Determining the Effects of Mistranslating Transfer RNA Variants on Drosophila Melanogaster
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.