Thesis Format
Monograph
Degree
Master of Science
Program
Biochemistry
Supervisor
O'Donoghue, Patrick
Abstract
Nonsense mutations disrupt protein synthesis by introducing a premature termination codon (PTC) into messenger RNA (mRNA), leading to truncated protein products and severe diseases. Transfer RNAs (tRNAs) can be mutated at their anticodon, allowing them to suppress these nonsense mutations, restoring full-length proteins to the cell. I demonstrate that three human tRNAArgUCG isodecoders, bearing the same engineered anticodons (G36A) and amino acids but with differences in body sequence, can mediate significant and differential suppression in a live-cell fluorescent reporter and with a clinically relevant PTC. Experiments in multiple mammalian cell lines demonstrate that suppressor tRNAArgUCA (sup-tRNAs ) promotes PTC readthrough levels that depend on the sequence of the tRNA and the cell type. Suppressor tRNAArg variants produced wildtype progranulin (PGRN/GRN) from a frontotemporal dementia (FTD)-causative allele (R493X). The suppressor tRNAs significantly outperform the aminoglycoside nonsense suppressor (G418) in efficacy, tolerability to the cells, and in translation fidelity as validated by mass spectrometry. The data indicates that suppressor tRNAs are an important area for further therapeutic development with applications in FTD and other diseases caused by PTCs.
Summary for Lay Audience
During normal protein production in cells, messenger RNA (mRNA) has one stop codon that is reached when the full-length protein has been produced, triggering ribosome disassembly and protein release. However, genetic mutations can introduce a premature termination codon (PTC) through a nonsense mutation. Premature stop signals cause around 11% of all genetic diseases including lethal forms of cystic fibrosis, muscular dystrophy, cancers, and frontotemporal dementia (FTD). There are currently no effective treatments available that target the root cause of these genetic diseases.
Transfer RNA (tRNA) are molecules that carry amino acids to the ribosome during translation and they can be engineered to base pair with a pathogenic PTC, allowing the PTC to be suppressed to produce full-length protein from a disease-causing gene. These suppressor tRNAs (sup-tRNAs) show great therapeutic promise. I examined three human tRNAArgTCG variants under control of their endogenous regulatory regions for their ability to support PTC suppression. I found that each sup-tRNA mediated differential readthrough of a PTC based on the tRNA’s unique identity as well as the cell type it was being expressed in. I further demonstrate that the sup-tRNA effectively read through the progranulin-R493X mutation which is causative of FTD, inserting the correct amino acid without impairing cell growth. This therapeutic efficacy surpasses that of other potential therapeutic approaches such as the antibiotic derivate G418. My results are the first application of sup-tRNA as an effective therapy for FTD, showing great promise as a clinically relevant treatment option.
Recommended Citation
Beharry, Aruun, "Human transfer RNA anticodon variants suppress pathogenic nonsense mutations" (2024). Electronic Thesis and Dissertation Repository. 10132.
https://ir.lib.uwo.ca/etd/10132