The recent discovery that double-stranded RNA (dsRNA) can invoke a potent and sequence-specific gene silencing response in a variety of eukaryotic organisms has profoundly influenced both the practice of modern biology and our understanding of gene regulation in general. Several types of dsRNA-mediated gene regulation have now been described, but the hallmark of each is the production of small (~21-25 nucleotide) RNA “triggers” that are the functional units that guide silencing. RNA interference (RNAi) is the branch of silencing that directs the sequence-specific degradation of
homologousmRNA using ~21 nt small interfering RNAs (siRNAs) as guides. RNAi has been of special interest because of its broad applicability as a reverse genetic tool and because robust /in vitro/ model systems from Drosophila and humans have already taught us much about underlying mechanism. At present, our knowledge of the cellular machinery required to carry out RNAi as well as other related forms of RNA-mediated silencing is incomplete. Moreover, less is known about the assembly process of the multi-subunit RNA-induced silencing complex (RISC) that is the ultimate effector of RNAi in Drosophila. The ultimate goals of my graduate work are: (1) to characterize the earliest biochemical step that determines RISC’s sequence specificity during the initiation of RNAi and (2) to identify the proteins present during discrete stages of RISC assembly such that an ordered pathway can be precisely defined.