RNA interference (RNAi) was discovered unexpectedly during attempts to experimentally manipulate the expression of specific genes. Researchers tried to inhibit the expression of a gene in C. elegans by microinjecting a single-stranded, complementary RNA that would hybridize to the encoded mRNA and prevent its translation, a method called antisense inhibition. But in control experiments, a perfectly base-paired double-stranded RNA a few hundred base pairs long was much more effective at inhibiting expression of the gene than the antisense strand alone (see Figure 6-42). Similar inhibition of gene expression by introduced double-stranded RNA was soon observed in plants. In each case, the double-stranded RNA induced degradation of all cellular RNAs containing a sequence that was exactly the same as that of one strand of the double-stranded RNA. Because of the specificity of this technique in targeting mRNAs for destruction, it has become a powerful experimental tool for studying gene function.

Subsequent biochemical studies with extracts of Drosophila embryos showed that a long double-stranded RNA that mediates RNA interference is initially processed into a double-stranded short interfering RNA (siRNA). The strands in siRNAs contain 21–23 nucleotides hybridized to each other so that the two bases at the 3′ end of each strand are unpaired. Further studies revealed that the cytoplasmic double-stranded RNA–specific ribonuclease that cleaves long double-stranded RNA into siRNAs is the same Dicer enzyme involved in processing pre-miRNAs after their export to the cytoplasm (see Figure 10-29). This discovery led to the realization that RNA interference and miRNA-mediated repression of translation and target-mRNA degradation are related processes. In both cases, the mature short single-stranded RNAs, either siRNAs or miRNAs, are assembled into RISC complexes in which they are bound by an Argonaute protein. What distinguishes a RISC complex containing an siRNA from one containing an miRNA is that the siRNA base-pairs perfectly with its target RNA and induces its cleavage, whereas a RISC complex associated with an miRNA recognizes its target through imperfect base pairing and results in inhibition of translation and a slower form of target-mRNA degradation (see Figure 10-28).

AGO2 is the protein responsible for the cleavage of target RNA. One domain of the protein is homologous to the RNase H enzymes that degrade the RNA of an RNA-DNA hybrid (see Figure 8-14). When the 5′ end of the siRNA of a RISC complex base-pairs precisely with a target mRNA over a distance of one turn of an RNA helix (10–12 base pairs), this domain of AGO2 cleaves the phosphodiester bond of the target RNA across from nucleotides 10 and 11 of the siRNA (see Figure 10-28b). The cleaved RNAs are released and subsequently degraded by cytoplasmic exosomes and the XRN1 5′→3′ exoribonuclease. If base pairing is not perfect, the AGO2 domain does not cleave or release the target mRNA. Instead, if several miRNA-RISC complexes associate with a target mRNA, its translation is inhibited, and the mRNA becomes associated with P bodies, where, as mentioned earlier, it is degraded by a different and slower mechanism than the degradation pathway initiated by RISC cleavage of a perfectly complementary target RNA.

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When double-stranded RNA is introduced into the cytoplasm of eukaryotic cells, it enters the pathway for the assembly of siRNAs into a RISC complex because it is recognized by Dicer and TRBP (see Figure 10-29). This process of RNA interference is believed to be an ancient cellular defense against certain viruses and mobile genetic elements in both plants and animals. Plants with mutations in the genes encoding the Dicer and RISC proteins exhibit increased sensitivity to infection by RNA viruses and increased movement of transposons within their genomes. The double-stranded RNA intermediates generated during replication of RNA viruses are thought to be recognized by Dicer, inducing an RNAi response that ultimately degrades the viral mRNAs. During transposition, transposons are inserted into cellular genes in a random orientation, and their transcription from different promoters produces complementary RNAs that can hybridize with each other, initiating the RNAi system, which then interferes with the expression of transposon proteins required for additional transpositions.

In plants and C. elegans, RNA interference can be induced in all cells of the organism by introduction of double-stranded RNA into just a few cells. Such organism-wide induction requires production of a protein that is homologous to the RNA replicases of RNA viruses. It has been revealed that double-stranded siRNAs are replicated and then transferred to other cells in these organisms. In plants, the transfer of siRNAs might occur through plasmodesmata, the cytoplasmic connections between plant cells that traverse the cell walls between them (see Figure 20-42). Organism-wide induction of RNA interference does not occur in Drosophila or mammals, presumably because their genomes do not encode RNA replicase homologs.

In mammalian cells, the introduction of long RNA-RNA duplex molecules into the cytoplasm results in generalized inhibition of protein synthesis via the PKR pathway, discussed further below. This response greatly limits the use of long double-stranded RNAs to experimentally induce RNA interference against a specific targeted mRNA. Fortunately, researchers discovered that double-stranded siRNAs 21–23 nucleotides long with two-base 3′ single-stranded regions lead to the generation of single-stranded RNAs that are incorporated into functional siRNA RISC complexes without inducing the generalized inhibition of protein synthesis. This discovery has allowed researchers to use synthetic double-stranded siRNAs to knock down the expression of specific genes in human cells as well as in other mammals. This siRNA knockdown method is now widely used in studies of diverse processes, including the RNAi pathway itself!