Center for RNA Science and Therapeutics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA.
RNA. 2024 Jun 17;30(7):920-937. doi: 10.1261/rna.079890.123.
RNA-binding proteins (RBPs) are essential for RNA metabolism and profoundly impact health and disease. The subcellular organization of RBP interaction networks with target RNAs remains largely unexplored. Here, we develop colocalization CLIP (coCLIP), a method that combines cross-linking and immunoprecipitation (CLIP) with proximity labeling, to explore in-depth the subcellular RNA interactions of the RBP human antigen R (HuR). Using this method, we uncover HuR's dynamic and location-specific interactions with RNA, revealing alterations in sequence preferences and interactions in the nucleus, cytosol, or stress granule (SG) compartments. We uncover HuR's unique binding preferences within SGs during arsenite stress, illuminating intricate interactions that conventional methodologies cannot capture. Overall, coCLIP provides a powerful method for revealing RBP-RNA interactions based on localization and lays the foundation for an advanced understanding of RBP models that incorporate subcellular location as a critical determinant of their functions.
RNA 结合蛋白 (RBPs) 是 RNA 代谢所必需的，对健康和疾病有着深远的影响。RBP 与靶 RNA 相互作用网络的亚细胞组织在很大程度上仍未得到探索。在这里，我们开发了共交联 CLIP(coCLIP)，这是一种将交联和免疫沉淀 (CLIP) 与邻近标记相结合的方法，深入研究了 RBP 人抗原 R (HuR) 的亚细胞 RNA 相互作用。使用这种方法，我们揭示了 HuR 与 RNA 的动态和位置特异性相互作用，揭示了在核、细胞质或应激颗粒 (SG) 隔室中序列偏好和相互作用的改变。我们在亚砷酸盐应激期间揭示了 HuR 在 SG 内的独特结合偏好，阐明了常规方法无法捕捉到的复杂相互作用。总的来说，coCLIP 提供了一种基于定位揭示 RBP-RNA 相互作用的强大方法，并为将亚细胞位置作为其功能关键决定因素的 RBP 模型的深入理解奠定了基础。
