Trans regulatory evolution of a young microRNA during Drosophila spermatogenesis

Both cis and trans regulatory evolution underlies phenotypic innovation and diversification. However, how the nascent trans regulators escape negative pleiotropy, which may constrain their evolution and contribution to adaptation, remain unclear. Here, we compare the testis transcriptomes at single-cyst level between knockout lines of miRNA-983, an adaptively evolving young miRNA, in D. melanogaster and D. simulans, and the replacement line of dme-miR-983 with dsi-miR-983 in D. melanogaster. By distinguishing context effects and miR-983 effects in the framework of cis and trans regulation of gene expression, we demonstrate that miR-983 contributes to interspecific expression divergence in spite of its antagonism with context effects which is often predominant. Gain and loss of miR-983 effects involve distinct gene sets featured in different phases of spermatogenesis, which may help minimize pleiotropy. The magnitude of miR-983 effects is negatively associated with the variability of target gene expression dynamics during spermatogenesis. Moreover, knocking out dme-miR-983 in D. melanogaster affects sperm length and sperm competitive ability, which can be rescued partially by dsi-miR-983. Our results provide empirical evidence for the resolution of negative pleiotropy, and also have broad evolutionary implications for the biological function of trans regulators. mRNA profiles of testes and spermatogenic single cysts for miR-983 related strains (wild types, knockout strains and interspecific gene replacement line) in two sibling species, Drosophila melanogaster and Drosophila simulans