| RNA interference is the process of dsRNA-mediated sequence-specific silencing of gene expression that has been applied as a research tool to manipulate gene expression. One technique of expressing the dsRNA intermediates required for interference in mammalian systems is the introduction of short-interfering RNAs that direct the degradation of homologous target mRNA. Although RNAi strategies rely on a high degree of specificity, evidence shows that many nonspecific side effects can result from this technique. We found that transfection of siRNAs causes the upregulation of ISGs, an event mediated by the dsRNA-dependent protein kinase PKR. The interferon response induced by siRNAs is similar to the response initiated by synthetic dsRNA, but results in distinct gene expression patterns and can be sufficient to provide cellular protection from viral infection. While siRNAs synthesized from the T7-polymerase are very efficient interferon inducers, chemically synthesized siRNAs also induce interferon induction, especially if the cells are primed for the response by an initial stimulus.; To further characterize the siRNA-mediated IFN response, we identified a primary, IFN-independent response, as well as a secondary, IFN-dependent response. In Type I IFN-null cells, the ISG Stat1 is not activated by siRNA; however, the dsRNA-activated protein p56 is upregulated. While the interferon-independent response is not as robust as that in normal responding cells, global changes in gene expression are observed. In addition, we have shown that this primary response is mediated by interferon regulatory factor 3.; Independent of these studies, we were interested in learning more about the mechanism of RNAi in mammalian systems. In mammalian cells, introduction of long dsRNA molecules leads to the activation of the interferon-mediated antiviral response and results in the general inhibition of protein synthesis. Early studies suggested that this global response to dsRNAs may prevent any specific effects of RNAi from being detected. Alternatively, it is possible that dsRNA-responsive pathways are not only activated by siRNAs, but may also mediate the specific gene silencing effects of RNAi through the double-stranded siRNA molecules. However, we show by using cell lines deficient in specific components mediating IFN action that the RNAi mechanism is independent of the interferon system. |
