Systematic evaluation of competition between dsRNA binding proteins - PROJECT SUMMARY Double-stranded RNAs (dsRNAs) are ancient molecules with essential roles in early and modern life. The proteins that bind dsRNA, dsRNA binding proteins (dsRBPs), are ubiquitous and crucial for various cellular functions, including RNA processing, localization, translation, stress responses, and sensing of viral infection. While dsRBPs have many essential and diverse functions, the RNAs they bind are often similar. This along with the general inability of dsRBPs to recognize specific RNA sequences makes it likely that many dsRBPs compete with one another. This competition is important for normal homeostasis, as changes to the expression of individual dsRBPs can alter the competitive balance, and in some cases cause activation of dsRNA sensors and cell death. For human cells that express dozens of dsRBPs and orders of magnitude more dsRNAs, controlling competition between dsRBPs is major regulatory challenge. Although there are examples of competition between dsRBPs in the literature, the mechanisms that regulate competition are not fully understood. Given the diverse and essential functions of dsRBPs, understanding how competition between them is controlled could inform our understanding of multiple aspects of human health, and may lead to new therapies to treat diseases caused by dysregulation of dsRBPs and/or dsRNAs. The cell likely uses several mechanisms to regulate competition between dsRBPs, for instance changing the abundance of specific dsRBPs or dsRNAs, or altering the localization of dsRBPs or dsRNAs. Furthermore, intrinsic differences between dsRBPs, like their affinity for dsRNA or specificity for structures like mis-matches, bulges and loops within dsRNAs, may also influence competition. However, information regarding the affinity, specificity, interactomes and localization of many dsRBPs is not known. Additionally, there are likely undiscovered dsRNAs and dsRBPs. Without those pieces of information, it is impossible to understand how global competition between dsRBPs is regulated, which makes it hard to develop therapies that target dsRBPs to treat disease, or interventions to correct dysregulation of competition caused by mutations in dsRBPs. To address these essential knowledge gaps, our laboratory will use a systematic approach with a combination of low and high-throughput methods to study competition between dsRBPs from multiple angles. We will map dsRNA-dsRBP interaction networks with subcellular resolution and evaluate the affinity and specificities of dsRNA binding domains and dsRBPs. For specific dsRBPs we will study how their localization to specific cellular compartments is regulated, and elucidate their function within those compartments. The information generated through this work will fill in the existing knowledge gaps, and eventually provide the means to model competition between dsRBPs.
