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First published online 14 November 2006
doi: 10.1242/jcs.03288
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Research Article |
Radiation Effect Mechanisms Research Group, National Institute of Radiological Sciences, 4-9-1, Anagawa, Inage-ku, Chiba 263-8555, Japan
* Author for correspondence (e-mail: k_sugaya{at}nirs.go.jp)
Accepted 3 October 2006
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Summary |
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Key words: Alternative splicing, DNA synthesis, Intron retention, RNA interference, Temperature-sensitive mutation
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Introduction |
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). One ts mutant, tsTM18, carries a mutation that interferes with maintenance of chromosome integrity and progression through the cell cycle. At the nonpermissive temperature, mutant cells exhibit accumulation of single-stranded DNA in the nucleus, abnormal chromosome configuration with incomplete assembly of the spindle, failure of proper activation of cdc2 kinase during G2, and cell-cycle arrest at S and G2 phases with decreased DNA synthesis and chromosome instability (Tsuji et al., 1990; Sugaya et al., 2005).
As part of our effort to identify the gene(s) responsible for the ts defects of tsTM18 cells, we found that the ts phenotype of tsTM18 cells was caused by a G-to-A transition at nucleotide (nt) 1465 of Smu1 that results in a G-to-A substitution at amino acid 489 in the WD-repeat region of Smu1 (Sugaya et al., 2005). The smu-1 gene of C. elegans encodes a nuclear protein that contains five WD motifs and that is expressed ubiquitously (Spike et al., 2001). Genetic studies in C. elegans have suggested that smu-1 is involved in alternative splicing of unc-52, which is a homolog of mammalian perlecan (Spike et al., 2001).
Perlecan is a major heparan sulfate proteoglycan in basement membranes and connective tissues (reviewed by Iozzo et al., 1994; Jiang and Couchman, 2003). The core protein of perlecan is divided into five domains on the basis of sequence homology to other known proteins. Domain IV of perlecan is the largest domain and contains a long series of immunoglobulin (Ig)-like repeats. Complex patterns of alternative splicing of the Ig repeat region of perlecan/unc-52 have been observed in nematodes. Alternative splicing of exons 16, 17 and 18 is regulated by the mec-8 (Lundquist et al., 1996; Spike et al., 2002) and smu-1 genes (Spike et al., 2001). A presumptive null mutation in smu-1 (smu for suppressor of mec-8 and unc-52) suppresses nonsense mutations in exon 17 but not in exon 18, indicating that SMU-1 regulates the splicing of exon 17, which is enhanced in the absence of functional SMU-1 (Spike et al., 2001).
Although Spartz and colleagues reported (Spartz et al., 2004) that human SMU1 is a component of the spliceosome fSAP57 (Zhou et al., 2002), the function of Smu1, especially in mammalian species, is not well understood. It is possible that the mutation in Smu1 affects spliceosome function, resulting in generation of improper splice variants, which could explain the phenotypes of tsTM18 cells. To clarify the relation between mutation of Smu1 and the phenotypes of tsTM18 cells, we analysed splicing of several genes in tsTM18 cells by reverse transcription-polymerase chain reaction (RT-PCR). In this study, we describe a defect in splicing of the perlecan transcript in tsTM18 cells and discuss the conserved role of mammalian Smu1 in spliceosomes.
We also wanted to study the dynamics of Smu1 in living cells. Our strategy was to construct a hybrid gene encoding a fluorescently tagged form of Smu1 and to use this gene to correct the genetic defect in tsTM18 cells. Previously, we developed cells expressing functional RNA polymerase II tagged with green fluorescent protein (GFP) and used the same strategy against tsTM4 cells, which have a ts mutation in polymerase (Sugaya et al., 2000). With the use of this cell line, the dynamics of RNA polymerase II, especially the transcription cycle of polymerase in living cells, was revealed (Kimura et al., 2002). We hoped that introduction and expression of a hybrid Smu1 would enable tsTM18 cells to grow at 39°C. However, we failed several times using plasmids encoding a variety of fusion proteins. Therefore, we performed RNA interference (RNAi) knockdown experiments to confirm that the mutation in Smu1 underlies the ts defects in tsTM18 cells.
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). These cells can grow at 39°C because they express wild-type human SMU1. After transfection of cells with plasmid DNAs encoding siRNAs, cells were incubated at 34°C or 39°C with 5 µg ml-1 puromycin for the appropriate number of days. Colonies on dishes were stained with methylene blue, and the mean number of colonies from triplicate dishes was calculated (Fig. 1B,C). Most cells formed colonies after transfection with a plasmid encoding siRNA against GFP (control). Cells survived at 34°C after transfection of plasmids encoding siRNAs S2 or S3, confirming that hamster Smu1, which includes the ts mutation, is functional at the permissive temperature. However, few cells grew at 39°C and 34°C after transfection of siRNA S1 (Fig. 1B). These results are consistent with the sequence similarities between target molecules (i.e. human SMU1 and hamster Smu1) and siRNAs (Fig. 1C). These RNAi knockdown experiments confirmed that the mutation in Smu1 underlies the phenotype of tsTM18 cells.
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). The tsTM18 cells also display chromosome instability, including sister chromatid exchange and chromosomal aberrations, in which most aberrations are chromatid-type (Tsuji et al., 1990). These findings suggest that the mutant phenotype is associated with DNA replication. After 24 hours of incubation at 39°C, accumulation of single-stranded DNA was observed in nuclei of mutant cells (Sugaya et al., 2005), suggesting that the Smu1 mutation may be related to generation of single-stranded DNA. It is possible that the mutation in Smu1 causes a defect in DNA replication that in turn leads to generation of single-stranded DNA, resulting in chromosome instability and cell-cycle arrest.
Time course analyses of DNA synthesis showed that [3H]thymidine incorporation in both wild-type (CHO-K1) and mutant (tsTM18) cells increased gradually from 20 minutes to 60 minutes (Fig. 2A,B). The rate of DNA synthesis in CHOK1 cells at 39°C was 1.34 times faster than that at 34°C (Fig. 2A). However, the rate of DNA synthesis in tsTM18 cells after 4 hours of incubation at 39°C was 77% lower than that at 34°C, although the DNA synthesis activity at 34°C was similar to that of CHO-K1 cells (Fig. 2B). These results suggest that an early effect of the temperature shift is on DNA synthesis in tsTM18 cells. Incubation times of less than 4 hours at 39°C were not sufficient to detect the effect of temperature on DNA synthesis (data not shown).
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We next examined the distributions of nascent DNA at 34°C and 39°C. Cells were incubated with bromodeoxyuridine (BrdU) to label nascent DNA, fixed, and the resulting Br-DNA was indirectly immunolabeled with Alexa Fluor 488. Br-DNA staining yielded different patterns, suggesting that cells containing nascent Br-DNAs were in different stages of S phase (Fig. 2C-F). Patterns of indirect labeling for Br-DNA were similar at 34°C and 39°C in both wild-type and mutant cells. These results are consistent with our findings that DNA synthesis in tsTM18 cells was decreased but still active after 4 hours of incubation at the nonpermissive temperature.
RT-PCR analysis for splice variants of the hamster perlecan transcript
The Ig-like repeats six, seven and eight in domain IV of C. elegans unc-52 are encoded by exons 16, 17 and 18 (Rogalski et al., 1993) (Fig. 3A). Alternative splicing of exons 16, 17 and 18 is regulated by the mec-8 (Lundquist et al., 1996; Spike et al., 2002) and smu-1 genes (Spike et al., 2001). We analysed expression of the perlecan transcript in tsTM18 cells by RTPCR. Because the hamster perlecan sequence has not been reported in a public database, we used multiple sequence alignment analysis of the human perlecan, mouse perlecan and C. elegans UNC-52 proteins to design primers to amplify the hamster sequence that corresponds to the fifth to eleventh Ig-like repeats in domain IV of C. elegans unc-52 (f2, f3, f4, f6, r2, r3, r4 in Fig. 3C).
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; Sugaya et al., 1997). There was no significant change in the amount of perlecan transcript after 4 hours of incubation at 39°C in tsTM4 cells, suggesting that the defect in splicing of perlecan pre-mRNA is unique to tsTM18 cells (Fig. 3B). To analyse the perlecan size variants in tsTM18 cells, we extracted, cloned and sequenced the RT-PCR products with the primers listed in Fig. 3C. Sequence analysis of these RT-PCR products revealed that there is one full-length form of the transcript and at least six variants resulting from alternative splicing and retention of intron sequences (Fig. 3C). The 1661-nt partial sequence of the hamster perlecan transcript was deposited under Accession No. AB259765 in the DDBJ, GenBank and EMBL databases. An amino acid sequence deduced from this 1661-nt hamster perlecan sequence showed significant homology both to the region between the fourth and tenth Ig-like repeats in domain IV of mouse perlecan and the region between the eleventh and seventeenth Ig-like repeats in domain IV of human perlecan with 86.1% and 84.6% identities, respectively. The number of Ig repeats in domain IV varies among species, and sequence analysis revealed the probable insertion of seven Ig repeats after the third Ig repeat of human perlecan (Fig. 3A). We also found the conserved structure of mammalian perlecan genes. Exon/intron junctions were conserved in both the mouse and human perlecan genes; the sites within the corresponding sequences to the present 1661-nt hamster perlecan were identical between mouse and human. Therefore, on the basis of the structures of the mouse and human perlecan genes, we predicted the exon/intron junctions in the 1661-nt hamster perlecan sequence. Structures of the size variants are shown in Fig. 3C. All six variants identified in this study lacked exon 4, and four of the six lacked multiple exons (variants P36, P20, P22B and P38B in Fig. 3C). Moreover, three of the six contained at least part of intron 11; P36 and P27B contained full-length intron 11, and P38 contained a partial fragment of intron 11 (Fig. 3C). The exon/intron junctions identified through sequence analysis were consistent with those predicted from the comparison between mouse and human sequences described above. The complicated pattern of splice variants suggests that Smu1 participates in at least two steps of splicing, splice-site selection and accuracy of splicing. Interestingly, the splicing abnormality in the perlecan/unc-52 transcript caused by the Smu1/smu-1 mutation was identified again by genetics in a mammalian cell line.
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Accumulation of splice variants in tsTM18 cells after culture at 39°C
To clarify the effect of temperature on splicing, we analysed accumulation of splice variants of the perlecan gene in tsTM18 cells by RT-PCR. Total RNAs prepared from CHO-K1 and tsTM18 cells after various incubation times at 39°C were analysed by RT-PCR with the f2/r2 primer pair. The 1661-bp full-length perlecan transcript was detected in CHO-K1 and tsTM18 cells incubated at 34°C (Fig. 5). However, when cells were cultured at 39°C, the amount of the 1661-bp product decreased gradually, and a number of splice variants appeared in tsTM18 cells. The defect in splicing of the perlecan premRNA appears slightly earlier than the defect in DNA synthesis.
RT-PCR analysis of other transcripts
To clarify whether the splicing defect in the perlecan premRNA in tsTM18 cells at the nonpermissive temperature is specific to the perlecan gene, we examined transcripts of four other genes, Aptx, Dnaja1, Tnc and Smu1, by RT-PCR. Aptx encodes aprataxin, which is a member of the histidine triad superfamily, some members of which have nt-binding and diadenosine polyphosphate hydrolase activities. It was reported that aprataxin may play a role in single-stranded DNA repair; mutations in this gene appear to be associated with ataxia-ocular apraxia, and multiple splice variants encoding distinct isoforms have been identified (Date et al., 2001; Moreira et al., 2001). RT-PCR analysis of the coding sequence of Aptx revealed that at least three variants exist, and the shortest variant became the most prominent in both CHO-K1 and tsTM18 cells during culture at 39°C (Fig. 6A). Dnaja1 shows significant homology to the bacterial heat shock protein DnaJ (Hsp40) and may function in protein folding and transport (Chellaiah et al., 1993). DnaJ was originally isolated as one of the genes involved in DNA replication in bacteria. A single RT-PCR product was obtained from both CHO-K1 and tsTM18 cells incubated at 34°C and 39°C (Fig. 6B). Expression of Smu1 was uniform and confirmed to be lower than expression of the Aptx, Dnaja1, Tnc and perlecan genes (Fig. 6C). Tnc encodes extracellular matrix glycoprotein tenascin-C, is expressed in a variety of tissues and is thought to play a role in mesenchymal-epidermal interactions not only during organ development but also under conditions of tissue remodeling, such as tumorgenesis and wound healing (Erickson and Bourdon, 1989; Chiquet-Ehrismann et al., 1995). Tnc was chosen as an example of an extracellular matrix molecule that contains repeated sequences similar to perlecan. Mouse tenascin-C contains six alternatively spliced sites between its fifth and sixth type-III fibronectin domains, which generate a large number of Tnc isoforms in mammalian development and in cancer progression (Joester and Faissner, 1999). When the region between the fifth and sixth type-III fibronectin domains was amplified by RT-PCR, single products were obtained from all templates, even tsTM18 cells grown at 39°C (Fig. 6D). When the results of RT-PCR analysis of the perlecan gene were compared with those of the other four genes, the splicing defect in the perlecan gene of Smu1 mutant cells appeared to be unique. However, the present results are not sufficient to conclude that the splicing defect is specific to the perlecan gene in tsTM18 cells. Therefore, analyses of additional genes involved in replication and in maintenance of genomic integrity are needed. Development of a comprehensive procedure to assess alternative splicing of all genes in the genome would be helpful for such analyses.
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Regulated alternative splicing of pre-mRNA most often results in the synthesis of multiple (functional) proteins from the same pre-mRNA, but alternative splicing can also function as an on-off switch for gene function (Baker, 1989). Sequence analyses of amino acid sequences deduced from six perlecan splice variants revealed that five of the six encoded truncated forms of the protein due to generation of stop codons by alternative splicing (variants P36, P20, P38, P22B and P27B in Fig. 3C). Interestingly, one variant did not contain any nonsense codons and encoded a truncated form that lacked a single Ig-repeat (P38B in Fig. 3C).
Some important regulatory events are controlled by intron retention. For the Drosophila Msl2 transcript, an intron is retained in the final mRNA in female flies (Gebauer et al., 1998). Retention of this intron requires binding of Sxl protein to both the 5' and 3' splice sites. Another well-characterized system of intron retention is the P element transcript in Drosophila (Rio, 1991). In our RT-PCR and sequence analyses of the variant transcripts, it remains unclear whether the introns retained in perlecan due to Smu1 mutation have any physiologic significance.
Role of Smu1 in spliceosomes
RT-PCR analysis of transcripts of four other genes revealed that the splicing defect is restricted to the perlecan gene, suggesting that functional Smu1 is not required for basic splicing activity in mutant cells. Comprehensive proteomic analysis of human spliceosomes revealed that the human homologue of Smu1 is fSAP57 (Zhou et al., 2002). However, as Spike and colleagues suggested (Spike et al., 2001), there is no homolog of Smu1 in a Saccharomyces cerevisiae, a basic model organism, further supporting that Smu1 does not encode a core component of the spliceosome. On the basis of the facts described above, we believe that Smu1 is an essential RNA splicing factor for one or more specific genes. However, the role of Smu1 in the spliceosome may be very important because regulation of editing of the perlecan/unc-52 premRNA is evolutionarily conserved.
Enzymes involved in RNA metabolism and their ts mutants
Both ts mutant cell lines, tsTM4 and tsTM18, have mutations in enzymes involved in RNA metabolism (the largest subunit of RNA polymerase II in tsTM4, transcription; Smu1 in tsTM18, splicing) and exhibit similar phenotypes including abnormal induction of sister chromatid exchange and cell-cycle arrest at S phase with decreased DNA synthesis (Tsuji et al., 1990; Sugaya et al., 1997; Sugaya et al., 2005). Taken together with the results of previous and present analyses of two hamster ts mutants, enzymes involved in RNA synthesis and editing may have some connection with replication and the maintenance of chromosome integrity. A recent study established a link between splicing, transcription and the maintenance of genomic stability (Li and Manley, 2005). They showed that a significant function of SF2/ASF is to prevent the formation of R loop structures between nascent transcripts and template DNAs. SF2/ASF is a member of the SR splicing factors (Ge and Manley, 1990; Krainer et al., 1990), which are proteins necessary for an early step in spliceosome assembly and that can influence the selection of alternative splice sites (Fu, 1995; Manley and Tacke, 1996; Graveley, 2000). Members of this family have characteristic RNA-binding domains and Ser/Arg-rich (SR) motifs. In tsTM18 cells cultured at the nonpermissive temperature, the ts defect of Smu1 appears to alter localization of SF2/ASF, suggesting that there is a link between Smu1 and SF2/ASF (our unpublished results). Our ts-mutant cell lines should prove useful in the analysis of the function of enzymes involved in RNA metabolism in connection with replication and the maintenance of chromosome integrity.
We conclude that one of the earliest observable effects of temperature shift in tsTM18 cells occurs in DNA synthesis. We also found that the defect in splicing of the perlecan premRNA appears slightly earlier than the defect in DNA synthesis because 4 hours of incubation at 39°C is needed to observe the effect of temperature on DNA synthesis. However, we have no evidence to explain the connection between perlecan and replication. In tsTM4 cells, there was no significant change in perlecan expression after 4 hours of incubation at 39°C (Fig. 3B). This result suggests that both the alterations (defects) in replication and in splicing of perlecan pre-mRNA may be caused independently by the Smu1 mutation. Furthermore, the relation between the defects in replication (DNA synthesis) and splicing (RNA editing) in tsTM18 cells at the nonpermissive temperature remains unclear. Further studies to clarify the functions of Smu1 in replication and splicing are needed.
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).
Plasmids and transfection
Plasmid vectors expressing short interfering RNAs (siRNAs) were generated with piGENE PUR hU6 vector by iGENE Therapeutics (Tsukuba, Japan). Sequences were S1, 5'-ACCACAGAATGTTCAAATA-3'; S2, 5'-GGCCCAAGATAACTTTATG-3'; S3, 5'-GTCAGAAATCACATGTGGA-3' and EGFP, 5'-TACGGCAAGCTGACCCTGAAGTTCATCTGC-3'. Plasmids were introduced into 18H-1c30 and 18K-1c1 cells with FuGENE6 transfection reagent (Roche Diagnostics, Mannheim, Germany). After selection with 5 µg ml-1 puromycin (Sigma-Aldrich), colonies in dishes were stained with methylene blue and counted, and the average colony number for triplicate dishes was calculated.
Radiolabeling
Cells were grown on glass coverslips at 34°C overnight with 1.85 kBq ml-1 methyl-[14C]thymidine (American Radiolabeled Chemicals, St Louis, MO, USA) to label DNA uniformly. Cells were then grown at 34°C or 39°C for 4 hours and incubated with 1.11 MBq ml-1 methyl-[3H]thymidine (American Radiolabeled Chemicals) to label nascent DNA for the periods indicated in Fig. 2A,B. Incorporation was stopped by adding an equal volume of 0.5% sodium azide. Cells on coverslips were washed with phosphate-buffered saline (PBS), fixed with methanol-acetic acid (3:1) and air dried. After cells were washed with 5% trichloroacetic acid (TCA), radioactivity was measured by scintillation counting. 3H counts were normalized to 14C counts.
Indirect immunolabeling and microscopy
Cells were grown on glass coverslips at 34°C or 39°C for 4 hours, incubated with 50 µM bromodeoxyuridine (BrdU) for 20 minutes, washed with PBS and then fixed (20 minutes at 4°C) with 4% paraformaldehyde (PFA) in 250 mM HEPES. After acid denaturation (1 hour) with 2 N HCl, cells were incubated (20 minutes) with 0.2% Triton X-100 in PBS and blocked with PBS containing 40 mM glycine, 1% bovine serum albumin and 0.2% gelatin. Br-DNA was indirectly immunolabeled with Alexa Fluor 488 (Molecular Probes, Eugene, OR). The primary antibody was mouse anti-BrdU (clone MD5110; 1:800; Caltag, Burlingame, CA). Images were collected with a Yokogawa CSU10 confocal microscope and `contrast-stretched' with Adobe Photoshop 5.5 (Adobe Systems, San Jose, CA).
RT-PCR
Total RNA was isolated from CHO-K1 and tsTM18 cells with TRIzol reagent (Invitrogen). At least three independent samples of RNAs were prepared from wild-type and mutant cells. For analysis of retention of genomic sequences in the perlecan transcript, total RNA and genomic DNA were treated with DNase (RQ1 RNase-free DNase, Promega, Madison, WI).
According to the manufacturer's protocol (Takara Bio, Otsu, Shiga, Japan), 1 µg of total RNA was reverse transcribed in a 10 µl volume containing dNTPs, RNase Inhibitor, oligo(dT) primer and BcaBEST polymerase in the supplied buffer and incubated at 65°C for 1 minute, 40°C for 5 minutes, 65°C for 30 minutes and 98°C for 5 minutes. The following primers were used for subsequent amplification of the hamster perlecan transcript: f2, 5'-GGCGAGTATGTGTGCCGRGCC-3'; f3, 5'-ATCGAGACCTCTTCCTCCCGA-3'; f4, 5'-GCCCAGCCTATCTATATAGAG-3'; f6, 5'-AAGAAGCTGAGCTGACCTTGG-3'; r2, 5'-CGCAGCTTGGACCAGTGGATG-3'; r3, 5'-TGAGATTGACGACACTCTGGG-3' and r4, 5'-GAAACTGGCATCTTGGCCCTG-3'. After initial denaturation for 2 minutes at 94°C, 35 cycles of PCR were performed. Each cycle consisted of denaturation for 30 seconds at 94°C, annealing for 30 seconds at 63°C and extension for 3 minutes for the f2/r2 and 1 minute for the f6/r2 primer pairs at 72°C. After the last cycle, all samples were incubated for an additional for 3 minutes for the f2/r2 and 1 minute for the f6/r2 primer pairs at 72°C. PCR products were separated by 1.5% agarose (f2/r2 products) or 3% NuSieve 3:1 agarose (f6/r2 products) gel electrophoresis. Sequence confirmation of bands was performed by gel extraction, cloning into the pUC118 vector with a Mighty Cloning Kit (Takara Bio) and sequencing of vector inserts with the primers listed above.
RT-PCR of the hamster Aptx, Dnaja1, Smu1 and Tnc transcripts was carried out with the following primer pairs: Aptx, 5'-ATGMARGACCCCAAAATGCAGGTT-3' and 5'-CYSKSYCCAGTGCTTCCTGAGRTG-3'; Dnaja1, 5'-ATGGTGAAAGAAACMACTTACTAC-3' and 5'-AGAGGTCTGACACTGAACRCCACC-3'; Smu1, 5'-GGGGTACCATGTCGATCGAAATC-3' and 5'-GCGGATCCGCGWGGTTTCCAGAG-3' and Tnc, 5'-GCACGTGTGAAGGCATCCAC-3' and 5'-CCCATGGCTGTTGTTGCTAT-3'. After initial denaturation for 2 minutes at 94°C, 30 cycles of PCR were performed. Each cycle consisted of a denaturing period (30 seconds at 94°C), an annealing phase (30 seconds at 50°C for Aptx and Smu1, 30 seconds at 55°C for Dnaja1, 30 seconds at 65°C for Tnc), and an extension period (1 minute for Aptx, 2 minutes for Dnaja1 and Tnc, 3 minutes for Smu1 at 72°C). After the last cycle, all samples were incubated at 72°C for an additional extension period shown above. Amplified products were separated by 1.5% agarose gel electrophoresis.
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