Thesis Format

Integrated Article

Determining the Effects of Mistranslating Transfer RNA Variants on Drosophila Melanogaster

Author

Joshua R. Isaacson, Western UniversityFollow

Degree

Doctor of Philosophy

Program

Biology

Supervisor

Moehring, Amanda J

2nd Supervisor

Brandl, Chris J

Co-Supervisor

Abstract

Transfer RNAs (tRNAs) play a central role in translation as adaptor molecules between mRNA and protein. Variant tRNAs can cause the misincorporation of an amino acid into a growing polypeptide. Mistranslating tRNA variants are surprisingly common in humans but the effects of mistranslating tRNA variants on eukaryotic biology are poorly understood. My thesis aimed to create a model of tRNA-induced mistranslation using the fruit fly Drosophila melanogaster and characterize the effects of mistranslating tRNA variants on eukaryotic biology.

I first integrated a gene encoding a serine tRNA variant that induced proline-to-serine (P>S) mistranslation into the fly genome. Proteins isolated from lines containing the mistranslating tRNA variant contained significantly more P>S substitutions than control lines. Flies containing the mistranslating tRNA variant presented with extended development, developmental lethality, more anatomical deformities, and impaired climbing performance compared to control flies. Interestingly, females presented with more deformities than males and experienced a more rapid decline in performance as they aged, indicating that females may be more susceptible to mistranslation.

To identify cellular pathways that may be affected by mistranslation, I next performed RNA-sequencing on flies containing the P>S mistranslating tRNA variant. This tRNA variant caused both sexes to downregulate metabolic genes and upregulate genes associated with gametogenesis. Males downregulated genes associated with development whereas females downregulated aerobic respiration. Females upregulated genes associated with DNA maintenance and the cell cycle, indicating that mistranslation may affect female genome integrity.

Finally, I tested whether different types of mistranslation have different effects on flies. I integrated genes encoding two new mistranslating serine tRNA variants, one that substitutes serine for valine (V>S) and another that substitutes serine for threonine (T>S). Both tRNA variants cause mistranslation at significantly higher levels than control flies. I observed extended development, developmental lethality, and increased prevalence of anatomical deformities similar to flies containing the mistranslating P>S variants. Surprisingly, both mistranslating tRNA variants extended female lifespan and improved climbing ability in both sexes. My work shows that mistranslation has multifaceted and sex-specific effects on flies. The model I created will aid investigations into how mistranslating tRNA variants impact fitness and disease.

Summary for Lay Audience

Translation describes the process by which cells produce proteins from expressed genes. Translation requires special molecules known as transfer RNAs (tRNAs) to convert the genetic code into proteins. Because of their central role in translation, if tRNA genes are mutated, then these variant tRNAs can cause proteins to be synthesized incorrectly. These translation errors are known as mistranslation.

Variant tRNAs are common in humans and associated with several diseases, but there is currently no model to study the effects of mistranslating tRNA variants in multicellular organisms. The goal of my thesis was to create a multicellular model of tRNA-induced mistranslation in the fruit fly and determine what effects mistranslating tRNA variants have on fly biology. The fruit fly is an ideal model for this research as it has a nervous system similar to humans, short generation times, and many available genetic tools.

I began by integrating a mistranslating tRNA variant into flies and characterizing its effects. This tRNA variant extended development time, increased developmental lethality, and caused anatomical deformities in adults such as missing wings or legs. Fly climbing ability, a common proxy for neurological function, was also decreased in flies containing the mistranslating tRNA variant. I was surprised to find that females were more strongly affected by mistranslation, indicating that females are particularly susceptible to translation errors. The mistranslating tRNA variant also affected gene expression of flies, causing metabolic genes to be expressed less and genes associated with reproductive processed to be expressed more, though males and females also showed different gene expression profiles.

I also tested two new mistranslating tRNA variants that induce different translation errors to see if they have different effects. These two tRNA variants replicated several of my original results, but both variants increased female lifespan and improved male and female climbing performance. My results demonstrate that I have successfully created a multicellular model of tRNA-induced mistranslation and that mistranslation has both positive and negative effects on fly biology. Variant tRNAs are surprisingly common in humans, and the model I created represents a powerful tool to study their effect on human health.

Recommended Citation

Isaacson, Joshua R., "Determining the Effects of Mistranslating Transfer RNA Variants on Drosophila Melanogaster" (2024). Electronic Thesis and Dissertation Repository. 10043.
https://ir.lib.uwo.ca/etd/10043

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