Max Delbrück Center for Molecular Medicine, Berlin Institute for Medical Systems Biology, 13125 Berlin, Germany.
Mol Cell. 2012 Jun 8;46(5):674-90. doi: 10.1016/j.molcel.2012.05.021.
Protein-RNA interactions are fundamental to core biological processes, such as mRNA splicing, localization, degradation, and translation. We developed a photoreactive nucleotide-enhanced UV crosslinking and oligo(dT) purification approach to identify the mRNA-bound proteome using quantitative proteomics and to display the protein occupancy on mRNA transcripts by next-generation sequencing. Application to a human embryonic kidney cell line identified close to 800 proteins. To our knowledge, nearly one-third were not previously annotated as RNA binding, and about 15% were not predictable by computational methods to interact with RNA. Protein occupancy profiling provides a transcriptome-wide catalog of potential cis-regulatory regions on mammalian mRNAs and showed that large stretches in 3' UTRs can be contacted by the mRNA-bound proteome, with numerous putative binding sites in regions harboring disease-associated nucleotide polymorphisms. Our observations indicate the presence of a large number of mRNA binders with diverse molecular functions participating in combinatorial posttranscriptional gene-expression networks.
蛋白质与 RNA 的相互作用是核心生物过程的基础，例如 mRNA 的剪接、定位、降解和翻译。我们开发了一种光反应核苷酸增强的 UV 交联和 oligo(dT) 纯化方法，用于使用定量蛋白质组学鉴定 mRNA 结合蛋白质组，并通过下一代测序展示 mRNA 转录本上的蛋白质占有率。将其应用于人类胚胎肾细胞系，鉴定出近 800 种蛋白质。据我们所知，其中近三分之一以前未被注释为 RNA 结合蛋白，约 15%的蛋白质不能通过计算方法预测与 RNA 相互作用。蛋白质占有率分析为哺乳动物 mRNA 上的潜在顺式调控区提供了一个全转录组范围的目录，并表明 3'UTR 中的大片段可以与 mRNA 结合蛋白质接触，在含有与疾病相关核苷酸多态性的区域中存在许多假定的结合位点。我们的观察结果表明，存在大量具有不同分子功能的 mRNA 结合蛋白，参与组合性转录后基因表达网络。
