Induction of RNA interference genes by double-stranded RNA; implications for susceptibility to RNA interference

Gene silencing by RNA interference (RNAi) can be a useful reverse genetics tool in eukaryotes. However, some species appear refractory to RNAi. To study the role of the differential expression of RNAi proteins in RNAi, we isolated partial dicer-2, argonaute-2 translin, vasa intronic gene (VIG) and tudor staphylococcus/micrococcal nuclease (TSN) genes from the tobacco hornworm, Manduca sexta, a well-studied insect model which we have found to be variably sensitive to RNAi. We found that the RNAi gene, translin, was expressed at minimal levels in M. sexta tissue and that there is a specific, dose-dependent upregulation of dicer-2 and argonaute-2 expression in response to injection with dsRNA, but no upregulation of the other genes tested. Upregulation of gene expression was rapid and transient. In order to prolong the upregulation we introduced multiple doses of dsRNA, resulting in multiple peaks of dicer-2 gene expression. Our results have implications for the design of RNAi experiments and may help to explain differences in the sensitivity of eukaryotic organisms to RNAi.