Genetic analysis of the RNA silencing pathway in mouse embryonic stem cells

Genetic analysis in model systems have advanced our understanding of biological pathways by identifying key components involved in the pathway. Mouse embryonic stem (ES) cells are valuable tools to perform genetic analysis in a mammalian system using both forward and reverse genetic approaches. This thesis explores the use of mouse ES cells for genetic analysis of the RNA silencing pathway.;A major goal of this thesis was to confirm the feasibility of performing a recessive genetic screen for components of the siRNA-directed RNA silencing (RNAi) pathway in a Bloom-deficient ES cell line. We successfully developed a screen to identify components of the RNAi pathway using a simple drug selection strategy. The screen isolated Argonaute2 (Ago2), a key component of the RNAi pathway. The Ago2 mutant created from our screen allowed us to further investigate the role of two conserved phenylalanines in Ago2 and corroborated their importance for Ago2 function in siRNA-directed mRNA cleavage. Analysis of the Ago2-deficient ES cell line also confirmed that loss of Ago2 had no effect on the miRNA-directed RNA silencing pathway.;The second goal of this thesis was to take a reverse genetic approach to examine the roles of the four mammalian Argonautes (Agos). Using gene-targeting in mouse ES cells, we created an inducible Ago-deficient cell line. We found that all Agos perform redundant roles in miRNA-directed silencing and can repress target mRNAs equally well using a bulged substrate, while Ago1 and Ago2 show slightly better repression when provided with a perfect substrate. In addition, we show that Agos have a critical and redundant role in stem cell maintenance. We identified two miRNA targets that were upregulated upon inducing Ago-deficiency, Bim and PTEN. Bim is an apoptotic protein that induces death in multiple cell types, including ES cells, while PTEN inhibits cell growth. Therefore, Agos regulate ES cell survival through miRNA repression.;The mechanisms by which Ago proteins use miRNAs to repress target mRNAs are not well understood. Studies have found that miRNA repression can occur through several different mechanisms including: mRNA degradation, deadenylation, inhibition of initiation, and sequestering to GW/P bodies. To understand the components of miRNA repression, we employed a third approach using an shRNA library to perform a genome-wide screen. The screen was developed to isolate components that act downstream of Ago2 binding to the mRNA target. This screening strategy pulled out several candidates. One of the candidates, Zcrb1, was chosen for further characterization and was confirmed as a newly identified component of the miRNA pathway.;Our understanding of the RNA silencing pathway has been greatly enriched by genetic analysis performed in non-mammalian model organisms. By utilizing novel techniques for genetic manipulation in ES cells, we examined the components of the mammalian RNA silencing pathway. Our strategies are not only valuable for increasing our understanding of this pathway but can be applied to genetic analysis for furthering our understanding of other pathways in mouse ES cells.