Ras signaling is required for multiple cell fate decisions during C. elegans development including specification of the excretory duct cell. Strong mutations in
let-60 ras result in distinct rod-like larval lethal phenotype owing to loss of the excretory duct cell. Mutations in positive regulators
ksr-1 and
cnk-1 display little or no rod-like lethality (<1%), but
cnk-1;
ksr-1 double mutants display >90% rod-like larval phenotype (1). To identify additional positive regulators of Ras signaling, we performed a RNAi screen for enhancers of
ksr-1 rod-like lethality. From a screen of chromosome I we identified eleven ekl genes ( e nhancers of k sr-1 l ethality). Several observations suggest that at least a subset of the ekl genes may function together in the generation or function of small regulatory RNAs. First, the molecular identities of three strong enhancers
ego-1 (a putative RNA-dependent RNA polymerase),
ekl-1 (a tudor domain protein), and
ekl-3 (a Dicer-related helicase), are consistent with an RNA based mechanism. Second, two additional ekl genes may be co-regulated with one or more of the above genes based on the microarray topology map (2), and Gene Recommender algorhythm (3). Third, RNAi of
ekl-4 (a conserved SANT domain protein) has embryonic lethal and heterochronic related phenotypes similar to those described for
alg-1/alg-2 and
dcr-1 (4), suggestive of a role in microRNA processing. These cumulative observations suggest that small regulatory RNAs may be cooperating with Ras signaling in specification of the excretory duct cell fate. We are in the process of confirming that
ekl-4(RNAi) phenotypes are due to heterochronic defects and if
ekl-4 is required for miRNA processing. In addition, we are determining if the other ekl genes have heterochronic phenotypes and if loss of
alg-1/alg-2 can enhance the
ksr-1 ras-like lethal phenotype. The results of these experiments will be presented at the meeting. 1. see poster by Rocheleau and Sundaram 2. Kim et al., Science (2001) 3. Owen et al., Genome Biology (2003) 4. Grishok et al., Cell (2001)