PROJECT SUMMARY Eukaryotic cells contain within them a myriad of spatially distinct sites that serve a variety of functions. To facilitate this organization, eukaryotic gene expression is routinely spatially regulated through the trafficking and sequestration of thousands of different RNA molecules to distinct cellular locations. Misregulation of this process leads to detrimental phenotypes in a wide range of systems, from developmental defects in Drosophila to neurological disease in humans. Despite this importance, our knowledge of the regulation of RNA localization is quite limited. For other modes of post-transcriptional regulation like splicing, our understanding of how the interactions of RNA binding proteins (RBPs) and RNA motifs lead to specific outcomes is much more mature. This relies on many years of work by many groups that have defined the regulatory language of splicing and allows us to make predictive and combinatorial models about how splicing is regulated across conditions and cellular environments. We lack such an ability with regards to RNA localization, in large part because we lack the analogous “parts list” that defines the language of localization regulation. The overall goal of the Taliaferro lab is to learn this language and be able to build predictive models that explain how RNAs are trafficked to their intracellular destinations. This includes identification of sequences within RNAs that govern their transport as well as RNA-binding proteins that bind these sequences and mediate the process. Historically, these features have been identified for one RNA at a time through a time consuming and laborious process. Although these studies form the foundation of the RNA localization field, the amount of effort they require has limited their application to a large number of RNAs. Further, it is often hard to take findings from these single-transcript experiments and generalize them into larger principles that underlie rules that govern the transport of thousands of RNAs. The Taliaferro lab aims to approach this problem from the opposite direction by isolating subcellular transcriptomes and identifying features shared in common among RNAs localized to a particular destination. This strategy identifies elements associated with the transport of hundreds of RNAs at once, parallelizing the one-transcript-at-a-time approaches used in the past. During the course of the proposed experiments, over the next five years, the Taliaferro lab will use a variety of high-throughput approaches to both discover new sequence elements that regulate RNA localization as well as deeply characterize previously identified elements in order to discern how they work. Additionally, we will develop new technologies that will facilitate the definition of RNA localization mechanisms in organisms and cell types that thus far have been intractable. We expect that results from these studies will be a positive step toward our goal of understanding and eventual...