A molecular-level understanding of regulatory networks has driven biomedical progress, but has mostly focused on protein networks. Non-coding RNAs (ncRNAs), including large ncRNAs (lncRNAs), also play important roles. For example, deletion of the Xist lncRNA causes cancers in mice. Yet the scope of lncRNA functions, their mechanisms of action, and their impact on therapeutically important pathways remain largely unknown. Approaches that identify high-stoichiometry or stable RNA-protein interactions have provided powerful insight into RNA, but miss important interactions, such as RNA helicase interactions with their substrates, that are transient or low stoichiometry. Similarly lncRNAs expressed at low abundance are important to regulation, yet the proteins they interact with are largely unknown. To address these needs, we are developing a platform to engineer covalent RNA modifications leading to new methodologies. First, the development of a covalent RNA tag will facilitate discovery of proteins that interact with low abundant lncRNAs. Second, this same platform is being used to develop a covalent-tracking strategy where a protein of interest is engineered to leave a covalent mark on transiently bound RNAs. This covalent modification will serve as a purification handle to isolate and identify RNAs that interacted with the protein of interest. As some lncRNAs are important regulators of chromatin, this approach will be applied to study RNAs that interact with specific chromatin proteins. These covalent-tagging strategies are designed to be broadly applicable, complement existing techniques, and accelerate our integration of regulatory RNAs into important regulatory networks.
Large non-coding RNAs and the proteins they interact with are important for human health and disease, but our understanding of the regulatory roles of RNA is still underdeveloped. By engineering new RNA-modifying enzymes, we will provide the tools necessary to integrate non-coding RNAs into networks of regulatory proteins. These projects will provide insight into specific RNA and RNA-binding proteins, and also establish general methodology to disseminate to assist the field connect RNAs with their roles in biomedically important regulation.