RNAi inDictyostelium:The Role of RNA-directed RNA Polymerases and Double-stranded RNase

Abstract

We show that in Dictyostelium discoideum an endogenous gene as well as a transgene can be silenced by introduction of a gene construct that is transcribed into a hairpin RNA. Gene silencing was accompanied by the appearance of sequence-specific RNA ∼23mers and seemed to have a limited capacity. The threeDictyostelium homologues of the RNA-directed RNA polymerase (RrpA, RrpB, and DosA) all contain an N-terminal helicase domain homologous to the one in the dicer nuclease, suggesting exon shuffling between RNA-directed RNA polymerase and the dicer homologue. Only the knock-out of rrpA resulted in a loss of the hairpin RNA effect and simultaneously in a loss of detectable ∼23mers. However, ∼23mers were still generated by theDictyostelium dsRNase in vitro with extracts from rrpA−, rrpB−, and DosA−cells. Both RrpA and a target gene were required for production of detectable amounts of ∼23mers, suggesting that target sequences are involved in ∼23mer amplification.