We are investigating the mechanism of RNA interference (RNAi) in C. elegans. Upon introduction of double-stranded RNA (dsRNA), homologous mRNA targets are degraded through a process that requires the RNA-dependent RNA polymerases (RdRPs) Rrf-1 and Ego-1 (1, 2). Other players that presumably negatively regulate RNAi have been identified; the RdRP Rrf-3, and the exonuclease Eri-1 (3, 4). We have undertaken a study of the roles of the RdRPs during RNAi and development of the worm.Transitive RNAi refers to the process by which RNAi spreads in the 5' direction from the initial primary trigger dsRNA. This spreading is reflected in the pool of secondary short interfering RNAs (siRNAs) and is dependent on
rrf-1 in the soma. To understand the precise nature of this process, we are cloning siRNAs from worms undergoing RNAi with the expectation that the structure and distribution of these siRNAs may provide some insight into the mechanism of transitive RNAi. In addition, we would like to understand the endogenous role of
ego-1 in germ line function or protection. Finally, we are investigating possible roles of the RdRPs in other RNA-triggered silencing phenomena.(1) Sijen, T., Fleenor, J., Simmer, F., Thijssen, K.L., Parrish, S., Timmons, L., Plasterk, R.H.A., and Fire, A. 2001. On the role of RNA amplification in dsRNA-triggered gene silencing. Cell 107:465-476(2) Smardon, A., Spoerke, J.M., Stacey, S.C., Klein, M.E., Mackin, N., and Maine, E.M. 2000. EGO-1 is related to RNA-directed RNA polymerase and functions in germ-line development and RNA interference in C. elegans Curr Biol 10:169-178(3) Kennedy, S., Wang, D., and Ruvkun, G. 2004. A conserved siRNA-degrading RNase negatively regulates RNA interference in C. elegans. Nature 427:645-649(4) Simmer, F., Moorman, C., van der Linden, A. M., Kuijk, E., van den Berghe, P. V. E., Kamath, R. S., Fraser, A. G., Ahringer, J., and Plasterk, R. H. A. 2003. Genome-wide RNAi of C. elegans using the hypersensitive
rrf-3 strain reveals novel gene functions. PLOS Biology 1:77-84