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1 Horizontal Medical Research Organization (HMRO), Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
2 Department of Functional Genomics, Graduate School of Biomedical Science, Tokyo Medical and Dental University, Tokyo 113-8510, Japan
3 Medical Top Track (MTT) Program, Medical Research Institute, Graduate School of Biomedical Science, Tokyo Medical and Dental University, Tokyo 113-8510, Japan
4 Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan
5 Division of Regeneration and Advanced Medical Science, Graduate School of Medicine, Gifu University, Gifu 501-1193, Japan
6 Molecular Probe & Drug Design Laboratory, Molecular Imaging Research Program, RIKEN, Kobe 650-0047, Japan
7 Laboratory of Gene Expression, Graduate School of Biomedical Science, Tokyo Medical and Dental University, Tokyo 113-8510, Japan
8 Cell Biology Group, Kansai Advanced Research Center, National Institute of Information and Communications Technology, Kobe 651-2492, Japan
9 Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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Several kinases have been reported to phosphorylate SR proteins, including SR protein kinases (SRPKs; Gui et al. 1994; Kuroyanagi et al. 1998; Wang et al. 1998), Cdc2-like kinases (Clks; Ben-David et al. 1991; Howell et al. 1991; Johnson & Smith 1991; Nayler et al. 1997; Duncan et al. 1998), pre-mRNA processing 4 (PRP4; Alahari et al. 1993; Kojima et al. 2001), topoisomerase I (Rossi et al. 1996) and dual-specificity tyrosine-regulated kinases (DYRKs; Alvarez et al. 2003; de Graaf et al. 2004). SRPK1 is the first SR kinase purified and cloned on the basis of its ability to phosphorylate SR proteins in vitro and localizes to cytoplasm (Gui et al. 1994). Two other structurally related proteins, SRPK2 and SRPK3 are also shown to phosphorylate an SR protein, splicing factor 2/alternative splicing factor (SF2/ASF; Kuroyanagi et al. 1998; Wang et al. 1998; Nakagawa et al. 2005). PRP4 is a Ser/Thr kinase identified by genetic screening to have defects in pre-mRNA splicing in Schizosaccharomyces pombe (Alahari et al. 1993). The human counterpart hPRP4 phosphorylates SF2/ASF in vitro and is concentrated in nuclear speckles, where SR proteins are enriched (Kojima et al. 2001). Clk family kinases, consisting of four members (Clk1/Sty and Clk2–4) and harboring a consensus amino acid sequence LAMMER, are shown to be dual-specificity kinases that autophosphorylate on Ser, Thr and Tyr residues in over-expression systems and in vitro (Ben-David et al. 1991; Howell et al. 1991; Nayler et al. 1997). When over-expressed in mammalian cells, their catalytically inactive mutants localize to the nuclear speckles, whereas the wild-type enzymes distribute throughout the nucleus and cause the nuclear speckles to dissolve (Colwill et al. 1996b; Duncan et al. 1998; Sacco-Bubulya & Spector 2002; Muraki et al. 2004). DYRK1A, a member of DYRK family, phosphorylates RS-domain containing proteins and is concentrated in the nuclear speckles (Alvarez et al. 2003; de Graaf et al. 2004). These SR kinases have distinct subcellular localizations and substrate specificities (for a review, see Hagiwara 2005), so that they could individually have specific functions in RNA metabolism through phosphorylation of distinct RS domains. However, their differential and/or coordinated roles in SR phosphorylation remain largely unknown (Nikolakaki et al. 2002; Ngo et al. 2005; Velazquez-Dones et al. 2005).
In order to examine the role of SR kinases on alternative splicing, we compared the effect of four different SR kinase families, such as SRPKs, Clks, Prp4 and DYRKs, on alternative splicing of Adenovirus E1A pre-mRNA. Among them, Clks most efficiently promoted the use of distal 5' splice site to produce 9S isoform. Under the same conditions, 75 kDa SR protein was hyperphosphorylated only by Clks. Complementarily, when mouse P19 cells were treated by a Clk-specific inhibitor, TG003 (Muraki et al. 2004), the phosphorylation level of 75 kDa SR protein was drastically and rapidly reduced. Live cell imaging with mRFP-SRp75 revealed that SRp75 was rapidly accumulated in nuclear speckles upon Clk inhibition by TG003. Finally, the over-expression of Clks, but not the other kinases, induced hyperphosphorylation of HA-tagged SRp75 and affected the splice site selection of Adenovirus E1A pre-mRNA. These results indicate that SRp75 function on alternative splicing can be controlled through its hyperphosphorylation by Clks. Our results strongly suggest that alternative splicing is regulated by a combination of SR protein and its kinase(s).
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Results |
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We first systematically analyzed the effect of the over-expression of several SR kinases on the splice site selection of Adenovirus E1A minigene (Fig. 1A), which produces three major splice isoforms (9S, 12S and 13S) depending on different 5' splice sites (Caceres et al. 1994; Duncan et al. 1997). The E1A minigene was transfected into HeLa cells with the expression vector of either of kinases, Clks, SRPKs, Prp4 and DYRKs, tagged with HA or enhanced green fluorescent protein (EGFP) (Fig. 1A). The production of different isoforms of Adenovirus E1A mRNA was analyzed by RT-PCR. As shown in Fig. 1A (lanes 4–7), Clk family kinases caused the increase of 9S and decrease of 13S isoforms in a kinase-activity dependent manner, since their kinase inactive KR mutants, in which the active site Lys (K) is substituted to Arg (R), had little effect on the splice site selection. This result is consistent with the previous studies using COS cells (Duncan et al. 1997, 1998; Muraki et al. 2004). In contrast, other kinases tested here did not show such an effect under the same experimental conditions, whereas the expression level of each wild-type kinase except DYRK2 was almost comparable (Fig. 1A,C). KR mutants of Prp4 and DYRK1A were expressed at lower levels, but their wild-type proteins had anyhow little effect (Fig. 1A,C). These results indicate that the selection of 9S 5' splice site is specifically stimulated by Clk activity. Since this effect is likely mediated through phosphorylation of SR protein(s), we next checked the phosphorylation state of SR proteins by using anti-phospho SR protein antibodies. The antibody mAb1H4 is known to specifically recognize phosphorylated RS repeats in SR proteins (Neugebauer & Roth 1997). Immunoblotting with mAb1H4 showed several signals derived from SR proteins as expected. Among them, we could detect the most significant difference in 75 kDa protein. The signal of this protein became more intense and its mobility slightly slower when Clks were expressed (Fig. 1B; lanes 4, 6 and 7), consistent with our previous data by COS cells (Muraki et al. 2004), in which the effect was much more evident probably due to the high level of protein expression. Similar results were also seen by immunoblotting with another anti-phospho SR protein antibody mAb104 (Roth et al. 1991; Fig. 1B), supporting the view that the 75 kDa SR protein is hyperphosphorylated by Clks. In addition to the 75 kDa protein, the other SR proteins could also become hyperphosphorylated, but exhibited little difference by one-dimensional gel electrophoresis used here. For example, the mobility of SF2/ASF remained unchanged by the expression of any kinases (Fig. 1B), whereas its mobility shifts were observed when phosphorylated by SRPKs and Clks in vitro (Gui et al. 1994; Nayler et al. 1997; Kuroyanagi et al. 1998; Wang et al. 1998; Muraki et al. 2004), suggesting that the endogenous SF2/ASF is already highly phosphorylated.
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We next investigated the effects of Clk inhibition on the phosphorylation of SR proteins using a Clk-specific inhibitor, TG003 (Muraki et al. 2004). P19 cells that express relatively high levels of Clk1/Sty mRNA were incubated in TG003 for 0–120 min and SR phosphorylation was analyzed by immunoblotting using mAb1H4. As shown in Fig. 2A, the most drastic change was observed in 75 kDa protein among the several SR proteins. Within 5–10 min after the administration of TG003, the indications of dephosphorylation, such as weakened signal and slightly faster migration, appeared in the 75 kDa SR protein (lanes 1–3). The signal became much weaker in 30 min and later (lanes 5–7). Similar results were obtained with another phospho-SR specific antibody, mAb104 (data not shown). As the mobility shifts of the same protein were also detected with the anti-SR antibody (16H3) that recognizes RS domains regardless of the phosphorylation state (Fig. 2B), the weakened signals found with phospho-specific antibodies should represent the dephosphorylation but not the protein degradation. In contrast, TG003 did not affect the mobility of SF2/ASF (Fig. 2C). From these results and the complementary data showing the hyperphosphorylation of 75 kDa SR protein in cells over-expressing Clk1/Sty (Fig. 1; Muraki et al. 2004), it is likely that SRp75 is one of the targets of Clk family kinases in vivo.
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SR proteins like SC35 and SF2/ASF are usually concentrated in nuclear speckles, or splicing factor compartments (Misteli 2000). The expression of the active Clks induces the disassembly of speckles and redistribution of SR proteins to diffuse (Colwill et al. 1996b; Duncan et al. 1997, 1998; Sacco-Bubulya & Spector 2002). The localization of Clks themselves also depends on their kinase activity; when the tagged protein is expressed, the kinase-active form distributes nearly homogenously in the nucleoplasm but the kinase-negative mutant is concentrated in the speckles (Colwill et al. 1996b; Duncan et al. 1997, 1998; Sacco-Bubulya & Spector 2002; Muraki et al. 2004). As SRp75 was a good candidate for a target of Clks in vivo (Figs 1 and 2; Muraki et al. 2004), we analyzed the localization of SRp75 in cells over-expressing Clk in response to TG003. We first determined the subcellular localization of SRp75 tagged with EGFP in HeLa cells. As shown in Fig. 3A, EGFP-SRp75 was co-localized with SF2/ASF tagged with the monomeric red fluorescent protein (mRFP) in the speckles. The mRFP-SRp75 protein also exhibited co-localization with SC35 in HeLa cell nuclei (data not shown; see below). In cells expressing Clk, mRFP-SRp75 distributed nearly homogeneously throughout the nucleoplasm (Fig. 3B, –5 min), and began to accumulate in speckles within 5 min after the addition of TG003. The accumulation at the speckles became more prominent in 20 min and the speckles continued to grow-up during the observation period of 60 min (Fig. 3B,C). The SRp75 speckles after TG003 treatment contained SC35 (Fig. 3D), indicating that SRp75 behaves similarly to other SR proteins. These results indicate that the inhibitory effect of TG003 on Clk activity emerges within several minutes in living cells, consistent with the immunoblotting data showing the dephosphorylation kinetics of SRp75 (Fig. 2). Taken together, it is highly likely that SRp75 is hyperphosphorylated specifically by Clks and the phosphorylation state is controlled under the dynamic balance between the phosphorylation and dephosphorylation in vivo.
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To test whether SRp75 is hyperphosphorylated by Clks in vivo more directly, HA-tagged SRp75 (HA-SRp75) was expressed in HeLa cells in combination with Clks, SRPKs or their kinase-negative (KR) mutants, and the phosphorylation level was analyzed by immunoblotting. Compared to the untransfected cells (Fig. 4A, lane 1), the expression of HA-SRp75 alone gave a strong, broad signal at around 75 kDa in blots with anti-HA (lane 2). When HA-Clks were co-expressed, the mobility of HA-SRp75 retarded (Fig. 4A, lanes 4, 6 and 7), and the signal in the mAb1H4 blot became much intense (Fig. 4B, lanes 4, 6 and 7). In contrast, although SRPK1 and SRPK2 were able to phosphorylate HA-SRp75 to some extent, they failed to cause the retardation of HA-SRp75 (Fig. 4A,B, lanes 9–11). KR mutants of Clks and SRPK2 had no effect on HA-SRp75 signal (Fig. 4A,B, lanes 5 and 8). We also determined the effect of Clks or SRPKs on another SR protein, SF2/ASF. When myc-SF2/ASF was over-expressed alone, it migrated as smear with a top band over 35 kDa (Fig. 4C, lane 2), suggesting the presence of differentially phosphorylated forms by over-expression; the endogenous SF2/ASF appears highly phosphorylated (Figs 1B and 2C), but the responsible kinases may not be present in large excess so the over-expressed proteins are underphosphorylated. The expression of both Clks and SRPKs caused myc-SF2/ASF signals concentrated into the top band by its hyperphosphorylation (Fig. 4C, lanes 4–10). Whereas the effect of Clks was more obvious than SRPKs on myc-SF2/ASF, the difference between two kinases was modest compared with that on HA-SRp75. These results support the above notion that SRp75 is a good substrate of Clks in vivo.
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RS) was used as a negative control. If SRp75 mediates the phosphorylation signals by Clks, its over-expression could enhance the effect of Clks to increase shorter isoforms while SRp75RS should have little effect. Alternatively, SRp75 could behave like another SR protein SF2/ASF, which is known to promote the use of 13S 5' splice site (Caceres et al. 1994). E1A minigene was transfected into HeLa cells with the expression vector of mRFP-tagged SR protein in combination with the wild-type HA-Clk4 or the KR mutant. The production of three splice isoforms was detected by RT-PCR and gel electrophoresis (Fig. 5A) and their average molar ratio from four independent experiments was plotted (Fig. 5B). As reported previously with myc-SF2/ASF (Caceres et al. 1994), mRFP-SF2/ASF promoted the production of 13S, and the ratio of 12S to 13S (12S : 13S) was decreased from 1.10 (mRFP; lane 1) to 0.40 (lane 2; P = 0.007). In contrast, the 12S : 13S ratio was slightly increased with the expression of mRFP-SRp75 (lane3; from 1.10 to 1.60; P = 0.020). The RS domain of SRp75 appears to be required for promoting the production of 12S, as mRFP-SRp75RS did not increase the 12S : 13S ratio (lane 4; 0.96; P = 0.067). When Clk4 was expressed in addition to mRFP, 9S and 12S isoforms were increased (lane 5; the 12S : 13S was 1.41), confirming the previous results (Fig. 1; Duncan et al. 1997, 1998; Muraki et al. 2004). When Clk4 was co-expressed with mRFP-SRp75, the 12S : 13S was further increased to 2.55 (lane 7; P = 0.002). Co-expression of mRFP-SRp75RS with Clk4, however, had no additive effect on the 12S : 13S (lane 8; 1.40; P = 0.89). These data suggest that Clk4-mediated phosphorylation on the RS domain of SRp75 promotes the selection of 12S 5' splice site. The kinase activity of Clk4 was indeed essential for promoting the function of SRp75, as the KR mutant of Clk4 did not increase 12S isoform compared to the single expression of SR proteins (lanes 9–12). The expression of each tagged protein was confirmed by immunoblotting using anti-RFP antibody (Fig. 5C). As found in HA-SRp75 (Fig. 4), the retardation of mRFP-SRp75 band was also observed when Clk4 was co-expressed (Fig. 5C, lanes 7 and 8), although the retardation appeared less obvious due to the higher molecular weight of mRFP-tagged version. On the other hand, the mobility of mRFP-SRp75RS did not change by Clk4 expression, indicating that the RS domain of mRFP-SRp75 is required for being phosphorylated. From these results, we concluded that the function of SRp75 to promote the 12S 5' splice site selection is stimulated by Clk-mediated hyperphosphorylation. However, SRp75 does not appear to mediate the production of 9S isoform (lanes 1 and 3; 5 and 7). The data also indicated that mRFP-SF2/ASF counteracts the effect of Clk4 (lanes 5 and 6). These results are consistent with the view that different SR proteins have different phosphorylational regulation and distinct functions on the splice site selection (Valcarcel & Green 1996).
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Discussion |
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Making use of the Clk-specific inhibitor, TG003 (Muraki et al. 2004), we investigated the kinetics of SRp75 dephosphorylation. Immunoblotting analysis using mAb1H4 showed that the phosphorylation level of SRp75 apparently decreased within several minutes after the administration of TG003. TG003 also induced rapid alteration of mRFP-SRp75 localization, which reflects the kinase activity of Clks (Colwill et al. 1996b; Duncan et al. 1998; Sacco-Bubulya et al. 2002). In EGFP-Clk4-expressing cells, mRFP-SRp75 distributed nearly-homogenously in nucleoplasm but it became concentrated into nuclear speckles within several minutes after the addition of TG003. As SR proteins are continuously exchanging between the nuclear speckles and nucleoplasm (Phair & Misteli 2000), it is reasonable to observe that the dephosphorylation of SR proteins by Clk inhibition results in the rapid accumulation of SRp75 into the nuclear speckles. The hyperphosphorylation of SRp75 is also rapidly restored when TG003 was removed from the medium (Muraki et al. 2004; data not shown). These data indicates that Clk-dependent SRp75 hyperphosphorylation is mediated through the balance between the kinase and phosphatase activities. The dephosphorylation may be mediated by protein phosphatase 1, which modulates alternative splicing in vitro (Cardinali et al. 1994), dephosphorylates SR proteins, and causes rounding up of speckles in permeabilized cells (Misteli & Spector 1996). Once Clk kinase is activated, SRp75 would become hyperphosphorylated and spread throughout the nucleus, and when Clk activity is down-regulated, both the phosphorylation and nuclear localization would soon be restored to the steady-state. These processes could occur within several minutes and such dynamic balance of SRp75 phosphorylation may be involved in the regulation of alternative splicing responding to outer stimuli. Similarly, phosphorylation of Sam68, a nuclear RNA-binding protein, has been shown to control the inclusion of alternative exon v5 of CD44 (Matter et al. 2002); when ERK (extracellular signal-regulated kinase) is activated by phorbor ester, Sam68 phosphorylation occurs within 1 min.
The dynamic balance of Clk-mediated hyperphosphorylation is consistent with the biological function of Clks, which has been suggested to have regulatory roles during development and differentiation rather than in constitutive splicing reaction (e.g. Rabinow et al. 1993; Myers et al. 1994; Yun et al. 1994; Du et al. 1998; Kim et al. 2001; Muraki et al. 2004). Whereas the activity of Clks is not essential for cell growth (Kim et al. 2001; Muraki et al. 2004), Drosophila Clk homologue doa (darkener of apricot) is required for segmentation and development of the nervous system (Rabinow et al. 1993; Yun et al. 1994) and the over-expression of xClk in Xenopus embryo results in abnormal development (Muraki et al. 2004). As for a target pre-mRNA of Clk-mediated regulation, it has been shown that mRNA production of Clk1/Sty gene is regulated by negative feedback through Clk kinase activity (Duncan et al. 1997). In this study, we demonstrated that SRp75 promotes 12S 5' splice site selection of Adenovirus E1A and this function is stimulated by hyperphosphorylation through Clks. As the mutant lacking RS domain, SRp75RS, did not increase 12S, RS domain or its phosphorylation appears to be essential for the splice site selection by SRp75. The phosphorylation of the tagged SRp75 at lower levels by endogenous kinases, detected by anti-phospho-SR antibody mAb1H4 (Fig. 4), may contribute to the subtle increase of 12S isoform without the additional expression of Clks. SRp75 may bind to cis elements on the pre-mRNA and the hyperphosphorylation of its RS domain by Clks may increase the affinity of SRp75 to the favorite site in target RNA and/or to the other splicing machineries. Although we expected that the production of 9S isoform might also be promoted by SRp75, the expression of SRp75 did not affect the production of 9S isoform. In addition, 9S isoform was still increased when the wild-type Clk is over-expressed in cells pre-treated with siRNA against SRp75 (data not shown). Therefore, it is more likely that the Clk signal to 9S 5' site selection is mediated through the other protein(s). Further identification of pre-mRNAs regulated by SRp75 will shed a light on the mechanism of Clk–SRp75-mediated alternative splicing pathway.
Recent global studies on cDNA sequences or microarray data predicted that as many as two thirds of human genes have multiple isoforms of mature mRNAs (Modrek & Lee 2002), and utilization of alternative splicing microarrays revealed that many alternative splicing events are controlled in tissue- and cell-type and/or developmental-stage dependent manners (Johnson et al. 2003; Kampa et al. 2004; Relogio et al. 2005). General cis-acting enhancer and silencer elements and trans-acting splicing factors including SR proteins have been characterized by analyzing model genes. However, these cis- and trans-acting factors are not sufficient to regulate alternative splicing of so many genes in metazoans. Recently, conditional knockouts of SR proteins revealed that alternative splicing of only a few target genes are crucially dependent on a specific protein in cardiac muscles, even though much more genes expressed in this tissue have typical cis-elements (Ding et al. 2004; Xu et al. 2005). These facts indicate that unidentified cellular codes underlie regulation mechanisms of alternative splicing. The combinations of a specific SR protein and the kinase(s), such as the Clk–SRp75 pathway and SRPK–SF2/ASF complex (Koizumi et al. 1999; Ngo et al. 2005), may carry the part of cellular codes. In this study, we showed evidence linking Clk-mediated hyperphosphorylation and alternative splicing through SRp75. The results we presented here strongly suggest that the regulation of alternative splicing depends on the specific combination of a kinase and SR protein. To understand the complicated regulatory mechanism of alternative splicing in multi-cellular organism, further identification of unknown kinase-SR–RNP pathways will be required. The reverse chemical genetics with specific protein kinase inhibitors may be one of the powerful approaches to tie together the substrate protein(s) of the kinase, the splicing patterns of specific genes, and their functions in vivo.
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Experimental procedures |
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Mouse SRp75 cDNA (database accession number BC019437) was amplified by PCR (High-Fidelity PCR Master; Roche, Basel, Switzerland; 1 cycle of 95 °C for 5 min, 33 cycles of 95 °C for 30 s, 65 °C for 30 s and 68 °C for 90 s, and 1cycle of 72 °C for 10 min) using 2-µg mouse A9 cell cDNA with a set of primers. The forward primer containing SalI site (5'-ATATGTCGACAT GCCGCGGGTGTACATCGGCCGCC-3') was used together with the reverse primer, either (5'-ATGGATCCTTAGGAC CTTGAGTGGGACCTGGAT-3') containing BamHI site for pEGFP- and pmRFP-mSRp75, or (5'-ATTTAGCGGCCGCT TAGGACCTTGAGTGGGACCTGGAT-3') containing NotI site for pME-HA-mSRp75. The 1.5 kb PCR products digested with SalI/BamHI or SalI/NotI were ligated into SalI/BamHI-digested pEGFP-C1 (Clontech, Palo Alto, CA), pmRFP-C1 (a monomeric RFP expression vector harboring the same backbone as pEGFP-C1 constructed using pRSETB-mRFP1; Campbell et al. 2002), or SalI/NotI-digested pME-HA vector from pME-HA-mClk4 (Katsu et al. 2002). SRp75RS, a mutant lacking the RS domain (encoding N-terminal 1–179 amino acids), was generated by PCR (1 cycle of 95 °C for 5 min, 33 cycles of 95 °C for 15 s, 60 °C for 15 s and 68 °C for 30 s and 1cycle of 72 °C for 10 min) with the forward primer described above and a reverse primer (5'-TTTGGATCCTCAACCTGGCTTGTCTTCAACTA-3') containing BamHI site using pmRFP-SRp75 as a PCR template. The expression vector of pmRFP-SF2/ASF was constructed by ligating BamHI-digested human SF2/ASF fragment from pQE-30-SF2/ASF and BamHI digested mRFP-C1. To construct pME-HA-mClk2, mouse Clk2 fragment excised from pBluescript II-SK(+)-Clk2 (Nayler et al. 1997) by XhoI/NotI digestion was inserted into SalI/NotI-digested pME-HA. All the resulting plasmids were verified by nucleotide sequencing. The other plasmid constructs were described previously; pME-c-Myc-SF2/ASF (Koizumi et al. 1999); pME-HA-mClk1/Sty, -mClk1/Sty K190R, -hPrp4, -hPrp4 K717R (Kojima et al. 2001); pME-HA-mClk4, -mClk4 K189R, pEGFP-mClk4 (Katsu et al. 2002); pME-HA-mSRPK1, -mSRPK2, -mSRPK2 K108R (Kuroyanagi et al. 1998); pEGFP-DYRK1A, -DYRK1A K188R, -DYRK2 (Becker et al. 1998); and pEGFP-DYRK1B (Leder et al. 2003).
Cell culture and transfection
Cells were maintained in Dulbecco's modified Eagle's medium (Sigma, St Louis, MO) supplemented with antibiotics (100 U/mL streptomycin and 100 µg/mL penicillin; Sigma) and 10% fetal calf serum at 37 °C with 5% CO2. For RNA extraction and immunoblotting, 4 x 105 HeLa cells were plated in each well in a 6-well plate, and the next day plasmid DNA (2 µg) was transfected using Lipofectamin 2000 (10 µL; Invitrogen, Carlsbad, CA) according to the manufacturer's instruction. The transfected cells were incubated for further 24 h before lysis. For immunofluorescence or live cell imaging, 1.5 x 105 HeLa cells were plated in each well containing a coverslip in a 12-well plate (Fig. 3A,D) or in a 35 mm glass bottom dish (MatTek, Ashland, MA; P35G-1.5-10-C; Fig. 3B,C). The next day, plasmid DNA (0.5 or 1 µg) was transfected using GeneJuice (1.5 or 3 µL; Novagen, Madison, WI). The localization of tagged proteins was essentially the same by using different transfection reagents including GeneJuice & Lipofectamin 2000, but the expression level was lower when GeneJuice was used; we chose low expressers for live imaging. TG003 (10 mM in DMSO; Muraki et al. 2004) was added to the medium at a final concentration of 10 µM.
Analysis of splice isoforms
Total RNA was extracted using Sepasol(R) RNA I super (Nacalai Tesque, Kyoto, Japan) according to the manufacturer's instruction. After DNase treatment (RNase-free DNase; Promega, Madison, WI), cDNA was synthesized by reverse transcription (RT) using oligo-dT primer and ReverTra Ace (TOYOBO, Osaka, Japan; 42 °C for 90 min and inactivated at 98 °C for 5 min) with 2 µg of RNA template in 20 µL reaction volume. An aliquot (2 µL) was then used for PCR reaction (30 µL) with rTaq (TAKARA, Shiga, Japan) or Blend Taq (TOYOBO). The E1A splice isoforms (Figs 1A and 5A) were detected by PCR using E1A primers (5'-TGAGTGCCAGCGAGTAGAGTTTTCT-3' and 5'-TCT GGCTCGGGCTCAGGCTCAGGTT-3') with following PCR steps: 1 cycle of 95 °C for 5 min, 20 cycles of 95 °C for 30 s, 55 °C for 30 s and 68 °C for 30 s, and 1 cycle of 72 °C for 10 min. The amounts of three isoforms were measured using a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and expressed as the molar ratio. The averages and SEs were calculated from four independent experiments, and the statistical analysis (Student's t-test) was performed using Excel (Microsoft).
Immunoblotting
Cells on a 6-well plate were lysed in 500 µL of 2x SDS-gel loading buffer (200 mM dithiothreitol, 100 mM Tris–HCl, pH6.8, 4% SDS and 20% glycerol) and the lysate was boiled for 10 min. The total proteins were separated in 8% SDS-polyacrylamide gels and transferred to PVDF membranes (Pall). Membranes were blocked using 5% skimmed milk in TBST (10 mM Tris–HCl, pH8.0, 150 mM NaCl and 0.05% Tween 20) for 30 min at room temperature and incubated with a primary antibody diluted in Can-Get-Signal (TOYOBO) at 4 °C overnight for mAb1H4 (Neugebauer & Roth 1997; ATCC; CRL-2383; 1 : 5 dilution of hybridoma culture supernatant), mAb104 (Roth et al. 1991; ATCC; CRL-2067; 1 : 2 dilution of hybridoma culture supernatant) and mouse anti-SR proteins (clone 16H3; Zymed; 1 : 2000). Alternatively, blocked membranes were incubated at room temperature for 2 h for mouse anti-GFP (peroxidase-conjugated; Nacalai tesque; 1 : 2000), mouse anti-c-Myc (Sigma, 1 : 4000), mouse anti-β-actin (MBL, Woburn, MA; 1 : 1000) mouse anti-SF2/ASF (mAb103; 1 : 5000; Hanamura et al. 1998), rabbit anti-RFP (MBL; 1 : 1000) and rat anti-HA (Roche; 1 : 500). After washing 4 times with TBST over 1 h, membranes were incubated with peroxidase-conjugated secondary antibody (anti-mouse or rabbit Ig; GE Healthcare, Little Chalfont, UK; 1 : 500, or anti-rat IgG (H + L); Jackson Immunoresearch, Mulkiteo, WA; 1 : 20 000) in Can-Get-Signal for 1 h at room temperature. After washing as above, the chemiluminescent signals developed by ECL reagents (GE Healthcare) were detected using a LAS-3000 image analyzer (Fujifilm) or a ChemiDoc XRS (Bio-Rad, Hercules, CA).
Imaging of fixed and living cells
To obtain images of fixed cells (Fig. 3A,D), cells were fixed with 4% paraformaldehyde in 250 mM Hepes-KOH (pH 7.4) for 10 min. The fluorescence images of Fig. 3A were captured using a confocal microscope (Carl Zeiss; LSM510 Meta; 4x zoom) with a Plan-apochromat 63x/1.4 oil immersion objective lens using sequential scanning (488 nm laser excitation, 505–530 emission filter, and 139 µm pinhole for EGFP, and 543 nm laser excitation, 560–615 emission filter, and 134 µm pinhole for mRFP). For Fig. 3D, immunostaining was performed as described (Muraki et al. 2004) using mouse monoclonal anti-SC35 (Sigma; 1 : 4000) and Cy5-conjugated AffiniPure donkey anti-mouse IgG (H + L) (Jackson Immunoresearch; 1 : 200). The fluorescence images were captured using an Olympus FV1000 (8x zoom) with a Plan-Apo 60x/1.4 oil immersion objective lens using sequential scanning (543 nm laser excitation, 555–625 nm emission filter for mRFP-SRp75, and 633 nm excitation, 650-nm long path filter for Cy5-SC35, with 150 µm pinhole).
For live cell imaging, the culture medium was replaced with CO2-independent medium (Invitrogen) supplemented with antibiotics and 10% fetal calf serum, before setting the glass-bottom dish on to a DeltaVision microscope system (Applied Precision, Issaquah, WA; Haraguchi et al. 1997) equipped with an Olympus IX-70 inverted microscope with a 60x/1.40 PlanApo objective lens in a hot room at 37 °C. Multi-channel images were sequentially captured every 1 min for 1 h 10 min (0.1 s exposures with 32% ND filter) using CH350 camera (Roper Scientific, Trenton, NJ). TG003 was added to the medium 10 min after starting the image acquisition.
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Acknowledgements |
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Footnotes |
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aPresent address: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, CA 92093, USA.
* Correspondence: E-mail: hkimura{at}fbs.osaka-u.ac.jp |
References |
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Received: 21 June 2007
Accepted: 25 November 2007
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