RNAi in Drosophila
RNAi has also been observed in Drosophila. Although a strategy in which yeast were engineered to produce dsRNA and then fed to fruit flies failed to work, microinjecting Drosophila embryos with dsRNA does effect silencing (2). Silencing can also be induced by "shooting" dsRNA into Drosophila embryos with a "gene gun" or by engineering flies to carry DNA containing an inverted repeat of the gene to be silenced. Over the last few years, these RNAi strategies have been used as reverse genetics tools in Drosophila organisms, embryo lysates, and cells to characterize various loss-of-function phenotypes (2, 19-23).

So how does injection of dsRNA lead to gene silencing? Many research groups have diligently worked over the last few years to answer this important question. A key finding by Baulcombe and Hamilton provided the first clue. They identified RNAs of ~25 nucleotides in plants undergoing cosuppression that were absent in non-silenced plants. These RNAs were complementary to both the sense and antisense strands of the gene being silenced (24).

Further work in Drosophila — using embryo lysates and an in vitro system derived from S2 cells — shed more light on the subject (3, 25, 26). In one notable series of experiments, Zamore and colleagues found that dsRNA added to Drosophila embryo lysates was processed to 21-23 nucleotide species. They also found that the homologous endogenous mRNA was cleaved only in the region corresponding to the introduced dsRNA and that cleavage occurred at 21-23 nucleotide intervals (26). Rapidly, the mechanism of RNAi was becoming clear.

Current Models of the RNAi Mechanism
Both biochemical and genetic approaches (see "The Genes and Enzymes Involved in PTGS and RNAi" below for a discussion of genetic approaches used to undersand RNAi) have led to the current models of the RNAi mechanism. In these models, RNAi includes both initiation and effector steps (27, see also a Flash animation of "How Does RNAi Work?", from reference 3).

In the initiation step, input dsRNA is digested into 21-23 nucleotide small interfering RNAs (siRNAs), which have also been called "guide RNAs" (reviewed in 3, 18, 27). Evidence indicates that siRNAs are produced when the enzyme Dicer, a member of the RNase III family of dsRNA-specific ribonucleases, processively cleaves dsRNA (introduced directly or via a transgene or virus) in an ATP-dependent, processive manner. Successive cleavage events degrade the RNA to 19-21 bp duplexes (siRNAs), each with 2-nucleotide 3' overhangs (27, 28).

In the effector step, the siRNA duplexes bind to a nuclease complex to form what is known as the RNA-induced silencing complex, or RISC. An ATP-depending unwinding of the siRNA duplex is required for activation of the RISC. The active RISC then targets the homologous transcript by base pairing interactions and cleaves the mRNA ~12 nucleotides from the 3' terminus of the siRNA (3, 18, 27, 29). Although the mechanism of cleavage is at this date unclear, research indicates that each RISC contains a single siRNA and an RNase that appears to be distinct from Dicer (27).

Because of the remarkable potency of RNAi in some organisms, an amplification step within the RNAi pathway has also been proposed. Amplification could occur by copying of the input dsRNAs, which would generate more siRNAs, or by replication of the siRNAs themselves (see "Possible Role for RNA-dependent RNA Polymerase" below). Alternatively or in addition, amplification could be effected by multiple turnover events of the RISC (3, 18, 27).

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