School of Life Science and Technology, ShanghaiTech University, Shanghai, China.
University of Chinese Academy of Sciences, Beijing, China.
Nucleic Acids Res. 2020 May 21;48(9):e52. doi: 10.1093/nar/gkaa143.
No RNA is completely naked from birth to death. RNAs function with and are regulated by a range of proteins that bind to them. Therefore, the development of innovative methods for studying RNA-protein interactions is very important. Here, we developed a new tool, the CRISPR-based RNA-United Interacting System (CRUIS), which captures RNA-protein interactions in living cells by combining the power of CRISPR and PUP-IT, a novel proximity targeting system. In CRUIS, dCas13a is used as a tracker to target specific RNAs, while proximity enzyme PafA is fused to dCas13a to label the surrounding RNA-binding proteins, which are then identified by mass spectrometry. To identify the efficiency of CRUIS, we employed NORAD (Noncoding RNA activated by DNA damage) as a target, and the results show that a similar interactome profile of NORAD can be obtained as by using CLIP (crosslinking and immunoprecipitation)-based methods. Importantly, several novel NORAD RNA-binding proteins were also identified by CRUIS. The use of CRUIS facilitates the study of RNA-protein interactions in their natural environment, and provides new insights into RNA biology.
没有一种 RNA 是从出生到死亡完全没有蛋白质结合的。RNA 的功能与其结合的一系列蛋白质有关，并受到这些蛋白质的调节。因此，开发用于研究 RNA-蛋白质相互作用的创新方法非常重要。在这里，我们开发了一种新的工具，即基于 CRISPR 的 RNA 联合相互作用系统（CRUIS），该系统通过结合 CRISPR 和 PUP-IT 的力量，即一种新的接近靶向系统，在活细胞中捕获 RNA-蛋白质相互作用。在 CRUIS 中，dCas13a 被用作靶向特定 RNA 的追踪器，而邻近酶 PafA 被融合到 dCas13a 上，以标记周围的 RNA 结合蛋白，然后通过质谱法对其进行鉴定。为了确定 CRUIS 的效率，我们将 NORAD（DNA 损伤激活的非编码 RNA）作为靶标，结果表明，CRUIS 可以获得与基于 CLIP（交联和免疫沉淀）的方法相似的 NORAD 互作组图谱。重要的是，CRUIS 还鉴定了几种新的 NORAD RNA 结合蛋白。CRUIS 的使用促进了在其自然环境中研究 RNA-蛋白质相互作用，并为 RNA 生物学提供了新的见解。
