Zhe, Q., Yiu, W.C., Yip, H.Y., Nong, W., Yu, C.W.C., Lee, I.H.T., Wong, A.Y.P., Wong, N.W.Y., Cheung, F.K.M., Chan, T.F., Lau, K.F., Zhong, S., Chu, K.H., Tobe, S.S., Ferrier, D.E.K., Bendena, W.G., Hui, J.H.L. (2020). Micro-RNA Clusters Integrate Evolutionary Constraints on Expression and Target Affinities: The miR-6/5/4/286/3/309 Cluster in Drosophila.  Mol. Biol. Evol. 37(10): 2955--2965.

FBrf0246873

Research paper

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.