Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
Chembiochem. 2018 Jan 18;19(2):171-176. doi: 10.1002/cbic.201700458. Epub 2017 Dec 18.
RNA-binding proteins recognizing unique sequences within large transcriptomes serve as a powerful tool to control RNA metabolism. Pumilio and fem-3 mRNA-binding factor (PUF) proteins are considered good candidates for such tools, because they are typically composed of eight highly homologous repeat segments and can be designed to recognize arbitrary 8 nt RNA sequences. However, a specific 8 nt RNA sequence is found at multiple sites in various RNAs in the transcriptome, making it difficult to specifically target a single RNA. Designer PUF proteins recognizing longer RNA sequences should achieve more selective binding. Here, we propose an approach for creating 16-repeat PUFs capable of targeting a single, unique mRNA in the transcriptome. Our design is simple and involves either the tandem alignment of two PUF segments or the nesting of one PUF segment within another. Designed 16-repeat PUFs bound to the target RNA sequence without partial recognition derived from the original 8-repeat PUF. Furthermore, based on our strategy, expression of an endogenous mRNA was selectively and effectively modulated, demonstrating the applicability of 16-repeat PUF proteins for regulating endogenous RNA metabolism.
RNA 结合蛋白识别大转录组内的独特序列，可作为控制 RNA 代谢的有力工具。Pumilio 和 fem-3 mRNA 结合因子（PUF）蛋白被认为是此类工具的良好候选物，因为它们通常由八个高度同源的重复片段组成，并且可以设计为识别任意 8nt RNA 序列。然而，在转录组中的各种 RNA 中，多个位点都存在特定的 8nt RNA 序列，这使得难以特异性靶向单个 RNA。识别更长 RNA 序列的设计 PUF 蛋白应实现更具选择性的结合。在这里，我们提出了一种创建能够靶向转录组中单一独特 mRNA 的 16 重复 PUF 的方法。我们的设计简单，涉及两个 PUF 片段的串联排列或一个 PUF 片段嵌套在另一个 PUF 片段内。设计的 16 重复 PUF 与靶 RNA 序列结合，而不会产生源自原始 8 重复 PUF 的部分识别。此外，根据我们的策略，内源性 mRNA 的表达被选择性和有效地调节，证明了 16 重复 PUF 蛋白在调节内源性 RNA 代谢中的适用性。
