Thesis Format

Integrated Article

Transfer RNA Missense Suppressors in Nature and Disease

Author

Rasangi Tennakoon, Western UniversityFollow

Degree

Master of Science

Program

Biochemistry

Supervisor

O'Donoghue, Patrick

Abstract

Transfer RNAs (tRNAs) serve as the bridge between the genome and proteome by recognizing the three-nucleotide codons in messenger RNAs and incorporating the correct amino acids into growing polypeptide chains. My goal was to assess the impact of human missense suppressor tRNAs that cause mistranslation, and their therapeutic potential in neurodegenerative disease. First, I created synthetic tRNA^Ala and tRNA^Ser anticodon mutants that mis-incorporate alanine or serine in expanded polyglutamine tracts in cellular models of Huntington’s disease. I found that tRNA^Ala_CUG significantly reduced disease-causing protein aggregates without inducing cytotoxicity. Then, I characterized three tRNA^Ala genes with anticodon variants that occur naturally in the human population. Expression of each variant in cells caused misincorporation of alanine at different codons and led to significant defects in protein production and cell proliferation. These findings demonstrate that mistranslation of alanine by tRNA missense suppressors can differentially affect protein synthesis, aggregation, and growth of human cells.

Summary for Lay Audience

The central dogma of molecular biology describes how genetic information moves through cells, starting with DNA's instructions for building proteins. These instructions are transcribed into messenger RNA (mRNA), which is then translated into proteins, ultimately governing the structure and function of living organisms. Transfer RNAs (tRNAs) are integral to this process, as they are responsible for carrying amino acids, which are the building blocks of proteins, to the protein synthesis machinery. There, they decode genetic information in mRNA, stored as three-nucleotide codons, and incorporate an amino acid into a growing protein. Attachment of the correct amino acid to the correct tRNA is facilitated through aminoacyl-tRNA synthetases (aaRSs). Most aaRSs recognize the appropriate tRNA through the anticodon, a sequence in the tRNA body that is also responsible for mRNA codon recognition. However, the aaRSs for alanine tRNA (tRNA^Ala)and serine tRNA (tRNA^Ser) do not recognize the anticodon and rely on other elements in the tRNA body for identification. Thus, mutations to the anticodon sequence of these tRNAs could lead to attachment of the intended amino acid, but recognition of the wrong codon on mRNA. This could cause misincorporation of alanine or serine at non-alanine or non-serine codons, also known as mistranslation. Here, I assessed the impact of missense suppressor tRNA^Ala that causes mistranslation in nature and investigated therapeutic potential of tRNA mutants in Huntington’s disease. Missense suppressor tRNAs function to incorporate a tolerable amino acid at the site of a mutation. I created synthetic tRNA^Ala and tRNA^Ser anticodon mutants that misincorporate alanine or serine in expanded polyglutamine tracts in cellular models of Huntington’s disease. I found that tRNA^Alawith the glutamine-decoding anticodon (CUG) significantly reduced Huntington’s disease-causing protein aggregates without inducing cytotoxicity. Then, I characterized three tRNA^Ala genes with anticodon variants that occur naturally in the human population. Expression of each variant in cells caused misincorporation of alanine at non-alanine codons, and led to significant defects in protein production and cell growth. These findings demonstrate that mistranslation of alanine by tRNA missense suppressors can differentially affect protein synthesis, aggregation, and cell growth in normal cells and in models of disease.

Recommended Citation

Tennakoon, Rasangi, "Transfer RNA Missense Suppressors in Nature and Disease" (2024). Electronic Thesis and Dissertation Repository. 10112.
https://ir.lib.uwo.ca/etd/10112