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• W2118039400 abstract "Article16 June 2003free access Truncated product of the bifunctional DLST gene involved in biogenesis of the respiratory chain Takashi Kanamori Takashi Kanamori Department of Biochemistry and Cell Biology, Institute of Development and Aging Sciences, Graduate School of Medicine, Nippon Medical School, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki, Kanagawa, 211-8533 Japan Search for more papers by this author Kiyomi Nishimaki Kiyomi Nishimaki Department of Biochemistry and Cell Biology, Institute of Development and Aging Sciences, Graduate School of Medicine, Nippon Medical School, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki, Kanagawa, 211-8533 Japan Search for more papers by this author Sadamitsu Asoh Sadamitsu Asoh Department of Biochemistry and Cell Biology, Institute of Development and Aging Sciences, Graduate School of Medicine, Nippon Medical School, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki, Kanagawa, 211-8533 Japan Search for more papers by this author Yoshitomo Ishibashi Yoshitomo Ishibashi Department of Biochemistry and Cell Biology, Institute of Development and Aging Sciences, Graduate School of Medicine, Nippon Medical School, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki, Kanagawa, 211-8533 Japan Search for more papers by this author Iichiro Takata Iichiro Takata Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, Tsukuba Science City, 305-8562 Japan Search for more papers by this author Tomoko Kuwabara Tomoko Kuwabara Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, Tsukuba Science City, 305-8562 Japan Search for more papers by this author Kazunari Taira Kazunari Taira Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, Tsukuba Science City, 305-8562 Japan Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Bunkyo-ku, Tokyo, 113-8656 Japan Search for more papers by this author Haruyasu Yamaguchi Haruyasu Yamaguchi Gunma University, School of Health Science, Maebashi, 371-8514 Japan Search for more papers by this author Shiro Sugihara Shiro Sugihara Department of Pathology, Gunma Cancer Center, Ohta, 373-0828 Japan Search for more papers by this author Tsuneo Yamazaki Tsuneo Yamazaki Department of Neuropathology, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, 113-0033 Japan Search for more papers by this author Yasuo Ihara Yasuo Ihara Department of Neuropathology, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, 113-0033 Japan Search for more papers by this author Kyoko Nakano Kyoko Nakano Department of Biochemistry, Kagoshima Women's Junior College, Kagoshima, 890-8565 Japan Search for more papers by this author Sadayuki Matuda Sadayuki Matuda Department of Biology and Health Science, Kanoya National Institute of Fitness and Sports, Kanoya, Kagoshima, 891-2393 Japan Search for more papers by this author Shigeo Ohta Corresponding Author Shigeo Ohta Department of Biochemistry and Cell Biology, Institute of Development and Aging Sciences, Graduate School of Medicine, Nippon Medical School, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki, Kanagawa, 211-8533 Japan Search for more papers by this author Takashi Kanamori Takashi Kanamori Department of Biochemistry and Cell Biology, Institute of Development and Aging Sciences, Graduate School of Medicine, Nippon Medical School, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki, Kanagawa, 211-8533 Japan Search for more papers by this author Kiyomi Nishimaki Kiyomi Nishimaki Department of Biochemistry and Cell Biology, Institute of Development and Aging Sciences, Graduate School of Medicine, Nippon Medical School, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki, Kanagawa, 211-8533 Japan Search for more papers by this author Sadamitsu Asoh Sadamitsu Asoh Department of Biochemistry and Cell Biology, Institute of Development and Aging Sciences, Graduate School of Medicine, Nippon Medical School, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki, Kanagawa, 211-8533 Japan Search for more papers by this author Yoshitomo Ishibashi Yoshitomo Ishibashi Department of Biochemistry and Cell Biology, Institute of Development and Aging Sciences, Graduate School of Medicine, Nippon Medical School, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki, Kanagawa, 211-8533 Japan Search for more papers by this author Iichiro Takata Iichiro Takata Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, Tsukuba Science City, 305-8562 Japan Search for more papers by this author Tomoko Kuwabara Tomoko Kuwabara Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, Tsukuba Science City, 305-8562 Japan Search for more papers by this author Kazunari Taira Kazunari Taira Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, Tsukuba Science City, 305-8562 Japan Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Bunkyo-ku, Tokyo, 113-8656 Japan Search for more papers by this author Haruyasu Yamaguchi Haruyasu Yamaguchi Gunma University, School of Health Science, Maebashi, 371-8514 Japan Search for more papers by this author Shiro Sugihara Shiro Sugihara Department of Pathology, Gunma Cancer Center, Ohta, 373-0828 Japan Search for more papers by this author Tsuneo Yamazaki Tsuneo Yamazaki Department of Neuropathology, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, 113-0033 Japan Search for more papers by this author Yasuo Ihara Yasuo Ihara Department of Neuropathology, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, 113-0033 Japan Search for more papers by this author Kyoko Nakano Kyoko Nakano Department of Biochemistry, Kagoshima Women's Junior College, Kagoshima, 890-8565 Japan Search for more papers by this author Sadayuki Matuda Sadayuki Matuda Department of Biology and Health Science, Kanoya National Institute of Fitness and Sports, Kanoya, Kagoshima, 891-2393 Japan Search for more papers by this author Shigeo Ohta Corresponding Author Shigeo Ohta Department of Biochemistry and Cell Biology, Institute of Development and Aging Sciences, Graduate School of Medicine, Nippon Medical School, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki, Kanagawa, 211-8533 Japan Search for more papers by this author Author Information Takashi Kanamori1, Kiyomi Nishimaki1, Sadamitsu Asoh1, Yoshitomo Ishibashi1, Iichiro Takata2, Tomoko Kuwabara2, Kazunari Taira2,3, Haruyasu Yamaguchi4, Shiro Sugihara5, Tsuneo Yamazaki6, Yasuo Ihara6, Kyoko Nakano7, Sadayuki Matuda8 and Shigeo Ohta 1 1Department of Biochemistry and Cell Biology, Institute of Development and Aging Sciences, Graduate School of Medicine, Nippon Medical School, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki, Kanagawa, 211-8533 Japan 2Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, Tsukuba Science City, 305-8562 Japan 3Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Bunkyo-ku, Tokyo, 113-8656 Japan 4Gunma University, School of Health Science, Maebashi, 371-8514 Japan 5Department of Pathology, Gunma Cancer Center, Ohta, 373-0828 Japan 6Department of Neuropathology, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, 113-0033 Japan 7Department of Biochemistry, Kagoshima Women's Junior College, Kagoshima, 890-8565 Japan 8Department of Biology and Health Science, Kanoya National Institute of Fitness and Sports, Kanoya, Kagoshima, 891-2393 Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:2913-2923https://doi.org/10.1093/emboj/cdg299 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Dihydrolipoamide succinyltransferase (DLST) is a subunit enzyme of the α-ketoglutarate dehydrogenase complex of the Krebs cycle. While studying how the DLST genotype contributes to the pathogenesis of Alzheimer's disease (AD), we found a novel mRNA that is transcribed starting from intron 7 in the DLST gene. The novel mRNA level in the brain of AD patients was significantly lower than that of controls. The truncated gene product (designated MIRTD) localized to the intermembrane space of mitochondria. To investigate the function of MIRTD, we established human neuroblastoma SH-SY5Y cells expressing a maxizyme, a kind of ribozyme, that specifically digests the MIRTD mRNA. The expression of the maxizyme specifically eliminated the MIRTD protein and the resultant MIRTD-deficient cells exhibited a marked decrease in the amounts of subunits of complexes I and IV of the mitochondrial respiratory chain, resulting in a decline of activity. A pulse-label experiment revealed that the loss of the subunits is a post-translational event. Thus, the DLST gene is bifunctional and MIRTD transcribed from the gene contributes to the biogenesis of the mitochondrial respiratory complexes. Introduction α-ketoglutarate dehydrogenase complex is a rate-limiting enzyme of the Krebs cycle in the mitochondrial matrix. The complex is composed of three major protein subunits, one of which is the structural core protein dihydrolipo amide succinyltransferase (DLST). The human DLST gene is located on chromosome 14 at q24.2–q24.3 (Nakano et al., 1993) and consists of 15 exons (Nakano et al., 1994). A genetic association has been reported between polymorphisms of the DLST gene and Alzheimer's disease (AD) in both Japanese and Caucasian populations (Nakano et al., 1997; Sheu et al., 1999a,b). Interestingly, the polymorphisms are also seen in Swedish patients with familial AD (Sheu et al., 1998). However, the association remains controversial (Kunugi et al., 1998; Matsushita et al., 2001), perhaps because the risk is not great and, additionally, because controls and AD patients were not matched for age and gender. In any case, the polymorphisms do not change amino acid residues of the DLST protein. Thus, the reason why the nucleotide substitutions in the DLST gene contribute to the pathogenesis of AD is not clear. Therefore, it is important to elucidate the role of the DLST gene when investigating the molecular mechanism involved in AD. AD is a complex neurodegenerative disease caused by multiple genetic and environmental factors. The accumulation of amyloid β protein is considered a main cause of neuronal cell death. Additionally, mitochondrial dysfunction may be associated with the pathogenesis of AD (Bonilla et al., 1999; Schon and Manfredi, 2003). In particular, a deficiency of cytochrome c oxidase of the mitochondrial respiratory chain in the brains of AD patients has been reported from several laboratories (Kish et al., 1992; Parker and Parks, 1995; Cottrell et al., 2001). On the other hand, amyloid β protein inhibits the activity of cytochrome c oxidase and other key enzymes of mitochondria (Caseley et al., 2002). Damage to mitochondria causes a decline in ATP synthesis and an increase in the generation of reactive oxygen species (ROS). ROS damage various molecules, including DNA, protein and lipid, and induce apoptosis. Evidence has recently emerged that oxidative stress is involved in the pathogenesis of various neurodegenerative diseases such as AD and Parkinson's disease (Schulz et al., 2000; Smith et al., 2000; Ohsawa et al., 2003). Moreover, it has been shown that amyloid β protein generates hydrogen peroxide (Behl et al., 1994; Harris et al., 1995). In the present study, we found a novel truncated mRNA that is transcribed starting from intron 7 of the DLST gene, in addition to full-length mRNA. The amount of this truncated mRNA is significantly reduced in the brain of patients with AD. In addition, selective digestion of the truncated mRNA lowered the steady-state level of the subunits of complexes I and IV of the mitochondrial respiratory chain. These results strongly suggest that the truncated DLST protein transcribed from the bifunctional DLST gene contributes to the biogenesis of the respiratory chain. Results Haplotype analysis of the DLST gene We have reported that polymorphisms at nucleotide 19116 in intron 13 and nucleotide 19183 at the third codon base in exon 14 of the DLST gene are associated with AD (Figure 1; Nakano et al., 1997). The gene was classified into three haplotypes, designated ac, at and gc depending on the nucleotides 19116 (A or G) and 19183 (C or T), in which the homozygous ac genotype was associated with AD (Nakano et al., 1997). Since these polymorphisms do not change any amino acid residues, we searched for pathogenic mutations that are in linkage disequilibrium with the haplotype. Figure 1.Schematic presentation of the structure of the DLST gene. Nucleotide residues are numbered on the basis of the revised sequence starting from the transcription initiation site of DLST mRNA at position 1 (Nakano et al., 1994). Haplotypes are designated by two nucleotide variants at 19116 (A or G) and at 19183 (C or T). Nucleotides specific to haplotype ac are listed. The transcription initiation site of MIRTD mRNA is at 10555. Download figure Download PowerPoint The nucleotide sequence of the entire DLST gene including ∼1000 bp of the upstream region was directly determined in six volunteers, two each with the homozygous ac, at and gc genotypes, by using PCR products from total DNA isolated from peripheral blood. On comparing the three haplotypes, we found an additional six polymorphisms at 10578, 11043, 11103, 13844, 15455 and 17106 in linkage disequilibrium specific to haplotype ac (19116A and 19183C haplotype) (Figure 1). A11043G lies at the third codon base in exon 8, while the other polymorphisms lie in introns. As a result, we found no polymorphism that leads to a change in amino acid in the haplotype ac. Truncated transcript from intron 7 of the DLST gene with poly(A)+ RNA We have reported the existence of a truncated DLST protein in muscle (Matuda et al., 1997). We hypothesized that this kind of unusual protein derived from the DLST gene contributes to cell death, that mRNA transcribed from an intron generates a truncated protein and that a polymorphism in the intron contributes to the expression of the truncated mRNA. Therefore, we tried to find mRNA that is transcribed from an intron of the DLST gene. We extensively performed PCR coupled with reverse transcription (RT) of poly(A)+ RNA from a human neuroblastoma cell line SH-SY5Y using various sets of primers, one of which corresponds to the sequence of an intron and the other to that of an exon. The primer sets covered most regions of the DLST gene. When a pair of primers corresponding to intron 7 and exon 9 was used (Figure 2A), a fragment was clearly amplified (Figure 2B, lane 1). The length was ∼240 bp, which agrees with a fragment length covering intron 7 to exons 8 and 9. The amplified RT–PCR product was sequenced to confirm that the fragment indeed consisted of intron 7, exon 8 and exon 9 of the DLST gene. The absence of the sequence of intron 8 strongly suggests that the RNA is derived from mRNA. Figure 2.Existence of a truncated mRNA transcribed from intron 7. (A) Schematic diagram of the primers (a–d) and TaqMan probe (e) used in this study. Arrows represent positions of primers. To detect the truncated mRNA (MIRTD mRNA), PCR was performed by preheating for 10 min at 95°C, followed by 35 cycles of 15 s at 95°C and 1 min at 57°C. Primers a, b, c and d represent the MIRTD forward primer 5′-ctcatttcagacagtgccagtg-3′ (intron 7), MIRTD reverse primer 5′-tggctcagctagtggtggg-3′ (exon 9), DLST forward primer 5′-acctacagcagcggcagt-3′ (exon 8) and DLST reverse primer 5′-ctcctggctcagctagtggt-3′ (exon 9), respectively. (B) Poly(A)+ RNA was isolated from the SH-SY5Y cells. Complementary DNA was synthesized with oligo(dT) primer and then PCR was carried out using primers a and b (lane 1) or primers c and d (lane 2). The poly(A)+ RNA fraction was untreated with reverse transcriptase, but subjected to PCR (lane 3). Lane 4 shows a marker of φX174 DNA digested with HincII. Arrows indicate each amplified fragment. (C) Initiation site of MIRTD mRNA was determined by an oligonucleotide-capping method (Yamabe et al., 1998) with Cap Site cDNA™ of human brain (Nippon Gene, Toyama, Japan) as described in Materials and methods. Download figure Download PowerPoint Next, its transcription initiation site was determined by an oligonucleotide capping method (Yamabe et al., 1998). This method confirmed the presence of the cap structure at the initiation site, and revealed the initiation site to be A at 10555 (Figure 2C). Thus we concluded the existence of a novel mRNA, with the cap structure, which is transcribed from intron 7 of the DLST gene. We designated the truncated protein derived from the novel mRNA as MIRTD (MItochondrial Respiration Generator of Truncated DLST) in consideration of its function as described later. Expression of MIRTD mRNA is significantly reduced in the brains of patients with AD As a first step, we examined whether the truncated mRNA is associated with AD. Relative amounts of MIRTD mRNA in AD and control brains were compared using poly(A)+ RNA prepared from cortex. The amounts of MIRTD mRNA were measured using real-time quantitative RT–PCR and normalized with the amounts of DLST or glyceraldehyde-3-phospate dehydrogenase (GAPDH) mRNA. When mRNA of DLST was not detected, we omitted the samples from analysis. The ratio of MIRTD mRNA to DLST mRNA was not correlated with age (correlation coefficient 0.097) or post-mortem period (correlation coefficient 0.22) of the control brains. In control brains, the ratios of MIRTD mRNA to DLST and GAPDH mRNAs were (0.0621 ± 0.0505) and (1.62 ± 1.47) × 10−4, respectively. In the AD brains, they were (0.0248 ± 0.0368) and (0.713 ± 1.15) × 10−4, respectively (Figure 3). The differences were apparently significant (p = 0.004 for MIRTD/DLST mRNA and p = 0.018 for MIRTD/GAPDH mRNA by Student's t-test). Strikingly, MIRTD mRNA was not detected in half of the AD brains (Figure 3). In addition, the ratio of MIRTD mRNA to DLST mRNA from carriers with the ac haplotype tended to be lower than that from non-carriers irrespective of the clinical diagnosis (0.041 ± 0.040 versus 0.055 ± 0.047), despite showing no statistical significance (p = 0.23). Thus, these results suggest that a decrease in MIRTD mRNA is associated with AD. Figure 3.Decreased amounts of MIRTD mRNA in brains of patients with AD. Amounts of MIRTD mRNA in the brains of AD patients or controls were measured by real-time quantitative RT–PCR and normalized against those of (A) DLST or (B) GAPDH mRNA as described in Materials and methods. Numbers of samples, mean values with standard deviations (± SD) and p values as judged by Student's t-test are given in the figure. Download figure Download PowerPoint Regulation of expression of MIRTD mRNA by haplotypes of the DLST gene Around the initiation site of MIRTD mRNA, there is a polymorphism (10578T) that is specific to haplotype ac (Figure 1). Therefore, we investigated whether haplotypes of the DLST gene influence the expression of MIRTD mRNA in vitro. For this purpose, we used the rat cell line PC12, because the nucleotide sequence of intron 7 of the rat DLST gene is distinguishable from the human counterpart. We isolated two independent cosmid clones carrying the full-length human DLST genes of each haplotype (the ac and gc haplotypes) and then transfected the human genes into PC12, where the endogenous rat DLST gene is present. The amount of exogenous human MIRTD mRNA was measured by real-time quantitative RT–PCR and normalized to that of human DLST mRNA (Figure 4). Each primer set specific to the human DLST gene was selected to detect human DLST mRNA and human MIRTD mRNA. In fact, only exogenous human DLST and MIRTD mRNAs, and no endogenous mRNAs derived from the host, were detected (Figure 4A and B). The ratios of human MIRTD mRNA to DLST mRNA in the cells carrying 10578T (haplotype ac) were 3.6 ± 1.9% and 4.6 ± 2.3%, compared with 8.7 ± 2.4% and 9.7 ± 3.2% in the cells carrying 10578A (haplotype gc) in each of two independent clones (Figure 4C). The difference was statistically significant, as shown in the figure legend. Therefore, expression of human MIRTD mRNA depends on the haplotype of the DLST gene. Figure 4.Decreased expression of human MIRTD in rat PC12 cells carrying the human DLST gene with haplotype ac. PC12 cells were cotransfected with the cosmid containing the ac- or gc-type DLST gene and the geneticin-resistance gene using Lipofectin (Invitrogen). Transfectants were enriched by selecting geneticin-resistant cells for 14 days and then poly(A)+ RNA was prepared from the transfectant pools. Amounts of human MIRTD and DLST mRNAs were measured by real-time quantitative RT–PCR. (A) Profiles of the real-time quantitative RT–PCR showing increases of PCR products derived from human DLST and MIRTD mRNAs. (B) Profiles of the real-time quantitative RT–PCR using poly(A)+ RNA from untransfected PC12 cells, detecting neither DLST nor MIRTD mRNA. (C) Relative values of MIRTD to DLST mRNA are shown with the mean ± SD obtained from four independent experiments. The difference was significant for 1 versus 3 (p = 0.039), 1 versus 4 (p = 0.049), 2 versus 3 (p = 0.016) and 2 versus 4 (p = 0.016). Download figure Download PowerPoint Location of the MIRTD protein To obtain evidence for the existence of the MIRTD protein, its location was determined using rat liver, which is more suitable for isolating intact organella for a subcellular fractionation experiment. Since a short RNA corresponding to the MIRTD mRNA was detected in the total rat liver RNA fraction by northern blotting (Figure 5A), there should be MIRTD protein in rat liver. Figure 5.Identification and location of the MIRTD protein. (A) Total RNA (50 μg) isolated from rat liver was analyzed by northern blotting. DLST and MIRTD mRNAs were detected with 32P-labeled rat DLST cDNA. The positions of 28S and 18S for rRNAs are indicated on the right. Arrowheads indicate mRNAs corresponding to DLST and MIRTD. (B) Fractionation of rat liver was carried out as described in Materials and methods. Post-nuclear supernatant was centrifuged at 10 000 g. The pellet fraction (P10 000) contained most of the mitochondria. The supernatant was centrifuged at 100 000 g. The pellet (P100 000) and the supernatant (S100 000) contained microsomes and cytosol, respectively. Samples were analyzed by western blotting with anti-DLST monoclonal antibody. (C) Isolated rat liver mitochondria were diluted with isotonic (lanes 1–3) or hypotonic (lanes 4–6) buffer to prepare mitoplast. Samples were divided into three aliquots and treated with 0 (lanes 1, 4), 10 (lanes 2, 5) or 50 μg/ml (lanes 3, 6) proteinase K. Samples were analyzed by western blotting with anti-DLST monoclonal, anti-Tom40 polyclonal or Tom20 polyclonal antibody. Asterisk, degraded product of DLST; PK, proteinase K; M, mitochondria; MP, mitoplast. Download figure Download PowerPoint To find the location of the MIRTD protein, rat liver was fractionated as usual and then western blotting was performed (Figure 5B). In the P10 000 fraction (the mitochondrial fraction), a 55 kDa product and an additional 30 kDa product were detected (Figure 5B, lane 1). The 55 kDa product corresponds to the full-length DLST protein. If the 30 kDa polypeptide is translated from the first AUG codon located in exon 8 of the DLST gene through the same reading frame with the full-length DLST protein, the molecular weight is estimated as 29.7 kDa, which corresponds to the apparent molecular weight of 30 kDa of the product. MIRTD was detected mainly in the P10 000 fraction (Figure 5B, lanes 2 and 3), suggesting that the protein belonged to the mitochondrial fraction. To determine the location of MIRTD more precisely, mitochondria isolated from rat liver were treated with external proteinase K. Tom20, Tom40 and the full-length DLST (a matrix protein) were used as internal controls. Tom40 and Tom20 are components of the translocation complex for transporting the nuclear-encoded mitochondrial precursor proteins. Tom40 is exposed to the intermembrane side, while Tom20 is exposed outside mitochondria (Pfanner and Geissler, 2001). Tom20 was easily digested with 10 μg/ml proteinase K, whereas Tom40, DLST and the MIRTD protein were resistant (Figure 5C, lanes 1–3). On the other hand, when mitochondria were converted to mitoplast, the MIRTD band disappeared (Figure 5C, lane 4), whereas Tom40 and Tom20 remained in the mitoplast. These results indicate that the MIRTD protein is located in the intermembrane space of mitochondria. Construction of cell lines for digesting the MIRTD mRNA To investigate the function of MIRTD, we tried to eliminate MIRTD from cultured cells. In order to reduce the amount of specific mRNA in general, antisense oligonucleotides covering initiation codons are introduced into the cell. However, in this case, the sequence of MIRTD mRNA is identical to that of the full-length DLST mRNA. If the antisense oligonucleotides for the MIRTD mRNA are introduced into the cell, the full-length DLST mRNA should be reduced together with the MIRTD mRNA. Thus, we used maxizyme technology to digest the MIRTD mRNA with high specificity. Recently, a new type of ribozyme that specifically digests a target RNA has been developed (Kuwabara et al., 1998). Maxizyme is a heterodimeric ribozyme and has sensor arms that can recognize target sequences. In the presence of a specific target sequence, it can form a cavity that can capture catalytically indispensable magnesium ions. To express the maxizyme under the control of a strong promoter in vivo, we embedded each monomeric unit downstream of the sequence of a human tRNAVal promoter (Kawasaki et al., 1996) that is recognized by RNA polymerase III (Perriman and de Feyter, 1997) to generate MzL (maxizyme left) and MzR (maxizyme right). The specific design of the tRNAVal constructs was based on previous success in attaching a ribozyme sequence to the 3′-modified side of the tRNAVal portion of the human gene to be exported into cytosol (Koseki et al., 1999). In order to obtain good substrate activity, we designed sequences of the maxizyme that can adopt an active conformation only in the presence of the intron 7–exon 8 junction of the DLST gene (Figure 6A). Indeed, the maxizyme digested the MIRTD mRNA with high specificity, but not the DLST mRNA in vitro as had been reported (Tanabe et al., 2000). Figure 6.Decreased amounts of MIRTD mRNA by introducing the maxizyme. (A) The sequences of MIRTD mRNA and the maxizyme heterodimer MzR and MzL are shown. The maxizyme heterodimer can form an active conformation only in the presence of the junction between intron 7 and exon 8 in the DLST gene. A triangle indicates the site of cleavage by the maxizyme. (B) Neuroblastoma SH-SY5Y cells were cotransfected with DNA of the maxizyme and the geneticin-resistance gene. Independent clones stably expressing the maxizyme (Z5, Z7 and Z21) or monomeric controls (L1 and R2) were isolated and then relative values of MIRTD mRNA to DLST mRNA were obtained as described in Materials and methods. (C) Upper panel: protein (50 μg) from each sample was subjected to SDS–PAGE followed by western blotting using antibody against the C-terminal region of the DLST protein. Lower panel: the amount of the MIRTD protein was quantified and normalized to that of the DLST protein. Results are shown as the average ± SD of three experiments. Download figure Download PowerPoint The DNA fragments that code MzL and MzR for MIRTD mRNA were transfected into human neuroblastoma SH-SY5Y cells and then the cells expressing the maxizyme were selected. Poly(A)+ RNA was isolated from the transfectants and the amount of MIRTD mRNA was measured by real-time quantitative RT–PCR. As expected, amounts of MIRTD mRNA were markedly reduced in the maxizyme-expressing cells (Figure 6B). The DLST and MIRTD proteins were semiquantified by western blotting (Figure 6C). Amounts of the DLST protein were not affected by the introduction of the maxizyme, whereas the MIRTD protein markedly decreased (Figure 6C, lanes Z5 and Z21). Thus, we concluded that the maxizyme specifically digested the MIRTD mRNA and that the 30 kDa protein is the product translated from the MIRTD mRNA. Phenotypes of the MIRTD-deficient cell lines To elucidate the role of MIRTD, we tried to find phenotypes that appeared on specific digestion of the MIRTD mRNA. First, we exposed the cells expressing the maxizyme or monomer (inactive form) to hydrogen peroxide, stained their nuclei with a mixture of fluorescent dyes [Hoechst 33342 (blue) and propidium iodide (PI) (pink) as indicators of living and dead cells, respectively] and then counted the cells stained with each dye to calculate viability. Cell viability against hydrogen peroxide was significantly reduced compared with the controls in a dose- and time-dependent manner (Figure 7A and B). When the transfectants expressing the maxizyme and monomeric controls differentiated into putative neurons on treatment with retinoic acid, the transfectants with the maxizyme were also more sensitive to hydrogen peroxide than the controls (Figure 7C). Figure 7.Sensitivity to oxidative stress in the MIRTD-deficient cells. (A) Maxizyme-expressing cells (Z5, Z7 and Z21) and control cells (L1 and R2) were exposed to medium containing 0.2, 0.4 or 0.6 mM H2O2. After 24 h, the cells were stained with" @default.
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• W2118039400 title "Truncated product of the bifunctional DLST gene involved in biogenesis of the respiratory chain" @default.
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