RNA molecules play special roles in cells because they encode information both in the linear sequence of nucleobases (abbreviated A, C, G, and U) that make up an RNA strand and by forming intra-strand interactions. These intra-strand interactions result in "secondary" structures stabilized primarily by Watson-Crick base pairing, which can now be readily measured in many cases, and "tertiary" structures that result in complex and compact three-dimensional shapes, which have been very difficult to measure. Measuring tertiary structures is important, however, as they affect how RNA molecules function in cells, including in synthesis of proteins, in regulation of how much protein is made from a particular gene, and in other important tasks. Until recently, it was nearly impossible to detect most RNA tertiary structures, especially in living cells. Through this project, a new technology will be created to identify sites of RNA tertiary structures. Within the context of this project, this technology will be used to understand how tertiary structures in RNA molecules influence their functions in human cells. This project also incorporates undergraduate researchers with the goal of inspiring these students to become informed about how research creates knowledge and enriches our understanding of cellular processes, both of which are important for scientific advances, economic development, and innovations in technology and quality of life. Novel RNA pockets will be explored through a completely undergraduate-driven research endeavor, the Undergraduate Transcriptome Project. Both undergraduate and graduate students who participate in this research are expected to achieve leadership roles in STEM fields in industry and academics.
The Intellectual Merit and overarching vision of this research project is to create a rigorous, easily implemented technology that specifically detects true higher order tertiary structures in large RNAs in living cells. Although it is clear that many RNAs form complex structures and that these structures have important consequences for cellular function, there are only a handful of known distinct classes of motifs that form true tertiary structures. By discovering RNA tertiary structures in living cells, this project will define numerous new functions of RNA molecules. This research project will use a recently developed chemical reagent that specifically detects higher order tertiary interactions in RNA molecules. These conformations only occur at sites where multiple RNA helices pack together to create an electronegative RNA pocket. These rare but highly diagnostic sites are detected efficiently by the small, positively charged reagent, revealing sites of tertiary interactions (termed T-sites). This technology will be used to examine T-sites across the human transcriptome in living cells and to understand a preliminary finding that relationships exist between T-sites and translational regulation. The technology will be advanced to the point where it can be widely used by non-experts. Results of this work are expected to be multi-fold. First, T-sites are expected to be strongly correlated with nexuses of gene regulation. Second, many new classes of RNA motifs will be discovered, substantially extending the important, but relatively few, classes of RNA tertiary structures currently known. Third, subsequent biophysical and functional studies will support numerous new discoveries regarding the fundamental principles of RNA structure and folding and their impacts on processes that regulate gene expression. This project is supported by the Molecular Biophysics and Genetic Mechanism Clusters of the Molecular and Cellular Biosciences Division in the Biological Sciences Directorate.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
