Date of Award

5-1-2022

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biological Sciences

Content Description

1 online resource (vii, 135 pages) : color illustrations.

Dissertation/Thesis Chair

Thomas J Begley

Committee Members

Morgan Sammons, Cara Pager, J. Andres Melendez, Scott Tenenbaum

Keywords

Arsenic, Detecting the epitranscriptome, Epitranscriptomic marks, Epitranscriptomics, Queuine/Queuosine, tRNA biology, Transfer RNA, Genetic transcription, Genetic regulation

Subject Categories

Biology

Abstract

Transfer ribonucleic acid (tRNA) is a non-coding RNA which interacts with the messenger RNA (mRNA) to facilitate protein synthesis. tRNA is also one of the most heavily modified RNAs with the collection of modifications, termed as the tRNA epitranscriptome, ranging from simple methylation to complex hypermodification catalyzed by RNA epitranscriptomic writers. Detecting tRNA modifications is an evolving field and the state-of-the-art technologies that include next generation sequencing and mass spectrometry of intact and derivatized RNAs is described in the chapter one. Liquid chromatography coupled with mass spectrometry has previously been used to show that arsenic promotes increases in the levels of three tRNA modifications, queuosine (Q), N6-methyl, N6-threonylcarbamoyladenosine (m6t6A) and 5-methoxycarbonylmethyluridine (mcm5U). The three tRNA modifications are added by three corresponding epitranscriptomic writers, Queuine tRNA ribosyltransferase (QTRT, Q), tRNA methyltransferase O (TRMO, m6t6A) and Alkylation repair homolog 8 (ALKBH8, mcm5U). In chapter 2, we show that epitranscriptomic writer knockdown cells have an altered growth phenotype, increased cellular reactive oxygen species (ROS) levels, and increased mitochondrial mass. When the epitranscriptomic writer knockdown cells were treated with arsenic, the cells have increased cellular reactive oxygen species (ROS) levels and decreased mitochondrial mass. Additionally, the epitranscriptomic writer knockdown cells have a uniform down-regulation in the genes for development and morphogenesis. The study, detailed in chapter two has demonstrated that writer deficiencies lead to global reprogramming of the transcriptome, which could be driven by changes in ROS. In addition, we have linked writers to mitochondrial physiology, providing insight into mitochondrial health and potentially diseases. Chapter 3 describes an exhaustive analysis of codon usage in human and mouse genomes, and links genes involved in stress responses and developmental programs to having distinct codon patterns. We highlight methodology to link codon usage patterns to distinct biological pathways, with application to Q, m6t6A, and mcm5U decoded codons. The analysis detailed in chapter three identifies codons bias in the genes and suggests a potential role of codon-bias in translational regulation. Additionally, the codon analysis points towards some conserved codon-usage patterns across humans and mice, supporting the idea of codon-dependent regulation of gene expression. My thesis further connects tRNA epitranscriptomic writers and codon usage to the cellular ROS response and mitochondrial physiology. Further, it highlights writers linked to the growing field of Environmental Epitranscriptomics and adds details to our understanding of codon-biased gene regulation.

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