Insights into the functions of RNA post-transcriptional modifications gained through studies in cellular stress
Creators: name: Baldridge, Kevin Charles
name: 0000-0003-1829-7714
Publisher: name: The University of Texas at Austin
Year: 2017
Subjects: Post transcriptional modification
Air pollution
Chemical RNA modification
RNA biology
Cellular stress
Genetic code expansion
Synthetic biology
Oxidative stress

description: In recent years, the explosion of knowledge regarding functional roles for RNA in biology have uncovered the need for detailed molecular understanding of the relationship between the chemistry of RNA and its function. Post-transcriptional modifications in RNA are changes to the chemistry of the four basic RNA nucleotides, which can be thought of as expansions to the genetic alphabet of RNA. By subtly changing the chemistry of the standard RNA nucleotides, post-transcriptional modifications (PTMs) can act to modulate RNA function through a variety of mechanisms ranging from altering the basic physicochemical properties of an RNA molecule to fine-tuning complex regulation processes in higher organisms such as humans. While a number of functions have been ascribed to RNA post-transcriptional modifications, we still lack a comprehensive picture of how subtle changes in RNA chemistry can alter cellular function. Therefore, my dissertation focuses on applying cellular stress models as a way to interrogate how RNA post-transcriptional modifications can modulate RNA function. First, I provide a thorough discussion of how ribosomal RNA methylations help Escherichia coli adapt to stressful environmental conditions by protecting the vital protein translation process. Furthermore, by performing a targeted meta-analysis of publicly available stress-induced gene expression data, I identify specific ribosomal RNA methylations which may be important for E. coli adaptation to particular stresses including oxidative, heat, and cold stresses. Next, I examined the question of how RNA chemistry is affected directly by an external stress using a model system for simulated air pollution exposure with human lung cell cultures to demonstrate that cellular RNA oxidation is a reliable consistent indicator of oxidative stress from air pollution exposure. Building upon this air pollution exposure model system, I further explored how chemical oxidation of RNAs can be specifically enriched in certain RNA transcripts under oxidative stress induced by air pollution exposure. This work highlights specific oxidation of mRNAs involved in regulatory pathways as a possible mechanism for air-pollution related disease, suggesting that accumulation of oxidized mRNAs might interfere with processing or gene regulation. Additionally, examination of specific RNA oxidation associated with pre- and post-stress differential gene expression after exposure to air pollution highlights the potential that the oxidized RNA base 8-oxoguanosine might function as an epitranscriptomic mark affecting transcription. Lastly, I characterized how RNA modification machinery can contribute to cellular stress associated with expression of engineered transfer RNAs for applications in expanding the genetic code in E. coli. Using a suite of suppressor tRNAs designed by directed evolution, I demonstrated that the evolved lower expression level minimizes the impact of stress caused by interactions with E. coli post-transcriptional modification machinery, resulting in high performing tRNAs that remain functional regardless of their interactions with host PTM machinery. Collectively, the studies described in this dissertation demonstrate the power of employing cellular stress models to interrogate functional roles of RNA post-transcriptional modifications and highlight the myriad roles that PTMs can play in responding to and contributing to cellular stress.