Isaacson, J. R. | Search bioRxiv

bioRxiv2021

A Novel Mistranslating tRNA Model in Drosophila melanogaster has Diverse, Sexually Dimorphic Effects

Transfer RNAs (tRNAs) are the adaptor molecules required for reading of the genetic code and the accurate production of proteins. tRNA variants can lead to genome-wide mistranslation, the misincorporation of amino acids not specified by the standard genetic code into nascent proteins. While genome sequencing has identified putative mistranslating tRNA variants in human populations, little is known regarding how mistranslation affects multicellular organisms. Here, we create a Drosophila melanogaster model for mistranslation by integrating a serine tRNA variant that mistranslates serine for proline (tRNASerUGG, G26A) into the fly genome. Using mass spectrometry, we find that tRNASerUGG, G26A misincorporates serine for proline at a frequency of [~] 0.6% per codon. We find that mistranslation extends development time and decreases the number of flies that reach adulthood. Adult flies containing tRNASerUGG, G26A present with more morphological deformities and worse climbing performance than flies expressing only wild type tRNA. Female flies with the serine tRNA variant have more deformities and experience a faster decline in climbing performance than males, suggesting sex-specific effects. This model will enable studies into the synergistic effects of mistranslating tRNA variants and disease-causing alleles.

molecular biology↗

bioRxiv2021

Regulating expression of mistranslating tRNAs by readthrough RNA polymerase II transcription

Transfer RNA (tRNA) variants that alter the genetic code increase protein diversity and have many applications in synthetic biology. Since the tRNA variants can cause a loss of proteostasis, regulating their expression is necessary to achieve high levels of novel protein. Mechanisms to positively regulate transcription with exogenous activator proteins like those often used to regulate RNA polymerase II (RNAP II) transcribed genes are not applicable to tRNAs as their expression by RNA polymerase III requires elements internal to the tRNA. Here, we show that tRNA expression is repressed by overlapping transcription from an adjacent RNAP II promoter. Regulating the expression of the RNAP II promoter allows inverse regulation of the tRNA. Placing either Gal4 or TetR-VP16 activated promoters downstream of a mistranslating tRNASer variant that mis-incorporates serine at proline codons in Saccharomyces cerevisiae allows mistranslation at a level not otherwise possible because of the toxicity of the unregulated tRNA. Using this inducible tRNA system, we explore the proteotoxic effects of mistranslation on yeast cells. High levels of mistranslation cause cells to arrest in G1 phase. These cells are impermeable to propidium iodide, yet growth is not restored upon repressing tRNA expression. High levels of mistranslation increase cell size and alter cell morphology. This regulatable tRNA expression system can be applied to study how native tRNAs and tRNA variants affect the proteome and other biological processes. Variations of this inducible tRNA system should be applicable to other eukaryotic cell types.

molecular biology↗