The specifics of small interfering RNA specificity.

T he discovery of transgene silencing in plants and doublestranded RNA (dsRNA) interference in the worm Caenorhabditis elegans has led to the latest revolution in molecular biology, RNA interference (RNAi). Over 10 years ago it was noted that several transgenic plant lines each containing the same ectopic transgene not only failed to be expressed but also inhibited the expression of the endogenous gene (1). Similarly, a determined Craig Mello and Andy Fire (2), attempting to reduce gene function using antisense RNA in the worm, discovered a minor contaminant in their antisense RNA preparations effectively and repeatedly reduced expression of the endogenous gene. In both cases, dsRNA homologous to the gene of interest was responsible for these observations. In the last 4 years, these discoveries have been extended to include protozoa, fungi, and mammals. What is RNAi? RNAi is a highly conserved mechanism found in almost all eukaryotes and believed to serve as an antiviral defense mechanism. The molecular details are becoming clear from combined genetic and biochemical approaches (reviewed in refs. 3 and 4). On entry into the cell, the dsRNA is cleaved by an RNase III like enzyme, Dicer, into small interfering (21to 23nt) RNAs (siRNAs) (5–8) (Fig. 1). Biochemical evidence indicates the siRNAs are incorporated into a multisubunit protein complex, the RNAi-induced silencing complex (RISC), which directs the siRNA to the appropriate mRNA. New data suggest that the RISC complex may unwind the siRNA to help interactions with the target mRNA (9). Mismatches 1–2 bp within the 21to 23-nt siRNA effectively disrupt proper degradation of the target mRNA (10, 11). In worms, interaction between the siRNA and mRNA can lead to immediate cleavage by Dicer, liberating a new siRNA, and degradation of the mRNA by endoand exonucleases (Fig. 1). Alternatively, the siRNA can serve as a primer for an RNA-dependent RNA polymerase (RdRP), creating many more siRNAs. The action of an RdRP could explain the catalytic mechanism of RNAi, because only a few dsRNA molecules are required to degrade a much larger population of mRNAs, and RNAi is inheritable (2). Two important pieces of data support the idea that an RdRP plays an important role in RNAi. One, worms with mutations in novel RdRPs are not able to perform RNAi (12, 13). Two, elegant studies using fusion genes of unc-22 and GFP showed that dsRNA directed against GFP could effectively lead to the degradation of the endogenous unc-22 (13) (Fig. 2). These experiments also showed the polarity of the RdRP must be 5 to 3 on the antisense strand because unc-22 fused to the 5 end of GFP caused degradation of the endogenous unc-22, but fusion to the 3 end did not. These experiments displayed a new level of specificity not previously appreciated outside the plant kingdom (14). Mainly, siRNA targeting is very specific; however, elongation of the siRNA using the mRNA as a template could lead to nonspecific interference by sequences homologous to other genes, known as transitive RNAi. At present, this phenomenon is particular to plants and nematodes and does not appear to be a concern for mammalian systems, because there is no easily identifiable mammalian RdRP homolog. Additionally, in vitro reconstitution of mammalian RNAi activity does not re-