Efficient RNA Interference in C. Elegans Requires the Putative RNA Helicase RDE-12 and Early Exposure to Double-Stranded RNA

Abstract

RNA interference (RNAi) is a phenomenon in which double stranded RNA (dsRNA) triggers silencing of cognate genes. In the nematode C. elegans, RNAi involves an amplifications step in which abundant secondary short-interfering RNAs (secondary siRNA) are produced, as well as nuclear processes, in which genes are transcriptionally silenced in a process involving histone modifications. In this thesis, I describe my research on RDE-12, a protein essential for the secondary siRNA amplifications step, as well as my discovery that nuclear RNAi has a critical period. I identified and characterized the gene rde-12. Although originally found in a screen for genes required for dsRNA movement, I show that RDE-12 functions cell- autonomously. rde-12 encodes a putative DEAD-box helicase, and I found that the ATPase domain of RDE-12 is required for its function. Furthermore, I found that RDE- 12 is required for amplifications or maintenance of secondary siRNA. Investigating why we found rde-12 in a screen for dsRNA transport mutants, I found that secondary siRNA amplification is not required for RNAi in the pharyngeal muscle. I found that the pharyngeal muscle is resistant to RNAi when first exposed to dsRNA, but is sensitive when exposed to dsRNA for two generations. Investigating this further, I found that pharyngeal RNAi requires nuclear RNAi genes, and that there is a critical period for nuclear RNAi, with early exposure to dsRNA triggering stronger silencing than late exposure. Finally, I found that genes in the Rb pathway extend this critical period. Rb pathway genes encode transcriptional repressors and chromatin modifying enzymes. I find that the known enhanced RNAi phenotype of Rb pathway mutants requires nuclear RNAi. Furthermore, by examining a pharynx::GFP transgene integrated at different loci, I found that local chromatin may influence silencing efficiency. I hypothesize that loss of Rb in humans, which promotes tumor progression, may do so by affecting chromatin compaction and thereby facilitate stochastic gene silencing.