RNA interference (RNAi) is a biological process in which the introduction of double-stranded RNA (dsRNA) into a cell results in targeted post-transcriptional gene silencing.
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.
