Riboswitches and their application to RNA visualization and transcription factor interactions with the transcriptome - Project Summary A broadly distributed means of bacterial gene regulation is an mRNA element called a riboswitch. These cis-acting elements are found in the 5′-leader regions and regulate expression through their ability to directly bind a specific cellular metabolite with a highly structured receptor called the aptamer domain. Metabolite binding is then communicated to a downstream secondary structural switch in the expression platform that instructs the expression machinery. Genes that are essential for survival or virulence are regulated by riboswitches in numerous medically important pathogens, making riboswitches attractive and novel targets for antimicrobial therapeutics. A long-term goal of my Research Program is to develop a molecular understanding of how mRNA interacts with small molecules and how these interactions drive biological processes. These overarching questions include: (1) How does RNA create small molecule binding pockets that recognize a spectrum of compounds with high affinity and varying degrees of specificity? (2) How can structural plasticity be exploited to by RNA-targeting compounds? (3) What are the mechanisms by which ligand binding drive changes in RNA structure to effect gene regulation? To address these questions, we will use a combination of structural, biophysical, and biochemical approaches as well as in vitro and cell-based functional assays. As our Research Program has developed a detailed understanding of RNA-small molecule interactions, we have begun to leverage this knowledge towards the design of research tools that facilitate basic research and therapeutic application. In this proposal, we are developing novel platforms for tagging RNAs with fluorophores for imaging in live mammalian cells, removing a technical barrier that has limited the study of RNA function. Specifically, we hypothesize that the robust folding and ligand binding properties of riboswitch aptamer domains will provide superior imaging performance over in vitro selected aptamers in cells. Thus, we are developing a set of riboswitch-based tags that bind modular chemical probes and a set of approaches to assess and benchmark their performance in mammalian cells. Recently, we launched a new direction in our Research Program to investigate how classical transcription factors (TFs) interact with the transcriptome as part of their regulatory function. We demonstrated that Sox2, a pioneer TF critical for maintenance of pluripotency and neural differentiation, directly interacts with RNA in mouse embryonic stem cells and binds various RNAs in vitro with affinities that rival its consensus DNA promoter site. We observed similar RNA interactions for other Sox and TCF/LEF family proteins, suggesting that RNA binding may be a general property of HMGB proteins that is critically linked to their function. In this proposal, we seek to extend these initial findings to address a set of key questions: (1) What is the full spectrum of RNAs bound by Sox2? (2) How does RNA binding impact chromosomal localization? (3) What is the structural basis for RNA recognition by HMGB proteins?
