Elucidating the Mechanisms of RNA-Induced Transcriptional Silencing Machinery in a HIV-1 Therapeutic

Abstract

RNA interference (RNAi) is an evolutionarily conserved mechanism that modulates gene expression through homology-dependent short interfering RNA (siRNA). The process consists of two distinct pathways whereby Argonaute proteins i) transiently degrade messenger transcripts or ii) silence genomic DNA via heritable epigenetic modifications. The latter offers artificial control of the human genome. It presents revolutionary therapeutic interventions for currently incurable genetic conditions and viral infections. This process however, is highly controversial. Fundamental mechanisms, including the components of functional epigenetic silencing machinery, are unknown. In order to harness the full potential of RNAi for gene therapy, it is crucial to understand the mechanics of the pathway.

This thesis explores RNAi-induced epigenetic silencing in the context of HIV-1 infection. To date, no treatment has been sufficient to surmount the proviral reservoir, making the virus a prime candidate for gene therapy. Using a promoter targeted siRNA developed by our lab, Chapter 3 investigates the intracellular trafficking of the silencing machinery. Through live-cell immunofluorescent microscopy, we show that the actin cytoskeleton is required for dynamic transport of siRNA loaded Argonaute 1 and subsequent nuclear import.

In Chapters 4-6, we perform a range of biochemical interaction studies to examine Argonaute 1 complexes and identify potential components of the core machinery. Lead candidates were further explored in Chapter 7, where we generated Argonaute 1 mutants, CRISPR knockout cultures and computational protein models to assess possible RNAi interplay. We demonstrate that HNRNPU and to a lesser extent, CBX3, are involved in siRNA-mediated gene silencing. Further, in a manner that mimics alternate systems, HSP90 may offer a mechanism of regulating the formation of the silencing machinery.

This is the first study to identify proteins, additional to the actin cytoskeleton, including HSP90, HNRNPU and CBX3, that are involved in human RNAi-induced epigenetic silencing. This knowledge provides unique avenues for enhanced RNAi therapeutic delivery and warrants further studies to elucidate the precise mechanisms of a pathway that was prior to this study, completely unknown.