Impact of tRNA-induced proline-to-serine mistranslation on the transcriptome of Drosophila melanogaster.

Mistranslation is the misincorporation of an amino acid into a polypeptide. Mistranslation has diverse effects on multicellular eukaryotes and is implicated in several human diseases. In Drosophila melanogaster, a serine transfer RNA (tRNA) that misincorporates serine at proline codons (P→S) affects male and female flies differently. The mechanisms behind this discrepancy are currently unknown. Here, we compare the transcriptional response of male and female flies to P→S mistranslation to identify genes and cellular processes that underlie sex-specific differences. Both males and females downregulate genes associated with various metabolic processes in response to P→S mistranslation. Males downregulate genes associated with extracellular matrix organization and response to negative stimuli such as wounding, whereas females downregulate aerobic respiration and ATP synthesis genes. Both sexes upregulate genes associated with gametogenesis, but females also upregulate cell cycle and DNA repair genes. These observed differences in the transcriptional response of male and female flies to P→S mistranslation have important implications for the sex-specific impact of mistranslation on disease and tRNA therapeutics.

Mistranslating tRNA variants have anticodon- and sex-specific impacts on Drosophila melanogaster.

Transfer RNAs (tRNAs) are vital in determining the specificity of translation. Mutations in tRNA genes can result in the misincorporation of amino acids into nascent polypeptides in a process known as mistranslation. Since mistranslation has different impacts, depending on the type of amino acid substitution, our goal here was to compare the impact of different mistranslating tRNASer variants on fly development, lifespan, and behaviour. We established two mistranslating fly lines, one with a tRNASer variant that misincorporates serine at valine codons (V→S) and the other that misincorporates serine at threonine codons (T→S). While both mistranslating tRNAs increased development time and developmental lethality, the severity of the impacts differed depending on amino acid substitution and sex. The V→S variant extended embryonic, larval, and pupal development whereas the T→S only extended larval and pupal development. Females, but not males, containing either mistranslating tRNA presented with significantly more anatomical deformities than controls. Mistranslating females also experienced extended lifespan whereas mistranslating male lifespan was unaffected. In addition, mistranslating flies from both sexes showed improved locomotion as they aged, suggesting delayed neurodegeneration. Therefore, although mistranslation causes detrimental effects, we demonstrate that mistranslation also has positive effects on complex traits such as lifespan and locomotion. This has important implications for human health given the prevalence of tRNA variants in humans.

Impact of tRNA-induced proline-to-serine mistranslation on the transcriptome of Drosophila melanogaster.

Mistranslation is the misincorporation of an amino acid into a polypeptide. Mistranslation has diverse effects on multicellular eukaryotes and is implicated in several human diseases. In Drosophila melanogaster, a serine transfer RNA (tRNA) that misincorporates serine at proline codons (P→S) affects male and female flies differently. The mechanisms behind this discrepancy are currently unknown. Here, we compare the transcriptional response of male and female flies to P→S mistranslation to identify genes and cellular processes that underlie sex-specific differences. Both males and females downregulate genes associated with various metabolic processes in response to P→S mistranslation. Males downregulate genes associated with extracellular matrix organization and response to negative stimuli such as wounding, whereas females downregulate aerobic respiration and ATP synthesis genes. Both sexes upregulate genes associated with gametogenesis, but females also upregulate cell cycle and DNA repair genes. These observed differences in the transcriptional response of male and female flies to P→S mistranslation have important implications for the sex-specific impact of mistranslation on disease and tRNA therapeutics.

A novel mistranslating tRNA model in Drosophila melanogaster has diverse, sexually dimorphic effects.

Transfer RNAs (tRNAs) are the adaptor molecules required for reading the genetic code and producing proteins. Transfer RNA 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 transfer RNA variants in human populations, little is known regarding how mistranslation affects multicellular organisms. Here, we create a multicellular model of mistranslation by integrating a serine transfer RNA variant that mistranslates serine for proline (tRNAUGG,G26ASer) into the Drosophila melanogaster genome. We confirm mistranslation via mass spectrometry and find that tRNAUGG,G26ASer misincorporates serine for proline at a frequency of ∼0.6% per codon. tRNAUGG,G26ASer extends development time and decreases the number of flies that reach adulthood. While both sexes of adult flies containing tRNAUGG,G26ASer present with morphological deformities and poor climbing performance, these effects are more pronounced in female flies and the impact on climbing performance is exacerbated by age. This model will enable studies into the synergistic effects of mistranslating transfer RNA variants and disease-causing alleles.

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 misincorporates 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 the 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.

The amino acid substitution affects cellular response to mistranslation.

Mistranslation, the misincorporation of an amino acid not specified by the "standard" genetic code, occurs in all organisms. tRNA variants that increase mistranslation arise spontaneously and engineered tRNAs can achieve mistranslation frequencies approaching 10% in yeast and bacteria. Interestingly, human genomes contain tRNA variants with the potential to mistranslate. Cells cope with increased mistranslation through multiple mechanisms, though high levels cause proteotoxic stress. The goal of this study was to compare the genetic interactions and the impact on transcriptome and cellular growth of two tRNA variants that mistranslate at a similar frequency but create different amino acid substitutions in Saccharomyces cerevisiae. One tRNA variant inserts alanine at proline codons whereas the other inserts serine for arginine. Both tRNAs decreased growth rate, with the effect being greater for arginine to serine than for proline to alanine. The tRNA that substituted serine for arginine resulted in a heat shock response. In contrast, heat shock response was minimal for proline to alanine substitution. Further demonstrating the significance of the amino acid substitution, transcriptome analysis identified unique up- and down-regulated genes in response to each mistranslating tRNA. Number and extent of negative synthetic genetic interactions also differed depending upon type of mistranslation. Based on the unique responses observed for these mistranslating tRNAs, we predict that the potential of mistranslation to exacerbate diseases caused by proteotoxic stress depends on the tRNA variant. Furthermore, based on their unique transcriptomes and genetic interactions, different naturally occurring mistranslating tRNAs have the potential to negatively influence specific diseases.

Chemical-Genetic Interactions with the Proline Analog L-Azetidine-2-Carboxylic Acid in Saccharomyces cerevisiae.

Non-proteinogenic amino acids, such as the proline analog L-azetidine-2-carboxylic acid (AZC), are detrimental to cells because they are mis-incorporated into proteins and lead to proteotoxic stress. Our goal was to identify genes that show chemical-genetic interactions with AZC in Saccharomyces cerevisiae and thus also potentially define the pathways cells use to cope with amino acid mis-incorporation. Screening the yeast deletion and temperature sensitive collections, we found 72 alleles with negative chemical-genetic interactions with AZC treatment and 12 alleles that suppress AZC toxicity. Many of the genes with negative chemical-genetic interactions are involved in protein quality control pathways through the proteasome. Genes involved in actin cytoskeleton organization and endocytosis also had negative chemical-genetic interactions with AZC. Related to this, the number of actin patches per cell increases upon AZC treatment. Many of the same cellular processes were identified to have interactions with proteotoxic stress caused by two other amino acid analogs, canavanine and thialysine, or a mistranslating tRNA variant that mis-incorporates serine at proline codons. Alleles that suppressed AZC-induced toxicity functioned through the amino acid sensing TOR pathway or controlled amino acid permeases required for AZC uptake. Further suggesting the potential of genetic changes to influence the cellular response to proteotoxic stress, overexpressing many of the genes that had a negative chemical-genetic interaction with AZC suppressed AZC toxicity.

Digest: Male and female beetle genitalia are under stabilizing selection.

The effect of selection on female genitalia is poorly studied, but such selection could affect mating success. House et al. (2020) studied the form and strength of selection acting on male and female Tribolium castaneum. They found that the sex organs of both male and female T. castaneum are under multivariate stabilizing selection and that both male and female genitalia interact to influence mating success.

Digest: How do nonnative frugivorous birds adapt to life in O'ahu?

Species adapt to the selective pressures of novel environments. Here, Gleditsch and Sperry tested whether four nonnative frugivorous bird species on the Hawaiian island of O'ahu diverged morphologically from their ancestral populations. They found that all four species significantly diverged from populations in their native ranges, with a general trend of smaller body size and larger bills. These differences were likely due to a combination of adaptive and nonadaptive processes.