Department of Microbiology, Center for Cell Signaling, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
Nucleic Acids Res. 2011 Apr;39(8):e53. doi: 10.1093/nar/gkq1316. Epub 2011 Feb 7.
The diverse localization of transcripts in cells suggests that there are many specific RNA-protein interactions that have yet to be identified. Progress has been limited, however, by the lack of a robust method to detect and isolate the RNA-binding proteins. Here we describe the use of an RNA aptamer, scaffolded to a tRNA, to create an affinity matrix that efficiently pulls down transcript-specific RNA-binding proteins from cell lysates. The addition of the tRNA scaffold to a Streptavidin aptamer (tRSA) increased binding efficiency by ∼ 10-fold. The tRSA system with an attached G-quartet sequence also could efficiently and specifically capture endogenous Fragile X Mental Retardation Protein (FMRP), which recognizes this RNA sequence. An alternative method, using biotinylated RNA, captured FMRP less efficiently than did our tRSA method. Finally we demonstrate the identification of novel RNA-binding proteins that interact with intron2 or 3'-UTR of the polarity protein Crumbs3 transcript. Proteins captured by these RNA sequences attached to the tRNA scaffold were identified by mass spectrometry. GFP-tagged versions of these proteins also showed specific interaction with either the Crb3 intron2 or 3'-UTR. Our tRSA technique should find wide application in mapping the RNA-protein interactome.
细胞中转录物的多样化定位表明，还有许多尚未被识别的特定 RNA-蛋白质相互作用。然而，由于缺乏一种强大的方法来检测和分离 RNA 结合蛋白，进展受到了限制。在这里，我们描述了使用 RNA 适体（支架连接到 tRNA）来创建亲和基质的方法，该方法可从细胞裂解物中有效提取转录本特异性的 RNA 结合蛋白。将 tRNA 支架添加到链霉亲和素适体（tRSA）中，将结合效率提高了约 10 倍。带有附着的 G-四联体序列的 tRSA 系统也能够高效且特异性地捕获内源性脆性 X 智力低下蛋白（FMRP），FMRP 可识别该 RNA 序列。另一种方法，使用生物素化 RNA，比我们的 tRSA 方法捕获 FMRP 的效率要低。最后，我们证明了鉴定与极性蛋白 Crumbs3 转录物的内含子 2 或 3'-UTR 相互作用的新 RNA 结合蛋白。通过质谱鉴定与 tRNA 支架相连的这些 RNA 序列捕获的蛋白质。这些蛋白质的 GFP 标记版本也显示出与 Crb3 内含子 2 或 3'-UTR 的特异性相互作用。我们的 tRSA 技术应该在绘制 RNA-蛋白质互作组图谱方面得到广泛应用。
