Department of Life Sciences, Tokyo Gakugei University, Koganei, Tokyo 184-8501, Japan.
Nucleic Acids Res. 2010 Jun;38(10):3489-501. doi: 10.1093/nar/gkq027. Epub 2010 Feb 15.
Many well-characterized examples of antisense RNAs from prokaryotic systems involve hybridization of the looped regions of stem-loop RNAs, presumably due to the high thermodynamic stability of the resulting loop-loop and loop-linear interactions. In this study, the identification of RNA stem-loops that inhibit U1A protein binding to the hpII RNA through RNA-RNA interactions was attempted using a bacterial reporter system based on phage lambda N-mediated antitermination. As a result, loop sequences possessing 7-8 base complementarity to the 5' region of the boxA element important for functional antitermination complex formation, but not the U1 hpII loop, were identified. In vitro and in vivo mutational analysis strongly suggested that the selected loop sequences were binding to the boxA region, and that the structure of the antisense stem-loop was important for optimal inhibitory activity. Next, in an attempt to demonstrate the ability to inhibit the interaction between the U1A protein and the hpII RNA, the rational design of an RNA stem-loop that inhibits U1A-binding to a modified hpII was carried out. Moderate inhibitory activity was observed, showing that it is possible to design and select antisense RNA stem-loops that disrupt various types of RNA-protein interactions.
许多来自原核系统的经过充分研究的反义 RNA 例子涉及到茎环 RNA 环区的杂交，这可能是由于形成的环-环和环-线性相互作用具有很高的热力学稳定性。在这项研究中，尝试使用基于噬菌体 lambda N 介导的抗终止的细菌报告系统，通过 RNA-RNA 相互作用来鉴定抑制 U1A 蛋白与 hpII RNA 结合的 RNA 茎环。结果，鉴定到了与框 A 元件 5' 区域具有 7-8 个碱基互补的环序列，该区域对于功能性抗终止复合物的形成很重要，但与 U1 hpII 环不互补。体外和体内突变分析强烈表明，所选的环序列与框 A 区域结合，并且反义茎环的结构对于最佳抑制活性很重要。接下来，为了尝试证明抑制 U1A 蛋白与 hpII RNA 之间相互作用的能力，对抑制 U1A 结合修饰后的 hpII 的 RNA 茎环进行了合理设计。观察到适度的抑制活性，表明可以设计和选择破坏各种类型的 RNA-蛋白相互作用的反义 RNA 茎环。
