In eukaryotes, microRNAs are small non-coding RNAs which have the role of regulating genes essential for development and cellular differentiation. Beside the RNAse III family members (Drosha and Dicer) and the Argonaute proteins ALG-1 and ALG-2, essential components of this new gene regulation pathway are still not uncovered. To identify new cellular factors essential for the microRNA pathway, we use the synthetic lethality feature of
alg-2 and
alg-1 genes. When both are not functional simultaneously, the animal can not survive (Grishok and al., Cell, 2001). We base our method on the synthetic lethal screen which led to identification of mutations that interact with
lin-35/Rb (Fay and al., Genes & Dev., 2001). Our screen is performed with a transgenic animal carrying an extrachromosomic array containing the
alg-2 gene linked to a broadly expressed GFP gene and in which the genomic copy of
alg-2 is deleted. Therefore, it will be easy to determine, by GFP expression, if the extrachromosomal copy of
alg-2 gene is essential for the survival of worms. This worm line has been mutagenized with EMS and candidates that express GFP protein have been collected. We have conducted a clonal screen by picking four GFP positive F2 animals per isolated GFP positive F1 (one thousand total). From this initial screen, we have isolated 10 positives candidates. We are currently using various mapping techniques to identify genetic lesions carried by these candidates. We will discuss at the meeting about our progress. We believe that this synthetic lethal screen will allow us to identify new genes that work in synergy with
alg-2, like
alg-1, and this will uncover new components of the microRNA pathway. This work is supported by the Canadian Institutes of Health Research (MOP-81186).