RNA−protein interactions play a pivotal role in cell homeostasis and disease, but current approaches to study them require a considerable amount of starting material, favor the recovery of only a subset of RNA species or are complex and time-consuming. We recently developed orthogonal organic phase separation (OOPS): a quick, efficient and reproducible method to purify cross-linked RNA−protein adducts in an unbiased way. OOPS avoids molecular tagging or the capture of polyadenylated RNA. Instead, it is based on sampling the interface of a standard TRIzol extraction to enrich RNA-binding proteins (RBPs) and their cognate bound RNA. OOPS specificity is achieved by digesting the enriched interfaces with RNases or proteases to release the RBPs or protein-bound RNA, respectively. Here we present a step-by-step protocol to purify protein–RNA adducts, free protein and free RNA from the same sample. We further describe how OOPS can be applied in human cell lines, Arabidopsis thaliana, Schizosaccharomyces pombe and Escherichia coli and how it can be used to study RBP dynamics.

RNA-蛋白质相互作用在细胞稳态和疾病中起着举足轻重的作用，但目前研究它们的方法需要大量的起始材料，有利于仅恢复一部分 RNA 种类，或者复杂且耗时。我们最近开发了正交有机相分离 (OOPS)：一种快速、高效且可重现的方法，以无偏见的方式纯化交联的 RNA-蛋白质加合物。OOPS 避免了分子标记或多腺苷酸化 RNA 的捕获。相反，它基于对标准 TRIzol 提取的界面进行采样，以丰富 RNA 结合蛋白 (RBP) 及其同源结合 RNA。OOPS 特异性是通过用 RNase 或蛋白酶消化富集的界面来分别释放 RBP 或蛋白质结合 RNA 来实现的。在这里，我们提出了一个逐步纯化蛋白质-RNA 加合物的方案，来自同一样品的游离蛋白质和游离 RNA。我们进一步描述了 OOPS 如何应用于人类细胞系，拟南芥、粟酒裂殖酵母和大肠杆菌及其如何用于研究 RBP 动力学。