Functional characterization of atlas, a putative de novo evolved gene essential for Drosophila male fertility

Unlike many genes that arise from duplication, de novo evolved genes arise from previously noncoding DNA. Many de novo genes are expressed in the male reproductive tract, but their functions remain uncharacterized. We used an RNAi screen to identify several testis-expressed, putative de novo genes that are essential for male reproduction. Both the RNAi knockdown and the CRISPR-mediated knockout (KO) of one such gene, atlas, resulted in almost complete male sterility. Cytological analyses of spermatogenesis in atlas null mutants revealed two defects. First, mutant male testes showed few sperm in the seminal vesicle (SV) and a distended basal end of the testis, suggesting that sperm in atlas mutants are not transferred to the SV during spermatogenesis and, thus, to females during mating. Second, phalloidin staining of whole testes revealed that atlas KO males inefficiently initiate the process of spermatid individualization. To understand the normal role of atlas in spermatogenesis, we used CRISPR to scarlessly insert GFP at the end of the gene’s protein-coding sequence, at the endogenous locus. This atlas-GFP allele fully rescued the fertility defect of atlas null males. We then observed a nuclear-localized GFP signal in the post-meiotic stages of the testes. This signal overlapped partially with fully condensed sperm nuclear bundles marked by protamine-dsRed, but the GFP-positive bundles were not as close as the dsRed-positive bundles to the basal end. These data, along with the biochemical properties of the atlas protein, suggest that atlas may encode a transition protein that facilitates the replacement of histones with protamines in late spermiogenesis. These results demonstrate how a putatively de novo evolved protein can acquire an essential reproductive function.