Investigating how inosine-containing dsRNA downregulates gene expression. (360G-Wellcome-084403_Z_07_Z)

We recently showed that I-dsRNA specifically binds a stress-complex , which comprises various stress granule (SG) components. Furthermore, we demonstrated that I-dsRNA in cells downregulates both endogenous and reporter gene expression, in trans. We provided evidence that this was the result of both reduced mRNA levels and impaired translation initiation. We therefore proposed a model in which hyper-editing of dsRNAs by ADARs results in downregulation of gene expression via SG formation. W e now plan to carry out experiments aimed at further understanding the proposed model, and to elucidate the mechanistic details. The I-dsRNA stress-complex will be further characterized. We will investigate whether SGs assemble in response to I-dsRNA, and which SG components are important for downregulating gene expression in the presence of I-dsRNA. We will explore the pathway by which I-dsRNA results in degradation of reporter mRNAs, and will identify the nuclease(s) responsible. The mechanism by which I-dsRNA inhibits translation initiation will also be investigated. We will analyze the effect of I-dsRNA on endogenous gene expression. Finally we will look at the potential relationship between ADARs and SGs. A key part of the model predicts that editing of an unknown substrate(s) by ADARs will lead to SG assembly.