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

Abstract

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.

Keywords: Drosophila melanogaster; RNA sequencing; mistranslation; proteotoxicity; stress response; tRNA; translation.

Fig. 1.

Differentially expressed genes in male or female flies containing tRNA^Ser_{UGG, G26A} (P→S). a) MA plot visualizing the relationship between transcript abundance and the difference in fold change of expression between male tRNA^Ser_{UGG, G26A} (P→S) and control tRNA^Ser_UGA samples. Blue points represent genes that are significantly differentially expressed between mistranslating tRNA^Ser_{UGG, G26A} (P→S) and control tRNA^Ser_UGA samples, whereas gray points represent genes where the expression change was not statistically significant. Triangular points at the edge of the y-axis indicate genes that have a fold change exceeding the limits of the y-axis. b) MA plot visualizing the relationship between transcript abundance and fold change of expression difference between female tRNA^Ser_{UGG, G26A} (P→S) and control tRNA^Ser_UGA samples. Red points represent genes that are significantly differentially expressed between mistranslating tRNA^Ser_{UGG, G26A} (P→S) and control tRNA^Ser_UGA samples. c) Venn diagram showing the number of significantly upregulated (FDR-adjusted P < 0.05) genes unique to tRNA^Ser_{UGG, G26A} (P→S) males, females, or genes upregulated in both sexes. d) Venn diagram showing the number of significantly downregulated (FDR-adjusted P < 0.05) genes unique to tRNA^Ser_{UGG, G26A} (P→S) males, females, or genes downregulated in both sexes. e) Scatterplot showing male vs female relative expression for all 1,705 genes that were identified as differentially expressed and not filtered out from analysis in either sex. Blue points represent genes that have higher relative expression in mistranslating males compared to females (log₂ fold change difference > 0.5); red points represent genes with higher relative expression in mistranslating females compared to males. Genes that demonstrate sex-biased patterns of relative expression (log₂ fold change difference > 1) in response to tRNA^Ser_{UGG, G26A} (P→S) are labeled. CG12057 is also labeled due to its strong downregulation in both sexes. The dashed line represents identical fold changes in expression for both males and females.

Fig. 2.

The top 10 significantly enriched GO terms in the list of genes a) downregulated or b) upregulated in male or female flies containing tRNA^Ser_{UGG, G26A} (P→S) compared to control tRNA^Ser_UGA flies. Higher enrichment ratios indicate that the set of genes associated with that GO term were more highly represented in our gene set. Note the differences in scale. Lists were produced using WebGestalt (Liao et al. 2019). A list of significantly enriched GO terms and associated statistics is found in Supplementary File 1.

Fig. 3.

Heatmap of enriched GO terms from the differentially expressed genes in male or female flies containing tRNA^Ser_{UGG, G26A} (P→S). a) Heatmap of enriched GO terms in the list of downregulated genes in male and female flies containing tRNA^Ser_{UGG, G26A} (P→S). Each horizontal bar represents a GO term identified as significantly enriched in male and/or female data. GO terms were clustered by semantic similarity according to ViSEAGO using Wang's method (Wang et al. 2007; Brionne et al. 2019). Dendrogram clades of the same color represent semantically similar GO terms. Darker bars within the heatmap represent lower P-values as determined through Fisher's exact test. Notable groups of enriched processes are labeled in blue if enriched in males, red if enriched in females, or purple if enriched in both sexes. b) The same as a) but using the list of upregulated genes. A full list of enriched GO terms and their associated genes can be found in Supplementary File 1.

Fig. 4.

Clustering proline-to-serine mistranslation-induced transcriptome changes with transcriptome changes due to various other physiological or environmental conditions. a) Z-score normalized gene expression changes in tRNA^Ser_{UGG, G26A} (P→S) males relative to tRNA^Ser_UGA (wild-type) males clustered with normalized male gene expression changes from Zhou et al. (2012). Genes with fewer than 10 normalized reads or fold changes > |5| for any condition were excluded from analysis. Clustering was performed using the “ComplexHeatmap” R package using Ward's method (Ward 1963; Gu et al. 2016). The P→S mistranslation condition is highlighted in green. b) The same as a) but clustering female data.