84. RNA Silencing: Role of Interferon System Components in Non_Specific Effects Induced by Chemically Synthesized Small Interfering RNAs, Enzymatically Synthesized Small Interfering RNAs and Short Hairpin RNAs

Abstract

In mammalian cells, long double-stranded RNAs (dsRNAs) activate interferon (IFN) signaling as well as interferon inducible RNA-dependent enzymes that affect translational activity including the protein kinase (PKR) and 2',5'-oligoadenylate synthetases (2',5'-OAS). DsRNA also is the mediator of RNA interference (RNAi), a widely used gene silencing strategy with utility both as a basic research tool and potential therapeutic. RNAi mediated by short dsRNA molecules (|[sim]|20-bp) was originally thought to circumvent the IFN response to achieve specific target silencing without triggering potentially problematic non-specific effects. However, reports suggest that some small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) produced from transfected plasmids can induce cytokine signaling and cause other non-specific effects. We therefore undertook a further combined genetic and biochemical examination of the involvement of IFN system components in RNAi to directly address the potential involvement of IFN-regulated gene products in non-specific responses to siRNAs. We examined the contribution of IFN signaling, PKR and RNase L to both on-target and non-specific gene silencing as well as induction of cytokines induced by three RNAi strategies. We show, through the use of cell lines genetically deficient in signaling and action components of the IFN system, that chemically synthesized siRNAs, T7 RNA polymerase-synthesized siRNAs, and shRNAs differentially activate IFN signaling and non-specific gene silencing. ShRNAs with up to 50 nucleotides complementary to a target gene mediated specific silencing and did not trigger nonspecific gene silencing. While the plasmids that delivered the shRNA constructs induced IFN-|[beta]| in a wild type cell line sensitive to CpG DNA, there was no differential induction of IFN-|[beta]| caused by the shRNAs in this cell type or other cell types not sensitive to CpG DNA. By contrast, both 20-bp and 50-bp T7 synthesized siRNAs caused significant non-specific gene silencing and induced higher levels of IFN-|[beta]| in wild type cells than chemically synthesized siRNAs. The IFN-|[beta]| induction capacity of the T7 synthesized siRNAs varied between preparations and could be reduced by gel purification of the siRNA. Chemically synthesized siRNAs caused non-specific gene silencing only at extremely high concentration and did not induce significant IFN-|[beta]| production in any cell type tested. Non-specific siRNA-mediated silencing was independent of PKR and RNase L, and only partially dependent upon IFN receptormediated signaling. We also found a relationship between the nonspecific gene silencing and the induction of apoptosis by the different RNAi mediators in different cell lines.