RNA Silencing Complex Assembly and Function

DESCRIPTION (provided by applicant): Cells from diverse eukaryotes silence cognate gene expression in response to small RNAs called siRNAs (short interfering RNAs) and miRNAs (microRNAs). These RNA silencing mechanisms can govern the expression of cellular genes, specify the functions of specific chromatin domains, and protect cells from viral infection and transposon invasion. One form of RNA silencing, RNA interference (RNAi), directs the specific degradation of mRNA transcripts that share sequence identity with an siRNA. Transcript destruction is executed by the RNA-induced silencing complex (RISC). The goal of the proposed research is to determine the biochemical pathway of RISC assembly and function, using Drosophila melanogaster embryo lysates that are active for RNAi in vitro. Drosophila RISCs of differing sizes have been reported, but the relationships between them are unclear, and almost nothing is known about how they assemble. Furthermore, the identity of the siRNA-directed endonuclease remains unknown. We have developed a novel native gel electrophoresis assay that we can use to monitor protein complexes that form on radiolabeled siRNA. We have used this assay to identify at least three complexes, two of which (called R1 and R2) are intermediates in a RISC assembly pathway. The other complex, R3, is a very large (80S) and potentially ribosome-associated form of RISC that can specifically recognize and cleave targeted mRNAs. This project aims to further define the RNAi pathway in three ways: 1) By characterizing the intermediate complexes and determining the requirements for their assembly into higher-order silencing complexes; 2) By purifying the R3 complex to identify proteins present within this novel form of RISC; and 3) By employing site-specific photocrosslinking to identify RISC components that contact functionally distinct regions of the target mRNA (including the cleavage site) during RISC assembly, activation and function. Six years after the in