chapter  1
Gene Silencing by RNA Interference and the Role of Small Interfering RNAs
Pages 14

Metazoan and Protozoan species, for example, Planarian, Hydra, and Trypanosoma; and more complex organisms, particularly Caenorhabditis elegans, Drosophila, and most recently mammalian cells both human and mouse.1-17 The term PTGS has been used mostly to indicate epigenetic post-transcriptional gene-silencing in plants and fungi (also known as co-suppression and quelling, respectively), whereas RNAi has been mostly applied to a similar epigenetic gene-silencing effect seen in invertebrates and mammals, though it has been suggested that the collective term of RNA silencing is more appropriate for all of these processes.18 Central to PTGS and RNAi in all species studied to date is the role of small RNA molecules (20 to 25 nts in length) and the formation of a ribonucleoprotein complex called the RNA-induced silencing complex or RISC.19-22 In plants, RNA silencing can be observed following the introduction of multiple copies of a transgene and in cells infected with cytoplasmically replicating RNA viruses.23 Plants defective for PTGS, show increased susceptibility to infection and viruses produce proteins that block PTGS, leading to the hypothesis that PTGS acts as an antiviral response in plants.24,25 The term RNAi was first used to describe the inhibition of gene expression seen in C. elegans following the direct injection into embryos of double-stranded RNA (dsRNA) of a sequence cognate to the gene that was silenced.6 As in plants and fungi the silencing induced by dsRNA in C. elegans is highly potent, suggesting that an intracellular process both amplifies and spreads this effect.6,7,26 Double-stranded RNA was quickly shown to mediate a similar inhibition of gene expression in other invertebrate species, including Drosophila.8,9

Though dsRNA generated through aberrant transcription from multiple transgenes (particularly those arranged as an inverted repeat), or from a viral intermediate, or introduced exogenously appeared to be the triggers of both PTGS and RNAi, it was unclear for some time how degradation of the target RNA was mediated. The first evidence suggesting a role for small RNA molecules in PTGS and RNAi emerged from a plant study that showed that in cells where PTGS had been triggered (by a transgene or virus) there was an accumulation of a small species of RNA estimated to be approximately 25 nucleotides (nts) in size.19 Subsequent in vitro analysis of RNAi in Drosophila cell extracts showed that a small species of RNA, now termed small or short interfering RNAs (siRNAs), are enzymatically derived from the input doublestranded RNA.20,22,27-29 Cleavage of the target mRNA was observed at a site that corresponded to approximately the middle of the siRNA, suggesting that the siRNA acts as guide for an enzyme-containing complex that leads to degradation of the target mRNA.22,30 Since the identification of siRNAs, their central role in PTGS and RNAi has been more fully defined and their relationship to endogenous small non-coding RNAs has begun to be elucidated.