Elucidating the function of ribonucleoprotein condensates in living cells - PROJECT SUMMARY Ribonucleoprotein complexes (RNPs) are essential for cellular function and their dysregulation has been implicated in various cancers and neurological disorders. These complexes are dynamic assemblies formed by interactions between RNAs and RNA-binding proteins (RBPs), which regulate numerous fundamental processes, including RNA biogenesis, RNA editing, stress response, and genome stability maintenance. Recent research has shown that RNPs can form membraneless organelle condensates through liquid-liquid phase separation for their functional output. However, the molecular mechanisms underlying the formation, disassembly, and regulation of these RNP condensates are poorly understood. The overall goal of my research program is to elucidate how RNP condensates contribute to cellular function and disease pathogenesis. We develop and apply novel methods to unravel the dynamics and composition of RNP condensates. We recently developed a new approach for readily tracking RNA dynamics in living cells and a novel method for identifying the RBPs of modular RNA motifs within RNPs. In this application, we propose to utilize and further expand these methods to address three major challenges in RNP condensate research, including: (1) Accurately identifying RBP composition of RNPs ─ A major challenge in understanding RNP condensate formation and regulation is a lack of robust methods to accurately identify the RBPs that trigger these changes. This proposal aims to address this major problem by developing and using an RNA tag with ultrahigh affinity for accurately identifying RBPs in RNPs, thus accelerating our understanding in the composition of RNP condensates; (2) Artifact-free characterizing RBP function in RNPs ─ Dynamic RBP-RNA interactions are essential for the formation and disassembly of RNP condensates. Being able to selectively induce and disrupt these interactions are critical to dissect the functional role of RBPs. However, current methods for RBP characterization lack temporal control and are prone to overexpression artifact. This proposal aims to address this limitation by using a small molecule- regulated RNA-protein tethering approach to functionally characterize RBPs in living cells; (3) Perturbation- free tracking RNP dynamics ─ A major question in RNP research is to understand how intermolecular RNA- RNA interactions contribute to condensate assemblies in response to cellular cues. This proposal seeks to address this longstanding question by applying a fluorogenic RNA imaging system to understand how long noncoding RNA-mRNA interactions contribute to RNP condensate formation during stress response. Together, the proposed research will provide an unprecedented view of the composition and dynamics of RNPs, thus laying the foundation for uncovering novel mechanism governing health and disease.
