RNAi and antisense mediated gene silencing in Dictyostelium

Based on an endogenous regulatory antisense system in Dictyostelium, we try to identify the cellular components involved in the mechanisms controlling gene regulation by experimentally introduced and natural antisense transcripts and to compare these mechanisms with RNA interference (RNAi).

We have identified and characterized a nuclease which specifically digests double stranded (ds)RNA and produces ~21mers, similar to the products of RNAi. Gene constructs expressing dsRNA can mediate RNAi in Dictyostelium, i.e. posttranscriptional gene silencing accompanied by the production of sequence specific ~23mers. Similar to Neurospora, C. elegans and others, an RNA dependent RNA polymerase (RdRP) is essential for efficient RNAi in Dictyostelium. We have identified three RdRP homologs in the Dictyostelium genome but only the disruption of one of them (rrpA) abolishes RNA interference.

In Drosophila, the RNase III related gene "dicer" has been shown to generate ~21mers from dsRNA. We have identified two dicer homologs in Dictyostelium and are investigating their involvement in RNAi. Interestingly, no ~21mers are found when rrpA is knocked out, even though the dsRNase activity is still functional in vitro. Likewise, no ~21mers are detectable when the RNAi construct is introduced into cells which do not contain a target gene (e.g. b -galactosidase). We conclude that small amounts of ~21mers are produced from the original dsRNA (RNAi construct). These are unwound and serve as primers for RdRP on the target mRNA thus generating a secondary double strand.