In a number of organisms, the introduction of double-stranded RNA into cells causes the post-transcriptional silencing of the corresponding gene. This experimental phenomenon is called RNA interference (RNAi). We are particularly interested in the molecular mechanism of this phenomenon. In order to study RNAi we screened for rde (RNAi deficient) strains and identified mutants that define at least seven complementation groups (
rde-1,-2,-3,-4,-5,-6,-7). Among these genes
rde-1(Tabara et al., 1999a) and
rde-4 have been cloned and analyzed further. RDE-1 encodes a novel protein with PAZ and PIWI domains found in numerous other proteins implicated in gene silencing and development. RDE-4 encodes a protein with two double-stranded RNA binding motifs. Genetic analysis by (Grishok et al., 2000) suggested that the functions of both
rde-1 and
rde-4 are required at an upstream step in the RNAi pathway. Recently we have shown through Immuno-precipitation experiments that RDE-4 associates with at least three other proteins in vivo, RDE-1, DCR-1 and DRH-1. DCR-1 is a conserved RNase III related protein implicated in RNAi in Drosophila and C. elegans. DRH-1 is a conserved DExH box helicase that appears to be required for RNAi in both the soma and the germline in C. elegans. Our data suggests that RDE-4 binds to foreign dsRNA and brings this dsRNA to DCR-1 to initiate RNAi. RDE-1 appears to be necessary for RDE-4 to interact with the long-trigger dsRNA in vivo. However in other organisms RDE-1 homologs appear to interact with siRNA products that function downstream in mRNA destruction. We therefore decided to ask if other RDE-1 homologs in C. elegans could provide this more downstream activity. To date, we have identified a total 26 homologues of
rde-1 in C. elegans genome. Two of these,
alg-1 and
alg-2, are implicated in the heterochronic pathway (Grishok et al. 2001). Interestingly, we have found that
alg-1 and
alg-2 are also required for RNAi (see the poster by Simard et al.). In order to analyze the potential function of other
rde-1 homologs in RNAi pathway, we have injected dsRNA targeting all 26 genes. We have found that in addition to
alg-1 and
alg-2, three more subclasses within the
rde-1gene family appear to be required for RNAi. We are currently investigating other cellular components that interact with these proteins and we are trying to determine if these RDE-1 homolog function redundantly or at distinct steps in the mechanism.