RNA İNTERFERANS (RNAİ)

Yıl 2009, Cilt: 25 Sayı: 1, 34 - 47, 01.02.2009

Ramazan Gündoğdu Venhar Çelik

Öz

RNA interferans, uygun çift zincirli RNA’nın hücreye girdiği zaman, endojenik komplementer mRNA dizisinin parçalanmasına yol açan, transkripsiyon sonrası gen susturma mekanizmasıdır. RNA interferans, Dicer adı verilen bir RNase III enzimi tarafından çift zincirli RNA’nın küçük engelleyici RNA’lara (siRNA) kesilmesi ile başlamaktadır. Bu siRNA’lar daha sonra, bir multiprotein-RNA nükleaz kompleksi olan, RNA- indükleyici baskılama kompleksine (RISC) bağlanır. RISC, siRNA’ları komplementer mRNA’yı bulmak için kullanır ve hedef mRNA’yı endonükleolitik olarak keser. Neticede spesifik mRNA’nın azalması uygun protein(ler)in azalmasına yol açar. RNA interferans ve RNA baskılamanın diğer formları olan transkripsiyon sonrası gen baskılama/co-supresyon ve quelling bitkiler, hayvanlar, fungus ve protozoa gibi geniş bir organizma çeşidinde gözlenmiştir. RNA interferans doğal bir mekanizma olmakla birlikte in vitro olarak sentezlenen siRNA’lar kullanılarak endojenik genlerin ekspresyonu baskılanabilmektedir. Bu yüzden RNA interferans moleküler biyolojide gen fonksiyonu analizinde ve aynı zamanda gen terapisinde geniş bir uygulama alanına sahiptir. Bu derlemede RNA interferans ve onun spesifik varyantları olan transkripsiyon sonrası gen baskılama/co-supresyon ve quelling’den, RNA interferansın mekanizmasından ve onun tedavi alanındaki uygulamalarından bahsedilmektedir.

Anahtar Kelimeler

RNA İnterferans, RNAi, siRNA, Gen baskılama

RNA INTERFERENCE (RNAi)

Öz

RNA interference is a post-transcriptional gene silencing process during which endogenous complementary messenger RNA is destroyed upon introduction of the corresponding double-stranded RNA into the cell. The initation of RNA interference occurs with the processing of double-stranded RNA into small interfering RNAs (siRNAs), by an RNase III enzyme called Dicer. These siRNAs are then incorporated into the RNA-Induced Silencing Complex (RISC), a multiprotein-RNA nuclease complex. RISC uses these siRNAs to find complementary mRNA and endonucleolytically cleave target mRNA. Consequently, the reduction of specific mRNAs leads to the reduction of the corresponding protein(s). RNA interference and other forms of RNA silencing such as posttranscriptional gene silencing/co-suppression, quelling have been observed in a wide variety of organisms such as plants, animals, fungi, and protozoa. Although RNA interference is a natural phenomenon, it can be used to silence expression of endogenous genes using in vitro synthesized siRNAs. Therefore, RNA interference is promptly utilized as a gene function analysis tool in molecular biology and also in gene therapy. This review describes RNA interference and its specific variants, post-transcriptional gene silencing/co-suppression and quelling, mechanism of RNA interference and applications of RNA interference as therapeutic.

Anahtar Kelimeler

RNA İnterferans, RNAi, siRNA, Gen baskılama

Kaynakça

• Kurreck J., Antisense Technologies Improvement Through novel Chemical Modifications, European Journal of Biochemistry, 270, 1628- 1644, 2003.
• Gewirtz A.M., On Future’s Doorstep: RNA Interference and The Pharmacopeia of Tomorrow, The Journal of Clinical Investigation, 117 (12), 3612- 3614, 2007.
• RNA Interference, 2005/2006 Dharmacon Applications Handbook and Catalog, pp. 3- 5, 2005.
• Mallery C., RNAi. Interference RNA, Department of Biology, University of Miami, Coral Gables, FL 33124, http://fig.cox.miami.edu/~cmallery/150/gene/siRNA.htm, March. 2007.
• Atalay A., RNA İnterferans Kursu, s. 25, Ankara Üniversitesi Biyoteknoloji Enstitüsü Yaşam Bilimleri Kursları Serisi, Ankara, 2007.
• Agrawal N., et al., RNA Interference: Biology, Mechanism, and Applications, Microbiology and Molecular Biology Reviews, 67 (4), 657- 685, 2003.
• Zeng Y., Cullen B.R., RNA Interference in Human Cells is Restricted to The Cytoplasm, RNA, 8, 855- 860, 2002.
• Reddy L.S., Sarojamma, V., Ramakrishna, V., Future of RNAi in Medicine. A Review, World Journal of Medical Sciences, 2 (1), 1- 14, 2007.
• Karagüzel A., Kalay E., Celep F., RNA İnterferans (RNAi): Gen Sessizleştirilmesi ve Tedavi Edici Uygulamaları, Uludağ Üniversitesi Tıp Fakültesi Dergisi, 33 (1), 41- 44, 2007.
• Fire A., et al., Potent and Specific Genetic Interference by Double-stranded RNA in Caenorhabditis elegans, Nature, 391, 806- 811, 1998.
• Khanna D., Balgir P.P., Gurlovleen K., RNA Interference: An Ancient Mechanism For Novel Therapeutics, The Internet Journal of Genomics and Proteomics, 2 (2), 1- 45, 2007.
• Couzin J., Breakthrough of The Year: Small RNAs Make Big Splash, Science, 298 (5602), 2296- 2297, 2002.
• Napoli C., Lemieux C., Jorgensen R., Introduction of A Chimeric Chalcone Synthase Gene Into Petunia Result in Supression of homologous Revesible Co-supression of Homologous Genes in Trans, The Plant Cell, 2, 279- 289, 1990.
• Alexander R., et al., An Anti-sense Chalgone Synthase Gene in Transgenic Plants Inhibits Flower Pigmentation, Nature, 333, 866- 869, 1988.
• Jorgensen R.A., Altered Gene Expression in Plants Due to Trans Interactions Between Homologous Genes, Trends Biotechnology, 8, 340- 344, 1990.
• Van Blokland R., et al., Transgene-Mediated Supression of Chalcone Synthase Expression in Petunia hybridia Results From An Increase in RNA Turnover, Plant Journal, 6, 861- 877, 1994.
• Metzlaff,M, et al., RNA-Mediated RNA Degradation and Chalcone Synthase A Silencing in Petunia. Cell, 88, 845- 854, 1997.
• Ruiz M.T., et al., Initiation and Maintenance of Virus- induced Gene Silencing, Plant Cell, 10, 937- 946, 1998.
• Angell S.M., Baulcombe D.C., Consistent Gene Silencing in Transgenic Plants Expressing A Replicating Potato Virus X RNA, EMBO Journal, 16, 3675- 3684, 1997.
• Dougherty W.G. et al., RNA-Mediated Virus Resistance in Transgenic Plants: Exploitation of A Cellular Pathway Possibly Involved in RNA Degradation, Molecular Plant Microbe Interactions, 7, 544- 552, 1994.
• Kumagai M.H. et al., Cytoplasmic Inhibition of Carotenoid Biosynthesis With Virus-Derived RNA, Proceedings of the National Academy of Sciences of the United States of America, 92, 1679- 1683, 1995.
• Hannon G.J., RNA Interference, Nature, 418, 244- 251, 2002.
• Betz N., RNAi: RNA Interference, Promega Notes, 83, 33- 36, 2003.
• Romano N., Macino, G., Quelling: Transient Inactivation of Gene Expression in Neurospora crassa by Transformation With Homologous Sequences, Molecular Microbiology, 6, 3343- 3353, 1992.
• Fire A., et al., Production of Antisense RNA Leads to Effective and Specific Inhibition of Gene Expression in C. elegans Muscle, Development, 113, 503- 514, 1991.
• Guo S., 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.
• Szweykowska- Kulinska Z., et al., RNA Interference and Its Role in The Regulation of Eucaryotic Gene Expression, Acta Biochimica Polonica, 50 (1), 217- 229, 2003.
• Bernstein E., et al., Role For A Bidentate Ribonuclease in The Initation Step of RNA Interference, Nature, 409, 363- 366, 2001.
• Zamore P.D., et al., RNAi: Double-Stranded RNA Directs The ATP Dependent Cleavage of mRNA at 21 to 23 Nucleotide Intervals, Cell, 101, 25- 33, 2000.
• Zhang H., Biochemistry and Modeling of Human Dicer, A Key Protein Involved in RNA Interference, Ph.D. Thesis, Philosophisch-Naturwissenschaftlichen Fakultaet der Universität Basel, 2004.
• Filipowicz W., RNAi: The Nuts and Bolts of The RISC Machine, Cell, 122, 17- 20, 2005.
• Black D., Newbury S., RNA Interference: What It is and What It Does, A Potent Gene Regulator, The Biochemist, 26 (5), 7- 10, 2004.
• Elbashir S.M., et al., RNA Interference is Mediated by 21- and 22- Nucleotide RNAs, Genes & Development, 15, 188- 200, 2001.
• Ambion, Applied Biosystem, http://www.ambion.com/techlib/resources/RNAi/overview/2.html, 2008.
• Kim K., et al., Conversion of Pre-RISC to Holo-RISC by Ago2 During Assembly of RNAi Complexes, RNA, 13, 22- 29, 2007.
• Blaszczyk J., et al., Crystallografic and Modeling Studies of RNase III Suggest A Mechanism For Double- Stranded RNA Cleavage, Structure (Camb.), 9, 1225- 1236, 2001.
• Zhang H., et al., Human Dicer Preferentially Cleaves dsRNAs at Their Termini Without A Requirement For ATP, EMBO Journal, 21, 5875- 5885, 2002.
• Hutvagner G., Small RNA Asymmetry in RNAi: Function in RISC Assembly and Gene Regulation, FEBS Letters, 579 (26), 5850- 5857, 2005.
• Rand T.A., et al., Argonaute2 Cleaves The Anti-Guide Strand of siRNA During RISC Activation, Cell, 123, 621- 629, 2005.
• Gregory R.I., et al., Human RISC Couples MicroRNA Biogenesis and Posttranscriptional Gene Silencing, Cell, 123 (4), 631- 640, 2005.
• Downward J., RNAi and Cancer Research, The Biochemist, 26 (5), 12- 14, 2004.
• Eggleston A., RNAi Therapy Collection, Nature Collections, p. 54, Sept. 2005.
• Çelik V., Analysis of Active Site Loop Amino Acids of Enzyme Plasmodium falciparum Lactate Dehydrogenase by Site-Directed Mutagenesis Studies, Fırat Üniversitesi Fen Bilimleri Enstitüsü, 2004.