Generation ofshRNA libraries and the elucidation of the RNAi pathway

The introduction of double stranded RNA into cells results in the sequence specific degradation of its homologous mRNA. This process is known as RNA interference. Specifically, double stranded RNA is cleaved into 21-23 nucleotide fragments known as small interfering RNAs (siRNAs). These siRNAs are recognized by the RNA induced silencing complex (RISC), composed minimally of Argonaute 2 (Ago2), which guides the siRNA to its target mRNA. Upon binding of the siRNA to target mRNA, Ago2 cleaves the message, resulting in its degradation. This thesis focuses on two aspects of the RNAi field. One is the development of novel technologies for using RNAi to silence genes in mammalian cells and the other is to further elucidate where RNAi occurs inside cells.; Previous reports have shown that siRNAs could be delivered to mammalian cells via plasmid based constructs. The process of generating these constructs was expensive and time consuming as each individual construct had to be tailor made. In the first part of the thesis, a novel technology is described to generate these siRNA constructs in a cheap and fast manner. This technology termed R&barbelow;estriction E&barbelow;nzyme G&barbelow;enerated s&barbelow;iRNAs (REGS) allows for the generation of genome wide siRNA libraries from sequenced or unsequenced genomes in a matter of a few days which is not possible using traditional methods.; The second part of the thesis elucidates further the RNAi pathway. One area of debate was whether siRNA mediated mRNA cleavage was dependent on active translation. Evidence for both sides exist. Experiments were done to address this. SiRNA mediated cleavage of a target mRNA was assayed in the presence or absence of translation for that particular mRNA. The results demonstrate that RNAi is independent of translation.; The next question was to determine where RNAi was occurring within cells. To do this, Ago2, the main component of RISC, was fused to eGFP. The results show that Ago2 is localized to the cytoplasm as well as mRNA decay centers. Further experiments demonstrate that Ago2 is dependent upon UPF1, a critical nonsense mediated decay (NMD) factor, for transport to decay centers. Ago2 and UPF1 also regulate similar sets of RNA indicating that Ago2 may also be part of the NMD pathway.