Expression of De Novo Open Reading Frames in Natural Populations of Drosophila melanogaster

Abstract

De novo genes, which originate from noncoding DNA, are known to have a high rate of turnover over short evolutionary timescales, such as within a species. Thus, their expression is often lineage- or genetic background-specific. However, little is known about their levels and breadth of expression as populations of a species diverge. In this study, we utilized publicly available RNA-seq data to examine the expression of newly evolved open reading frames (neORFs) in comparison to non- and protein-coding genes in Drosophila melanogaster populations from the derived species range in Europe and the ancestral range in sub-Saharan Africa. Our datasets included two adult tissue types as well as whole bodies at two temperatures for both sexes and three larval/prepupal developmental stages in a single tissue and sex, which allowed us to examine neORF expression and divergence across multiple sample types as well as sex and population. We detected a relatively large proportion (approximately 50%) of annotated neORFs as expressed in the population samples, with neORFs often showing greater expression divergence between populations than non- or protein-coding genes. However, differential expression of neORFs between populations tended to occur in a sample type-specific manner. On the other hand, neORFs displayed less sex-biased expression than the other two gene classes, with the majority of sex-biased neORFs detected in whole bodies, which may be attributable to the presence of the gonads. We also found that neORFs shared among multiple lines in the original set of inbred lines in which they were first detected were more likely to be both expressed and differentially expressed in the new population samples, suggesting that neORFs at a higher frequency (i.e. present in more individuals) within a species are more likely to be functional.

Keywords: de novo genes; gene expression; genome evolution; innovations; novelty; population genetics.

Figure 1

Differentially expressed (DE) genes in adult whole body. Shown are the (A, B) magnitude of expression changes (i.e. absolute value of the LFC, see Methods) for DE genes and the (C, D) proportion of DE (FDR < 0.05) protein coding genes (pc), ncRNAs (nc), and neORFs (ne) between populations or sexes at (A, C) 15°C or (B, D) 28°C. Boxes at the bottom of the figure indicate if a comparison is between populations (left in each panel) or between sexes (right in each panel). (A, B) Significance was assessed with a t‐test. (C, D) Dashed lines indicate the expected proportion of DE genes based on the total number of DE genes among all analyzed genes. Significance was assessed with a χ² test. ***BH‐corrected p < 0.001, **p < 0.01, *p < 0.05, ns not significant after multiple test correction. p > 0.05 before multiple test correction not shown.

Figure 2

Differentially expressed (DE) genes in somatic tissues. Shown are the (A, B) magnitude of expression changes (i.e. absolute value of the LFC, see Methods) for DE genes and the (C, D) proportion of DE (FDR < 0.05) protein coding genes (pc), ncRNAs (nc), and neORFs (ne) between populations or sexes in the (A, C) Malpighian tubule or (B, D) brain. Boxes at the bottom of the figure indicate if a comparison is between populations (left in each panel) or between sexes (right in each panel). (A, B) Significance was assessed with t‐test. (C, D) Dashed lines indicate the expected proportion of DE genes based on the total number of DE genes among all analyzed genes. Significance was assessed with a χ² test. ***BH‐corrected p < 0.001, **p < 0.01, *p < 0.05, ns not significant after multiple test correction. p > 0.05 before multiple test correction not shown.

Figure 3

Differentially expressed (DE) genes in the larval/prepupal fat body and overlapping neORFs among datasets. Shown are the (A) magnitude of expression changes for DE genes and the (C) proportion of DE (FDR < 0.05) protein coding genes (pc), ncRNAs (nc), and neORFs (ne) between populations in the early and late wandering larval or prepupal (prepup) stages. Boxes at the bottom of the figure indicate the stage of a population comparison. (A) Significance was assessed with a t‐test. (C) Dashed lines indicate the expected proportion of DE genes based on the total number of DE genes among all analyzed genes. Significance was assessed with a χ² test. ***BH‐corrected p < 0.001, **p < 0.01, *p < 0.05, ns not significant after multiple test correction. p > 0.05 before multiple test correction not shown. Shown are the number and percentage of (B) expressed and (D) differentially expressed neORFs between populations in all examined sample types.

Figure 4

Expression of neORFs in the whole body (WB), brain (BR), Malpighian tubule (MT), and larval/prepupal fat body (FB), and their frequency in the initial lines used for annotation. (A) Proportion of neORFs expressed in each sample type binned by the number of lines in which the respective neORF was present in the original data set (Grandchamp et al. 2023). (B) Proportion of differentially expressed neORFs between populations in relation to the number of lines containing the neORF in the original data set.