RNAs fold into intricate three-dimensional (3D) structures and undergo conformational changes to perform a myriad of essential biological functions. As such, an important goal for biomedical research it to understand RNA folding, dissect the dynamic conformational landscape of functional RNAs, and ultimately use this knowledge to understand RNA biology. Despite decades of research and significant progress, we are far from a predictive understanding of RNA folding and 3D conformation, stemming from the small number of RNA 3D structures solved to date and the difficulty of structurally characterizing dynamic functional RNAs using traditional methods. For example, X-ray crystallography is a powerful tool that provides atomic level information but is limited to samples that are conformationally homogeneous. NMR can solve 3D structures of dynamic RNAs but is limited to small specimens that typically fall below the size of fully functional RNAs. Thus, the scientific premise that underlays this proposal is the critical need for a better understanding of dynamic functional RNA structures, which can be gained by applying the powerful method of cryo-EM. Recent developments have turned cryo-EM into a premier structural biology tool and allow visualization of distinct conformational states under a variety of solution conditions, potentially to high resolution. As such, cryo-EM promises 3D assessment of RNA folding and conformational landscapes in a way not previously possible. However, cryo-EM studies of discrete folded RNAs are sparse and most studies have focused on large RNA- protein complexes or on a single conformational state. I propose to develop a methodology to employ cryo-EM for the study of RNA folding and for the dissection of dynamic RNA 3D conformations. To accomplish this, I will use cryo-EM, biochemistry, and chemical probing to answer long-standing questions about two canonical RNA model systems: group I introns and viral tRNA-like structures (TLS). These RNAs reflect diversity in function, size, amount of prior knowledge, dynamics, and predicted architecture and will test the applicability of cryo-EM to solving diverse RNA biological questions. Thus, my studies will have a double impact: They will answer important questions about these functional RNAs, while providing new techniques for the study of RNA folding and dynamic RNA conformations. Given the diversity of functional RNAs in nature, the ability to directly visualize dynamic RNA 3D conformations may spark an explosion in newly solved 3D structures and in our predictive understanding of RNA folding and conformational dynamics.
RNA molecules fold into complex three-dimensional (3D) structures to perform essential functions in the cell and, therefore, an important goal for biomedical research is to understand the RNA folding process, visualize the 3D structure of biologically important RNAs, and ultimately use this knowledge to understand RNA function in biology, health, and disease. Despite significant progress, we still lack a predictive understanding of RNA folding and 3D structure, stemming from the difficulty of visualizing dynamic RNA structures using traditional experimental techniques. I propose to test the applicability of cryo-EM towards the study of RNA folding and dynamic RNA structures by answering long-standing questions about two diverse types of functional RNAs: catalytic self-splicing group I introns, and viral tRNA-like structures.
