Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, 02139, USA.
ACS Chem Biol. 2010 Sep 17;5(9):851-61. doi: 10.1021/cb100070j.
The importance and pervasiveness of naturally occurring regulation of RNA function in biology is increasingly being recognized. A common mechanism uses inducible protein-RNA interactions to shape diverse aspects of cellular RNA fate. Recapitulating this regulatory mode in cells using a novel set of protein-RNA interactions is appealing given the potential to subsequently modulate RNA biology in a manner decoupled from endogenous cellular physiology. Achieving this outcome, however, has previously proven challenging. Here, we describe a ligand-responsive protein-RNA interaction module, which can be used to target a specific RNA for subsequent regulation. Using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method, RNA aptamers binding to the bacterial Tet Repressor protein (TetR) with low- to subnanomolar affinities were obtained. This interaction is reversibly controlled by tetracycline in a manner analogous to the interaction of TetR with its cognate DNA operator. Aptamer minimization and mutational analyses support a functional role for two conserved sequence motifs in TetR binding. As an initial illustration of using this system to achieve protein-based regulation of RNA function in living cells, insertion of a TetR aptamer into the 5'-UTR of a reporter mRNA confers post-transcriptionally regulated, ligand-inducible protein synthesis in E. coli. Altogether, these results define and validate an inducible protein-RNA interaction module that incorporates desirable aspects of a ubiquitous mechanism for regulating RNA function in Nature and can be used as a foundational interaction for functionally and reversibly controlling the multiple fates of RNA in cells.
RNA 功能的自然调控在生物学中的重要性和普遍性正日益得到认识。一种常见的机制是利用诱导型蛋白-RNA 相互作用来塑造细胞 RNA 命运的多个方面。鉴于有可能以与内源性细胞生理学解耦的方式随后调节 RNA 生物学，在细胞中使用一组新的蛋白-RNA 相互作用来再现这种调节模式是很有吸引力的。然而，实现这一结果以前一直具有挑战性。在这里，我们描述了一个配体响应的蛋白-RNA 相互作用模块，可用于靶向特定的 RNA 进行后续调控。我们使用指数富集的配体系统进化（SELEX）方法获得了与细菌 Tet 阻遏蛋白（TetR）结合的低至亚纳摩尔亲和力的 RNA 适体。这种相互作用可以通过四环素以类似于 TetR 与其同源 DNA 操纵子相互作用的方式进行可逆控制。适体最小化和突变分析支持 TetR 结合的两个保守序列基序在功能上的作用。作为在活细胞中使用该系统实现基于蛋白的 RNA 功能调控的初始说明，将 TetR 适体插入报告 mRNA 的 5'UTR 赋予了在大肠杆菌中具有转录后调控、配体诱导的蛋白质合成能力。总的来说，这些结果定义并验证了一个可诱导的蛋白-RNA 相互作用模块，该模块结合了调节 RNA 功能的普遍机制的理想方面，可作为功能性和可逆地控制细胞中 RNA 多种命运的基础相互作用。
