Mapping RNA Binding Protein Interactions Within Cellular Microenvironments

PROJECT SUMMARY RNA binding proteins (RBPs) play critical roles in all aspects of RNA metabolism, with significant implications in physiological and pathological settings. A crucial factor in these processes is the exact location of these RBP:RNA interactions within the cell. These interactions are pivotal when cells adapt to stress and are often dysregulated in disease. Our current tools fall short in identifying RBP targets with subcellular resolution. This proposal introduces a groundbreaking approach to bridge this knowledge gap. We aim to develop and apply innovative tools designed to capture subcellular information for individual RBP:RNA binding events at a systems scale within living cells. Using cellular models that mimic halted protein synthesis, we aim to build a comprehensive spatial map of RBP:RNA interactions, shedding light on the remodeling of these networks during changes in post-transcriptional and translational control. At the level of individual RBPs, we will explore the temporal sequence of events by which specific RBPs regulate the localization, stability, and translation of their target RNAs, especially during stress-induced relocalizations. Using cutting-edge proximity labeling and isolation techniques, we will map RBP targets across subcellular space and integrate them within the broader landscape of cellular RNA networks and interactions. These data will be analyzed using state-of-the-art computational approaches and integrated with other high-throughput datasets such as Riboseq and RNAseq. This integrative analysis will enable us to construct and iteratively test predictive models of RBP activity based on their spatial distribution. Building off of this, we will apply these innovative methods to discern RBP:RNA regulatory programs in difficult to isolate membrane-less organelles, using static and oscillating mammalian stress granules (SGs) as our investigative platform. Overall, this proposal lays the foundation for an advanced understanding of RBP models that incorporate subcellular location as a critical determinant of their functions. Our comprehensive approach, bridging experimental and computational analyses, promises to uncover novel mechanisms of RBP action during changing cell states, providing insights that may guide future therapeutic strategies against an array of RBP-associated diseases that impact human health.