Investigating Evolutionary Constraints on Regulatory RNA - Project Summary/Abstract One of RNA’s chief biological functions is control of gene expression. However, unlike RNA’s roles in information transfer (e.g., mRNA, rRNA, tRNA), the mechanisms by which RNA functions to regulate gene expression are diverse across the tree of life and show evidence of recent and ongoing evolution. Despite the important role played by RNA in gene regulation, very little is known about how such mechanisms arise, the selective pressures that maintain them in genomes, or the biophysical constraints that underly their functional evolution. Bacterial cis-regulatory RNAs provide a platform to examine the parameters influencing the evolution of regulatory RNA. Such RNAs consist of complex structural domains that induce ligand-dependent RNA folding changes, which in turn alter downstream gene expression. Furthermore, bacterial RNA regulators employ diverse mechanisms of action, and display evidence of diverse evolutionary histories. In the study of protein evolution, computational, theoretical, and experimental studies have connected biophysical understanding with observations from comparative genomics and laboratory experimentation, yet these have only been applied in limited contexts to the study of RNA. The goal of my research program is to understand how concepts developed in the context of protein evolution such as robustness, plasticity, promiscuity and epistasis apply to the evolution of RNA regulators. We recently published a study quantifying the impact of a series of RNA cis-regulators on S. pneumoniae fitness in culture and in vivo. In the next five years we will leverage this series of regulators to map the relationships between RNA sequence, regulatory functionality (including expression level and dynamic range, as well as ligand sensitivity and specificity) and organism fitness both in vitro and within mouse infection models. In particular, we anticipate separating the sequence à function relationship into its component sequence à function and function à fitness mappings in order to understand both biophysical constraints of the RNA, as well as those that originate from the robustness of the organism’s homeostatic mechanisms. We also plan to go beyond characterization of the sequence à fitness mapping to probe natural mechanisms for the creation, and adaptation, of RNA regulators in S. pneumoniae. Our work will enable connection of biophysical models of RNA folding with functional regulatory parameters, as well as a provide an appreciation for the degree to which fitness is sensitive or robust to regulatory changes, and how organisms adapt to deleterious regulatory changes. By understanding both the sequence à function and functionà fitness mappings, we will not only start to understand the constraints on RNA regulator evolution by providing large datasets for training and testing biophysical models, but also inform development of therapeutics that target such RNAs, the creation of synthetic RNA regulatory systems for biotechnological applications.
