RNA interference：The short answer

One way of seeing what a gene does is to block its messenger RNA and note the effects. New work should make the approach more broadly applicable.

RNA interference (RNAi) was discovered only a few years ago1, but many scientists find it hard to imagine life without it. Once the sequence of a gene is known, RNAi offers a quick and easy way to determine its function, and the technique is accessible to a scientist in a small lab, as well as to a consortium attempting to assign function to the genes of an entire chromosome2,3. But although RNAi is now routine in laboratories studying a wide range of organisms, its use in mammalian cells has been problematic. On page 494 of this issue4 Tuschl and colleagues describe research that paves the way for successful RNAi in mammalian cells.

The basic idea behind RNAi is shown in the right-hand part of Fig. 1 — this is the sequence-specific pathway indicated by blue arrows. A double-stranded RNA (dsRNA) matching a gene sequence is synthesized in vitro and introduced into a cell. The dsRNA feeds into a natural, but poorly understood, biological pathway, and is broken into short pieces called short interfering (si) RNAs5. With the help of cellular enzymes that have not yet been well characterized6, the siRNA triggers the degradation of the messenger RNA that matches its sequence. This often leads to adverse consequences for the organism, evident in an aberrant phenotype, that allow the gene’s function to be identified.

Figure 1Mammalian cells have at least two pathways that compete for double-stranded RNA (dsRNA). In the RNAi, or sequence-specific, pathway (blue arrows), the initiating dsRNA is first broken into short interfering (si) RNAs. siRNAs have sense and antisense strands of about 21 nucleotides that form 19 base pairs to leave overhangs of two nucleotides at each 38 end. siRNAs are thought to provide the sequence information that allows a specific messenger RNA to be targeted for degradation. The nonspecific pathway (red arrow) is triggered by dsRNA of any sequence, as long as it is at least 30 base pairs long. The nonspecific effects occur because dsRNA activates two enzymes: PKR, which in its active form phosphorylates the translation initiation factor eIF2a to shut down all protein synthesis, and 28, 58 oligoadenylate synthetase (28, 58-AS), which synthesizes a molecule that activates RNase L, a nonspecific enzyme that targets all mRNAs. The nonspecific pathway represents a host response to stress or viral infection; in the second case, the activating dsRNA is thought to derive from viral replication.
 

RNAi was first discovered in the nematode worm Caenorhabditis elegans1 , but is present in many other organisms (the fruitfly Drosophila , certain parasitic protozoa, and plants, for instance), and so seems to represent an ancient pathway7. Nonetheless, researchers have always been pessimistic about applying RNAi to mammalian cells, because exposing such cells to dsRNA, of any sequence, triggers a global shut-down of protein synthesis8. This nonspecific pathway is indicated on the left of Fig. 1 by a red arrow. The lore has been that this pathway would mask any sequence-specific effects that might occur from the RNAi pathway.

But it almost always pays to consider how one’s own research fits in with previous observations. Tuschl and colleagues were, it seems, being especially diligent in this respect. Their earlier work showed that the siRNA intermediates themselves could initiate RNAi, at least in non-mammalian cells5. However, the nonspecific pathway requires longer dsRNA, and will not occur with dsRNAs shorter than around 30 base pairs8–10. They don’t say as much in the paper, but one presumes that Tuschl’s group began with the idea that, because of this size discrimination, siRNAs might be able to bypass the more global, nonspecific response. They turned out to be right.