Abstract
New miRNAs are evolutionarily important but their impact on existing biological networks remains unclear. We report the evolution of a microRNA cluster, mir-972C, that arose de novo and the subsequently rewired gene regulatory networks in Drosophila. Molecular evolution analyses revealed that mir-972C originated in the common ancestor of Drosophila where it comprises five old miRNAs. It subsequently recruited five new members in the melanogaster subgroup after conservative evolution for at least 50 million years. Population genetics analyses reveal that young and old mir-972C miRNAs evolved rapidly under positive selection in both seed and non-seed regions. Combining target prediction and cell transfection experiments, we find that sequence changes in individual mir-972C members resulted in extensive gene regulatory network divergence among D. melanogaster, D. simulans, and D. virilis, whereas the target pool of the cluster as a whole remains relatively conserved. Our results suggest that clustering of young and old miRNAs at the same locus broadens target repertoires, resulting in the gain of new targets without losing many old ones. This may facilitate the establishment of new miRNAs within existing regulatory networks.