Micro-RNA Clusters Integrate Evolutionary Constraints on Expression and Target Affinities: The miR-6/5/4/286/3/309 Cluster in Drosophila

摘要

A striking feature of micro‐RNAs 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 micro‐RNA cluster miR-6/5/4/286/3/309 that is conserved across drosophilid lineages. Small RNA sequencing revealed expression of this micro‐RNA cluster in Drosophila melanogaster leg discs, and conditional overexpression of the whole cluster resulted in leg appendage shortening. Transgenic overexpression lines expressing different combinations of micro‐RNA cluster members were also constructed. Expression of individual micro‐RNAs from the cluster resulted in a normal wild-type phenotype, but either the expression of several ancient micro‐RNAs together (miR-5/4/286/3/309) or more recently evolved clustered micro‐RNAs (miR-6-1/2/3) can recapitulate the phenotypes generated by the whole-cluster overexpression. Screening of transgenic fly lines revealed downregulation of leg-patterning gene cassettes in generation of the leg-shortening phenotype. Furthermore, cell transfection with different combinations of micro‐RNA cluster members revealed a suite of downstream genes targeted by all cluster members, as well as complements of targets that are unique for distinct micro‐RNAs. Considered together, the micro‐RNA targets and the evolutionary ages of each micro‐RNA in the cluster demonstrate the importance of micro‐RNA clustering, where new members can reinforce and modify the selection forces on both the cluster regulation and the gene regulatory network of existing micro‐RNAs. Key words: micro‐RNA, cluster, evolution.