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. 2007;35(13):4322-30.
doi: 10.1093/nar/gkm437. Epub 2007 Jun 18.

A systematic analysis of the effect of target RNA structure on RNA interference

Ellen M Westerhout  1 , Ben Berkhout
Affiliations

A systematic analysis of the effect of target RNA structure on RNA interference

Ellen M Westerhout et al. Nucleic Acids Res. 2007.

Abstract

RNAi efficiency is influenced by local RNA structure of the target sequence. We studied this structure-based resistance in detail by targeting a perfect RNA hairpin and subsequently destabilized its tight structure by mutation, thereby gradually exposing the target sequence. Although the tightest RNA hairpins were completely resistant to RNAi, we observed an inverse correlation between the overall target hairpin stability and RNAi efficiency within a specific thermodynamic stability (DeltaG) range. Increased RNAi efficiency was shown to be caused by improved binding of the siRNA to the destabilized target RNA hairpins. The mutational effects vary for different target regions. We find an accessible target 3' end to be most important for RNAi-mediated inhibition. However, these 3' end effects cannot be reproduced in siRNA-target RNA-binding studies in vitro, indicating the important role of RISC components in the in vivo RNAi reaction. The results provide a more detailed insight into the impact of target RNA structure on RNAi and we discuss several possible implications. With respect to lentiviral-mediated delivery of shRNA expression cassettes, we present a DeltaG window to destabilize the shRNA insert for vector improvement, while avoiding RNAi-mediated self-targeting during lentiviral vector production.

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Figures

Figure 1.
Figure 1.
Target RNA structure influences RNAi efficiency. (A) The HIV-based target sequences (54 nt) were cloned downstream of the firefly luciferase gene in the pGL3 reporter plasmid. These reporter constructs were co-transfected into C33A cells with the shRNA expressing plasmid pSUPER-shRNA-Pol. The respective H1 (polymerase III) and SV40 (polymerase II) promoter units are indicated by a black box, the arrow marks the transcription initiation site. (B) The predicted RNA structures (Mfold program) of the wild-type (wt) shPol and mutated hairpins (T1–T7). The 19-nt target sequence is highlighted as a gray box and the mutated nucleotides are encircled. The thermodynamic stability (ΔG in kcal/mol) of the target hairpins is indicated (54 nt total; CCCC + indicated hairpin + UUU). (C) Luciferase expression upon transfection of the reporter constructs with increasing amounts of pSUPER-shRNA-Pol. The firefly luciferase activity was normalized to that of the Renilla luciferase to correct for variation in transfection efficiency. The level of expression observed in the absence of shRNA-Pol was set at 100% for each reporter construct. This level did not vary significantly for the different constructs. The mean values of six independent experiments are shown (± SD). (D) The thermodynamic stability of the target hairpins is plotted against the level of luciferase expression as observed in Figure 1C with 10 ng pSUPER-shRNA-Pol. (E) Semi-quantitative RT-PCR on RNA isolated from cells transfected with 100 ng pGL3-target hairpin variants with or without 10 ng pSUPER-shRNA-Pol. Actin levels serve as a control.
Figure 2.
Figure 2.
Target RNA structure influences siRNA binding. (A) Radioactively labeled oligonucleotide simulating the siRNA antisense strand (processed from shRNA-Pol) was incubated with increasing amounts of hairpin target RNA variants (wt, T1–T7). SiRNA/target RNA duplex formation was analyzed by EMSA. (B) Free and bound siRNA oligonucleotide was quantified to calculate the level of duplex formation (bound siRNA/free + bound siRNA). (C) The thermodynamic stability of the target hairpins is plotted against the level of duplex formation with 0.2 µM target RNA.
Figure 3.
Figure 3.
Position-specific destabilization of target hairpin triggers RNAi differentially. (A) Predicted RNA structures of the wt and mutant hairpins A–G. The target sequence is highlighted in the gray box and the mutated nucleotides are encircled. The thermodynamic stability (ΔG in kcal/mol) of the target hairpins is provided for each structure (54 nt total; CCCC + indicated hairpin + UUU). (B) Luciferase expression observed after transfection of the reporter constructs with 10 ng pSUPER-shRNA-Pol is plotted against the thermodynamic stability of the target hairpins. The mean values of five independent experiments are shown (± SD). The gray dotted line represents the trend line observed for the initial set of mutants in Figure 1D. The right graph zooms in on a smaller ΔG segment.
Figure 4.
Figure 4.
In vitro siRNA binding does not prefer an accessible 3′ end of the target. (A) Radioactively labeled oligonucleotide simulating the siRNA antisense strand (processed from shRNA-Pol) was incubated with increasing amounts of target variants A–G. SiRNA/target RNA duplex formation was analyzed by EMSA. (B) Free and bound siRNA oligonucleotide was quantified to calculate the level of duplex formation (bound siRNA/free + bound siRNA). (C) The thermodynamic stability of the target hairpins is plotted against the level of duplex formation with 0.2 µM target RNA. The gray dotted line represents the trendline observed in Figure 2C in the experiments with the initial set of mutants.
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References

    1. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998;391:806–811. - PubMed
    1. Vance V, Vaucheret H. RNA silencing in plants-defense and counterdefense. Science. 2001;292:2277–2280. - PubMed
    1. Waterhouse PM, Wang MB, Lough T. Gene silencing as an adaptive defence against viruses. Nature. 2001;411:834–842. - PubMed
    1. Galiana-Arnoux D, Dostert C, Schneemann A, Hoffmann JA, Imler JL. Essential function in vivo for Dicer-2 in host defense against RNA viruses in drosophila. Nat. Immunol. 2006;7:590–597. - PubMed
    1. Kalmykova AI, Klenov MS, Gvozdev VA. Argonaute protein PIWI controls mobilization of retrotransposons in the Drosophila male germline. Nucleic Acids Res. 2005;33:2052–2059. - PMC - PubMed

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