Each time RISC cleaves a new RNA molecule, the RISC is released; thus, as we saw for miRNAs, a single RNA molecule can act catalytically to destroy many complementary RNAs. Some organisms employ an additional mechanism that amplifies the RNAi response even further. In these organisms, RNA-dependent RNA polymerases use siRNAs as primers to produce additional copies of double-strand RNAs which are then cleaved into siRNAs. This amplification ensures that, once initiated, RNA interference can continue even after all the initiating double-stranded RNA has been degraded or diluted out. For example, it permits progeny cells to continue carrying out the specific RNA interference that was provoked in the parent cells.
In some organisms, the RNA interference activity can be spread by the transfer of RNA fragments from cell to cell. This is particularly important in plants (whose cells are linked by fine connecting channels, as discussed in Chapter 19), because it allows an entire plant to become resistant to an RNA virus after only a few of its cells have been infected. In a broad sense, the RNAi response resembles certain aspects of the animal immune system; in both, an invading organism elicits a customized response, and—through amplification of the “attack” molecules—the host becomes systemically protected.
Figure 7–76 Human Argonaute protein carrying an miRNA. the protein is folded into four structural domains, each indicated by a different color. the mirnA is held in an extended form that is optimal for forming rnA–rnA base pairs. the active site of Argonaute that “slices” a target rnA, when it is extensively base-paired with the mirnA, is indicated in red. Many Argonaute proteins (three out of the four human proteins, for example) lack the catalytic site and therefore bind target rnAs without slicing them. (Adapted from C.D. Kuhn and l. Joshua-tor, Trends Biochem. Sci. 38:263–271, 2013. with permission from Cell press.)
We have seen that although miRNAs and siRNAs are generated in slightly different ways, they rely on the same proteins and seek out their targets in a fundamentally similar manner. Because siRNAs are found in widespread species, they are believed to be the most ancient form of RNA interference, with miRNAs being a later refinement. These siRNA-mediated defense mechanisms are crucial for plants, worms, and insects. In mammals, a protein-based system (described in Chapter 24) has largely taken over the task of fighting off viruses.
The siRNA interference pathway just described does not necessarily stop with the destruction of target RNA molecules. In some cases, the RNA interference machinery can also selectively shut off synthesis of the target RNAs. For this to occur, the short siRNAs produced by the Dicer protein are assembled with a group of proteins (including Argonaute) to form the RITS (RNA-induced transcriptional silencing) complex. Using single-stranded siRNA as a guide sequence, this complex binds complementary RNA transcripts as they emerge from a transcribing RNA polymerase II (Figure 7–77). Positioned on the genome in this way, the RITS complex attracts proteins that covalently modify nearby histones and eventually direct the formation of heterochromatin to prevent further transcription initiation. In some cases, an RNA-dependent RNA polymerase and a Dicer enzyme are also recruited by the RITS complex to continually generate additional siRNAs in situ. This positive feedback loop ensures continued repression of the target gene even after the initiating siRNA molecules have disappeared.