Investigating the Contributions of Staufen Domains to Granule Formation

Abstract

RNA-binding proteins (RBPs) and their target RNAs assemble to form microscopic RNA granules. These subcellular compartments act as hubs of post-transcriptional regulation, coordinating the localization, translation, and degradation of RNA. One component commonly found in granules is Staufen (Stau), a conserved RBP with an intrinsically disordered region (IDR) and five double-stranded RNA-binding domains (dsRBDs)—one of which (dsRBD5) is noncanonical because it lacks RNA-binding ability. While roles of Stau in regulating development, neurogenesis, and synaptic plasticity have been established, the ability of Stau to form granules as a result of its sequence and structure has yet to be evaluated. This thesis sought to understand the role of Stau dsRBDs and the Stau IDR in granule formation. I established an assay for Stau-mediated granule formation in Drosophila Schneider (S2R+) cells and used this assay to test the ability of GFP-Stau proteins containing single deletions at the IDR and each of the dsRBDs to form granules. Among these domains, only dsRBD5 is necessary for Stau granule formation. DNA analysis and western blotting confirmed that the muGFP-StauΔdsRBD5 construct and protein contained the correct deletion, and a novel granule quantification approach demonstrated a significant lack of granules for this protein. I further demonstrated that dsRBD5 is sufficient among the dsRBDs to form granules because a deletion of a region containing the first four dsRBDs did not diminish granule formation. Cells with a construct containing only dsRBD5 fused to GFP produced granules, albeit variably, indicating that isolated dsRBD5 is capable of forming granules. This thesis demonstrates an important function of noncanonical dsRBDs in forming granules, broadening our knowledge of how dsRBDs contribute to granule assembly, and ultimately leading to advancements in treating pathological granules.