PDF(Pubmed)
Abstract：
The RNA recognition motif (RRM) is the most common RNA-binding protein domain identified in nature. However, RRM-containing proteins are only prevalent in eukaryotic phyla, in which they play central regulatory roles. Here, we engineered an orthogonal post-transcriptional control system of gene expression in the bacterium Escherichia coli with the mammalian RNA-binding protein Musashi-1, which is a stem cell marker with neurodevelopmental role that contains two canonical RRMs. In the circuit, Musashi-1 is regulated transcriptionally and works as an allosteric translation repressor thanks to a specific interaction with the N-terminal coding region of a messenger RNA and its structural plasticity to respond to fatty acids. We fully characterized the genetic system at the population and single-cell levels showing a significant fold change in reporter expression, and the underlying molecular mechanism by assessing the in vitro binding kinetics and in vivo functionality of a series of RNA mutants. The dynamic response of the system was well recapitulated by a bottom-up mathematical model. Moreover, we applied the post-transcriptional mechanism engineered with Musashi-1 to specifically regulate a gene within an operon, implement combinatorial regulation, and reduce protein expression noise. This work illustrates how RRM-based regulation can be adapted to simple organisms, thereby adding a new regulatory layer in prokaryotes for translation control.
摘要：
RNA识别基序（RRM）是自然界中最常见的RNA结合蛋白结构域。然而,含RRM的蛋白质仅在真核生物门中普遍存在，他们在其中发挥着核心监管作用。这里,我们用哺乳动物RNA结合蛋白Musashi-1设计了一个在大肠杆菌中基因表达的正交转录后控制系统，Musashi-1是一种具有神经发育作用的干细胞标记，包含两个规范的RRM。在电路中，Musashi-1在转录上受到调节，并由于与信使RNA的N端编码区的特异性相互作用及其对脂肪酸的结构可塑性而作为变构翻译阻遏物起作用。我们在群体和单细胞水平上充分表征了遗传系统，显示出报告表达的显着倍数变化，以及通过评估一系列RNA突变体的体外结合动力学和体内功能来评估潜在的分子机制。自下而上的数学模型很好地概括了系统的动态响应。此外，我们应用了Musashi-1的转录后机制来特异性调节操纵子内的基因，实施组合调节，并降低蛋白质表达噪声。这项工作说明了基于RRM的调控如何适应简单的生物体，从而在原核生物中增加了一个新的调节层用于翻译控制。
