Project summary / Abstract The forefront of current RNA research centers on understanding the amazing complexity of this molecule. There is an expanding appreciation that the roles of RNA are multifaceted and extend far beyond its coding potential. RNA has the capacity for diverse structures, numerous modifications, and combinatorial interactions, and thus RNA is a foundational component for many complex regulatory networks. It is crucial to understand these networks from the level of individual molecular RNA interactions up through their effects on disease and development. In particular, many chromatin-associated proteins (CAPs) additionally interact with RNA, and may function in regulating gene expression by way of these RNA interactions. Previous studies, limited to identifying the RNAs bound by individual CAPs, did not consider combinatorial RNA binding. Here, I reveal that the CAP- RNA network contains clusters of RNAs that bind multiple proteins. Notably, Polycomb group and Trithorax group CAPs (EZH2 and WDR5, respectively) seem to interact with similar RNAs despite their opposing roles in chromatin modification. This suggests that EZH2- and WDR5- RNA interactions may affect or counter one another, and that this could manifest in altered chromatin states. I aim to elucidate how different CAP-RNA interactions affect one another and to characterize the relationship among RNA-binding of multiple CAPs. I hypothesize that RNA contains multiple elements, or modules, that interact with specific proteins, and that the functional readout of combinatorial interactions in a network can be understood by modular design principles of RNA. I propose the following aims to investigate this idea, particularly focusing on EZH2- and WDR5- RNA interactions: (1) development of a massively-parallel RNA assay (MPRNA) to identify RNA elements that bind to a particular protein, (2) investigating the modular principle using combinatorial synthesis of multiple RNA elements, (3) uncovering the underlying mechanism of RNA element organization and protein interaction. This approach will establish an interdisciplinary framework for studying RNA-centric networks, using both experimental and computational methods. It will reveal interaction principles and mechanisms of protein-RNA interactions, and examine functional consequences of these interactions. It will increase our understanding of RNA-based regulatory networks, particularly in the context of chromatin and CAP-mediated gene regulation. It may even provide insight into the control of Polycomb- and Trithorax- group complexes at sites of bivalent chromatin.