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?
