Max-Delbrück-Center for Molecular Medicine, Berlin Institute for Medical Systems Biology, Robert-Rössle Str. 10, 13125 Berlin, Germany.
Max-Delbrück-Center for Molecular Medicine, Berlin Institute for Medical Systems Biology, Robert-Rössle Str. 10, 13125 Berlin, Germany.
Methods. 2014 Feb;65(3):302-9. doi: 10.1016/j.ymeth.2013.09.017. Epub 2013 Oct 1.
A key prerequisite to understand how gene regulatory processes are controlled by the interplay of RNA-binding proteins and ribonucleoprotein complexes with RNAs is the generation of comprehensive high-resolution maps of protein-RNA interactions. Recent advances in next-generation sequencing technology accelerated the development of various crosslinking and immunoprecipitation (CLIP) approaches to broadly identify RNA regions contacted by RNA-binding proteins. However these methods only consider single RNA-binding proteins and their contact sites, irrespective of the overall cis-regulatory sequence space contacted by other RNA interacting factors. Here we describe the application of protein occupancy profiling, a novel approach that globally displays the RNA contact sites of the poly(A)+ RNA-bound proteome. Protein occupancy profiling enables the generation of transcriptome-wide maps of protein-RNA interactions on polyadenylated transcripts and narrows the sequence search space for transcript regions involved in cis-regulation of gene expression in response to internal or external stimuli, altered cellular programs or disease.
理解基因调控过程如何受到 RNA 结合蛋白和核糖核蛋白复合物与 RNA 相互作用的控制，一个关键的前提条件是生成蛋白质-RNA 相互作用的全面高分辨率图谱。下一代测序技术的最新进展加速了各种交联和免疫沉淀 (CLIP) 方法的发展，这些方法广泛地识别 RNA 结合蛋白结合的 RNA 区域。然而，这些方法仅考虑单个 RNA 结合蛋白及其结合位点，而不考虑其他 RNA 相互作用因子所接触的整体顺式调控序列空间。在这里，我们描述了蛋白质占据谱分析的应用，这是一种新的方法，可以全局显示多聚（A）+ RNA 结合蛋白质组的 RNA 结合位点。蛋白质占据谱分析能够生成多聚腺苷酸化转录物上蛋白质-RNA 相互作用的全转录组图谱，并缩小了参与顺式调控基因表达的转录区域的序列搜索空间，以响应内部或外部刺激、改变的细胞程序或疾病。
