Overview of RNA Interference
Allison O'Brien, PhD
RNA interference (RNAi) is a well-recognized pathway involved in post-transcriptional regulation, transposon expansion, and cellular defense against viral invasion.[1-13] This evolutionarily conserved phenomenon is observed in nearly every eukaryote studied thus far, and represents a unique form of post-transcriptional gene silencing (PTGS). As a novel biological pathway, RNAi has quickly distinguished itself as a valuable reverse genetic tool. The functional intermediates in the RNAi pathway, small interfering RNAs (siRNAs), may be synthesized by a variety of means and introduced intracellularly to effect changes in gene function. The potency and specificity of siRNA-mediated gene silencing exploits a naturally occurring pathway, thus distinguishing it from other nucleic acid-based silencing technologies such as antisense oligonucleotides and ribozymes (Figure 1).
History
The original phenotypic observations of RNAi were made over a decade ago by researchers working in plant and fungal genetics.[14] In the late 1990’s, Fire, et al. observed in C. Elegans that the injection of double-stranded RNA (dsRNA) induced sequence-specific reduction of the target mRNA. Moreover, the authors documented that only a few molecules were required to reduce the population of target mRNA, thus suggesting an extremely potent mechanism of action.[15]
Since these early observations, several groups have contributed to elucidating the short RNA-mediated pathway, diagrammed in Figure 2.[16-24] In nature, the siRNA pathway is initiated when the host cell encounters a long dsRNA transcribed from an invading virus or from an endogenous source such as a mobilized transposon, or an inappropriately transcribed sequence.[25-28] Conversely, the miRNA pathway begins with an endogenously expressed transcript that is exported from the nucleus as dsRNA. (For further information on microRNAs, please read the following Tech Review.) The appearance of these dsRNAs induces a cascade of events involving a cytoplasmic RNase III-like protein known as Dicer, and the RNA Induced Silencing Complex (RISC). The small RNAs (both siRNA and miRNA) are incorporated into RISC, whereupon a RISC-associated, ATP-dependent helicase activity unwinds the duplex, thus enabling either of the two strands to independently guide target mRNA recognition.[29-32] The degree of complementarity between the guide strand and target mRNA determines whether mRNA silencing is achieved via site-specific cleavage of the message in the region of the siRNA-mRNA duplex[33, 34] or through an microRNA-like mechanism of translational repression[35-37]. For siRNA-mediated silencing, the cleavage products are released and degraded, leaving the siRNA-programmed RISC to survey and further deplete the available pool of target mRNA.
While siRNA-mediated gene regulation is commonly used for functional gene studies in plants, C. elegans, and preliminary attempts to induce this response using long dsRNA in mammalian cell lines initially met with limited success.[16, 38, 39] This was due, in part, to induction of the interferon response, which results in a general inhibition of protein synthesis and eventual cell death.[40, 41] In 2001, two groups independently demonstrated that short, synthetic duplexes mimicking natural 19-25 bp siRNAs could be introduced into cultured mammalian cells to elicit sequence-specific inhibition of target mRNA without the induction of the interferon response.[42, 43] The synthetic siRNAs functioned catalytically at nanomolar concentrations and were capable of cleaving up to 95% of the target mRNA in the cell.
Applications
The endogenous RNAi process has now expanded as a tool into a wide range of molecular biological applications including gene mapping, pathway dissection, and functional gene analysis. In the field of gene mapping, Li et al. replaced standard approaches for gene localization with an siRNA-based methodology to identify and characterize the vitamin K epoxide reductase (VKOR) gene.[44] The rapid identification of the VKOR gene by this group stands in contrast to an accompanying report that identified VKOR using more traditional, cumbersome mapping strategies.[45] In the field of gene function analysis, Harborth and colleagues employed RNAi to distinguish the essential (or non-essential) nature of a host of genes encoding structural proteins involved in cytoskeletal assembly and disassembly during the cell cycle.[46] Still others have used RNAi to assess the contribution of genes to cellular processes such as apoptosis[47], cell differentiation[48], and insulin signaling[49]. On a more global scale, the ability to stably suppress gene expression using large-scale or genome-wide screens with expressed versions of siRNAs (short hairpin RNAs or shRNAs) has also been demonstrated.[50, 51] Finally, in more focused analyses of specific biological pathways, RNAi has simplified the ability to study the interrelationship of multiple genes.[52, 53]
Future Applications
In addition to the current applications of RNAi, thought leaders in the field foresee expanded utility of this new technology in research, therapeutic, and diagnostic venues. In academic and pharmaceutical settings, siRNAs are envisioned to serve as invaluable tools for elucidating the function of the roughly 10,000 human genes by both individual application of RNAi technologies on each gene of unknown function [54-56] as well as broad phenotypic screens dedicated to identifying new genes involved in known biological functions.[48, 57] In therapeutics, siRNAs hold promise as precise, target-specific silencers of disease-related genes. As demonstrated by Miller et al., siRNAs have been used to target mutant alleles that contain linked, single nucleotide polymorphisms (SNPs).[58] Furthermore, studies by several laboratories have demonstrated that siRNA can be used to target human pathogens.[59, 60] In addition, siRNAs can serve as useful tools in dissecting the mechanisms by which prospective drugs adversely affect cell function and, in conjunction with microarray analysis, can be used to validate potential drug targets. Applications such as these will minimize the costs associated with new drug development and enhance the quality of patient medical care.[61, 62]
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