PROJECT SUMMARY/ABSTRACT A major mechanism of post-transcriptional gene expression regulation occurs through the binding of RNA transcripts with RNA-binding proteins (RBPs), a large class of cellular proteins consisting of over 1500 members. Identifying the native substrates of RBPs and characterizing the function of these interactions remains a significant challenge in biology. Further, oligonucleotides and small molecules that target RNA-protein interactions and modulate protein expression have been recently deployed in the clinic and promise to provide a new therapeutic modality for the treatment of human diseases. We have developed TRIBE-ID, an RNA editing approach to interrogate dynamic RNA-protein interactions in a small molecule-dependent manner. We applied our approach to profile the RNA substrates of the RBP G3BP1, a central component of stress granule condensates. Here, we propose to further develop and apply TRIBE-ID as a general platform to investigate RNA-binding events in cells. We will advance the efficiency and generality of RNA editing for interactomic studies and investigate the trafficking and post-transcriptional regulation of RNA stability/translation by cytoplasmic biomolecular condensates. Further, we will develop RNA editing approaches to investigate small-molecule RNA interactions. Our project has the following specific aims: Aim 1. Develop an RNA editing platform with improved generality, efficiency, and molecular resolution. Aim 2: Characterize regulation of RNA stability/translation by biomolecular condensate-forming RBPs. Aim 3: Develop an RNA editing approach to investigate RNA-binding small molecule therapeutics. Our findings will provide new approaches for studying dynamic RNA-protein interactions and RNA-binding small molecules and reveal new insights into the formation and function of phase-separated biomolecular condensates. These studies should advance our understanding of fundamental RNA regulatory mechanisms and accelerate the development and characterization of RNA-targeted therapeutics.