and pmtTFS. (A) PCR products were visualized by ethidium bromide staining of an agarose gel, Left lane shows Hind Ill-digested ~,phage DNA (size marker).
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BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages 567-573
I N H I B I T I O N OF M I T O C H O N D R I A L G E N E E X P R E S S I O N B Y A N T I S E N S E R N A OF M I T O C H O N D R I A L T R A N S C R I P T I O N F A C T O R A (mtTFA)
IHidetoshi Inagaki, Shigetomo Kitano, Kong Hua Lin, Sumio Maeda, and Takao Saito. Department of Chemistry, National Industrial Research Institute of Nagoya. 1 Hirate-Cho, Kita-ku, Nagoya, 462, Japan. Received March 17, 1998 Received after revision,March 20, 1998 Summary: Mitochondrial transcription factor A (mtTFA) plays an important role in regulating the expression of mitochondrial genes. To gain a better understanding of the relationships between mitochondrial gene expression and mtTFA, the mtTFA gene was inserted into a mammalian expression vector, both in the sense orientation and in the antisense orientation. After construction, these plasmids were transfected into COS-7 cells. In antisense-transformed cells, the expression of the cytochrome c oxidase subunit I and subunit III genes encoded by mitochondrial DNA was inhibited and there was an accompanying reduction of the level of mtTFA protein. These results provide direct evidence that the expression of mitochondrial genes is under the control of mtTFA. K e y w o r d s : mtTFA/mitochondrial gene expression/antisense/COS-7 Introduction
Human mitochondrial DNA encoding 2 rRNA, 13 mRNA and 22 tRNA genes (1), are transcribed with the coordination of mtRNA polymerase and transcription factors transloeated from the nucleus. One of the factors, mtTFA, was found and the human (2), mouse (3) and Xenopus (4) mtTFA cDNAs have been isolated. It was shown that mtTFA is an essential factor for accurate and efficient mitochondrial transcription in vitro (1, 3). Sequence data for mtTFA showed that it has two HMG boxes (DNA binding motifs) that are common in UBF (upstream binding factor) (5) and LEF-1 (lymphoid enhancer factor 1) (6) and so on. Garstka et al. reported that following thyroid hormone treatment, the expression of both mitochondrial genes and the mtTFA gene in liver and muscle changed at the same time (7). In the case of fatal mitochondrial myopathy patients, Larsson et al. indicated that both the mtTFA protein level and the amount of mitochondrial DNA were reduced, and that replication is closely linked with mitochondrial gene expression (8). However these results are circumstantial evidence that mtTFA contributes to mitochondrial gene
Abbreviations: DMEM, Dulbecco's modified Eagle medium; PCR, polymerase chain reaction. 1 To whom correspondence should be addressed.: Hidetoshi Inagaki, PhD Department of Chemistry, National Industrial Research Institute of Nagoya. 1 Hirate-Cho, Kita-ku, Nagoya, 462, Japan. 1039-9712/98/090567-07505.00/0
Copyright 9 1998 by Academic Press Australia. All rights of reproduction in any form reserved.
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expression in vivo. To gain a better understanding of the relationship between mitochondrial gene expression and mtTFA, the antisense technique was used. Antisense inhibition of gene expression has become a widely used method for interfering with specific gene function. Inhibition of particular target genes has been achieved using injected RNAs, expressed RNAs, and oligonueleotides (9). In this paper, we report that the level of mtTFA protein in COS-7 cells was decreased after transfection of the plasmid to produce antisense mtTFA RNA. The reduction of this protein seemed to lead to the reduction of mitochondrial gene transcription. These results provide direct evidence that the expression of mitochondrial genes is under the control of mtTFA. Materials and M e t h o d s
Materials. Materials for the cell culture were obtained from the following sources: DMEM from Nissui Pharmaceutical Co (Tokyo, Japan), FCS from JRH Biosciences (Lenexa, KS, U.S.A.) and culture dishes from Ntmc (Roskilde, Demnark). Trans IT-LT1 was purchased from Pan Vera Co (Madison, WI, U.S.A). New Zealand White rabbits for the production of antibody were purchased from Nihon SLC (Shizuoka, Japan). An ECL direct nucleic acid labeling and detection system for Northern hybridization and an ECL Western blotting analysis system were purchased from Amersham (Buckinghamshire, England). Other materials were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Cell culture. COS-7 and HeLa cells were cultivated in DMEM, supplemented with 10% fetal calf serum. To maintain the proliferation of antisense-transformed cells, 110 ~xg/ml of pyruvate and 50 ~tg/ml uridine were added to the DMEM after transfection (10). These cell lines were cultured under humidified 95% air and 5% CO2 at 3 7 ~ . Isolation o f m t T F A c D N A and p l a s m i d construction. Total RNA isolated from the HeLa cells (11) was reverse transcribed to cDNA and subjected to PCR amplification. The oligonucleotides used in the PCR amplification were 5' CATCCACCGGAGCGATGGCGTTTCTCCGAAGCA-3' (sense), and 5' CACTCCTCAGCACCATAqTFT-3' (antisense). PCR amplification was conducted for 30 cycles of denaturation (94~C, 1 min), annealing (55~C, 2 rain), and elongation (72~C, 2 min). After cloning PCR products into the Sma I site of pBK-CMV (Stratagene, La Jolla, CA, U.S.A.), the inserts were sequenced by the method of Sanger et d (12). N o r t h e r n H y b r i d i z a t i o n . The ECL direct nucleic acid labeling and detection system was used in the Northern analysis. The PCR products (corresponding to 5943-6301) of human mtDNA, encoding the gene cytochrome c oxidase subunit I and the Pst I -Sac I fragment (corresponding to 9020-9643) (1) of human mtDNA, encoding the gene cytochrome c oxidase subunit III, were used for the Northern hybridization probes (13). Cell transfection. Trans IT LT-1 was used in the transfection. Each 100 mm dish of COS-7 cells (5.0x 105 cells/dish) was transfected with 16 ~tg of plasmid DNAs. Five hours after transfection, the cell monolayers were washed with PBS and were replaced with serum-containing medium. At 48 h posttransfection, total RNA and cellular lysate were extracted from each transformant. To estimate the transfection efficiencies, pENL encoding [3-galactosidase was transfected to COS-7 cells simultaneously (14). The transfection efficiencies of the different experiments ranged from 30% to 40%. Preparation of anti-mtTFA antibody and Western blqtting. To express mtTFA protein in E coli, a Sal I-Sal I fragment of mtTFA cDNA corresponding to amino acids 28-246 was cloned into the Sal I site of the bacterial expression vector pQE30 (Qiagen, Chatsworth, CA, U.S.A.). We termed this plasmid pQE-TF. A bacterial culture expressing this plasmid was grown in LB mediun~ containing 100 ~xg/ml ofampicillin and induced with 0.1 mMIPTG for 2 hr. Since the protein expressed by this plasmid is fused with 6 histidine residues at the N-terminus, we
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could easily purif.v the fusion protein with a Ni-NTA column as described by the manufacturer (Qiagen, Chatsworth, CA, U.S.A.). Immunizations of New Zealand White rabbits were initiated by intramuscular injections of 300 ~tg of purified fusion protein emulsified in complete Freund' s adjuvant, followed by boosting with the same amount of protein in incomplete Freund' s adjuvant. Collection of sera was begun 4 weeks after the initial injection (15). The antibody was affinity purified from antiserum (16). The specificity and titer of the sera were examined by Western blotting (15). R e s u l t s and D i s c u s s i o n
Vector c o n s t r u c t i o n . In order to isolate the human mtTFA eDNA, PCR amplification was
carried out. As shown in Fig: 1A, a single 750-bp DNA fragment was detected and was subcloned into pBK-CMV (Fig. I-A). Plasmid DNAs from individual colonies were first characterized by a restriction enzyme analysis and then by DNA sequence determination. Some of the DNA sequences were identical to the mtTFA sequence previously reported (2). We termed two resulting vectors, that expressed sense mtTFA RNAs or antisense mtTFA RNAs, pmtTFS and pmtTFAS respectively (Fig. l-B). pBK-CMV, pmtTFS, and pmtTFAS, were individually introduced into COS-7 cells by Trans IT-LT1, and transient transformants were analyzed, pBK-CMV without the mtTFA sequence was used as a control for the nonspecific effects of transformation. The growth properties of antisense-transformed cells were normal in the presence of pyruvate and uridine. In the present study, we used COS-7 cells, that has the viral T antigen, pBK-CMV, that has an SV40 replication origin and an efficient promoter CMV, massively replicated in COS-7 cells (100,000 copies per cell). On the other hand, based on a purification ofmtTFA, Parisi et al. estimated that there are 150,000 mtTFA molecules per cell (18). Taken together, it was postulated that the number of antisense mtTFA RNA molecules was enough to depress the mtTFA protein level in COS-7 cells. To estimate the levels of mtTFA protein and mitochondrial gene expression, total cellular lysate and total RNA was isolated from transient transformed COS-7 cells 2 days after transfection, and then subjected to Western blotting or Northern analysis. To estimate the transfection efficiencies of each type of plasmid, pENL encoding [~galactosidase was cotransfected to COS-7 cells independently. There was no difference in the transfection efficiencies among each type of plasmid. The transfection efficiencies of the different experiments ranged from 30% to 40% COS-7 cells. Preliminary, cells from the human cell lines HeLa, KB, A431 and Saos-2, were transfected to estimate the transfection efficiencies. However, the transfection efficiencies, which were less than 10%, were not enough to detect the reduction of the mtTFA protein level in these cell lines. m t T F A protein l e v e l in a n t i s e n s e - t r a n s f o r m e d cells. To produce the anti-mtTFA
antibody, recombinant mtTF protein was overexpressed in E. coli and purified using a Ni-NTA column. As shown in Fig. 2, a 29-kDa fusion protein was effectively purified. After injection of recombinant mtTFA protein, an anti-mtTFA antibody was prepared. As shown in Fig. 3-A, the
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F I G . 1. P C R a m p l i f i c a t i o n o f m t T F A eDNA and p l a s m i d c o n s t r u c t i o n o f p m t T F A S and p m t T F S .
(A) PCR products were visualized by ethidium bromide staining of an agarose gel, Left lane shows Hind Ill-digested ~,phage DNA (size marker). (13) Conslruction of both pmtTFAS and pmtTFS, mtTFA cDNA fragment (corresponding to 119869) cloned into the Sma I site of the transformation vector pBK-CMV. Open arrow indicates the direction of transcription. The promoter (CMV) and selection marker gene (G418) are noted.
(kDa 66 45 36 recombinant
FIG. 2. Purification of recombinant mtTFA protein.
Purified recombinant protein was analyzed by SDS-PAGE. Molecular weights for SDS-PAGE are indicated on the left : 94, 66, 45, 36, 29 and 22 kDa.
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66 45 36 29 24 20
FIG. 3. The level o f mtTFA protein in COS-7 cells after transformation o f pBKC M V , p m t T F A S and p m t T F S .
(A) Ten ixg of total cellular protein, isolated from HeLa and COS-7 cells, was electrophoresed, the gel was blotted on the membrane, and the membrane was subjected to Amido Black staining or immunostaining using mtTFA antibodies. Molecular weights are indicated on the left. (B) Ten ~g of total cellular protein, isolated from COS-7 cells that had been transfected with the plasmids, pBK-CMV, pmtTFAS and pmtTFS respectively, was electrophoresed, and the gel was blotted on the membrane, and the membrane was subjected to Amido Black staining or immunostaining using mtTFA antibodies. Molecular weights are indicated on the left.
anti-mtTFA antibody recognized only a 25-kDa protein in HeLa cells. This size corresponds to a previously reported mtTFA protein (2). However, two major bands were detected in COS-7 cells. One of the detected bands seemed to be derived from alternative splicing products (3) or a specific, modified form of mtTFA in COS-7 cells (17). To determine whether mtTFA protein level was decreased in antisense-transformed cells, we carried out Western blotting. The mtTFA protein level in the cells transfected by mtTFAS was decreased to 70% compared with the level in the cells transfected by pBK-CMV, as shown in Fig. 3-B.
Inhibition o f mitochondrial gene expression in COS-7 cells transformed with pmtTFAS. The mitochondrial gene of vertebrates are so compact structure that there are two promoters (HSP and LSP) for both the light and heavy strands of rnitochondrial DNA. Transcription from these promoters gives rise to long polycistronic messages that are cleaved to produce individual RNA species by mtRNase. Science we used the cytochrome c oxidase subunit I and subunit III
mRNAs for the representation for mitochondrial RNA species. To test whether
the mitochondrial gene expression could be inhibited by antisense mtTFA RNA, we examined the
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COX 1 COX U
F I G . 4. I n h i b i t i o n o f m i t o c h o n d r i a l g e n e e x p r e s s i o n in a n t i s e n s e - t r a n s f o r m e d c e l l s . Total RNA was extracted from COS-7 cells, which were transfected with pmtTFS, pmtTFAS and pBK-CMV respectively. Ten ~tg of total RNA was electrophoresed on an agarose gel and transblotted on a Nylon membrane. A Northern analysis was carried out with the cDNAs of the human mitochondrial genes corresponding to cytochrome c oxidase subunit I and subunit lII. The corresponding total RNAs were electrophoresed and stained with ethidium bromide to compare the anaounts of RNAs used for the Northern analysis.
expression of antisense RNA and mitochondrial genes by Northern blotting. The amount of the cytochrome c oxidase subunit I and subunit III mRNAs encoded by a mitochondrial DNA in antisense-transformed cells were reduced to 70% of that in sense-transfomaed cells (Fig. 4). This inhibition level closely paralleled the reduction of the level of mtTFA protein in the cells transformed by pmtTFAS. Considering the fact that the transfection efficiencies ranged from 30% to 40%, the expression of cytochrome c oxidase subunit I and subunit III genes, was completely suppressed in an each antisense-transformed cell. Recently Antoshechkin et al. purified Xenopus mtTFB (4), which is another transcription factor for mitochondria. In vitro transcription studies showed that Xenopus mtTFB was required for basal transcription, while, in contrast mtTFA resulted in a 2- to 3- fold increase in basal transcription for Light strand promoter activity and a 10-fold increase for heavy strand promoter activity (4). Hence, cytochrome c oxidase subunit I and subunit III genes, which are encoded by the heavy strand, might be sensitive to the reduction of mtTFA.
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To understand mitochondrial gene regulation, we focused on mtTFA in this study. Eventually we hope to isolate another transcription factors (4, 19). Isolation of these factors will help to more precisely understand the overall mechanism of mitochondrial gene regulation. References
(1) Anderson, S., Bankier, A. T., Barrel, B. G., De Bruijin, M. H. L., Coulson, A. R., Drouin, J., Eperon, I. C., Nierlich, D. P., Roe, B. A., Sanger, F., Schrerier, P. H., Smith, A. J. H., Staden, R., and Young, I. G. (1981) Nature 290, 457-465. (2) Parisi, M. A., and Clayton, D. A. (1991) Science 252, 965-969. (3) Larsson, N. -G., Garman, J. D., Oldfors, A., Barsh, G. S.; and Clayton, D. A. (1996) Nature genetics 13, 296-302. (4) Antoshechkin, I., and Bogenhagen, D. F. (1995) Mol. Cell. Biol. 15, 7032-7042. (5) Jantzen, H. -M., Admon, A., Bell, S. P., and Tjian, R. (1990) Nature 344, 830-836. (6) Giese, K., Amsterdam, A., and Grosschedl, R. (1991) Genes Dev. 5, 2567-2578. (7) Garstka, H. L., F~icke,M., Escribano, J. R., and Wiesner, R. J. (1994) Biochem. Biophys. Res. Comm. 200, 619-626. (8) Larsson, N. -G., Oldfors, A., Holme, E.,and Clayton, D. A. (1994) Biochem. Biophys. Res. Comm. 200, 1374-1381. (9) Izant, J. G., and Weintraub, H. (1985) Science 229, 345-352. (10) King, M. P., and Attardi, G. (1989) Science 246, 500-503. (11) Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156-159. (12) Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad Sci. U.S.A. 74, 5463-5467. (13) Kadowaki, T., and Kitagawa, Y. (1991) Exp. Cell Res. 192, 243-247. (14) Taniguchi, Y., and Kitagawa, Y. (1993) Cytotechnology 1 1, 175-82. (15) Harlow, E., and Lane, D. (1988) in Antibodies: A Laboratory Manual, pp. 498-510, Cold Spring Harbor Laboratory Press, New York. (16) Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) in Molecular Cloning: A Laboratory Manual, pp 18.17-18.18, Cold Spring Harbor Laboratory Press, New York. (17) Alami-ouahabi, N., Veilleux, S., Meistrich. M., and Bossonneault, G. (1996) Mol. Cell. Biol.16,3720-3729.
(18) Parisi, M. A., Xu, B., and Clayton, D. A. (1993) MoI. Cell. Biol. 13, 1951-1961. (19) Suzuki, H., Suzuki, S., Kumar, S., and Ozawa, T. (1995) Biochem. Biophys. Res_ Comm. 213, 204-210.