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Comparative genomics has enabled the identification of genes that potentially evolved de novo from non-coding sequences. Many such genes are expressed in male reproductive tissues, but their functions remain poorly understood. To address this, we conducted a functional genetic screen of over 40 putative de novo genes with testis-enriched expression in Drosophila melanogaster and identified one gene, atlas, required for male fertility. Detailed genetic and cytological analyses showed that atlas is required for proper chromatin condensation during the final stages of spermatogenesis. Atlas protein is expressed in spermatid nuclei and facilitates the transition from histone- to protamine-based chromatin packaging. Complementary evolutionary analyses revealed the complex evolutionary history of atlas. The protein-coding portion of the gene likely arose at the base of the Drosophila genus on the X chromosome but was unlikely to be essential, as it was then lost in several independent lineages. Within the last ~15 million years, however, the gene moved to an autosome, where it fused with a conserved non-coding RNA and evolved a non-redundant role in male fertility. Altogether, this study provides insight into the integration of novel genes into biological processes, the links between genomic innovation and functional evolution, and the genetic control of a fundamental developmental process, gametogenesis., Author summary Genomes are in flux, as genes are constantly added and lost throughout evolution. New genes were once thought to arise almost exclusively via the modification or duplication of existing genes. Recently, however, interest has grown in alternative modes of new gene origination, such as de novo evolution from genetic material that previously did not encode proteins. Many de novo genes are expressed in male reproductive tissues, but their significance for fertility is not well understood. We screened likely de novo genes expressed in the Drosophila testis for reproductive roles and found one gene, atlas, essential for male fertility. We leveraged genetic and cell biological experiments to investigate roles for Atlas protein in reproduction and found that it is required during sperm development for proper packaging of DNA in the sperm nucleus. Evolutionary analyses of this gene revealed a complicated history, including loss in some lineages, movement between chromosomes, and fusion with a non-protein-coding gene. Studying both the functions and evolutionary histories of new proteins illustrates how they might evolve critical roles in biological processes despite their relative novelty. Furthermore, the study of atlas identifies an essential genetic player in the fly testis, an important model system for understanding how gametes are produced.

Vps54 is a subunit of the Golgi-associated retrograde protein (GARP) complex, which is involved in tethering endosome-derived vesicles to the trans-Golgi network (TGN). In the wobbler mouse, a model for human motor neuron (MN) disease, reduction in the levels of Vps54 causes neurodegeneration. However, it is unclear how disruption of the GARP complex leads to MN dysfunction. To better understand the role of Vps54 in MNs, we have disrupted expression of the Vps54 ortholog in Drosophila and examined the impact on the larval neuromuscular junction (NMJ). Surprisingly, we show that both null mutants and MN-specific knockdown of Vps54 leads to NMJ overgrowth. Reduction of Vps54 partially disrupts localization of the t-SNARE, Syntaxin-16, to the TGN but has no visible impact on endosomal pools. MN-specific knockdown of Vps54 in MNs combined with overexpression of the small GTPases Rab5, Rab7, or Rab11 suppresses the Vps54 NMJ phenotype. Conversely, knockdown of Vps54 combined with overexpression of dominant negative Rab7 causes NMJ and behavioral abnormalities including a decrease in postsynaptic Dlg and GluRIIB levels without any effect on GluRIIA. Taken together, these data suggest that Vps54 controls larval MN axon development and postsynaptic density composition through a mechanism that requires Rab7., Summary: The retrograde vesicle tethering factor, Vps54, regulates development of the Drosophila larval NMJ. Moreover, presynaptic Vps54 interacts genetically with the small GTPase, Rab7, to control NMJ growth and postsynaptic density composition.

Vps54 is a subunit of the Golgi-associated retrograde protein (GARP) complex, which is involved in tethering endosome-derived vesicles to thetrans-Golgi network (TGN). In the wobbler mouse, a model for human motor neuron (MN) disease, reduction in the levels of Vps54 causes neurodegeneration. However, it is unclear how disruption of GARP-mediated vesicle transport leads to MN dysfunction and ultimately neurodegeneration. To better understand the role of Vps54 in MNs, we have disrupted expression of theVps54ortholog inDrosophilaand examined the impact on the larval neuromuscular junction (NMJ). Here, we show that both null mutants and MN-specific knockdown ofVps54leads to NMJ overgrowth. Reduction ofVps54partially disrupts localization of the t-SNARE, Syntaxin-16, to the TGN but has no impact on endosomal pools. Presynaptic knockdown ofVps54in MNs combined with overexpression of the small GTPases Rab5, Rab7, or Rab11 suppresses theVps54NMJ phenotype. Conversely, knockdown ofVps54combined with overexpression of dominant negative Rab7 causes axonal and behavioral abnormalities including a decrease in postysynaptic Dlg and GluRIIB levels without any effect on GluRIIA. Taken together, these data suggest thatVps54controls larval MN axon development and postsynaptic density composition by modulating Rab7-mediated endosomal trafficking.

Hypusination is a unique posttranslational modification by which lysine is transformed into the atypical amino acid hypusine. eIF5A (eukaryotic initiation factor 5A) is the only known protein to contain hypusine. In this study, we describe the identification and characterization of nero, the Drosophila melanogaster deoxyhypusine hydroxylase (DOHH) homologue. nero mutations affect cell and organ size, bromodeoxyuridine incorporation, and autophagy. Knockdown of the hypusination target eIF5A via RNA interference causes phenotypes similar to nero mutations. However, loss of nero appears to cause milder phenotypes than loss of eIF5A. This is partially explained through a potential compensatory mechanism by which nero mutant cells up-regulate eIF5A levels. The failure of eIF5A up-regulation to rescue nero mutant phenotypes suggests that hypusination is required for eIF5A function. Furthermore, expression of enzymatically impaired forms of DOHH fails to rescue nero clones, indicating that hypusination activity is important for nero function. Our data also indicate that nero and eIF5A are required for cell growth and affect autophagy and protein synthesis.