Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Biochemistry

Supervisor

O'Donoghue, Patrick

Abstract

Transfer RNAs (tRNAs) are essential for protein synthesis and translation fidelity. Some human tRNA variants may cause amino acid misincorporation: tRNAGly variants (tRNAGlyCCC, tRNAGlyGCC) have mutations that generate an alanine tRNA identity element (G3:U70), likely causing mis-aminoacylation of glycine tRNAs with alanine, while the tRNAAlaAGC G35C (tRNAAlaACC) variant may function similarly to mis-incorporate Ala at Gly codons by generating a Gly anticodon. I propose that these mistranslating tRNAs will disrupt protein homeostasis in mammalian cells. Although the tRNAGly and tRNAAla variants did not affect protein synthesis in normal growth conditions, tRNAGlyGCC A3G depressed protein synthesis following proteasome inhibition. Mass spectrometry confirmed Ala mistranslation at multiple Gly codons caused by the tRNAGlyGCC A3G and tRNAAlaAGC G35C variants. Multiple mistranslation events were also observed within the same peptide. These data reveal mistranslation of Ala at Gly codons that is caused by natural human tRNA variants and tolerated under normal conditions.

Summary for Lay Audience

Transfer RNAs (tRNAs) are responsible for bringing the correct amino acid to the site of protein production in cells. Each amino acid has its own set of tRNAs and an enzyme, called an aminoacyl-tRNA synthetase (aaRS) that specifically recognizes both the tRNA and amino acid to link them together. aaRS recognize tRNAs through sequences unique to tRNAs for that amino acid. Most tRNAs are recognized by their anticodon, a sequence in the tRNA that determines which amino acid goes where in the protein and is complementary to a codon or set of codons found in the messenger RNA (mRNA). However, some aaRS recognize features of the tRNA outside the anticodon. For example, to attach the amino acid alanine to its tRNAs, the alanine-aaRS recognizes the tRNA solely by the presence of a unique G3:U70 base pair close to the site of amino acid attachment in the tRNA and does not recognize the anticodon at all. For tRNAs that carry glycine, the anticodon is essential for attaching the amino acid to glycine tRNAs. Specific recognition requirements in tRNAs may allow for mutations to occur outside of unique recognition sequences that would cause them to mis-incorporate amino acids into proteins. Amino acid misplacement can cause proteins to misfold, be degraded, or build-up, leading to further problems within the cell. I studied three different tRNAs that could incorrectly place the amino acid alanine instead of glycine into proteins, a process called mistranslation. Through expression of a fluorescent protein alongside mistranslating tRNAs, I determined that glycine to alanine mistranslation does not have a significant negative impact on protein levels in mammalian cells under normal conditions. When cells were unable to degrade proteins, expression of one glycine to alanine mistranslating tRNA decreased protein levels compared to wild-type tRNA expression. I determined sites of amino acid mutations in protein isolated from mistranslating cells to conclude that mistranslating tRNA expression caused amino acid misincorporation. However, the mistranslation was conservative enough to not disrupt normal protein production in cells. My findings show that glycine to alanine mistranslation in mammalian cells is not toxic under normal conditions.

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