RNA Interference : Past , Present and Future 1

RNA interference (RNAi) is the sequence-specific gene silencing induced by double-stranded RNA. RNAi is mediated by 21-23 nucleotide small interfering RNAs (siRNAs) which are produced from long double-stranded RNAs by RNAse II-like enzyme Dicer. The resulting siRNAs are incorporated into a RNA-induced silencing complex (RISC) that targets and cleaves mRNA complementary to the siRNAs. Since its inception in 1998, RNAi has been demonstrated in organisms ranging from trypanosomes to nematodes to vertebrates. Potential uses already in progress include the examination of specific gene function in living systems, the development of anti-viral and anticancer therapies, and genome-wide screens. In this review, we discuss the landmark discoveries that established the contextual framework leading up to our current understanding of RNAi. We also provide an overview of current developments and future applications. Introduction RNA interference (RNAi), initially coined by Fire et al., (1998), is the sequence-specific gene silencing induced by double-stranded RNA (dsRNA; Figure 1; Wall and Shi, 2003). This phenomenon has generated an explosion of interest and enthusiasm by scientists, similar to that created by the entrance of the green fluorescent protein as a species-independent molecular reporter in the mid-1990s. Since its inception, RNAi has been induced in organisms ranging from trypanosomes (Ngo et al., 1998) to nematodes (Fire et al., 1998) to flies (Kennerdell and Carthew, 1998) to vertebrates (Wianny and Zericka-Goetz, 2000). Because of its specificity, efficiency, and cost-effectiveness, RNAi has drawn the bulk of attention away from previous antisense methods such as ribozymes and oligodeoxynucleotides (ODNs). Furthermore, technical expertise accumulated from previous approaches is now being applied to RNAi, thus rapidly advancing its application to the medicinal domain (Kim, 2003). Currently, three areas of human disease research have already strongly embraced the RNAi phenomenon: infectious disease, cancer, and dominantly inherited disorders (Kim, 2003; Wall and Shi, 2003). RNAi has also shown exceptional promise as a genome-wide screening tool (Kamath et al., 2003; Kiger et al., 2003; Berns et al., 2004; Paddison et al., 2004). In this review, we discuss the landmark discoveries that established the contextual framework leading up to our current understanding of RNAi. We also provide an overview of current developments and future applications. RNAi: Past Prior to the discovery of RNAi, several other previously characterized, homology-dependent gene silencing mechanisms had been described. In 1990, the Jorgensen laboratory introduced exogenous transgenes into petunias in an attempt to upregulate the activity of a gene for chalcone synthase, an enzyme involved in the production of specific pigments (Napoli et al., 1990; Agrawal et al., 2003). Unexpectedly, flower pigmentation did not deepen, but rather showed variegation with complete loss of color in some cases. This indicated that not only were the introduced trangenes themselves inactive, but that the added DNA sequences also affected expression of the endogenous loci (Hannon, 2002). This phenomenon was referred to as “cosuppression” (Napoli et al., 1990). Reports from other laboratories noted similar effects (Vander Krol et al., 1990; Ingelbrecht et al., 1994). Subsequently, cosuppression was renamed posttranscriptional gene silencing (PTGS) since all cases of cosuppression resulted in the degradation of endogene and transgene RNAs postnuclear transcription and this posttranscriptional RNA degradation was observed in a wide range of transgenes expressing the plant, bacterial, or viral sequences (Kooter et al., 1999; Agrawal et al., 2003). As more researchers corroborated PTGS results in plants, a similar phenomenon labelled “quelling” was reported in fungi. Two different laboratories demonstrated transgene-induced silencing of both transgenes and Figure 1. Proposed mechanism of RNA interference. Abbreviations include: dsRNA (double-stranded RNA), siRNA (small interfering RNA), RISC (RNAinduced silencing complex), and mRNA (messenger RNA). *For correspondence. Email tessac@uvic.ca. © Horizon Scientific Press. Offprints from www.cimb. org RNA Interference: Past, Present and Future Curr. Issues Mol. Biol. 7: 1-6. Online journal at www.cimb. org 2 Campbell and Choy RNA Interference: Past, Present and Future 3 endogenous genes in Neurospora crassa (Pandit and Russo, 1992; Romano and Macino, 1992). In 1996, a N. crassa strain was transformed with a plasmid containing a segment of the al1 gene to increase production of an orange pigment. The opposite, however, was observed, with a few stably quelled transformants showing albino phenotypes (Cogoni et al., 1996). These results substantiated earlier observations of transgene-induced gene silencing in N. crassa, but also demonstrated that DNA methylation was not obligatory for this process (Cogoni et al., 1996; Sweykowsa-Kulinska et al., 2003). Around the same time, Guo and Kemphues (1995) demonstrated that sense RNA was as effective as antisense RNA in suppressing gene expression in Caenorhabditis elegans. Fire et al., (1998) built upon these results, leading to the discovery of the RNAi phenomenon. The researchers targeted the unc22 gene, which encodes a nonessential myofilament protein. A decrease in unc22 activity produces a twitching phenotype. Results indicated that introduction of a dsRNA mixture directed at the unc22 gene was at least tenfold more effective than were sense or antisense RNA alone. Similar results were noted when other genes were targeted. This experiment paved the way for specific silencing of a functional gene by exogenous application of dsRNA in organisms ranging from worms (Fire et al., 1998) to trypanosomes (Ngo et al., 1998) to flies (Kennerdell and Curthew, 1998). Initial experiments in mammals proved disappointing, since introduction of dsRNA >30 base pairs triggered the interferon response, resulting in global shutdown of protein translation and, ultimately, dramatic alteration in cellular metabolism (Gil and Esteban, 2000). To avoid this general shutdown, Elbashir et al., (2001) chemically synthesized small RNAs which mimicked Dicer products, resulting in the desired gene-specific silencing in mammalian systems. These findings highlighted the possibility of RNAi-induced gene silencing as a therapeutic alternative. Within the same year, Caplen et al., (2001) demonstrated knockdown of enhanced green fluorescent protein (EGFP), a modified version of the Aequorea victoria wildtype GFP (Campbell and Choy, 2003; 2004; Figure 2). From this point on, EGFP has been employed as a popular positive control to ascertain successful siRNA delivery and to test system integrity.