Investigating Evolutionary Constraints on Regulatory RNA

One of RNAs chief biological functions is control of gene expression. However unlike RNAs roles in informationtransfer (e.g. mRNA rRNA tRNA) the mechanisms by which RNA functions to regulate gene expression arediverse across the tree of life and show evidence of recent and ongoing evolution. Despite the important roleplayed by RNA in gene regulation very little is known about how such mechanisms arise the selective pressuresthat maintain them in genomes or the biophysical constraints that underly their functional evolution. Bacterialcis-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 inturn alter downstream gene expression. Furthermore bacterial RNA regulators employ diverse mechanisms ofaction and display evidence of diverse evolutionary histories. In the study of protein evolution computationaltheoretical and experimental studies have connected biophysical understanding with observations fromcomparative genomics and laboratory experimentation yet these have only been applied in limited contexts tothe study of RNA. The goal of my research program is to understand how concepts developed in the context ofprotein evolution such as robustness plasticity promiscuity and epistasis apply to the evolution of RNAregulators. 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 mapthe relationships between RNA sequence regulatory functionality (including expression level and dynamic rangeas well as ligand sensitivity and specificity) and organism fitness both in vitro and within mouse infection models.In particular we anticipate separating the sequence function relationship into its component sequence function and function fitness mappings in order to understand both biophysical constraints of the RNA as wellas those that originate from the robustness of the organisms homeostatic mechanisms. We also plan to gobeyond characterization of the sequence fitness mapping to probe natural mechanisms for the creation andadaptation of RNA regulators in S. pneumoniae. Our work will enable connection of biophysical models of RNAfolding with functional regulatory parameters as well as a provide an appreciation for the degree to which fitnessis sensitive or robust to regulatory changes and how organisms adapt to deleterious regulatory changes. Byunderstanding both the sequence function and function fitness mappings we will not only start tounderstand the constraints on RNA regulator evolution by providing large datasets for training and testingbiophysical models but also inform development of therapeutics that target such RNAs the creation of syntheticRNA regulatory systems for biotechnological applications.