Figure 1. Double-stranded RNA (dsRNA) is a potent inducer of gene silencing. dsRNA can be introduced experimentally to silence target genes of interest. In plants, silencing can be triggered, for example, by engineered RNA viruses or by inverted repeat transgenes. In worms, silencing can be triggered by injection or feeding of dsRNA. In both these systems, silencing is systemic and spreads throughout the organism. (a) A silencing signal moving from the veins into leaf tissue. Green is GFP fluorescence and red is chlorophyll fluorescence that is seen upon silencing of the GFP transgene. Similarly, in (b), a C. elegans has been engineered to express GFP in nuclei. Animals on the right have been treated with a control dsRNA, while those on the left have been exposed to GFP dsRNA. Some neuronal nuclei remain fluorescent, correlating with low expression of a protein required for systemic RNAi (Winston et al., 2002). In (c), HeLa cells have been treated with an ORC6 siRNA and stained for tubulin (green) and DNA (red). Depletion of ORC6 results in accumulation of multinucleated cells. Stable silencing can also be induced by expression of dsRNA as hairpins or snap-back RNAs. In (d), adult Drosophila express a hairpin homologous to the white gene (left), which results in unpigmented eyes as compared to wild type (right).

Figure 2. Dicer and RNA-induced silencing complex (RISC) (a) RNAi is initiated by the Dicer enzyme, which processes dsRNA into ~22 nt siRNAs (Bernstein et al., 2001a). Based upon the known mechanisms for RNaseIII enzymes, Dicer is thought to work as a dimeric enzyme. Cleavage into precisely sized fragments is determined by the fact that one of the active sites in each Dicer protein is defective, shifting the periodicity of cleavage from ~9–11 nt for bacterial RNaseIII to ~22 nt for Dicer family members (Blaszczyk et al., 2001). The siRNAs are incorporated into a multicomponent nuclease, RISC, which uses the siRNA as a guide to substrate selection (Hammond et al., 2000). Recent reports suggest that RISC must be activated from a latent form, containing a double-stranded siRNA to an active form, RISC*, via unwinding of siRNAs (Nykanen et al., 2001). (b) Diagrammatic representation of Dicer binding and cleaving dsRNA. Mutations in the second RNaseIII domain inactivate the central catalytic sites, resulting in cleavage at 22 nt intervals.

Figure 3. Small interfering RNAs versus small temporal RNAs. siRNAs are produced by Dicer from dsRNA silencing triggers as dsRNAs of ~21–23 nt in length. Characteristic of RNaseIII products, these have 2 nt 3¢ overhangs and 5¢ phosphorylated termini. In order to work with maximum efficiency, siRNAs must have perfect complementarity to their mRNA target (with the exception of the two terminal nucleotides, which contribute only marginally to recognition). stRNAs, such as lin-4 and let-7, are transcribed from the genome as hairpin precursors. These are also processed by Dicer, but in this case, only one strand accumulates. Notably, neither lin-4 nor let-7 shows perfect complementarity to its targets. In addition, stRNAs regulate targets at the level of translation rather than RNA degradation. It remains unclear whether the difference in regulatory mode results from a difference in substrate recognition or from incorporation of siRNAs and stRNAs into distinct regulatory complexes.

Figure 4. Model for the mechanism of RNAi. Silencing triggers in the form of dsRNA may be presented in the cell as synthetic RNAs, replicating viruses or may be transcribed from nuclear genes. These are recognized and processed into siRNAs by Dicer. The duplex siRNAs are passed to RISC, and the complex becomes activated by unwinding of the duplex. Activated RISC complexes can regulate gene expression at many levels. Almost certainly, such complexes act by promoting RNA degradation and translational inhibition. However, similar complexes probably also target chromatin remodeling. Amplification of the silencing signal in plants may be accomplished by siRNAs priming RNA-dependent RNA polymerase (RdRP)-dependent synthesis of new dsRNA. This could be accomplished by RISC-mediated delivery of an RdRP or by incorporation of the siRNA into a distinct, RdRP-containing complex.