Arguello A Emilia, Srikumar Tharan, Kleiner Ralph E
Department of Chemistry, Princeton University, Princeton, New Jersey.
Proteomics and Mass Spectrometry Core, Princeton University, Princeton, New Jersey.
Curr Protoc Nucleic Acid Chem. 2018 Dec;75(1):e69. doi: 10.1002/cpnc.69. Epub 2018 Nov 8.
Post-transcriptional modifications play an important role in RNA biology. In particular, the addition of small chemical groups to the nucleobases of mRNA can affect how modified transcripts are processed in the cell, thereby impacting gene expression programs. In order to study the molecular mechanisms underlying these modifications, it is necessary to characterize their 'readers', that is, proteins that directly bind to these modifications to mediate their functional consequences; this is a major challenge because we lack approaches to precisely manipulate RNA chemistry in the cell and because protein-modified RNA interactions can be low affinity. In this unit, we describe in detail a photocrosslinking-based RNA chemical proteomics approach to profile the protein-modified RNA interactome modulated by N -methyladenosine (m A), the most abundant internal modification in eukaryotic mRNA. First, we present protocols for the synthesis and characterization of short, diazirine-containing synthetic RNA probes, followed by a description of their use in mass spectrometry-based proteomics with HeLa cell lysate and a short commentary on data analysis and result interpretation. © 2018 by John Wiley & Sons, Inc.
转录后修饰在RNA生物学中发挥着重要作用。特别是，向mRNA的核碱基添加小化学基团会影响修饰转录本在细胞中的加工方式，从而影响基因表达程序。为了研究这些修饰背后的分子机制，有必要对它们的“读取蛋白”进行表征，即直接结合这些修饰以介导其功能后果的蛋白质；这是一项重大挑战，因为我们缺乏在细胞中精确操纵RNA化学的方法，而且蛋白质与修饰RNA的相互作用可能具有低亲和力。在本单元中，我们详细描述了一种基于光交联的RNA化学蛋白质组学方法，用于分析由N-甲基腺苷（m⁶A）（真核mRNA中最丰富的内部修饰）调节的蛋白质-修饰RNA相互作用组。首先，我们介绍了含重氮丙啶的短合成RNA探针的合成和表征方案，随后描述了它们在基于质谱的蛋白质组学中与HeLa细胞裂解物一起使用的方法，以及对数据分析和结果解释的简短评论。© 2018 John Wiley & Sons, Inc.
