RNAi is a relatively new and rapidly developing technology for sequence-specific gene silencing (8-13). Studies have shown that siRNAs are more potent in gene silencing than different types of ODNs (14-19) and ribozymes (20-22). RNAi is a cellular mechanism by which double-stranded RNAs (dsRNAs) trigger the silencing of the corresponding gene that was first observed in the nematode worm, Caenorhabditi selegans (23), and plants (24). It is now known that RNAi is an evolutionarily conserved mechanism of dsRNA-mediated gene silencing in diverse species. siRNAs found in nature are derived from long dsRNAs that are expressed from viruses, transposons, experimentally introduced transgenes or endogenous genes. In plants, the RNAi pathway is used as a natural defense mechanism against viral infection. In mammalian cells, however, dsRNAs longer than 30 nucleotides will provoke the activation of dsRNA-acti-vated protein kinase (PKR), which causes nonspecific inhibition of protein translation and cell death (25,26). A major breakthrough in the application of RNAi technology in mammalian cells came from the observation that synthetic siRNAs of 21 nucleotides in length that mimic Dicer cleavage products efficiently induced sequence-specific gene silencing when transiently transfected into mammalian cells (27,28). However, one drawback of transient transfected synthetic siRNAs is that their effects are transient, as mammals apparently lack the mechanisms that amplify silencing in worms and plants (11). Therefore, this approach is not suitable for studies that require long-term gene silencing in mammalian cells. Another important technical advance came from the demonstration that dsRNAs of 19-29 nucleotides that are expressed endogenously using RNA polymerase III promoters, either as short hairpin RNAs (shRNAs) or as separate complementary RNAs, efficiently induced target gene silencing in mammalian cells (21,29-35). The endogenous expression of siRNAs from DNA templates has several advantages over the exogenous delivery of synthetic siRNAs (36,37). For example, it allows stable gene silencing both in vitro in cultured cells and in vivo in animals
(32,38-42). More recently, it was demonstrated that transgenic mice expressing shRNA can pass the RNAi to the next generation (43,44).
The long dsRNA or shRNA silencing triggers are cleaved to produce siRNAs by Dicer, which is a member of the RNase-III family of dsRNA-specific endonucleases (45). Dicer cleaves long dsRNA or shRNA into 21- to 28- nucleotide siRNA duplexes that contain 2-nucleotide 3'-overhangs with 5'-phosphate and 3'-hydroxyl termini (46,47) (see Fig. 1). This configuration is functionally important for siRNA incorporation into the RNA-induced silencing complex (RISC) (47,48). Components of the RNAi machinery specifically recognize the siRNA duplex and incorporate a single siRNA strand into RISC (49). The antisense strand of the siRNA in the RISC complex functions as a guide for target mRNA degradation (49,50) (see Fig. 1). Cleavage of target mRNA by RISC complex is endonucleolytic. RISC cleaves mRNAs containing perfectly complementary sequences, 10 nucleotides from the 5'-end of the incorporated siRNA strand (46).
Was this article helpful?