增加檸檬酸的產生被認為可以提供大量增生的癌細胞中特別是膜上的磷脂質等脂類的先驅物。最近的研究已經發現在很多人類癌症裡檸檬酸合成酶（Citrate synthase, CS）活性顯著提高，例如胰的導管和膀胱癌細胞，顯示檸檬酸合成酶的活性與癌細胞的大量分裂分化有關。因此，檸檬酸合成酶可能可以成為癌症的治療目標。檸檬酸合成酶是在產生能量的代謝途徑中重要的調控酵素之一，在檸檬酸循環中催化由草醯乙酸（oxaloacetate, OAA）和乙醯輔酶A（Acetyl-CoA）形成檸檬酸。人類的檸檬酸合成酶基因共有2萬8千6百個鹼基對，由12個外顯子（exon）透過交替剪接（alternative splicing）表現出兩種型：CSa以及CSb。我們針對兩種型分析在20種組織細胞中的表現量。雖然大家都認為CS是一個典型的粒線體蛋白質，但是目前還沒有研究利用分子生物的技術探討它的粒線體標的序列。為了詳細研究CS的粒線體標的序列，我們構築了一個CSa與綠色螢光融合蛋白的表現載體，同時構築了會特定表現在粒線體(mitochondron)與過氧化體(peroxisome)的紅色螢光表現載體，在共同轉染後，我們確認CSa只會單獨表現在粒線體中。同時我們透過融合不同大小片段的粒線體標的序列在綠色螢光蛋白的N端，研究不同大小片段的粒線體標的序列使綠色螢光蛋白進入粒線體的能力，結果發現CS的N端前27個胺基酸是讓CS進入粒線體必需的。另外我們證明序列中對於進入粒線體最重要的是鹼性胺基酸，當鹼性胺基酸全部突變時會明顯降低它進入粒線體的能力，反之即使所有的帶氫氧基的胺基酸(Serine、Theronine)都突變時，仍然不會影響蛋白質進入粒線體的能力。為了研究粒線體蛋白質進入粒線體的機制，我們將會進入粒線體的綠色螢光融合蛋白表現載體與針對粒線體外膜上的轉移蛋白(translocase of the outer membrane, TOM)所設計的shRNA進行共同轉染，結果發現的確會影響蛋白質進入粒線體。另外，為了研究CS 的生物機能，我們已經利用siRNA評估系統篩選出有效的shRNA，可以有效的抑制細胞內生性的CS表現量，導致CS活性降低，因此可能成為一個治療癌症的策略。

It is believed that the increasing production of citrate can provide lipid precursors for proliferating tumor cells especially for membrane lipid formation. In addition, recent studies have found that the citrate synthase (CS) activity is markedly overexpressed in many human cancers, such as pancreatic ductal and bladder carcinoma cells, strongly suggesting that the CS activity is associated with cell proliferation in cancer cells. Thus, CS may serve as a putative therapeutic target for cancers. Citrate synthase (CS) is one of the key regulatory enzymes in the energy-generating metabolic pathway. It catalyzes the condensation of oxaloacetate (OAA) and acetyl coenzyme A (acetyl-CoA) to form citrate in the tricarboxylic acid (TCA) cycle. The 28.6 kb of human CS gene consists of twelve exons that express two transcriptional variants, citrate synthase isoform a (CSa) and isoform b (CSb). In this study, we are interested in the expression level of different tissues between the two transcriptional variants. CSa is localized in mitochondrial matrix; however, its mitochondrial targeting sequence has not been analyzed by molecular technology. To dissect the mitochondrial targeting sequence of CSa, we have constructed a CSa-enhanced green fluorescence protein (EGFP) fusion protein expression vector and two red fluorescence protein (DsRed2) expression vectors that specifically targeted to mitochondria and peroxisomes. After co-transfection, the results confirmed that CSa is localized in mitochondria. Furthermore, we have expressed fusion constructs of truncated or site-mutated CSa and flurorescent proteins. The results indicated that the N-terminal 27 amino acid sequence in CSa is essential and sufficient for mitochondrial import. The reduction of the net positive charge in this segment decreased mitochondrial specificity and the mutants were distributed throughout the cytosol. However, when all serine and threonine in this segment were mutated, the mitochondrial specificity would not be affected. To dissect the mechanism of mitochondria import, we co-transfected the expression vector targeting to mitochondria and shRNAs against Tom complex, and found that these shRNAs have effect on the efficacy of motchondria import. In addition, to study the biological function of CS, we have used an efficient siRNA screening strategy to identify extremely potent siRNA molecules. These siRNAs could induce CS mRNA degradation and results in the reduction of CS activity. The effective siRNA may be a potential therapeutic strategy for the cancers that CS activity is significantly elevated.

Abbas-Terki, T., Blanco-Bose, W., Deglon, N., Pralong, W. and Aebischer, P. Lentiviral-mediated RNA interference. Hum. Gene Ther. 13, 2197-2201 (2002).
Abe, Y., Shodai, T., Muto, T., Mihara, K., Torii, H., Nishikawa, S., Endo, T. and Kohda, D. Structural basis of presequence recognition by the mitochondrial protein import receptor Tom20. Cell 100, 551–560 (2000).
Ahting, U., Thun, C., Hegerl, R., Typke, D., Nargang, F. E., Neupert, W. and Nussberger, S. The TOM core complex: the general protein import pore of the outer membrane of mitochondria. J. Cell. Biol. 147, 959–968 (1999).
Alberi, L., Sgado, P., and Simon, H.H. Engrailed genes are cell-autonomously required two prevent apoptosis in mesencephalic dopaminergic neurons. Development 131, 3229-3236 (2004).
Anderson, J., Banerjea, A., Planelles, V., and Akkina, R. Potent suppression of HIV type 1 infection by a short hairpin anti-CXCR4 siRNA. AIDS Res Hum Retroviruses 19, 699-706 (2003).
Ashrafi, K., Chang, F. Y., Watts, J. L., Fraser, A. Anderson, J., Banerjea, A., Planelles, V., and Akkina, R. Potent suppression of HIV type 1 infection by a short hairpin anti-CXCR4 siRNA. AIDS Res Hum Retroviruses 19, 699-706 (2003).
G., Kamath, R. S., Ahringer, J. and Ruvkun, G. Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature 421, 268-272 (2003).
Banerjee, D. and Slack, F. Control of developmental timing by small temporal RNAs: a paradigm for RNA-mediated regulation of gene expression. Bioessays 24, 119-129 (2002).
Bartel, D.P. MicroRNAs:genomics , biogenesis, mechanism, and function. Cell 116, 218-297 (2004).
Barton, G. M. and Medzhitov, R. Retroviral delivery of small interfering RNA into primary cells. Proc. Natl. Acad. Sci. U S A. 99, 14943-14945 (2002).
Becker, K., Guiard, B., Rassow, J., Sollner, T. and Pfanner, N. Targeting of a chemically pure preprotein to mitochondria does not require the addition of a cytosolic signal recognition factor. J. Biol. Chem. 267, 5637-5643 (1992).
Bitko, V., Musiyenko, A., Shulyayeva, O., and Barik, S. Inhibition of respiratory viruses by nasally administered siRNA. Nat. Med. 11, 50-55 (2005).
Blobel, G. Intracellular protein topogenesis. Proc. Natl. Acad. Sci. USA 77, 1496–1500 (1980).
Boden, D., Pusch, O., Silbermann, R., Lee, F., Tucker, L. and Ramratnam, B. Enhanced gene silencing of HIV-1 specific siRNA using microRNA designed hairpins. Nucleic Acids Res. 32, 1154-1158 (2004).
Bolliger, L., Junne, T., Schatz, G. & Lithgow, T. Acidic receptor domains on both sides of the outer membrane mediate translocation of precursor proteins into yeast mitochondria. EMBO J. 14, 6318–6326 (1995).
Braasch, D.A., Paroo, Z., Constantinescu, A., Ren, G., Oz, O.K. and Mason, R.P. Biodistribution of phosphodiester and phosphorothioate SiRNA. Bioorg Med Chem Lett 14, 1139-1143 (2004).
Brix, J., Dietmeier, K. & Pfanner, N. Differential recognition of preproteins by the purified cytosolic domains of the mitochondrial import receptors Tom20, Tom22, and Tom70. J. Biol. Chem. 272, 20730–20735 (1997).
Brix, J., Rüdiger, S., Bukau, B., Schneider-Mergener, J. and Pfanner, N. Distribution of binding sequences for the mitochondrial import receptors Tom20, Tom22, and Tom70 in a presequence-carrying preprotein and a noncleavable preprotein. J. Biol. Chem. 274, 16522–16530 (1999).
Brummelkamp, T. R., Bernards, R. and Agami, R. A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550-553 (2002).
Brummelkamp, T. R., Bernards, R. and Agami, R. Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2, 243-247 (2002).
Brusselmans, K., De Schrijver, E., Verhoeven, G. and Swinnen, J. V. RNA interference-mediated silencing of the acetyl-CoA-carboxylase-alpha gene induces growth inhibition and apoptosis of prostate cancer cells. Cancer Res. 65, 6719-25 (2005).
Caplen, N. J., Zheng, Z., Falgout, B. and Morgan, R. A. Inhibition of viral gene expression and replication in mosquito cells by dsRNA-triggered RNA interference. Mol. Ther. 6, 243-251 (2002).
Chalbos, D., Joyeux, C., Galtier, F., and Rochefort, H. Progestin-induced fatty acid synthetase in human mammary tumors: from molecular to clinical studies. J. Steroid Biochem. Mol. Biol. 43, 223–228 (1992).
Chiu, Y. L., and Rana, T. M. RNAi in human cells：basic structural and functional features of small interfering RNA. Mol. Cell 10, 549-561 (2002).
Chiu, Y. L. and Rana, T. M. siRNA function in RNAi: a chemical modification analysis. RNA 9, 1034-1048 (2003).
Churchill, J. D., Galvez, R., Colcombe, S., Swain, R. A., Kramer, A. F. and Greenough, W. T. Exercise, experience and the aging brain. Neurobiol Aging. 23, 941-955 (2002).
Cioca, D. P., Aoki, Y. and Kiyosawa, K. RNA interference is a functional pathway with therapeutic potential in human myeloid leukemia cell lines. Cancer Gene Ther. 10,125-133 (2003).
Clayton, J. RNA interference: the silent treatment. Nature 431, 599-605 (2004).
Davidson, B. L. and Paulson, H. L. Molecular medicine for the brain: silencing of disease genes with RNA interference. Lancet Neurol. 3, 145-149 (2004).
De Castro Silva Filho, M., Chaumont, F., Leterme, S., Boutry, M. Mitochondrial and chloroplast targeting sequences in tandem modify protein import specificity in plant organelles. Plant. Mol. Biol. 30, 769-780 (1996).
De Schrijver, E., Brusselmans, K., Heyns, W., Verhoeven, G. and Swinnen, J. V. RNA interference-mediated silencing of the fatty acid synthase gene attenuates growth and induces morphological changes and apoptosis of LNCaP prostate cancer cells. Cancer Res. 63, 3799-3804 (2003).
Dekker, P. J. et al. Preprotein translocase of the outer mitochondrial membrane: molecular dissection and assembly of the general import pore complex. Mol. Cell Biol. 18, 6515–6524 (1998).
Dorsett, Y. and Tuschl, T. siRNAs: applications in functional genomics and potential as therapeutics. Nat. Rev. Drug Discov. 3, 318-329 (2004).
Du, Q., Thonberg, H., Zhang, H. Y., Wahlestedt, C. and Liang, Z. Validating siRNA using a reporter made from synthetic DNA oligonucleotides. Biochem. Biophys. Res. Commun. 325, 243-249 (2004).
Duhl, D. M., Powell, T., Poyton, R. O. Mitochondrial import of cytochrome c oxidase subunit VIIa in Saccharomyces cerevisiae. Identification of sequences required for mitochondrial localization in vivo. J. Biol. Chem. 265, 7273-7277 (1990).
Dykxhoorn, D. M., Novina, C. D., and Sharp, P. A. Killing the messenger: short RNAs that silence gene expression. Nat. Rev. Mol. Cell Biol. 4, 457-467 (2003).
Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl, T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494-498 (2001).
Elbashir, S. M., Martinez, J., Patkaniowska, A., Lendeckel, W. and Tuschl, T. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20, 6877-6888 (2001).
Epand, R. M., Hui, S. W., Argan, C., Gillespie, L. L. and Shore, G. C. Structural analysis and amphiphilic properties of a chemically synthesized mitochondrial signal peptide. J. Biol. Chem., 261, 10017-10020 (1986).
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. and Mello, C. C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811 (1998).
Ge, Q., McManus, M. T., Nguyen, T., Shen, C. H., Sharp, P. A., Eisen, H. N. and Chen, J. RNA interference of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all viral RNA transcription. Proc. Natl. Acad. Sci. USA. 100, 2718-2723 (2003).
Giladi, H., Ketzinel-Gilad, M., Rivkin, L., Felig, Y., Nussbaum, O. and Galun, E. Small interfering RNA inhibits hepatitis B virus replication in mice. Mol. Ther. 8, 769-776 (2003).
Grishok, A., Pasquinelli, A. E., Conte, D., Li, N., Parrish, S., Ha, I., Baillie, D. L., Fire, A., Ruvkun, G. and Mello, C. C. Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106, 23-34 (2001).
Guo, S., and Kemphues, K.J. par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell 81: 611-620 (1995).
Hallermayer, G., Zimmermann, R. & Neupert, W. Kinetic studies on the transport of cytoplasmically synthesized proteins into the mitochondria in intact cells of Neurospora crassa. Eur. J. Biochem. 81, 523–532 (1977).
Hammond, S. M., Boettcher, S., Caudy, A. A., Kobayashi, R. and Hannon, G. J. Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293,1146-1150 (2001).
Hannon, G. J. and Rossi, J. J. Unlocking the potential of the human genome with RNA interference. Nature 431, 371-378 (2004).
Harborth, J., Elbashir, S. M., Bechert, K., Tuschl, T. and Weber, K. Identification of essential genes in cultured mammalian cells using small interfering RNAs. J. Cell Sci. 114, 4557-4565 (2001).
Harborth, J., Elbashir, S. M., Vandenburgh, K., Manninga, H., Scaringe, S. A., Weber, K. and Tuschl, T. Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing. Antisense Nucleic Acid Drug Dev. 13, 83-105 (2003).
Hatzivassiliou, G., Zhao, F., Bauer, D. E., Andreadis, C., Shaw, A. N., Dhanak, D., Hingorani, S. R., Tuveson, D. A. and Thompson, C. B. ATP citrate lyase inhibition can suppress tumor cell growth. Cancer Cell 8, 311-321 (2005).
Hay, R., Bohni, P. and Gasser, S. How mitochondria import proteins. Biochim. Biophys. Acta. 779, 65-87 (1984).
Henschel, A., Buchholz, F. and Habermann, B. DEQOR: a web-based tool for the design and quality control of siRNAs. Nucleic Acids Res. 32, W113-120 (2004).
Hill, K., Model, K., Ryan, M. T., Dietmeier, K., Martin, F., Wagner, R. and Pfanner, N. Tom40 forms the hydrophilic channel of the mitochondrial import pore for preproteins. Nature 395, 516–521 (1998).
Hollmann, W. amd Struder, H. K. Brain function, mind, mood, nutrition, and physical exercise. Nutrition. 16, 516-519 (2000)
Hommel, J. D., Sears, R. M., Georgescu, D., Simmons, D. L. and DiLeone, R. J. Local gene knockdown in the brain using viral-mediated RNA interference. Nat. Med. 9, 1539–1544 (2003).
Horwich, A. L., Kalousek, F., Mellman, I. and Rosenberg, L. E. A leader peptide is sufficient to direct mitochondrial import of a chimeric protein. EMBO J. 4, 1129–1135 (1985).
Horwich, A. L., Kalousek, F., Rosenberg, L. E. Arginine in the leader peptide is required for both import and proteolytic cleavage of a mitochondrial precursor. Proc. Natl. Acad. Sci. U S A. 82, 4930-4933 (1985).
Horwich, A. L., Kalousek, F., Fenton, W. A., Furtak, K., Pollock, R. A. and Rosenberg, L. E. The ornithine transcarbamylase leader peptide directs mitochondrial import through both its midportion structure and net positive charge. J. Cell. Biol. 105, 669-77 (1987).
Howard, K. Unlocking the money-making potential of RNAi. Nat Biotechnol 21: 1441-1446 (2003).
Huang, F., Khvorova, A., Marshall, W., and Sorkin, A. Analysis of clathrin-mediated endocytosis of epidermal groth factor receptor by RNA interference. J Biol Chem 279: 16657-16661 (2004).
Hurt, E. C., Pesold-Hurt, B. & Schatz, G. The cleavable prepiece of an imported mitochondrial protein is sufficient to direct cytosolic dihydrofolate reductase into the mitochondrial matrix. FEBS Lett. 178, 306–310 (1984).
Jacque, J. M., Triques, K. and Stevenson, M. Modulation of HIV-1 replication by RNA interference. Nature 418, 435-438 (2002).
Joost Haasnoot, P. C., Cupac, D. and Berkhout, B. Inhibition of virus replication by RNA interference. J. Biomed. Sci. 10, 607-616 (2003).
Kamath, R. S., Fraser, A. G., Dong, Y., Poulin, G., Durbin, R., Gotta, M., Kanapin, A., Le Bot, N., Moreno, S., Sohrmann, M., Welchman, D. P., Zipperlen, P. and Ahringer, J. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, 231-237 (2003).
Kang, P. J., Ostermann, J., Shilling, J., Neupert, W., Craig, E. A. and Pfanner, N. Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins. Nature 348, 137–143 (1990).
Kapadia, S. B., Brideau-Andersen, A. and Chisari, F. V. Interference of hepatitis C virus RNA replication by short interfering RNAs. Proc. Natl. Acad. Sci. USA. 100, 2014-2018 (2003).
Karagiannis, T. C. and El-Osta, A. RNA interference and potential therapeutic applications of short interfering RNAs. Cancer Gene Ther. (2005).
Kartasheva, N. N., Contente, A., Lenz-Stoppler, C., Roth, J. and Dobbelstein, M. p53 induces the expression of its antagonist p73 Delta N, establishing an autoregulatory feedback loop. Oncogene 21, 4715-4727 (2002).
Kasischke, K. A., Vishwasrao, H. D., Fisher, P. J.and Zipfel, W. R. Neural activity triggers neuronal oxidative metabolism followed by astrocytic glycolysis. Science 305, 99-103 (2004).
Kayser, B. Exercise starts and ends in the brain. Eur. J. Appl. Physiol. 90, 411-419 (2003).
Kennerdell, J. R. and Carthew, R. W. Heritable gene silencing in Drosophila using double-stranded RNA. Nat. Biotechnol. 18, 896-898 (2000).
Ketting, R. F., Haverkamp, T. H., van Luenen, H. G. and Plasterk, R. H. Mut-7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNaseD. Cell 99, 133-141 (1999).
Khvorova, A., Reynolds, A. and Jayasena, S. D. Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209-216 (2003).
Klein, C., Bock, C. T., Wedemeyer, H., Wustefeld, T., Locarnini, S., Dienes, H. P., Kubicka, S., Manns, M. P. and Trautwein, C. Inhibition of hepatitis B virus replication in vivo by nucleoside analogues and siRNA. Gastroenterology 125, 9-18 (2003).
Komiya, T., Rospert, S., Schatz, G. and Mihara, K. Binding of mitochondrial precursor proteins to the cytoplasmic domains of the import receptors Tom70 and Tom20 is determined by cytoplasmic chaperones. EMBO J. 16, 4267–4275 (1997).
Kubota, Y., Nash, R.A., Klungland, A., Schar, P., Barnes, D. E. and Lindahl, T. Reconstitution of DNA base excision-repair with purified human proteins: interaction between DNA polymerase beta and the XRCC1 protein. EMBO j. 15, 6662–6670. (1996).
Kuhajda, F. P. Fatty-acid synthase and human cancer: new perspectives on its role in tumor biology. Nutrition 16, 202–208 (2000).
Kumar, R., Conklin, D. S. and Mittal, V. High-throughput selection of effective RNAi probes for gene silencing. Genome Res. 13, 2333-2340 (2003).
Künkele, K. P. et al. The preprotein translocation channel of the outer membrane of mitochondria. Cell 93, 1009–1019 (1998).
Kusakabe, T., Nashimoto, A., Honma, K., and Suzuki, T. Fatty acid synthase is highly expressed in carcinoma, adenoma and in regenerative epithelium and intestinal metaplasia of the stomach. Histopathology 40, 71–79 (2002).
Lee, N. S., Dohjima, T., Bauer, G., Li, H., Li, M. J., Ehsani, A., Salvaterra, P. and Rossi, J. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat. Biotech. 20, 500-505 (2002).
Lee, Y., Ahn, C., Han, J., Choi, H., Kim, J., Yim, J., Lee, J., Provost, P., Radmark, O., Kim, S. and Kim, V. N. The nuclear RNase III Drosha initiates microRNA processing. Nature 425, 415-419 (2003).
Lipardi, C., Wei, Q. and Paterson, B. M. RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell 107, 297-307 (2001).
Lu, P. Y., Xie, F. Y. and Woodle, M. C. Modulation of angiogenesis with siRNA inhibitors for novel therapeutics. Trends Mol. Med. 11, 104-113 (2005).
Mao, J., Chirala, S. S., Wakil, S. J. Human acetyl-CoA carboxylase 1 gene: presence of three promoters and heterogeneity at the 5'-untranslated mRNA region. Proc. Natl. Acad. Sci. U S A. 100, 7515-7520 (2003).
Maccecchini,M.-L., Rudin,Y., Bobel,G. and Schatz,G. Import of proteins into mitochondria: precursor forms of the extramitochondrially made F1-ATPase subunits in yeast. Proc. Natl. Acad. Sci. USA 76, 343-347. (1979).
Marchenko, N. D., Zaika, A. and Moll, U. M. Death signal-induced localization of p53 protein to mitochondria. J Biol Chem. 275, 16202-16212 (2000).
Martinez, J., Patkaniowska, A., Urlaub, H., Luhrmann, R. and Tuschl, T. Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 110, 563–574 (2002).
Matsukura, S., Jones, P. A., and Takai, D. Establishment of conditional vectors for hairpin siRNA knockdowns. Nucleic Acid Res. 31, e77 (2003).
McCaffrey, A. P., Meuse, L., Pham, T. T., Conklin, D. S., Hannon, G. J. and Kay, M. A. RNA interference in adult mice. Nature 418, 38-39 (2002).
McCaffrey, A. P., Nakai, H., Pandey, K., Huang, Z., Salazar, F. H., Xu, H., Wieland, S. F., Marion, P. L., Kay, M. A. Inhibition of hepatitis B virus in mice by RNA interference. Nat. Biotechnol. 21, 639-644 (2003).
Meisinger, C., Ryan, M. T., Hill, K., Model, K., Lim, J. H., Sickmann, A., Muller, H., Meyer, H. E., Wagner, R. and Pfanner, N. Protein import channel of the outer mitochondrial membrane: a highly stable Tom40–Tom22 core structure differentially interacts with preproteins, small Tom proteins and import receptors. Mol. Cell Biol. 21, 2337–2348 (2001).
Mette, M. F., Aufsatz, W., van der Winden, J., Matzke, M. A. and Matzke, A. J. Transcriptional silencing and promoter methylation triggered by double-stranded RNA. EMBO J. 19, 5194-5201 (2000).
Miyagishi, M. and Taira, K. U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells. Nat. Biotechnol. 20, 497-500 (2002).
Myers, M., Mayorga, O. L., Emtage, J. and Freire, E. Thermodynamic characterization of interactions between ornithine transcarbamylase leader peptide and phospholipid bilayer membranes. Biochemistry, 26, 4309-4315 (1987).
Naito, Y., Yamada, T., Ui-Tei, K., Morishita, S. and Saigo, K. siDirect: highly effective, target-specific siRNA design software for mammalian RNA interference. Nucleic Acids Res. 32, W124-129 (2004).
Napoli, C., Lemieux, C., and Jorgensen, R. lntroduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. Plant Cell 2, 279-289 (1990).
Novina, C. D., Murray, M. F., Dykxhoorn, D. M., Beresford, P. J., Riess, J., Lee, S. K., Collman, R. G., Lieberman, J., Shankar, P. and Sharp, P. A. siRNA-directed inhibition of HIV-1 infection. Nat. Med. 8, 681-686 (2002).
Otterlei, M., Haug, T., Nagelhus, T. A., Slupphaug, G., Lindmo, T. and Krokan, H. E. Nuclear and mitochondrial splice forms of human uracil-DNA glycosylase contain a complex nuclear localisation signal and a strong classical mitochondrial localisation signal, respectively. Nucleic Acids Res. 26, 4611-46177 (1998).
Paddison, P. J., Caudy, A. A, Bernstein, E., Hannon, G. J. and Conklin, D. S. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 16, 948-958 (2002).
Paddison, P. J., Caudy, A. A. and Hannon, G. J. Stable suppression of gene expression by RNAi in mammalian cells. Proc. Natl. Acad. Sci. U S A. 99, 1443-1448 (2002).
Paddison, P. J., Silva, J. M., Conklin, D. S., Schlabach M, Li M, Aruleba S, Balija V, O'Shaughnessy A, Gnoj L, Scobie K, Chang K, Westbrook T, Cleary M, Sachidanandam R, McCombie WR, Elledge SJ, and Hannon, G. J. A resource for large-scale RNA-interference-based screens in mammals. Nature 428, 427-431 (2004).
Palmeri, D., Malim, M. H. Importin beta can mediate the nuclear import of an arginine-rich nuclear localization signal in the absence of importin alpha. Mol Cell Biol. 19, 1218-1225 (1999).
Paul, C. P., Good, P. D., Winer, I. and Engelke, D. R. Effective expression of small interfering RNA in human cells. Nat. Biotechnol. 20, 505-508 (2002).
Pfanner, N., Geissler, A. Versatility of the mitochondrial protein import machinery. Nat. Rev. Mol. Cell. Biol. 2, 339-349 (2001).
Piyathilake, C. J., Frost, A. R., Manne, U., Bell, W. C., Weiss, H., Heimburger, D. C. and Grizzle, W. E. The expression of fatty acid synthase (FASE) is an early event in the development and progression of squamous cell carcinoma of the lung. Hum. Pathol. 31, 1068–1073 (2000).
Qin, X. F., An, D. S., Chen, I. S. and Baltimore, D. Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. Proc. Natl. Acad. Sci. U S A. 100, 183-188 (2003).
Reich, S. J., Fosnot, J., Kuroki, A., Tang, W., Yang, X., Maguire, A. M., Bennett, J. and Tolentino, M. J. Small interfering RNA (siRNA) targeting VEGF effectively inhibits ocular neovascularization in a mouse model. Mol. Vis. 9, 210-216 (2003).
Reid, G. A. & Schatz, G. Import of proteins into mitochondria: extramitochondrial pools and posttranslational import of mitochondrial protein precursors in vivo. J. Biol. Chem. 257, 13062–13067 (1982).
Reinhart, B. J., Slack, F. J., Basson, M., Pasquinelli, A. E., Bettinger, J. C., Rougvie, A. E., Horvitz, H. R. and Ruvkun, G. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403, 901-906 (2000).
Reynolds, A., Leake, D., Boese, Q., Scaringe, S., Marshall, W. S. and Khvorova, A. Rational siRNA design for RNA interference. Nat. Biotechnol. 22, 326-330 (2004).
Roise, D., Horvath, S. J., Tomich, J. M., Richards, J. H. and Schatz, G. A chemically synthesized pre-sequence of an imported mitochondrial protein can form an amphiphilic helix and perturb natural and artificial phospholipids bilayers. EMBO J. 5, 1327–1334 (1986).
Roise, D., Theiler, F., Horvath, S. J., Tomich, J. M., Richards, J. H., Allison, D. S. and Schatz, G. Amphiphilicity is essential for mitochondrial presequence function. EMBO J. 7, 649-653 (1988).
Rosenkrantz, M., Alam, T., Kim, K. S., Clark, B. J., Srere, P. A., Guarente, L. P. Mitochondrial and nonmitochondrial citrate synthases in Saccharomyces cerevisiae are encoded by distinct homologous genes. Mol. Cell. Biol. 6, 4509-4515 (1986).
Rubinson, D. A., Dillon, C. P., Kwiatkowski, A. V., Sievers, C., Yang, L., Kopinja, J., Rooney, D. L., Ihrig, M. M, McManus, M. T., Gertler, F. B., Scott, M. L. and Van Parijs, L. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat. Genet. 33, 401-406 (2003).
Saksela, K. Human viruses under attack by small inhibitory RNA. Trends Microbiol. 11, 345-347 (2003).
Scherr, M., Battmer, K., Ganser, A. and Eder, M. Modulation of gene expression by lentiviral-mediated delivery of small interfering RNA. Cell Cycle 2, 251-257 (2003).
Schlichtholz, B., Turyn, J., Goyke, E., Biernacki, M., Jaskiewicz, K., Sledzinski, Z. and Swierczynski, J. Enhanced citrate synthase activity in human pancreatic cancer. Pancreas. 30, 99-104 (2005).
Schwarz, D. S., Hutvagner, G., Haley, B. and Zamore, P. D. Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathways. Mol. Cell 10, 537-548 (2002).
Schwarz, E., Neupert, W. Mitochondrial protein import: mechanisms, components and energetics. Biochim. Biophys. Acta. 1187, 270-274 (1994)
Semizarov, D., Kroeger, P., and Fesik, S. SiRNA-mediated gene silencing: a global genome view. Nucleic Acids Res 32: 3836-3845 (2004).
Shen, C., Buck, A. K., Liu, X., Winkler, M. and Reske, S. N. Gene silencing by adenovirus-delivered siRNA. FEBS Lett. 539, 111-114 (2003).
Shlomai, A. and Shaul, Y. Inhibition of hepatitis B virus expression and replication by RNA interference. Hepatology 37, 764-770 (2003).
Sijen, T., Fleenor, J., Simmer, F., Thijssen, K. L., Parrish, S., Timmons, L., Plasterk, R. H. and Fire, A. On the role of RNA amplification in dsRNA-triggered gene silencing. Cell 107, 465-76 (2001).
Siolas, D., Lerner, C., Burchard, J., Ge, W., Linsley, P. S., Paddison, P. J., Hannon, G. J. and Cleary, M. A. Synthetic shRNAs as potent RNAi triggers. Nat. Biotechnol. 23, 227-231 (2005).
Sioud, M. Therapeutic siRNAs. Trends Pharmacol. Sci. 25, 22-28 (2004).
Smart, N., Scambler, P. J. and Riley, P. R. A rapid and sensitive assay for quantification of siRNA efficiency and specificity. Biol. Proced. Online 7, 1-7 (2005).
Song, E., Lee, S. K., Dykxhoorn, D. M., Novina, C., Zhang, D., Crawford, K., Cerny, J., Sharp, P. A., Lieberman, J., Manjunath, N. and Shankar, P. Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages. J. Virol. 77, 7174-7181 (2003).
Sorensen, D. R., Leirdal, M. and Sioud, M. Gene silencing by systemic delivery of synthetic siRNAs in adult mice. J. Mol. Biol. 327, 761–766 (2003).
Soutschek, J., Akinc, A., Bramlage, B., Charisse, K., Constien, R., Donoghue, M., Elbashir, S., Geick, A., Hadwiger, P., Harborth, J., John, M., Kesavan, V., Lavine, G., Pandey, R. K., Racie, T., Rajeev, K. G., Rohl, I., Toudjarska, I., Wang, G., Wuschko, S., Bumcrot, D., Koteliansky, V., Limmer, S., Manoharan, M. and Vornlocher, H. P. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432, 173-178 (2004).
Stark, G. R., Kerr, I. M., Williams, B. R., Silverman, R. H. and Schreiber, R. D. How cells respond to interferons. Annu. Rev. Biochem. 67, 227-264 (1998).
Stewart, S. A., Dykxhoorn, D. M., Palliser, D., Mizuno, H., Yu, E. Y., An, D. S., Sabatini, D. M., Chen, I. S., Hahn, W. C., Sharp, P. A., Weinberg, R. A. and Novina, C. D. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA. 9, 493-501 (2003).
Stojanovski, D., Johnston, A. J., Streimann, I., Hoogenraad, N. J. and Ryan, M. T. Import of nuclear-encoded proteins into mitochondria. Exp. Physiol. 88, 57-64. (2003).
Sun, L., Deng, L., Ea, C.K., Sia, Z.P. and Chen, Z.J. The TRAF6 ubiquitin ligase and TAK1 kinase mediate IKK activation by BCL10 and MALT1 in T lymphocytes. Mol Cell 14: 289-301. (2004).
Swinnen, J. V., Roskams, T., Joniau, S., Van Poppel, H., Oyen, R., Baert, L., Heyns, W. and Verhoeven, G. Overexpression of fatty acid synthase is an early and common event in the development of prostate cancer. Int. J. Cancer. 98, 19–22 (2002).
Tabara, H., Sarkissian, M., Kelly, W. G., Fleenor, J., Grishok, A., Timmons, L., Fire, A. and Mello, C. C. The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99, 123-132 (1999).
Tijsterman, M., Ketting, R. F. and Plasterk, R. H. The genetics of RNA silencing. Annu. Rev. Genet. 36, 489-519 (2002).
Timmons, L., Tabara, H., Mello, C. C. and Fire, A. Z. Inducible systemic RNA silencing in Caenorhabditis elegans. Mol. Biol. Cell 14, 2972-2983 (2003).
Tiscornia, G., Singer, O., Ikawa, M. and Verma, I. M. A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA. Proc. Natl. Acad. Sci. U S A. 100, 1844-1848 (2003).
Thomas, C. E., Ehrhardt, A. and Kay, M. A. Progress and problems with the use of viral vectors for gene therapy. Nat. Rev. Genet. 4, 346-358 (2003).
Tuschl, T.¬, Zamore, P. D.¬, Lehmann, R.¬, Bartel, D. P. and Sharp, P. A. Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev. 13, 3191-3197 (1999).
Van den Haute, C., Eggermont, K., Nuttin, B., Debyser, Z. and Baekelandt, V. Lentiviral vector-mediated delivery of short hairpin RNA results in persistent knockdown of gene expression in mouse brain. Hum. Gene Ther. 14, 1799-1807 (2003).
Vanhecke, D. and Janitz, M. Functional genomics using high-throughput RNA interference. Drug Discov. Today 10, 205-212 (2005).
Wianny, F. and Zernicka-Goetz, M. Specific interference with gene function by double-stranded RNA in early mouse development. Nat. Cell Biol. 2, 70-75 (2000).
Wienhues, U. et al. Protein folding causes an arrest of preprotein translocation into mitochondria in vivo. J. Cell Biol. 115, 1601–1609 (1991).
Williams, D. A. and Baum, C. Medicine. Gene therapy--new challenges ahead. Science 302, 400-401 (2003).
Wilson, J. A., Jayasena, S., Khvorova, A., Sabatinos, S., Rodrigue-Gervais, I. G., Arya, S., Sarangi, F., Harris-Brandts, M., Beaulieu, S. and Richardson, C. D. RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells. Proc. Natl. Acad. Sci. USA. 100, 2783-2788 (2003).
Wu, M. T., Wu, R. H., Hung, C. F., Cheng, T. L., Tsai, W. H. and Chang, W. T. Simple and efficient DNA vector-based RNAi systems in mammalian cells. Biochem. Biophys. Res. Commun. 330, 53-59 (2005).
Xia, H., Mao, Q., Paulson, H. L. and Davidson, B. L. siRNA-mediated gene silencing in vitro and in vivo. Nat. Biotechnol. 20, 1006-1010 (2002).
Yu, J. Y., DeRuiter, S. L. and Turner, D. L. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci. U S A. 99, 6047-6052 (2002).
Yuan, B., Latek, R., Hossbach, M., Tuschl, T. and Lewitter, F. siRNA Selection Server: an automated siRNA oligonucleotide prediction server. Nucleic Acids Res. 32, W130-134 (2004).
Zamore, P. D., Tuschl, T., Sharp, P. A. and Bartel, D. P. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101, 25-33 (2000).
Zeng, Y., and Cullen, B. R. RNA interference in human cells is restricted to the cytoplasm. RNA 8, 855-860 (2002).
Zhang, L., Yang, N., Mohamed-Hadley, A., Rubin, S. C. and Coukos, G. Vector-based RNAi, a novel tool for isoform-specific knock-down of VEGF and anti-angiogenesis gene therapy of cancer. Biochem. Biophys. Res. Commun. 303, 1169-1178 (2003).
Zheng, L., Liu, J., Batalov, S., Zhou, D., Orth, A., Ding S. and Schultz, P. G. An approach to genomewide screens of expressed small interfering RNAs in mammalian cells. Proc. Natl. Acad. Sci. U S A. 101, 135-140 (2004).
Zimmermann, R. & Neupert, W. Transport of proteins into mitochondria: posttranslational transfer of ADP/ATP carrier into mitochondria in vitro. Eur. J. Biochem. 109, 217–229 (1980).