Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA 94305, USA.
Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
STAR Protoc. 2021 Apr 28;2(2):100477. doi: 10.1016/j.xpro.2021.100477. eCollection 2021 Jun 18.
The majority of the mammalian genome is transcribed into non-coding RNAs, many of which co-evolve with RNA-binding proteins (RBPs) to function as biochemically defined and tractable ribonucleoproteins (RNPs). Here, we applied icSHAPE- a robust and versatile RNA structural probing pipeline- to endogenous RNPs purified from nuclei, providing base-resolution structural rationale for RNP activity and subcellular localization. Combining with genetic and biochemical reconstitutions, structural and functional alternations can be directly attributed to a given RBP without ambiguity. For complete details on the use and execution of this protocol, please refer to Chen et al. (2018).
大多数哺乳动物基因组都被转录为非编码 RNA，其中许多与 RNA 结合蛋白 (RBP) 共同进化，作为生化定义明确且易于处理的核糖核蛋白 (RNP) 发挥作用。在这里，我们应用了一种强大而通用的 RNA 结构探测管道 icSHAPE- 对从核中纯化的内源性 RNP 进行了分析，为 RNP 活性和亚细胞定位提供了基于碱基的结构基础。通过与遗传和生化重建相结合，可以直接将结构和功能的改变归因于给定的 RBP，而不会产生歧义。有关该方案使用和执行的完整详细信息，请参阅 Chen 等人。(2018)。
