The Role of Caenorhabditis elegans dicer in endogenous small RNA silencing pathways

RNA interference (RNAi) was originally identified as a conserved biological response to exogenous double-stranded RNA (dsRNA). During this response, dsRNA is processed into 21-23 nucleotide small interfering RNAs (siRNAs) by the ribonuclease III enzyme Dicer. siRNAs produced by this pathway initiate the sequence specific degradation of their cognate messenger RNAs (mRNAs). While RNAi was originally discovered as a response to exogenous dsRNA, during the time of my dissertation research, it has become clear that endogenous dsRNA is responsible for regulating many biological processes. Dicer is also responsible for processing endogenous dsRNA in the form of micro- RNAs (miRNAs) and many endogenous-siRNAs (endo-siRNAs). The effects that Dicer dependent endo-siRNAs and miRNAs have on their cognate mRNAs was unclear prior to the work described in this dissertation. In Chapter 2, I describe my work to characterize how transcripts are misregulated in C. elegans harboring a mutation in the dcr-1 gene. I found that Dicer is responsible for regulating a large number of transcripts including known and predicted miRNA targets. Among the misregulated transcripts in dcr-1 (-/-) was an enrichment for genes involved in C. elegans innate immunity. This later finding prompted me to investigate the role of Dicer's helicase domain, particularly in light of this domain's similarity to RIG-I and MDA-5, two mammalian helicases involved in innate immunity. In Chapter 3,1 describe experiments in which I reintroduced the dcr-1 gene with targeted mutations in the helicase domain into dcr-1 (-/-) C. elegans. Reintroduction of either wild-type (WT) or helicase mutant dcr-1 rescued the fertility defects exhibited in dcr-1 (-/-) animals. Furthermore, I show that dcr-1 helicase mutants have a normal RNAi response following feeding of exogenous dsRNA. I present deep sequencing data that indicates that while dcr-1 helicase mutants process miRNAs to WT levels, they are defective for the production some, but not all, endo-siRNAs. In light of these data, I present a model for Dicer cleavage in which Dicer acts as a processive translocase to process long dsRNA to siRNAs. In Chapter 4, I propose experiments to test this model in light of current literature.