RNA-Protein Interactions Guided by and Identified with RNA Modifications

Abstract

Binding between protein and ribonucleic acid molecules leads to life-sustaining processes, and identification of these interactions is crucial to understand their biological functions. Over 1,000 proteins in human genome have been annotated asRNA-binding proteins (RBPs), and they regulate almost all aspects of life cycle of RNA molecules. However, many RNA-protein interactions are difficult to capture due to their transient and dynamic nature. Moreover, post-transcriptional modifications on RNA add another layer of regulation in RNA-protein binding, further complicating the holistic view of cellular binding events. Therefore, to fully elucidate their biological functions, RBPs and their binding events need to be detected and characterized with high sensitivity and in various contexts. Here, we characterize the effects of RNA modifications, RBP, and cellular localization on RNA-protein interactions. First, we discover and characterize RBPs that specifically recognize m1A-modified RNAs with chemical proteomics method and biochemical studies. We show that m1A recognition by one of the “reader” RBPs leads to destabilization of modified RNA molecules in living cells. Next, we present a facile and quantitative method to identify RNA-protein interactions with temporal control achieved by RNA deamination modification and chemically induced dimerization. We show that this method can detect changes in RNA-protein interactions during stress granule formation and small molecule-mediated inhibition of RBP-RNA binding. We next directly capture and measure RNA modifications in various cellular condensates via proximity labeling, and we show the presence of non-coding RNAs in stress granules. Finally, we discover small molecule modulators of stress granule formation. Collectively, we develop and apply multiple methods to systematically characterize RNA-protein interactions and their biological consequences.