Non-coding RNAs (ncRNA) are transcripts produced by the cell that are not translated into proteins yet play a fundamental role in maintaining proper regulation and function of cellular activities. The term encompasses a myriad of different types of RNAs, due to the different properties these molecules can be categorized by, such as size, structure or function. In general, ncRNAs can be split into two major groups: housekeeping and regulatory. Housekeeping ncRNAs encompass RNAs that are necessary to maintain fundamental cellular functions such as translation, hence includes ribosomal RNA (rRNA) and transfer RNA (tRNA). For example, tRNAs range from 70 nucleotides (nt) to 95 nt and are constitutively expressed at high levels in all cell types. Regulatory ncRNAs are typically categorized based on their size, those shorter than 200 nt classified as small non-coding RNAs (sncRNA) and long non-coding RNAs (lncRNAs) for those larger than 200 nt. The mechanism in which these molecules exert their regulatory function in maintaining cellular functions varies wildly, but many are typically studied within the context of affecting transcriptional or translational events.

The advancement in high throughput sequencing technology has allowed more encompassing and intricate studies of ncRNAs, such as through the detection and discovery of more elusive types, in depth quantification and characterization of previously characterized species, and even the profiling of RNA specific properties such as modifications. The types and properties of ncRNAs can vary wildly in between different types of bacteria as adaptations to stressors and environments exerts selective pressure in maintaining specific profile of these ncRNAs. Using different bacteria, I leverage the power of high throughput sequencing technology to investigate two facets of ncRNAs in prokaryotes: profiling of tRNA and tRNA modifications in response to stress conditions, and unbiased discovery and characterization of small RNAs.

In Chapters 2 and 3, I investigate the N1-methyladenosine (m¹A) tRNA modification profile in Streptomyces venezuelae using advanced tRNA sequencing technology. This study documents the dynamic nature of the m¹A tRNA signature in a mesophilic bacterium and how it responds to changes in the environment. Moreover, this investigation leads to the discovery and characterization of the first prokaryotic RNA modification eraser enzyme, one of two necessary maintenance proteins required for proper regulation of tRNA methylation levels. 

In Chapters 4 and 5, I continue the investigation of ncRNAs in Bacteroides fragilis, a commensal human gut bacteria capable of causing opportunistic infection. Using high throughput RNA sequencing technology, we document changes in the tRNA profiles for this bacterium in response to oxygen stress. Additionally, I utilize the same sequencing data as a case study for an unbiased approach in the identification and profiling of ncRNAs that can be applied to other organisms which represents a heuristic approach to screen potential ncRNAs of interest.