PT  - JOURNAL ARTICLE
AU  - Zhe Qu
AU  - Wing Chung Yiu
AU  - Ho Yin Yip
AU  - Wenyan Nong
AU  - Clare W.C. Yu
AU  - Ivy H.T. Lee
AU  - Annette Y.P. Wong
AU  - Nicola W.Y. Wong
AU  - Fiona K.M. Cheung
AU  - Ting Fung Chan
AU  - Kwok Fai Lau
AU  - Silin Zhong
AU  - Ka Hou Chu
AU  - Stephen S. Tobe
AU  - David E.K. Ferrier
AU  - William G. Bendena
AU  - Jerome H.L. Hui
TI  - MicroRNA clusters integrate evolutionary constraints on expression and target affinities: the miR-6/5/4/286/3/309 cluster in <em>Drosophila</em> leg development
AID  - 10.1101/714766
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 714766
4099  - http://biorxiv.org/content/early/2020/02/28/714766.short
4100  - http://biorxiv.org/content/early/2020/02/28/714766.full
AB  - 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 is conserved across drosophilid lineages. Small RNA sequencing revealed expression of this microRNA 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 microRNA cluster members were also constructed. Expression of individual microRNAs from the cluster resulted in a normal wild-type phenotype, but either the expression of several ancient microRNAs together (miR-5/4/286/3/309) or more recently evolved clustered microRNAs (miR-6-1/2/3) can recapitulate the phenotypes generated by the whole-cluster overexpression. Screening of transgenic fly lines revealed down-regulation of leg patterning gene cassettes in generation of the leg-shortening phenotype. Furthermore, cell transfection with different combinations of microRNA cluster members revealed a suite of downstream genes targeted by all cluster members, as well as complements of targets that are unique for distinct microRNAs. Considering together the microRNA targets and the evolutionary ages of each microRNA in the cluster demonstrates the importance of microRNA clustering, where new members can reinforce and modify the selection forces on both the cluster regulation and the gene regulatory network of existing microRNAs.