Dicer, a novel RNase III, is required for RNA interference and development

RNA interference (RNAi) is the process by which double stranded RNAs induce the silencing of homologous genes in a sequence-specific manner. Until recently, the mechanism and underlying biology of this phenomenon was a complete mystery. RNAi was discovered fortuitously by C. elegans researchers attempting to use an antisense approach to knock down gene function. Their function phenotype than either strand of RNA alone. RNAi has since been demonstrated in a wide variety of organisms, including flies, trypanosomes, hydra, planaria, and mammals and similar phenomena have been recognized in plants and fungi. These findings have revolutionized the capacity to create targeted gene knock-downs, and have revealed that the underlying biology of RNAi controls many aspects of genomic regulation.; Our laboratory and others were able to recapitulate the RNAi process in vitro, using Drosophila embryo and cell extracts. We identified a sequence-specific nuclease activity responsible for targeting mRNA for destruction after dsRNA corresponding to that message had been introduced into Drosophila cells. This large multi-component complex is referred to as RISC (RNA-induced silencing complex), and many protein components have since been characterized. One unexpected part of this complex was small RNAs of approximately 21--25 nucleotides in length, which had previously been identified in plants undergoing what is now recognized as a related form of homology-dependent gene silencing.; These small RNAs are now considered a hallmark feature of RNAi. They are referred to as small interfering RNAs (siRNAs) and are thought to guide the RISC complex to its target mRNA. It had been demonstrated that dsRNAs could be processed into siRNAs in vitro, suggesting that a nucleolytic activity was responsible for their production. In this thesis, I have identified the enzyme responsible for this activity. This ribonuclease is referred to as Dicer, and is a novel RNase III family member. I have characterized the activity of Dicer in the RNAi pathway. In addition, I have identified a C. elegans Dicer mutant (in collaboration with Ronald Plasterk's lab at the University of Utrecht, The Netherlands) and demonstrated that Dicer is not only required for RNA interference, but also plays a critical role in developmental timing in the worm. I have since created a Dicer knock-out mouse and, although this project it is at an early stage, the null mouse appears to have an early embryonic lethal phenotype. This strongly suggests that Dicer plays a critical role in mammalian development.