C. elegans has multiple related gene silencing pathways that mediate responses to exogenous double-stranded RNA (dsRNA), endogenous short interfering RNAs (siRNAs), transgenes and transposons. All involve dsRNA and/or siRNAs that direct post-transcriptional silencing and possibly transcriptional gene silencing. Most genetic screens have been aimed at identifying genes required for an RNAi response, such as
rde-1, which is required for RNAi triggered by exogenously administered dsRNA. We carried out a screen to identify negative regulators of RNAi and have identified mutations in two genes that generally enhance RNAi responses.
eri-1 (enhanced RNAi) encodes an siRNase1.
eri-2/rrf-3 was previously identified in a reverse genetic analysis of RNA-dependent RNA polymerase genes as a negative regulator of RNAi2. In addition to the Eri phenotype,
eri-1 and
rrf-3 mutants share other phenotypes, such as temperature-sensitive (ts) sterility and enhanced transgene silencing1,2. A second class of eri mutants from the screen does not show a ts-sterility phenotype. At least some of these mutants do show enhanced silencing of somatically expressed, simple transgene arrays.
mg411 silences a
rol-6 transgene and enhances RNAi. Upon
lin-1 RNAi by feeding E. coli, only 1.5% of wild type worms display a Lin-1 phenotype. In contrast, 24 % of
mg411 mutants and 72%
eri-1(
mg366) mutants are Muv.
mg411 mutants also display an Eri phenotype upon
hmr-1,
dpy-13 and
cel-1 RNAi. Using both the enhanced transgene silencing and Eri phenotypes, we are currently characterizing and mapping two mutations:
mg379 and
mg411. As a second approach to identify negative regulators of RNAi, we are doing a genome-wide RNAi screen for genes required for somatic transgene expression. We expect that some of the genes that we identify in this screen are negative regulators of RNAi and therefore may overlap with our eri mutants. References 1. Kennedy S, Wang D, Ruvkun G. (2004). Nature 427:645-9. 2. Simmer F, Tijsterman M, Parrish S, Koushika SP, Nonet ML, Fire A, Ahringer J, Plasterk RH (2002). Curr Biol. 12:1317-9.