Howard Hughes Medical Institute, Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27701, USA
Keywords: antiviral, RNA, interference
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Introducing silence |
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Current models of RNAi suggest that the pathway involves a five-step process (for an excellent review see reference 2). First, dsRNAs (>26 bp) are cleaved by an RNase III-like enzyme, termed Dicer, to generate 2123 nt fragments (step 1).57 These short duplexed siRNAs have two unusual properties that are critical for their function: they contain 2 nt 3'-overhangs and 5'-phosphate groups.8 It is presumed that an ATP-dependent RNA helicase recognizes these short duplexes and resolves the siRNA duplex into two single-stranded RNAs (step 2).9 One strand is then incorporated into a high-molecular-weight protein complex termed RISC (RNA-induced silencing complex) (step 3),9 where it serves as guide RNA to direct the cleavage of homologous RNA sequences by an as yet unidentified endonucleolytic component of RISC (step 4).8,10 Finally, RISC is liberated from the cleaved mRNA and is recycled to perform multiple rounds of catalysis (step 5). This property of RISC is responsible for the potent nature of the silencing effect.
An intriguing facet of the RNAi pathway is the tight link between RNAi and developmental timing in C. elegans.11 The enzyme Dicer is required for both induction of RNAi and processing of two small temporal RNAs (stRNAs), lin-4 and let-7 from 70 nt structured precursor RNAs.6,7 Interestingly, the 21 nt single-stranded stRNAs, also referred to as microRNAs (miRNAs), are identical in length to siRNAs; however, unlike siRNAs that trigger mRNA degradation, stRNAs bind to multiple sites within the 3' UTRs of target mRNAs and inhibit their translation by a mechanism that remains poorly understood.12 The finding that repressed mRNAs are associated with polysomes suggests that translation initiation is not affected.12
Additional observations have further strengthened the connection between the RNAi and miRNA pathways. Purification of miRNP complexes from HeLa extracts led to the isolation of >40 unique miRNAs.13 The miRNP complex isolated from human cells is the same approximate size as Drosophila RISC (500 kDa), and one member of the complex, eIF2C2, an orthologue of Argonaute-2, is a known component of Drosophila RISC.14 Secondly, and more importantly, the recent discovery that the endogenous human let-7 miRNA not only directs mRNA cleavage but also co-purifies with human RISC,15 implies that RISC mediates both silencing processes. The choice of RISC action is probably dependent on the degree of complementarity between the guide RNA and the target sequence, with mRNA cleavage activity requiring almost 100% complete base pairing between the siRNA and target sequence.15 Moreover, the finding that at least one plant miRNA can function as an siRNA, directing the cleavage of an endogenous mRNA in Arabidopsis,16 provides a clear demonstration that at least some miRNAs can enter the RNAi pathway. The discovery of this new class of RNA, with >200 distinct miRNAs now catalogued in nematodes,17,18 fruit flies,19 humans13 and plants,16,20 suggests that miRNAs and RNAi may play a more general role in regulating gene expression than hitherto imagined.
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RNAi-mediated inhibition of viral replication |
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One impediment to utilizing RNAi technology for therapeutic benefit in humans remains the development of efficient delivery systems for siRNAs. Previous methods relied on harsh lipid-based transfection reagents to introduce siRNAs into cells in culture and are either inefficient and/or unsuitable for use in animals. An additional caveat is that, unlike in plants and lower eukaryotes, RNAi-mediated gene silencing is not long lasting in mammals. In cell culture, gene silencing effects can disappear within three to four generations, with the targeted protein returning to normal levels quickly thereafter.4 An enormous step forward in addressing these complications is the finding that natural or designed siRNAs and miRNAs can be expressed in vivo.3033 Short hairpin and pre-miRNA-based precursor RNAs can be transcribed from either RNA polymerase III (H1 or U6)30,32 or RNA polymerase II promoters33 in vivo, and processed by Dicer to release functional siRNAs. Importantly, siRNAs expressed from DNA templates can silence gene expression as effectively as exogenously introduced synthetic siRNAs.30,31,33 As an extension of these studies, many groups have begun constructing the first generation of retroviral-,34 adenoviral-35 and lentiviral-based36 gene therapy vectors that are capable of expressing siRNAs in a stable manner in virtually any cell and tissue type. Already progress has been made, with the demonstration that murine retroviral vectors expressing siRNAs directed against a mutant allele of the human K-Ras proto-oncogene have the ability to reverse tumorigenicity in mice.34 The further development of these delivery systems will push siRNA technology quickly from the proof of principle phase into animal studies of important human diseases.
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Conclusions and future challenges |
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The appearance of a resistant strain of poliovirus in cultures treated with polio-specific siRNAs24 is a strong reminder that a single nucleotide mutation could render viruses immune to a single specific siRNA. Although it was concluded that the mutation was already present in the primary viral isolate, emergence of siRNA resistance is a major concern that will need to be addressed, particularly for viruses encoding error-prone polymerases such as HIV-1. Thus, in future, siRNA expression cassettes will require a higher degree of sophistication, probably encoding tandem arrays of highly expressed siRNAs that target several conserved viral sequences simultaneously.
Much remains to be accomplished, but RNAi will continue to play an important role in determining cellular gene function and shows a great deal of promise as a therapeutic agent. The next few years of research will indicate whether RNAi technology will realize its potential as the next wave of therapeutic molecules.3
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Footnotes |
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References |
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2 . Hutvágner, G. & Zamore, P. D. (2002). RNAi: nature abhors a double strand. Current Opinion in Genetics and Development 12, 22532.[CrossRef][ISI][Medline]
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Ketting, R. F., Fischer, S. E., Bernstein, E., Sijen, T., Hannon, G. J. & Plasterk, R. H. (2001). Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes and Development 15, 26549.
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Knight, S. W. & Bass, B. L. (2001). A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans. Science 293, 226971.
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Elbashir, S. M., Lendeckel, W. & Tuschl, T. (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes and Development 15, 188200.
9 . Nykänen, A., Haley, B. & Zamore, P. D. (2001). ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 30921.[ISI][Medline]
10 . Hammond, S. M., Bernstein, E., Beach, D. & Hannon, G. J. (2000). An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404, 2936.[CrossRef][ISI][Medline]
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Hammond, S. M., Boettcher, S., Caudy, A. A., Kobayashi, R. & Hannon, G. J. (2001). Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293, 114650.
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