An artificial riboswitch for controlling pre-mRNA splicing
An artificial riboswitch for controlling pre-mRNA splicing
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作者：
Kim, DS;Gusti, V;Gaur, RK
核糖开关，如前所述，是天然的RNA适体，其响应于小分子配体调节许多细菌代谢基因的表达。最近的研究表明，这些RNA遗传元件也存在于植物和真菌内含子的剪接位点连接处附近，从而提高了它们参与调节mRNA剪接的可能性。在这里，它是第一次显示，核糖开关可以在体外调控前mRNA剪接工程。我们表明，插入一个高亲和力的茶碱结合适体的3'剪接位点（T SS）区域的模型前mRNA（AdML-heo 29 AG），使其剪接被抑制的茶碱。我们的研究结果表明，3' SS AG在适体中的位置在赋予茶碱依赖性的前体mRNA剪接控制中起着至关重要的作用。我们还表明，茶碱介导的前体mRNA剪接的控制是高度特异性的，首先证明了在形状和大小上类似于茶碱的小分子配体对AdMIL-Theo 29 AG前体mRNA的剪接没有影响。第二，茶碱未能对不含其结合位点的前体mRNA的剪接产生任何影响。第三，茶碱特异性阻断剪接反应的步骤II。最后，我们提供的证据表明，茶碱依赖的前体mRNA剪接控制功能相关。
Riboswitches, as previously reported, are natural RNA aptamers that regulate the expression of numerous bacterial metabolic genes in response to small molecule ligands. It has recently been shown that these RNA genetic elements are also present near the splice site junctions of plant and fungal introns, thus raising the possibility of their involvement in regulating mRNA splicing. Here it is shown for the first time that a riboswitch can be engineered to regulate pre-mRNA splicing in vitro. We show that insertion of a high-affinity theophylline binding aptamer into the 3' splice site (T ss) region of a model pre-mRNA (AdML-heo29AG) enables its splicing to be repressed by the addition theophylline. Our results indicate that the location of 3' ss AG within the aptamer plays a crucial role in conferring theophylline-dependent control of pre-mRNA splicing. We also show that theophylline-mediated control of pre-mRNA splicing is highly specific by first demonstrating that a small molecule ligand similar in shape and size to theophylline had no effect on the splicing of AdMIL-Theo29AG pre-mRNA. Second, theophylline failed to exert any influence on the splicing of a pre-mRNA that does not contain its binding site. Third, theophylline specifically blocks the step II of the splicing reaction. Finally, we provide evidence that theophylline-dependent control of pre-mRNA splicing is functionally relevant.