IRI Life Sciences, Humboldt University, Philippstr. 13, 10115, Berlin, Germany.
Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125, Berlin, Germany.
Nat Commun. 2019 Mar 1;10(1):990. doi: 10.1038/s41467-019-08942-3.
Recent methodological advances allowed the identification of an increasing number of RNA-binding proteins (RBPs) and their RNA-binding sites. Most of those methods rely, however, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to non-adenylate RNA classes (tRNA, rRNA, pre-mRNA) as well as the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria). We have developed the Phenol Toluol extraction (PTex) protocol that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to RNA as short as 30 nt, RNPs directly from animal tissue and can be used to simplify complex workflows such as PAR-CLIP. Finally, we provide a global RNA-bound proteome of human HEK293 cells and the bacterium Salmonella Typhimurium.
最近的方法学进展使得能够鉴定越来越多的 RNA 结合蛋白 (RBP) 和它们的 RNA 结合位点。然而，这些方法大多依赖于捕获与多聚腺苷酸化 RNA 相关的蛋白质，而这些方法忽略了与非腺苷酸化 RNA 类 (tRNA、rRNA、前体 mRNA) 结合的 RBP，以及绝大多数缺乏多 A 尾巴的 mRNA 中的物种 (包括所有古菌和细菌)。我们开发了酚甲苯提取 (PTex) 方案，该方案不依赖于特定的 RNA 序列或基序来分离交联的核糖核蛋白 (RNP)，而是完全根据它们的物理化学性质来纯化它们。PTex 可以捕获与 RNA 结合的 RBP，这些 RBP 与 RNA 的结合长度短至 30 个核苷酸，可直接从动物组织中提取 RNP，并可用于简化复杂的工作流程，如 PAR-CLIP。最后，我们提供了人类 HEK293 细胞和沙门氏菌 Typhimurium 的全 RNA 结合蛋白质组。
