RNAi: Background Information
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Historically, RNAi has been used as a tool for functional genomics research in C. elegans and drosophila. Initial attempts to activate the RNAi pathway in mammalian cells were unsuccessful, since the introduction of dsRNA >30 nucleotides (nt) in length leads to non-specific suppression of gene expression. Much of this response is due to activation of the dsRNA-dependent protein kinase PKR, and the subsequent phosphorylation and inactivation of the translation factor eIF2a. As RNAi became better understood, scientists discovered that double stranded short interfering RNA (siRNA) oligos of 23 nt could be used to mediate a gene silencing effect in mammalian cells. The application of RNAi to mammalian cells has the potential to revolutionize the field of functional genomics. The ability to simply, effectively, and specifically down-regulate the expression of genes in mammalian cells holds enormous scientific, commercial, and therapeutic potential.
Discovery of RNAi
The origins of RNAi involved a number of scientists working in different research fields, who observed a phenomenon that they did not immediately understand. Plant biologists attempting to boost the activity of the gene for chalcone synthase in petunias by introducing a powerful promoter sequence into a transgene, observed that instead of the deep purple color they expected, flowers were variegated, or virgin white. The researchers concluded that the introduced chalcone synthase transgene had somehow muted both itself and the endogenous petunia gene, and so termed this phenomenon co-suppression (1).
Another research group who were expressing genes from the potato virus X in tobacco plants, hoped that viral proteins produced by the plants would stimulate a defense mechanism allowing the plants to resist subsequent attack by the virus. To their surprise, the plants with the strongest resistance to the virus were those in which the introduced gene was silent. The researchers concluded that the introduced gene was suppressing expression both of itself and the same gene in introduced virus (2).
In fungi, gene silencing was observed during attempts to boost the production of an orange pigment by the mold Neurospora crassa . Extra copies of a gene involved in making a carotenoid pigment were introduced into mold cells. However, rather than turning a deeper orange, a third of the engineered mold appeared yellow or white. Something had suppressed the pigment genes. They called the observed phenomenon of gene silencing in N. crassa quelling (3, 4).
Other scientists working with C. elegans obtained strange results in their antisense RNA experiments. The antisense approach to gene silencing involves injecting an organism with RNA sequence complementary to mRNA transcribed from a target gene. The antisense RNA and sense mRNA hybridize and block production of the encoded protein. However in one case, a sense strand, injected as a control, led to gene silencing (5). This effect was later explained by the presence in the antisense RNA preparation of very small amounts of the corresponding antisense strand. The presence of dsRNA duplex led to what we now recognize as an RNAi effect (6). Antisense experimental theory predicts that these small contaminants would have no effect on gene expression.
Using C. elegans , it was demonstrated that injection of double stranded RNA was more effective in gene silencing than injection of sense or antisense strands alone (7). Only a few molecules of injected double stranded RNA were required to shut down expression of protein in a cell. The dsRNA gene-silencing mechanism was found to be highly gene-specific and to be part of a complex biological regulation system. The phenomenon of gene silencing using dsRNA was termed “RNA interference” (RNAi) (7).
The Application of RNAi to Mammalian Cells
Initial attempts to activate the RNAi pathway in mammalian cells were unsuccessful, since the introduction of dsRNA leads to activation of protein kinase PKR and 2',5'-oligoadenylate synthetase (2',5'-AS). The activation of these two enzymes triggers a non-specific shutdown of protein synthesis and the non-specific degradation of mRNA. Consequently, some researchers were led to believe gene-specific RNAi was not possible in mammalian systems.
Elbashir et al. showed that 21–23 nt dsRNA fragments successfully trigger RNAi in an in vitro system using drosophila lysate. In addition, they demonstrated that chemically synthesized 21 nt siRNA duplexes specifically suppress the expression of endogenous and heterologeous genes in different mammalian cell lines, including human 293 and HeLa cells (8). A key discovery from these studies was that no non-specific gene-silencing effects were seen in mammalian cells by transfection of short dsRNA sequences (<30 nt). These results showed that 21 nt siRNA duplexes can be used as a new tool for studying gene in mammalian cells, and may eventually find a use as gene-specific therapeutics.
Work by Caplen et al., (9) confirmed and extended the reports of siRNA-mediated RNAi in mammalian cell extracts. They demonstrated that identically sized synthetic siRNAs can induce gene-specific inhibition of expression in C. elegans, human, and mouse cells. Consistent with this hypothesis, numerous studies have since shown that dsRNA-induced gene silencing occurs in a number of different eukaryotic species (7, 11–21). The finding that the size of al dsRNA fragments is conserved in plants and animals suggests a highly conserved mechanism in nature (10).
How Does RNAi Lead to Gene Silencing?
The basic mechanism of RNAi is thought to be a multi-step process:
In cultured mammalian cells, RNAi is mediated by 21nt RNA duplexes with symmetric 2-nt 3' overhangs. These siRNAs are introduced into a cell by transfection and lead to degradation of mRNA having the same sequence, thereby silencing gene expression. The specific pathways and mechanism of RNAi in mammalian cells are currently under intense investigation.
References
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2. Baulcombe, D. C. (1996) RNA as a target and an initiator of post-tranional gene silencing in transgenic plants. Plant Mol Biol. 32, 79.
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4. Cogoni, C., and Macino, G. (1997) Isolation of quelling-defective (qde) mutants impaired in posttranional transgene-induced gene silencing in Neurospora crassa. Proc. Natl Acad. Sci. USA. 94,10233.
5. Guo, S., and Kemphues, K. (1995) par-1, a gene required for establishing polarity in embryos, encodes a putative Ser/Thr kinase that is symmetrically disrupted. Cell 81, 611.
6. Parish, S., Fleenor, J., Xu, S., Mello, C., Fire, A. (2000). al anatomy of a dsRNA trigger: differential requirement for the two trigger strands in RNA interference. Mol Cell. 6, 1007.
7. Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., Mello, C. C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806.
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9. Caplen, N. J., Parrish, S., Imani, F., Fire, A., and Morgan, R.A. (2001) Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. USA 98, 9742.
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17. Misquitta, L., Paterson, B M. (1999) Targeted disruption of gene in Drosophila by RNA interference (RNA-i): a role for nautilus in embryonic somatic muscle formation. Proc. Natl. Acad. Sci. USA 96, 1451.
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