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.

中文翻译：

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MicroRNA 簇整合了对表达和目标亲和力的进化限制：果蝇中的 miR-6/5/4/286/3/309 簇。

micro-RNA 的一个显着特征是它们通常聚集在动物的基因组中。这种集群的功能和进化后果仍然不清楚。在这里，我们研究了在果蝇谱系中保守的 micro-RNA 簇 miR-6/5/4/286/3/309。小 RNA 测序揭示了这种微 RNA 簇在黑腹果蝇中的表达腿盘和整个簇的条件性过度表达导致腿附肢缩短。还构建了表达不同 micro-RNA 簇成员组合的转基因过表达系。来自簇的单个 micro-RNA 的表达导致正常的野生型表型，但是几个古老的 micro-RNA 一起表达（miR-5/4/286/3/309）或最近进化的簇状 micro-RNA (miR-6-1/2/3) 可以概括整个簇过表达产生的表型。对转基因蝇系的筛选揭示了在腿部缩短表型的产生过程中腿部图案化基因盒的下调。此外，用不同的 micro-RNA 簇成员组合进行细胞转染揭示了一组被所有簇成员靶向的下游基因，以及不同 micro-RNA 所特有的目标互补物。综合考虑，micro-RNA 靶标和簇中每个 micro-RNA 的进化年龄证明了 micro-RNA 聚类的重要性，新成员可以加强和修改簇调控和基因调控网络的选择力现有的微RNA。