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Mef2 is the key transcription factor for muscle development and differentiation in Drosophila. It activates hundreds of downstream target genes, including itself. Precise control of Mef2 levels is essential for muscle development as different Mef2 protein levels activate distinct sets of muscle genes, but how this is achieved remains unclear. Here, we have identified a novel heart- and muscle-specific microRNA, miR-92b, which is activated by Mef2 and subsequently downregulates Mef2 through binding to its 3′UTR, forming a negative regulatory circuit that fine-tunes the level of Mef2. Deletion of miR-92b caused abnormally high Mef2 expression, leading to muscle defects and lethality. Blocking miR-92b function using microRNA sponge techniques also increased Mef2 levels and caused muscle defects similar to those seen with the miR-92b deletion. Additionally, overexpression of miR-92b reduced Mef2 levels and …