observed in control siRNA-treated cells (Fig. ). To exclude the possibility that the observed effect is due to nonspecific suppression of off-target genes by the SRm160 siRNA, we tested a second siRNA to SRm160. Indeed, expression of this second siRNA also reduced the inclusion of CD44 v5 (data not shown), suggesting that the reduction is specific to SRm160 silencing. These observations indicate that the activity of SRm160 is important for v5 inclusion.
SRm160 interacts with Sam68.
The Src substrate and RNA binding protein Sam68 have been reported to be important for stimulation of v5 inclusion by Ras activation (23
). Since both SRm160 and Sam68 stimulate this inclusion, it is possible that these two proteins might act in the same pathway and could interact with each other. To test this possibility, immunoprecipitation experiments were performed. A plasmid containing Flag-tagged SRm160 was transfected into 293T cells. Proteins that interact with Flag-tagged SRm160 protein were precipitated using anti-Flag antibody-coupled beads. The presence of Sam68 in the immunoprecipitated fraction was detected by immunoblotting using a Sam68 antibody. Indeed, the result shows that SRm160 and Sam68 coimmunoprecipitate (Fig. ). The above observation was further confirmed by immunoprecipitation using lysates from cells that overexpressed HA-tagged Sam68 protein and anti-HA antibody-coupled beads (Fig. ). Again, we observed that SRm160 immunoprecipitated with Sam68. Moreover, this coimmunoprecipitation was resistant to extensive RNase treatment (data not shown), indicating that SRm160 and Sam68 association was not dependent on RNA bridging. We conclude that these two proteins can associate through protein-protein interactions. In addition, we observed that overexpression of Sam68 did not rescue the reduction of v5-luciferase inclusion caused by SRm160 siRNA treatment, suggesting that these two proteins may be functionally cooperative (data not shown).
FIG. 3. SRm160 and Sam68 coimmunoprecipitate. 293T cells were transfected with plasmids containing either Flag-tagged SRm160 (A) or HA-tagged Sam68 (B) or corresponding empty vectors. Cell lysates were immunoprecipitated using anti-Flag or anti-HA antibodies, (more ...) SRm160 regulates splicing of endogenous CD44 variable exons.
The above results, obtained using a transfected plasmid as a reporter for RNA splicing, suggest that SRm160 is important for inclusion of CD44 v5. Therefore, we wanted to determine whether SRm160 regulates the alternative splicing of endogenous CD44. First, the expression pattern of CD44 variants in Ras-activated HeLa cells was examined. As shown in Fig. set of 5′ primers that specifically base pair to individual variable exons and a common 3′ constitutive exon were used to detect CD44 variants using quantitative RT-PCR analysis. In this case, the length and sequences of the PCR products from each primer pair should indicate the composition of the CD44 variants containing the specific exon. Using this set of primers, we observed that expression of variants containing v3 to v10 was detectable in HeLa cells. We were not able to assay the levels of v2 since the amplification levels with v2 primers were very low, probably due to the length of the v2 PCR products. In addition, the mobility of the band amplified by the exon 4 primer suggested that the v4 and v5 exons were spliced together (this observation was also verified by sequencing the PCR products). Similarly, exons v7 to v10 are apparently spliced as one unit in these cells. The v6 exon is both joined to the v7 to v10 exons and spliced directly to the 3′ constitutive exon (Fig. ).
FIG. 4. SRm160 is required for splicing of endogenous CD44 variants. (A) Expression pattern of CD44 variants in HeLa cells. The relative locations of PCR primers are depicted at the top of the gels. To analyze different variants of CD44, PCRs were performed using (more ...)
Next, the effect of SRm160 knockdown by siRNA was examined. Following SRm160 siRNA treatment, the overall splice patterns of CD44 variants were largely unchanged; however, the levels of expression of most variants were reduced relative to the constitutive isoform (Fig. ). For v2, v3, v6, and v7 variants, a second set of PCR primers was used for quantitation. This set contained a 5′ primer complementary to the proximal 5′ constitutive exon and 3′ primers complementary to these individual variable exons (Fig. , v2r, v3r, v6r, and v7r). Each set of primers amplified one major band, corresponding to the joining of the variable exon to the 5′ constitutive exon (v2r, v3r, and v6r) or the joining of both v6 and v7 exons to the constitutive exon (v7r). For quantitation, all of the PCRs were monitored to ensure that measurement was done in a linear range (data not shown). Amplification of the constitutive spliced form of CD44, the product from the two flanking primers, was performed in parallel. Amplification of the DHFR gene was used as an internal control. To compare the expression levels of CD44 between samples (control siRNA- and SRm160 siRNA-treated cells), the CD44 PCR intensities were normalized to those of the DHFR products to obtain relative levels of CD44 expression. Interestingly, we observed that the CD44 constitutive isoform and v10 variant were expressed at similar levels in both groups of siRNA-treated cells. However, expression levels of variants v2 to v9 were reduced in SRm160 siRNA-treated cells. Inclusions of v4 and v5 were most dramatically affected, by five- to sixfold. Inclusion of v3 was decreased by fivefold. Inclusions of v7, v8, and v9 were reduced by three- to fourfold, and v2 and v6 inclusions were decreased by three- and twofold, respectively. As mentioned above, exons v4 and v5 and exons v7, v8, and v9 are spliced as units in HeLa cells (Fig. ). The similar degrees of downregulation of these exons provide evidence for the accuracy of the quantitations. Taken together, these results indicate that SRm160 is important for the inclusion of most of the endogenous CD44 variable exons.
Because of the involvement of Sam68 in CD44 alternative splicing and the interaction between Sam68 and SRm160 described above, it was important to determine whether Sam68 is important for inclusion of the same subset of CD44 variable exons as SRm160. To examine this, we knocked down Sam68 expression by siRNAs and examined the effect on CD44 inclusion. RT-PCR analysis showed about a fourfold reduction of Sam68 RNA expression in Sam68 siRNA-treated cells, and Western blot analysis showed a similar reduction of Sam68 protein in these cells (Fig. ). Notably, we observed very similar to CD44 inclusion patterns in cells treated with Sam68 siRNA those treated with SRm160 siRNA, with slight differences (Fig. ). For example, inclusion of v2 was more affected in Sam68 siRNA- than in SRm160 siRNA-treated cells, with a sevenfold versus a threefold reduction, respectively. Inclusion of v5 was also less affected in the same comparison, with a threefold versus a sixfold reduction, respectively. These results indicate that knockdown of Sam68 impaired the inclusion of endogenous CD44 variable exons to degrees similar to those found with the knockdown of SRm160. Together with the coimmunoprecipitation studies, these results indicate that SRm160 and Sam68 are important for regulation of alternative splicing of the endogenous CD44 gene in HeLa cells.
SRm160 and tumor cell invasion.
CD44 variants have been implicated in tumor invasion and metastasis (16
). Therefore, it is tempting to speculate that the activity of SRm160 could influence tumor cell invasion by regulating the inclusion of CD44 variable exons. Thus, we tested whether reducing SRm160 levels in cells would affect their invasiveness. In order to validate an invasion assay while performing an interesting novel experiment, we tested whether inhibition of CD44 variants by RNAi would affect the invasive properties of cells. We chose to target variants containing v5 exon because v5 inclusion is elevated in some metastatic cells and tissues (12
). We used human cervical carcinoma cells (HeLa) as these cells are active in the invasion assay and are highly transfectable (42
). An siRNA designed to specifically target the v5 exon was transfected into HeLa cells. As shown in Fig. , we observed a 3.5-fold knockdown of v5-containing variants in cells treated with this siRNA. A control siRNA, whose sequence does not match any known human genes, did not cause a reduction of v5 expression.
FIG. 5. Knockdown of SRm160 reduces tumor cell invasiveness. (A) Quantitative RT-PCR analysis of the v5 mRNA from HeLa cells transiently transfected with either control siRNA or v5 siRNA. Primers specific to v5 were used. Amplification of DHFR mRNA was used as (more ...)
At 48 h posttransfection, equal numbers of cells treated with either v5 siRNA or control siRNA were added to invasion chambers in the absence of serum. The bottom of the invasion chamber contained a membrane with a layer of reconstituted basement membrane matrix, which impedes noninvasive cells from migrating through the membrane. Invasive cells are able to digest and migrate through the matrix and thus the membrane. Serum-containing medium was placed in the well surrounding the chamber and acted as a chemoattractant for a growth factor-induced invasion. After 22 to 24 h of incubation, we fixed and counted cells that invaded through the matrigel membrane and accumulated in the lower chamber. Interestingly, v5 siRNA-treated cells were impaired in their invasive potential (Fig. ). The number of v5 siRNA-treated cells passing through the membrane was only approximately 20% of the control siRNA-treated cells. We observed similar results when the above-described experiments were repeated using a second v5 siRNA. These results demonstrate that reduction of the CD44 v5-containing variants inhibits tumor cell invasion. It substantiates the previous suggestion that CD44 variants are involved in cell invasiveness.
Since reduction of SRm160 expression by siRNA knockdown inhibited v5 inclusion, we tested whether treatment with SRm160 siRNA would alter the invasiveness of cells. HeLa cells were treated with siRNAs specific to either SRm160, v5, or nonspecific control siRNA and tested as described above for invasiveness. Interestingly, cells treated with SRm160 siRNA showed a dramatic fivefold reduction in invasion activity (Fig. ), a reduction in invasiveness similar to that seen with v5 siRNA-treated cells. These results suggested that SRm160 activity could have an important role in tumor cell invasion by altering the level of CD44 variants in cells.
We have previously observed that SRm160 siRNA-treated cells proliferate more slowly than control siRNA-treated cells (unpublished observations). To confirm that the impaired invasiveness by SRm160 was not due to slower cell proliferation, the proliferation rate of these cells was monitored in parallel to the invasion assay. In the invasion assay period, 22 to 24 h, the cell numbers from these two populations differ by less than 15% (data not shown). Therefore, the reduction in cell number in the lower chamber following SRm160 siRNA treatment is not due to the decrease in rate of proliferation.