SMALL RNA-MEDIATED REGULATION OF THE OXIDATIVE STRESS RESPONSE IN HALOARCHAEA

Small RNAs (sRNAs), a type of non-coding RNA, are ubiquitously found in the three domains of life where they play large-scale roles in gene regulation. While a substantial body of experimental work has been done to uncover function of sRNAs in Bacteria and Eukarya, the functional roles of sRNAs in Archaea are still poorly understood. Archaea are often found in the most extreme environments on Earth, suggesting that they have evolved unique adaptions to respond to these stressful conditions. Archaea remain understudied in their regulatory processes of the central dogma, which is a hybrid system between Bacteria and Eukarya together with features unique to the Archaea. The goal of this thesis was to characterize sRNAs and elucidate their regulatory mechanisms in response to oxidative stress in the model extremophilic archaeon, Haloferax volcanii. Oxidative stress was investigated because it is a universal environmental stressor that produces robust phenotypes. Using high-throughput RNA sequencing, I first characterized the transcriptional landscape of H. volcanii during oxidative stress. The oxidative stress response included upregulation of redox homeostasis, protein turnover, and DNA repair genes, and down regulation of metabolic genes. I also discovered thousands of novel sRNAs, comprising 25 to 30% of the total H. volcanii transcriptome, and found that hundreds of these sRNAs were differentially expressed during oxidative stress. Next, using ribosome profiling, I revealed the translational landscape, for the first time in H. volcanii, allowing for the validation of previously identified sRNAs. In addition, I discovered many novel translation start sites that may produce functionally important proteins during oxidative stress. Third, I elucidated the mechanism of action of the most up-regulated sRNA during oxidative stress in H. volcanii and its functional role in survival. Finally, I developed and applied a new sRNA annotation pipeline to an extremophilic microbial community inhabiting halite (salt) rocks in the Atacama Desert, Chile. I discovered sRNAs expressed from diverse microorganisms and provided evidence for their functional relevance in the natural environment. The implications of this thesis work are that large-scale gene regulation is essential for Archaea to thrive in extreme environments and that sRNAs play key roles in those regulatory processes