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RNA Interference
RNA interference (RNAi) is one of the most exciting discoveries of the past decade in functional genomics. RNAi is rapidly becoming an important method for analyzing gene functions in eukaryotes and holds promise for the development of therapeutic gene silencing [1].
RNAi is a post-transcriptional process triggered by the introduction of double-stranded RNA (dsRNA) which leads to gene silencing in a sequence-specific manner.
RNAi has been reported to naturally occur in organisms as diverse as nematodes, trypanosomes, plants and fungi [2]. It most likely serves to protect organisms from viruses, modulate transposon activity and eliminate aberrant transcription products.
The first evidence that dsRNA could achieve efficient gene silencing through RNAi came from studies on the nematode Caenorhabditis elegans. Fur ther analyses in the fruit fly Drosophila melanogaster have contributed greatly toward understanding the biochemical nature of the RNAi pathway [3].
Long dsRNAs are cleaved by the RNase III family member, Dicer, in 19-23 nucleotides (nt) fragments with 5’ phosphorylated ends and 2-nt unpaired and unphosphorylated 3’ ends.
These small dsRNAs were named small interfering RNAs (siRNAs). Each siRNA duplex is formed by a guide strand and a passenger strand. The endonuclease Argonaute 2 (Ago 2) catalyzes the unwinding of the siRNA duplex. Once unwound, the guide strand is incorporated into the RNA Interference Specificity Complex (RISC), while the passenger strand is released. RISC uses the guide strand to find the mRNA that has a complementary sequence leading to the endonucleolytic cleavage of the target mRNA [4] (see figure and legend).
Ago 2 is an essential component of RISC. Post-translation hydroxylation has been shown to regulate Ago 2 stability and is important for effective RNAi [5]. It has also been shown that codelivery of Ago 2 with siRNA duplexes provides a strategy to enhance the efficacy and the specificity of RNAi [6].
Endogenously expressed siRNAs have not been found in mammals. However, many micro RNAs (miRNAs) have been identified in various organisms and cell types [7]. These miRNAs are produced by Dicer cleavage of longer (~70 nt) endogenous precursors with imperfect hairpin RNA structures. The miRNAs bind to sites that have partial sequence complementarity in the 3’ untranslated region of the target mRNA, causing repression of translation and inhibition of protein synthesis.
Even though dsRNA were shown to induce gene-specific interference in early mouse embryos [8], preliminary attempts to use dsRNA in mammalian systems were not conclusive. These experiments employed long dsRNAs which instead of triggering RNAi generated an overall decrease in mRNA eventually leading to apoptosis, a response mediated by the interferonactivated dsRNA-dependent protein kinase [9].
Tuschl and colleagues [3] have demonstrated that this non-specific response can be bypassed by using chemically synthesized 19 to 23 nt dsRNAs [10-13].
Transfection of these siRNAs resulted in strong and sequence-specific suppression of gene expression in different mammalian cell lines. This discovery has led to the widespread use of this technology to study mammalian gene function including clinically relevant genes, alluding to the potential therapeutic applications of RNAi-based technologies [14].
Chemically synthesized siRNAs are expensive and they induce only transient gene silencing due to their short life length. Another limiting step for efficient gene silencing is cell transfectability. To overcome these limitations, InvivoGen has designed an efficient, highly transfectable and simple-to-use plasmid, called psiRNA™, that allows the production of siRNAs within the cells.
psiRNA is an RNA polymerase III-based plasmid that produces short hairpin RNAs. psiRNA is used to insert a DNA fragment of approximately 50 mer designed in such a way that after transcription from the human 7SK RNA polymerase III promoter it will generate short RNAs with a hairpin structure (shRNAs). shRNAs are more stable than synthetic siRNAs and since they are continuously expressed within the cells, this method permits long-lasting silencing of your gene of interest. In order to improve silencing efficiency, InvivoGen has developed an siRNA design algorithm, named siRNA Wizard, that reliably identifies shRNA sequences for any given gene. In addition, InvivoGen provides the psiTEST system, a rapid and simple method to screen for functional siRNA or shRNA sequences.
Pairing between the heptamer seed region of a small interfering RNA (siRNA) guide strand (nucleotides 2-8) and complementary sequences in the 3’UTR of the mature transcripts has been implicated as an important element in off-target gene regulation and false positive phenotypes.
Microarray analysis of cells transfected with siRNAs revealed that low seed complement frequencies (SCFs) generally induced fewer off-target phenotypes [15]. To avoid off target effects, Invivogen’s siRNA Wizard filters candidate siRNA sequences to remove sequences displaying a known miRNA seed recognition region at the 3' end. Fur thermore, from the sequences provided by siRNA Wizard, it is recommended to select sequences with low GC content in the central region (the middle 5-15 base pairs), as high GC content results in secondary structure which reduces target accessibility [16,17].
1. Cheng JC. et al., 2003. RNA interference and human disease. Mol Genet Metab. 80(1-2):121-8.
2. Derek M et al., 2003. Killing the messenger : shor t RNAs that silence gene expression. Nat. Rev Mol Cell. Biol 4: 457-467.
3. Elbashir S.M. et al., 2001. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev, 15(2):188-200.
4. Fuchs U et al., 2004. Silencing of disease-related genes by small interfering RNAs. Curr. Mol. Med 4:507-517.
5. Qi HH et al., 2008. Prolyl 4-hydroxylation regulates Argonaute 2 stability. Nature 455: 421-424.
6. Diederichs S. et al., 2008. Coexpression of Argonaute-2 enhances RNA interference toward perfect match binding sites. PNAS 105:9284-9289.
7. Moss EG., 2002. MicroRNAs : Hidden in the genome. Curr. Biol 12:R138-R140.
8. Wianny F. and M. Zernicka-Goetz, 2000. Specific interference with gene function by double-stranded RNA in early mouse development. Nat Cell Biol, 2(2):70-5.
9. Gil J. and M. Esteban, 2000. Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): mechanism of action. Apoptosis, 5(2):107-14.
10. Elbashir SM. et al., 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 411(6836):494-8.
11. Brummelkamp TR. et al., 2002. A system for stable expression of shor t interfering RNAs in mammalian cells. Science, 296(5567):550-3.
12. Lee NS. et al., 2002. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat Biotechnol, 20(5): 500-5.
13. Scherr M. et al., 2003. Gene silencing mediated by small interfering RNAs in mammalian cells. Curr Med Chem. 10(3):245-56.
14. Ryther RCC et al., 2005. SiRNA therapeutics : big potential from small RNAs. Gene Ther 12(1): 5-11.
15. Anderson EM. et al., 2008. Experimental validation of the importance of seed complement frequency to siRNA specificity. RNA 14: 853-861.
16. Tafer H. et al., 2008. The impact of target site accessibility on the design of effective siRNAs. Nature Biotechnol. 26; 578-583. 17. Shao Y. et al., 2007. Effect of target secondary structure on RNAi efficiency. RNA 13: 1631-1640.
1- Plasmid-expressed short hairpin RNA (shRNA) requires the activity of endogenous Exportin 5 for nuclear export [1].
2- Ago2 (Argonaute 2) is recruited by TRBP [2], that forms a dimer with Dicer [3], and then receives the shRNA [4, 5, 6].
3- The shRNA is cleaved in one step by Dicer generating a 19-23 nt duplex siRNA with 2 nt 3’ overhangs.
4- After identification of the “guide strand” in the siRNA duplex, the “passenger strand” is cleaved by Ago2 [4].
5- The “guide strand” is released.
6- The “guide strand” is integrated in the active RNA Interference Specificity Complex (RISC) that contains different argonautes and argonaute-associated proteins [7].
7- The siRNA guides RISC to the target mRNA.
8- RISC delivers the mRNA to cytoplasmic foci named processing bodies (P-bodies or GW-bodies) wherein mRNA decay factors are concentrated [8, 9].
9- The target mRNA is cleaved by Ago2 and degraded.
1. Yi R. et al., 2005. Overexpression of exportin 5 enhances RNA interference mediated by short hairpin RNAs and microRNAs. RNA. 11(2):220-6.
3. Haase AD. et al., 2005. TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing.EMBO Rep. 6(10):961-7.
2. Chendrimada TP. et al., 2005. TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature. 436(7051):740-4.
4. Matranga C. et al., 2005. Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell. 123(4):607-2
5. Gregory RI. et al., 2005. Human RISC couples microRNA biogenesis and posttranscriptional gene silencing. Cell. 123(4):631-40.
6. Rivas FV. et al., 2005. Purified Argonaute2 and an siRNA form recombinant human RISC. Nat Struct Mol Biol. 12(4):340-9.
7. Meister G. et al., 2005. Identification of novel argonaute-associated proteins. Curr Biol. 15(23):2149-55.
8. Liu J. et al., 2005. Arole for the P-body component GW182 in microRNA function. Nat Cell Biol. 7(12):1161-6.
9. Jakymiw A. et al., 2005. Disruption of GW bodies impairs mammalian RNA interference. Nat Cell Biol. 7(12):1167-74.