Critical roles of long noncoding RNAs in Drosophila spermatogenesis

Long noncoding RNAs (IncRNAs), a recently discovered class of cellular RNAs, play important roles in the regulation of many cellular developmental processes. Although IncRNAs have been systematically identified in various systems, most of them have not been functionally characterized in vivo in animal models. In this study, we identified 128 testis-specific Drosophila IncRNAs and knocked out 105 of them using an optimized three-component CRISPR/Cas9 system. Among the IncRNA knockouts, 33 (31%) exhibited a partial or complete loss of male fertility, accompanied by visual developmental defects in late spermatogenesis. In addition, six knockouts were fully or partially rescued by transgenes in a trans configuration, indicating that those lncRNAs primarily work in trans. Furthermore, gene expression profiles for five IncRNA mutants revealed that testis-specific IncRNAs regulate global gene expression, orchestrating late male germ cell differentiation. Compared with coding genes, the testis-specific IncRNAs evolved much faster. Moreover, IncRNAs of greater functional importance exhibited higher sequence conservation, suggesting that they are under constant evolutionary selection. Collectively, our results reveal critical functions of rapidly evolving testis-specific lncRNAs in late Drosophila spermatogenesis.