Aspects of Eukaryotic Transfer RNA Structurome, Interactome, and Expression

Transfer ribonucleic acid (tRNA) is an essential molecule for protein translation across kingdoms of life. Canonically, tRNA is responsible for carrying the amino acid to the ribosome during translation. Of all the RNA families, tRNA has the greatest number of nucleotides that are modified with post translational chemical modifications. Recently, it has been shown that these chemical modifications can significantly regulate expression, mediate RNA-protein interactions, and modulate RNA structure. To further study these specific aspects of eukaryotic tRNA, I and fellow colleagues conducted several studies, and the results of these studies will be presented in this thesis. In the Chapter 1 and Chapter 2, I will discuss the results of an analysis using a newly developed DM-DMS-MaPseq to determine the interactome and structome of chromosomally and mitochondrially encoded tRNA in vivo. This analysis provides the first in vivo evidence that human EF1A, an important protein for elongation and protein synthesis, interacts with tRNA and amino acids with the same interaction paradigm as EF-Tu. Using arsenite stress, we observed distinct structerome and interactome responses by the cytosolic and mitochondrial translation systems. In Chapter 3, we studied the selective packaging of host RNA in SARS-CoV-2 viral particles using RNA sequencing. We found six anticodon families of tRNA was selectively packaged during SARS-CoV-2 viral particle formation in addition to other small RNA such as Y RNA. We found evidence that packaging of specific tRNA isodecoders was modification and sequence dependent as well, suggesting that a specific sequences or modifications may be used as identity elements for selective packaging. In Chapter 4, we validated the expression of tRNA from a synthetic tRNA chromosome in yeast. During this study, we determined that the synthetic tRNA chromosome did not negatively impact endogenous tRNA expression.