Double stranded (ds) RNA induces gene silencing in many organisms. In C. elegans this process has been termed (ds) RNA mediated interference (RNAi). We have undertaken a forward genetic screen to identify enhancers of RNAi (eri) loci. We took advantage of the relative inefficiency of neuronal RNAi in C. elegans to screen for genes which when mutated cause enhanced sensitivity to dsRNA. Animals carrying an integrated
unc-47::GFP fusion gene show little or no decline in GFP fluorescence following exposure to GFP dsRNA. We screened ~40,000 haploid genomes for mutants that give a wild-type fluorescence pattern under normal growth conditions but exhibit a decrease in GFP fluorescence following exposure to GFP dsRNA. Candidate mutants were then screened for increased sensitivity to dsRNAs derived from endogenous chromosomal loci with known loss of function phenotypes. As expected, our screen identified an allele of the only previously identified enhanced RNAi loci, the RNA dependent RNA polymerase
rrf-3. Here we report on the identification and characterization of a novel enhanced RNAi loci;
eri-1.
eri-1 animals exhibit the strongest enhanced RNAi phenotype of all candidate mutants obtained from our screen.
eri-1 animals show an enhanced sensitivity to dsRNA targeted against multiple chromosomal loci. The enhanced RNAi phenotype of
eri-1 is accompanied by an ~10x increased accumulation of small interfering (si)RNAs following exposure to dsRNA. Genetic epistasis analysis places
eri-1 either upstream or in parallel to the RNAi defective mutants
rde-1,
rde-4,
sid-1, and
mut-16 and most likely in the same pathway with
rrf-3.
eri-1 encodes an evolutionarily conserved protein with sequence homology to DEDDh 3-5 exonuclease proteins and SAP/ SAF-BOX domain proteins. We have identified putative orthologues of ERI-1 in S. pombe and vertebrates. ERI-1 is expressed predominantly within the cytoplasm of a subset of head and tail neurons and asymmetrically within the spermatheca. While
eri-1 enhances the efficacy of RNAi,
eri-1 (like
rrf-3) is not sufficient to allow for the detection of most neuronal phenotypes in feeding RNAi assays. Double eri mutant combinations are being constructed in an attempt to create a strain that allows for efficient feeding neuronal RNAi to be performed. Progress on neuronal RNAi and the characterization of the mechanism of enhanced RNAi in
eri-1 will be discussed.