Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Faculty of Health Sciences , DK-2200 Copenhagen, Denmark.
Disease Systems Biology, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Faculty of Health Sciences , DK-2200 Copenhagen, Denmark.
J Proteome Res. 2017 Aug 4;16(8):2762-2772. doi: 10.1021/acs.jproteome.7b00042. Epub 2017 Jul 7.
RNA-binding proteins (RBPs) allow cells to carry out pre-RNA processing and post-transcriptional regulation of gene expression, and aberrations in RBP functions have been linked to many diseases, including neurological disorders and cancer. Human cells encode thousands of RNA-binding proteins with unique RNA-binding properties. These properties are regulated through modularity of a large variety of RNA-binding domains, rendering RNA-protein interactions difficult to study. Recently, the introduction of proteomics methods has provided novel insights into RNA-binding proteins at a systems level. However, determining the exact protein sequence regions that interact with RNA remains challenging and laborious, especially considering that many RBPs lack canonical RNA-binding domains. Here we describe a streamlined proteomic workflow called peptide cross-linking and affinity purification (pCLAP) that allows rapid characterization of RNA-binding regions in proteins. pCLAP is based upon the combined use of UV cross-linking and enzymatic digestion of RNA-bound proteins followed by single-shot mass spectrometric analysis. To benchmark our method, we identified the binding regions for polyadenylated RNA-binding proteins in HEK293 cells, allowing us to map the mRNA interaction regions of more than 1000 RBPs with very high reproducibility from replicate single-shot analyses. Our results show specific enrichment of many known RNA-binding regions on many known RNA-binding proteins, confirming the specificity of our approach.
RNA 结合蛋白（RBPs）使细胞能够进行前 RNA 加工和转录后基因表达调控，并且 RBP 功能的异常与许多疾病有关，包括神经紊乱和癌症。人类细胞编码具有独特 RNA 结合特性的数千种 RNA 结合蛋白。这些特性通过大量 RNA 结合域的模块性来调节，使得 RNA-蛋白相互作用难以研究。最近，蛋白质组学方法的引入为 RNA 结合蛋白提供了系统水平的新见解。然而，确定与 RNA 相互作用的确切蛋白序列区域仍然具有挑战性和繁琐，特别是考虑到许多 RBPs 缺乏典型的 RNA 结合域。在这里，我们描述了一种称为肽交联和亲和纯化（pCLAP）的简化蛋白质组学工作流程，该流程可快速表征蛋白质中的 RNA 结合区域。pCLAP 基于 UV 交联和 RNA 结合蛋白的酶消化的联合使用，然后进行单次质谱分析。为了基准测试我们的方法，我们确定了 HEK293 细胞中多聚腺苷酸化 RNA 结合蛋白的结合区域，使我们能够从重复的单次分析中以非常高的重现性绘制 1000 多个 RBPs 的 mRNA 相互作用区域。我们的结果显示，许多已知的 RNA 结合蛋白上的许多已知 RNA 结合区域特异性富集，证实了我们方法的特异性。
