This dissertation, "Roles of Human Double-stranded RNA Binding Proteins TRBP and PACT in RNA Interference" by Kin-hang, Kok, 郭健恆, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled ROLES OF HUMAN DOUBLE-STRANDED RNA BINDING PROTEINS TRBP AND PACT IN RNA INTERFERENCE submitted by Kok Kin Hang for the degree of Doctor of Philosophy at the University of Hong Kong in September 2006 The double-stranded RNA (dsRNA) binding proteins are a large family of proteins sharing a dsRNA-binding domain. These proteins play important roles in various biological processes such as translation and RNA interference (RNAi). Human dsRNA binding proteins TRBP and PACT are structurally related, but exert opposite effects on protein kinase PKR, a key regulator of dsRNA-mediated signaling and antiviral defense. Recent evidence indicates that Dicer, an endonuclease of the RNase III-type, interacts with TRBP or PACT to mediate RNAi and microRNA (miRNA) processing. However, their exact roles in RNAi and the underlying mechanisms are not understood. In this thesis I carry out a study to provide new insights into two aspects of TRBP and PACT function in human cells. Although both TRBP and PACT have been shown to interact with Dicer, it is not clear whether TRBP and PACT have redundant function or are simultaneously required for Dicer. In the first part of my thesis I seek to clarify this issue by investigating the interaction between TRBP and PACT. First I conduct protein affinity binding assays with recombinant TRBP and PACT purified from Escherichia coli and show that TRBP directly interacts with PACT in vitro. Then I also perform co-immunoprecipitation and confocal immunofluorescence microscopy to verify the association of TRBP and PACT in mammalian cells. Furthermore, I provide the evidence that TRBP and PACT form a triple complex with Dicer and facilitate the production of small interfering RNA (siRNA) by recombinant Dicer. Knockdown of both TRBP and PACT in cultured cells leads to significant inhibition of gene silencing mediated by short hairpin RNA (shRNA), but not by siRNA, suggesting that TRBP and PACT function primarily at the step of siRNA production. My findings indicate a direct interaction between human TRBP and PACT that has a role in facilitating the cleavage of shRNA to siRNA by Dicer. This work significantly alters the model for assembly and function of Dicer-containing complex that generates siRNA and miRNA in human cells. Several cellular and viral RNAs and proteins are known to function in protein translation and/or RNAi by interacting with TRBP or PACT. Influenza A virus NS1 protein is an inhibitor of PKR and has recently been shown to suppress RNAi in plants and Drosophila. NS1 has also been thought to inhibit RNAi in mammals as well as being an antagonist of interferon. In the second part of my thesis, I explore whether influenza NS1 might fulfill its role as an RNAi inhibitor by binding to PACT. While I can verify the binding of NS1 with PACT in vitro, I am unable to observe the suppressive effect of NS1 on RNAi in cultured human cells. I show that transiently or stably expressed NS1 from influenza virus strain A/WSN/33 is fully competent to inhibit the interferon pathway, but it does not suppress RNAi-mediated silencing of different reporter genes. These findings imply a significant difference in RNAi mechanism between mammals and plants. The biological significance of the interaction between PACT and NS1 remains to be elucidated. (An abstract of 499 words) DOI: 10.5353/th