miR-1/133a Clusters Cooperatively Specify the Cardiomyogenic Lineage by Adjustment of Myocardin Levels during Embryonic Heart Development

miRNAs are small RNAs directing many developmental processes by posttranscriptional regulation of protein-coding genes. We uncovered a new role for miR-1-1/133a-2 and miR-1-2/133a-1 clusters in the specification of embryonic cardiomyocytes allowing transition from an immature state characterized by expression of smooth muscle (SM) genes to a more mature fetal phenotype. Concomitant knockout of miR-1-1/133a-2 and miR-1-2/133a-1 released suppression of the transcriptional co-activator myocardin, a major regulator of SM gene expression, but not of its binding partner SRF. Overexpression of myocardin in the embryonic heart essentially recapitulated the miR-1/133a mutant phenotype at the molecular level, arresting embryonic cardiomyocytes in an immature state. Interestingly, the majority of postulated miR-1/133a targets was not altered in double mutant mice, indicating that the ability of miR-1/133a to suppress target molecules strongly depends on the cellular context. Finally, we show that myocardin positively regulates expression of miR-1/133a, thus constituting a negative feedback loop that is essential for early cardiac development.微小RNA（miRNAs）是一类通过对蛋白编码基因进行转录后调控，参与众多发育进程的小型RNA分子。我们揭示了miR-1-1/133a-2与miR-1-2/133a-1基因簇在胚胎心肌细胞特化中的全新功能：该基因簇可介导胚胎心肌细胞从以平滑肌（smooth muscle，SM）基因表达为特征的未成熟状态，过渡至更为成熟的胎儿表型。同时敲除miR-1-1/133a-2与miR-1-2/133a-1，可解除对转录共激活因子心肌素（myocardin）的抑制，该因子是平滑肌基因表达的核心调控因子，但不会影响其结合蛋白血清应答因子（serum response factor，SRF）。在胚胎心脏中过表达心肌素，可在分子层面基本重现miR-1/133a突变体的表型，使胚胎心肌细胞停滞于未成熟状态。值得注意的是，多数被推测为miR-1/133a靶标的分子在双突变小鼠中并未出现表达改变，这表明miR-1/133a对靶标分子的抑制能力强烈依赖于细胞背景。最后，我们证实心肌素可正向调控miR-1/133a的表达，由此构成一条对早期心脏发育至关重要的负反馈环路。