An Investigation of the RNA Induced Silencing Complex and its Therapeutic Implications.

Abstract

Small interfering RNAs (siRNAs) are regulatory non-coding RNA, which regulate gene expression at the post-transcriptional level through the highly conserved RNA interference (RNAi) pathway. RNAi is employed by most eukaryotes as a key component of their innate immune response to viruses and retrotransposons. As a countermeasure, many viruses have evolved viral RNA silencing suppressors (RSS) that thwart host anti-viral responses. Here, we show that the siRNA:p19 (RSS) interaction is reversible and p19 efficiently competes with recombinant Dicer and inhibits the formation of RISC (RNA Induced Silencing Complex)-related assembly complexes found in human cell extracts. Computational modeling of our results suggests the formation of a ternary complex between siRNA, human Dicer, and p19. Although siRNAs have given birth to a new field of gene therapeutics, several questions concerning their potency are still unanswered, necessitating an understanding of how short double-stranded RNAs are processed in the cellular milieu. We have developed a set of doubly-fluorophore labeled RNAs to probe in real-time the stability of siRNAs and related molecules by fluorescence resonance energy transfer (FRET). Our results suggest that RNAi mediating RNAs are specifically protected in the cellular environment and possibly provides an explanation for recent results, which show that unmodified siRNAs persist inside cells for extended periods of time. Despite its vast application, several outstanding questions about the composition of siRISC are still unanswered. Although it is known that the minimal RISC is composed of Dcr, TRBP and Ago2, the roles played by other Ago2 and RISC associated proteins are still under debate. Much of the complexity associated with assigning specific roles to the pathways components arises from the inability to develop a comprehensive system of study for RNAi. Our results demonstrate that the two systems we and others have employed, i.e., the HeLa whole cell extract and the HeLa S100 cytoplasmic extract, utilize siRNA duplexes with differential efficiency in cleaving a target mRNA. Additionally, we have shown that ATP has an inhibitory effect on target cleavage in HeLa S100 cytoplasmic extract. Taken together, the work presented in this thesis has furthered our understanding of the RNAi pathway and its therapeutics implications.