Atanasov Janina, Groher Florian, Weigand Julia E, Suess Beatrix
Department of Biology, Technical University Darmstadt, Darmstadt 64287, Germany.
Nucleic Acids Res. 2017 Dec 15;45(22):e181. doi: 10.1093/nar/gkx858.
Synthetic RNA-based systems have increasingly been used for the regulation of eukaryotic gene expression. Due to their structural properties, riboregulators provide a convenient basis for the development of ligand-dependent controllable systems. Here, we demonstrate reversible conditional control of miRNA biogenesis with an aptamer domain as a sensing unit connected to a natural miRNA precursor for the first time. For the design of the pre-miR switch, we replaced the natural terminal loop with the TetR aptamer. Thus, the TetR aptamer was positioned close to the Dicer cleavage sites, which allowed sterical control over pre-miR processing by Dicer. Our design proved to be highly versatile, allowing us to regulate the biogenesis of three structurally different miRNAs: miR-126, -34a and -199a. Dicer cleavage was inhibited up to 143-fold via co-expression of the TetR protein, yet could be completely restored upon addition of doxycycline. Moreover, we showed the functionality of the pre-miR switches for gene regulation through the interaction of the respective miRNA with its specific target sequence. Our designed device is capable of robust and reversible control of miRNA abundance. Thus, we offer a novel investigational tool for functional miRNA analysis.
基于合成RNA的系统越来越多地用于真核基因表达的调控。由于其结构特性，核糖调节因子为依赖配体的可控系统的开发提供了便利的基础。在此，我们首次展示了以适体结构域作为传感单元连接到天然miRNA前体上，对miRNA生物合成进行可逆的条件控制。为了设计前体miR开关，我们用TetR适体取代了天然末端环。因此，TetR适体被定位在靠近Dicer切割位点的位置，这使得能够通过空间位阻控制Dicer对前体miR的加工。我们的设计被证明具有高度的通用性，使我们能够调控三种结构不同的miRNA（miR-126、-34a和-199a）的生物合成。通过共表达TetR蛋白，Dicer切割被抑制高达143倍，但在添加强力霉素后可完全恢复。此外，我们通过各自的miRNA与其特定靶序列的相互作用，展示了前体miR开关在基因调控中的功能。我们设计的装置能够对miRNA丰度进行稳健且可逆的控制。因此，我们提供了一种用于功能性miRNA分析的新型研究工具。
