Rogell Birgit, Fischer Bernd, Rettel Mandy, Krijgsveld Jeroen, Castello Alfredo, Hentze Matthias W
European Molecular Biology Laboratory (EMBL), 69117 Heidelberg, Germany.
German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.
RNA. 2017 Aug;23(8):1290-1302. doi: 10.1261/rna.060798.117. Epub 2017 May 5.
RNA-binding proteins (RBPs) play essential roles in RNA biology, responding to cellular and environmental stimuli to regulate gene expression. Important advances have helped to determine the (near) complete repertoires of cellular RBPs. However, identification of RBPs associated with specific transcripts remains a challenge. Here, we describe "specific ribonucleoprotein (RNP) capture," a versatile method for the determination of the proteins bound to specific transcripts in vitro and in cellular systems. Specific RNP capture uses UV irradiation to covalently stabilize protein-RNA interactions taking place at "zero distance." Proteins bound to the target RNA are captured by hybridization with antisense locked nucleic acid (LNA)/DNA oligonucleotides covalently coupled to a magnetic resin. After stringent washing, interacting proteins are identified by quantitative mass spectrometry. Applied to in vitro extracts, specific RNP capture identifies the RBPs bound to a reporter mRNA containing the Sex-lethal (Sxl) binding motifs, revealing that the Sxl homolog sister of Sex lethal (Ssx) displays similar binding preferences. This method also revealed the repertoire of RBPs binding to 18S or 28S rRNAs in HeLa cells, including previously unknown rRNA-binding proteins.
RNA结合蛋白（RBPs）在RNA生物学中发挥着至关重要的作用，它们响应细胞和环境刺激来调节基因表达。重要进展有助于确定细胞RBPs的（近乎）完整清单。然而，鉴定与特定转录本相关的RBPs仍然是一项挑战。在这里，我们描述了“特异性核糖核蛋白（RNP）捕获”，这是一种在体外和细胞系统中确定与特定转录本结合的蛋白质的通用方法。特异性RNP捕获利用紫外线照射共价稳定在“零距离”发生的蛋白质-RNA相互作用。与靶RNA结合的蛋白质通过与共价偶联到磁性树脂上的反义锁核酸（LNA）/DNA寡核苷酸杂交来捕获。经过严格洗涤后，通过定量质谱鉴定相互作用的蛋白质。应用于体外提取物时，特异性RNP捕获可鉴定与含有性致死（Sxl）结合基序的报告mRNA结合的RBPs，揭示性致死同源物性致死姐妹（Ssx）表现出相似的结合偏好。该方法还揭示了HeLa细胞中与18S或28S rRNA结合的RBPs清单，包括以前未知的rRNA结合蛋白。
