Date of Award

Spring 2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular, Cellular, and Developmental Biology

First Advisor

Breaker, Ronald

Abstract

Noncoding RNAs (ncRNAs) were previously overlooked by molecular biology. But their importance is being increasingly recognized in science. Between 1989 and 2020, seven scientists shared three Nobel Prizes for their discovery and work in ncRNAs. I suspect that this trend will pick up over the following decades. Of ncRNAs, riboswitches stand out for their versatility and prospects for future discovery. Bacterial riboswitches consist of an aptamer region and an expression platform. The aptamer binds a ligand, usually a signaling molecule, coenzyme, inorganic ion, nucleotide derivative, amino acid, or other metabolite. This then causes a conformational change in the downstream expression platform, controlling the nearby gene usually through transcriptional termination or translational initiation. This means that riboswitch discovery is important not only to learn more about the processes of transcription and translation, but also for their potential for driving the discovery of novel signaling molecules. Perhaps most pertinently, riboswitches can be manipulated as antibiotic targets. The exciting part about this is that the power law, to which most natural phenomena adhere, predicts that there are thousands of novel riboswitch classes yet to be discovered. In chapter 1, you will learn more about riboswitch and other ncRNA importance, diversity, and discovery. Here, you will be introduced to the Breaker lab’s GC-IGR pipeline, which is used to discover riboswitches and other ncRNAs. In chapter two, I dive into how I use the GC-IGR analysis pipeline to discover novel ncRNA in Baumannia cicadellinicola, as part of a paper analyzing five bacterial genomes. I paid special attention to the discovery of the corA riboswitch candidate. This finding was particularly intriguing given that B. cicadellinicola is an obligate endosymbiont. As such, it has a minimalist genome, and was not expected to have something as intricate as a riboswitch candidate. In chapter three, I expound on the eight riboswitch candidates I found in eight genomes that I analyzed as part of a recently submitted manuscript encompassing 26 genomes. This research was a significant expansion from the initial study covering only five genomes. In chapter four, I describe the experimental validation of the pnuC motif, which I discovered among the eight genomes noted above, and which serves as the second class of NAD+ riboswitches. The resulting paper provided additional validation for the GC-IGR pipeline, and the riboswitch candidates I found using it. Finally, in chapter five, I highlight top riboswitch, ribozyme, protein-binding, and high-ranking candidates from my analysis of 50 bacterial genomes. This study represents the largest pool of novel bacterial ncRNAs uncovered to date, and substantially expands the previous analyses using this pipeline. Perhaps most exciting about these findings is that nearly one novel riboswitch candidate was found per bacterial genome analyzed. Of course, these computational findings must be verified by experimental analysis. As such, all of these candidates may not turn out to be new riboswitch classes. But I am over 75% confident that the strong riboswitch candidates are indeed riboswitches, and over 50% confident that the weak riboswitch candidates are true riboswitches. With 44,270 bacterial genomes in RefSeq80, it is easy to see how the power law prediction of hundreds of novel riboswitch classes is possible. The future outlook for riboswitch discovery is extremely bright. In addition to the precise discovery capabilities of this powerful GC-IGR pipeline, more automated systems such as the DIMPL program offer promise to further speed up the process of discovering novel riboswitches and ncRNAs. The treasure map has been printed for future bioinformaticians and molecular biologists to use for future discoveries. I am excited about the prospects of the discovery and uses of future riboswitches. Hopefully, after reading this thesis, you will be too.

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