Varying constraints impact the miR-6/5/4/286/3/309 microRNA cluster in Drosophila leg development.

A striking feature of microRNAs is that they are often clustered in the genomes of animals. The functional and evolutionary consequences of this clustering remain obscure. Here, we investigated a microRNA cluster miR-6/5/4/286/3/309 that arose and conserved in the drosophilid lineages. Small RNA sequencing revealed expression of this microRNA cluster in Drosophila melanogaster leg discs, and its overexpression caused leg shortening. Mutants are fertile and can mate, but males cannot efficiently grasp the female abdomen for courtship. Further mutants expressing different combinations of members in the microRNA cluster were constructed. Expression of individual ancient microRNA in the cluster resulted in normal phenotype, but either the expression of several ancient microRNAs together or recently evolved clustered microRNAs can recapitulate the phenotypes. Transcriptomic analyses revealed differentially expressed genes between mutants and wild-type, and screening of overexpression and loss-of-function mutants identified down-regulation of leg patterning gene cassettes. Further cell transfection with different combinations of cluster members revealed a suite of common and unique downstream target genes. This study demonstrates the importance of microRNA clustering where new members can reinforce the selection forces on both the flanking sequences (i.e. regulatory polycistronic promoter in controlling expression level) and the gene regulatory network of existing microRNA.