RNA interference (RNAi) has developed into one of the most exciting and important methods for analyzing gene function and metabolic pathways in the past fifteen years.
During the 1990's, this phenomenon of gene-specific silencing was described in a variety of model organisms before its recognition as a widespread, perhaps universal, mechanism. In plants, the RNA-mediated process was termed "co-suppression" (12) and post-transcriptional or homologous gene silencing (PTGS or HGS)(7). In fungi, it was defined as "quelling" (4,14), and in nematodes, it became known as RNAi (13,16). Further studies implicate the RNAi mechanism as an evolutionarily conserved ancient cellular line of defense directed against invading viral genomes or as a method to clear a cell of aberrant transcription products (7,15).Further analyses of the RNAi pathway revealed that it is triggered by double stranded RNA (dsRNA) molecules sharing sequence-specific homology to particular "target" mRNAs (3,6,13). Target mRNAs include transcription products of an invading viral genome, transfected transgenes, parasitic transposons or repetitive elements within the host genome itself (10). Subsequent biochemical studies in Drosophila cell-free lysates revealed that the mediators of RNA-dependent gene silencing are 21-25 nucleotide "small interfering" RNA duplexes (siRNAs). These siRNAs are derived from processing of the trigger dsRNA by an enzyme known as Dicer in an RNase III-like fashion (1,9,11). The mechanism involves the recruitment of siRNA duplex products into a multi-protein siRNA complex, known as the RISC (RNA-Induced Silencing Complex). The RISC is then thought to be guided to the target mRNA of interest where the siRNA duplex interacts in a sequence-specific manner to mediate catalytic cleavage (1,2,8).
Preliminary attempts to use RNAi in mammalian systems employed long dsRNAs as triggers, but led to the induction of a non-specific Type I interferon response rather than sequence-specific silencing (17). This interferon response resulted in widespread changes in protein expression, masking any sequence-specific effects and eventually leading to apoptosis. This global phenomenon prevented initial efforts to employ RNAi as a viable means to study individual gene function. In subsequent landmark studies using chemically synthesized siRNAs, investigators showed that siRNA could effectively bypass the mammalian interferon response and sequence-specifically silence gene expression (3,5). Clearly, the ability to assess gene function via siRNA-mediated methods represents an exciting and valuable tool that has already begun to accelerate critical investigations across a broad range of biomedical and biological research.
For more information, you may fill out a short questionairre to receive a FREE copy of Dharmacon's RNAi Technical Reference and Application Guide. This Guide provides comprehensive information on the background of RNAi, experimental design, transfection, use of controls, detection methods and other useful techniques for anyone interested in getting started in RNAi.
References
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2. Boutla, A., C. Delidakis, I. Livadaras, M. Tsagris and M. Tabler. 2001. Short 5'-phosphorylated double-stranded RNAs induce RNA interference in Drosophila. Curr Biol 11:1776-1780.
3. Caplen, N.J., S. Parrish, F. Imani, A. Fire and R.A. Morgan. 2001. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc Natl Acad Sci U S A 98:9742-9747.
4. Cogoni, C., N. Romano and G. Macino. 1994. Suppression of gene expression by homologous transgenes. Antonie Van Leeuwenhoek 65:205-209.
5. Elbashir, S.M., J. Harborth, W. Lendeckel, A. Yalcin, K. Weber and T. Tuschl. 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494-498.
6. Grishok, A., A.E. Pasquinelli, D. Conte, N. Li, S. Parrish, I. Ha, D.L. Baillie, A. Fire, G. Ruvkun and C.C. Mello. 2001. Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106:23-34.
7. Hamilton, A.J. and D.C. Baulcombe. 1999. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286:950-952.
8. Hammond, S.M., E. Bernstein, D. Beach and G.J. Hannon. 2000. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404:293-296.
9. Hutvagner, G., J. McLachlan, A.E. Pasquinelli, E. Balint, T. Tuschl and P.D. Zamore. 2001. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 293:834-838.
10. Ketting, R.F., T.H. Haverkamp, H.G. van Luenen and R.H. Plasterk. 1999. 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.
11. Knight, S.W. and B.L. Bass. 2001. A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans. Science 293:2269-2271.
12. Napoli, C., C. Lemieux and R. Jorgensen. 1990. Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. Plant Cell 2:279-289.
13. Parrish, S., J. Fleenor, S. Xu, C. Mello and A. Fire. 2000. Functional anatomy of a dsRNA trigger: differential requirement for the two trigger strands in RNA interference. Mol Cell 6:1077-1087.
14. Romano, N. and G. Macino. 1992. Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol Microbiol 6:3343-3353.
15. Sijen, T., J. Fleenor, F. Simmer, K.L. Thijssen, S. Parrish, L. Timmons, R.H. Plasterk and A. Fire. 2001. On the role of RNA amplification in dsRNA-triggered gene silencing. Cell 107:465-476.
16. Tabara, H., M. Sarkissian, W.G. Kelly, J. Fleenor, A. Grishok, L. Timmons, A. Fire and C.C. Mello. 1999. The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99:123-132.
17. Ui-Tei, K., S. Zenno, Y. Miyata and K. Saigo. 2000. Sensitive assay of RNA interference in Drosophila and Chinese hamster cultured cells using firefly luciferase gene as target. FEBS Lett 479:79-82.