Howard Hughes Medical Institute, RNA Therapeutics Institute, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, United States.
Howard Hughes Medical Institute, RNA Therapeutics Institute, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, United States.
Methods. 2014 Feb;65(3):320-32. doi: 10.1016/j.ymeth.2013.09.013. Epub 2013 Oct 2.
Development of high-throughput approaches to map the RNA interaction sites of individual RNA binding proteins (RBPs) transcriptome-wide is rapidly transforming our understanding of post-transcriptional gene regulatory mechanisms. Here we describe a ribonucleoprotein (RNP) footprinting approach we recently developed for identifying occupancy sites of both individual RBPs and multi-subunit RNP complexes. RNA:protein immunoprecipitation in tandem (RIPiT) yields highly specific RNA footprints of cellular RNPs isolated via two sequential purifications; the resulting RNA footprints can then be identified by high-throughput sequencing (Seq). RIPiT-Seq is broadly applicable to all RBPs regardless of their RNA binding mode and thus provides a means to map the RNA binding sites of RBPs with poor inherent ultraviolet (UV) crosslinkability. Further, among current high-throughput approaches, RIPiT has the unique capacity to differentiate binding sites of RNPs with overlapping protein composition. It is therefore particularly suited for studying dynamic RNP assemblages whose composition evolves as gene expression proceeds.
高通量方法的发展使得能够在转录组范围内绘制单个 RNA 结合蛋白 (RBP) 的 RNA 相互作用位点图谱，这迅速改变了我们对转录后基因调控机制的理解。在这里，我们描述了一种我们最近开发的核糖核蛋白 (RNP) 足迹分析方法，用于鉴定单个 RBP 和多亚基 RNP 复合物的占据位点。串联的 RNA:蛋白质免疫沉淀 (RIPiT) 通过两次连续纯化分离细胞 RNP ，得到高度特异性的细胞 RNA 足迹；然后可以通过高通量测序 (Seq) 来鉴定这些 RNA 足迹。RIPiT-Seq 广泛适用于所有 RBP，无论其 RNA 结合模式如何，因此为那些固有紫外 (UV) 交联能力差的 RBP 提供了一种绘制 RNA 结合位点的方法。此外，在当前的高通量方法中，RIPiT 具有独特的区分具有重叠蛋白组成的 RNP 结合位点的能力。因此，它特别适合研究随着基因表达进行而演变的动态 RNP 组装体。
