A live-cell assay for the detection of pre-microRNA–protein interactions,RSC Chemical Biology

当前位置： X-MOL 学术 › RSC Chem. Biol. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

A live-cell assay for the detection of pre-microRNA–protein interactions
RSC Chemical Biology ( IF 4.2 ) Pub Date : 2020-12-8 , DOI: 10.1039/d0cb00055h
Sydney L Rosenblum ₁ , Daniel A Lorenz ₁ , Amanda L Garner _{1,

2}

Affiliation

Recent efforts in genome-wide sequencing and proteomics have revealed the fundamental roles that RNA-binding proteins (RBPs) play in the life cycle and function of coding and non-coding RNAs. While these methodologies provide a systems-level view of the networking of RNA and proteins, approaches to enable the cellular validation of discovered interactions are lacking. Leveraging the power of bioorthogonal chemistry- and split-luciferase-based assay technologies, we have devised a conceptually new assay for the live-cell detection of RNA–protein interactions (RPIs), RNA interaction with Protein-mediated Complementation Assay, or RiPCA. As proof-of-concept, we utilized the interaction of the pre-microRNA, pre-let-7, with its binding partner, Lin28. Using this system, we have demonstrated the selective detection of the pre-let-7-Lin28 RPI in both the cytoplasm and nucleus. Furthermore, we determined that this technology can be used to discern relative affinities for specific sequences as well as of individual RNA binding domains. Thus, RiPCA has the potential to serve as a useful tool in supporting the investigation of cellular RPIs.

中文翻译：

用于检测 pre-microRNA-蛋白质相互作用的活细胞测定

最近在全基因组测序和蛋白质组学方面的努力揭示了 RNA 结合蛋白 (RBP) 在编码和非编码 RNA 的生命周期和功能中发挥的基本作用。虽然这些方法提供了 RNA 和蛋白质网络的系统级视图，但缺乏对已发现的相互作用进行细胞验证的方法。利用生物正交化学和基于分裂荧光素酶的检测技术的力量，我们设计了一种概念上新的检测方法，用于活细胞检测 RNA-蛋白质相互作用 (RPI)、RNA 与蛋白质介导的互补分析或 RiPCA 的相互作用。作为概念验证，我们利用了 pre-microRNA、pre-let-7 与其结合伙伴 Lin28 的相互作用。使用该系统，我们证明了细胞质和细胞核中 pre-let-7-Lin28 RPI 的选择性检测。此外，我们确定该技术可用于辨别特定序列以及单个 RNA 结合域的相对亲和力。因此，RiPCA 有潜力成为支持细胞 RPI 研究的有用工具。

更新日期：2020-12-09

点击分享查看原文

点击收藏

公开下载

阅读更多本刊最新论文