Biochemical determinants for the assembly of RNA induced silencing complexes in Drosophila melanogaster

In order to understand how a gene issues instructions to bring about life, we need to understand when, where, and to what extent it is functioning. The recent discovery of conserved small RNA-based silencing pathways has fostered a new appreciation for the startling complexity underlying the coordinated regulation of gene expression. In Drosophila, the RNA interference (RNAi) pathway and other small RNA-related gene silencing mechanisms assemble complex macromolecular machines to bring about mRNA degradation, heterochromatin rearrangement, translational repression, and possibly other forms of sequence-specific regulation. Here I focus on several key steps in the assembly process of the mRNA-degrading holo-enzyme of RNAi (called RISC) and also report the discovery of a new silencing factor that may be a core component of a previous unknown small RNA-related gene regulation machine that is essential for male gametogenesis.;Drosophila RNAi is initiation by the processing of long double-stranded RNA (dsRNA) into short, 21 nucleotide small interfering RNA (siRNA) duplexes by the double-stranded RNA endonuclease Dicer. I investigated the relationship between Dicer processing and selection of the siRNA guide strand that is loaded into the active site of RISC. I found that the generation of an siRNA from a long double-stranded precursor and the initiation of RISC assembly are two biochemically distinct events, despite a mutual requirement for the enzyme Dicer-2. Subsequently, I observed that ubiquitin-mediated signals influence the RNAi pathway. Specifically, RISC maturation is dependent upon a ubiquitin conjugation event that is independent of the ubiquitin-proteasome pathway. This observation is consistent with recent findings that RNAi components associate with endosomes and the multivesicular body, each of which utilize ubiquitin-mediated signals to sort cargo between intracellular membranes.;I also worked to characterize a novel pair of double-stranded RNA binding proteins that are critical for Drosophila spermatogenesis. These proteins contribute to RNA silencing in cell culture, and loss of expression in the germline leads to spermatogenic defects and sterility. A novel mRNA-derived small RNA population is dependent upon at least one of these proteins for their sustainability, and their sequences suggest they may play a direct role in regulating genes that are essential for spermiogenesis.