Foley Shawn W, Gregory Brian D
Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania.
Cell and Molecular Biology Graduate Program, University of Pennsylvania, Philadelphia, Pennsylvania.
Curr Protoc Mol Biol. 2016 Oct 10;116:27.5.1-27.5.15. doi: 10.1002/cpmb.21.
Every eukaryotic RNA transcript undergoes extensive post-transcriptional processing from the moment of transcription up through degradation. This regulation is performed by a distinct cohort of RNA-binding proteins which recognize their target transcript by both its primary sequence and secondary structure. Here, we describe protein interaction profile sequencing (PIP-seq), a technique that uses ribonuclease-based footprinting followed by high-throughput sequencing to globally assess both protein-bound RNA sequences and RNA secondary structure. PIP-seq utilizes single- and double-stranded RNA-specific nucleases in the absence of proteins to infer RNA secondary structure. These libraries are also compared to samples that undergo nuclease digestion in the presence of proteins in order to find enriched protein-bound sequences. Combined, these four libraries provide a comprehensive, transcriptome-wide view of RNA secondary structure and RNA protein interaction sites from a single experimental technique. © 2016 by John Wiley & Sons, Inc.
从转录开始到降解，每个真核生物RNA转录本都会经历广泛的转录后加工。这种调控由一群独特的RNA结合蛋白执行，这些蛋白通过其一级序列和二级结构识别靶转录本。在这里，我们描述了蛋白质相互作用谱测序（PIP-seq），这是一种利用基于核糖核酸酶的足迹分析，随后进行高通量测序，以全面评估蛋白质结合的RNA序列和RNA二级结构的技术。PIP-seq在没有蛋白质的情况下利用单链和双链RNA特异性核酸酶来推断RNA二级结构。这些文库还与在蛋白质存在下进行核酸酶消化的样品进行比较，以找到富集的蛋白质结合序列。综合起来，这四个文库从单一实验技术提供了RNA二级结构和RNA-蛋白质相互作用位点的全转录组范围的综合视图。© 2016 John Wiley & Sons, Inc.
