The long-term goal of the PI’s research program is to understand how RNAs exert their regulatory effects through interaction with RNA-binding proteins (RBPs) and identify the basis for their therapeutic effects in human diseases. RBPs interact with RNAs to form ribonucleoprotein complexes that that control the fate of nearly every transcript. These roles are essential for normal human physiology, as defects in RBP function are implicated in autoimmunity, neurodegeneration, cancer, and innate antiviral immune response. The molecular mechanisms of RNA-protein interactions (RPIs) that underpin gene expression and form the basis of numerous human diseases remain obscure. Consequently, RBPs are rapidly emerging as promising targets for the development of chemical probes and candidate therapeutic agents. The discovery of therapeutically tractable RPIs, however, remains a formidable challenge. This proposal describes an interdisciplinary program leveraging quantitative chemoproteomics, natural product synthesis, molecular biology, and biochemistry to exploit reactivity of proteinaceous lysines at the RPI interface that modulate the structural properties of ribonucleoprotein complexes. Preliminary findings from the PI demonstrate that phloroglucinol meroterpenoid natural products modulate RPIs through reversible-covalent modification of RNA-binding lysines in RBPs. The central hypothesis of this proposal is that high occurrence of reactive lysines at the RPI interface may provide an unprecedented opportunity in the development of precision chemical tools with specificity for RBPs and on-target potency gained through reversible-covalent modification. The proposed research program embodies three integrated projects that address specific challenges in targeting RBPs in human cells. First, the lack of traditional enzymatic pockets or functional epitopes renders RBPs as canonically intractable targets. This proposal aims to overcome this challenge through reversible-covalent modification of RNA-binding lysines at the RPI interface using phloroglucinol meroterpenoids. Second, the full complement of lysines with heightened reactivity at the RPI interface remains unexplored. This proposal aims to overcome this limitation by using innovative chemoproteomic methods to globally map lysine reactivity and ligandability directly in human cells. Last, the functional consequences of meroterpenoid-lysine interactions remain obscure. By leveraging integrative RNA pulldown and extraction methods with RNA-interactome capture and immunofluorescence imaging, the proposal aims to identify advanced tool compounds that perturb RPIs through site-selective modification of lysines in RBPs. Overall, the proposed research program is significant because over the next five years it will deliver innovative chemical tools and proteomic methods that enrich understanding of the pathophysiological functions of RPIs and identify the basis for their therapeutic effects in human diseases. Thi...