Medical Scientist Training Program, University of Chicago, Chicago, IL 60637, USA.
Department of Chemistry, University of Chicago, Chicago, IL 60637, USA.
Methods. 2017 Aug 15;126:105-111. doi: 10.1016/j.ymeth.2017.04.019. Epub 2017 Apr 26.
The reversible N-methyladenosine (mA) modification of eukaryotic messenger RNAs (mRNAs) is a widespread regulatory mechanism that impacts every step in the mRNA life cycle. The effect of mA on mRNA fate depends on the binding of "mA reader" proteins - RNA binding proteins that specifically bind to RNAs containing mA. Here, we describe an RNA pull-down method that can be used to identify novel mA reader proteins starting from a known mA-modified site in cellular or viral RNA. We further describe how a combination of immunoprecipitation-based sequencing methods can be used to identify mA-modified sites bound by an mA reader protein on a transcriptome-wide level. The discovery of new mA reader proteins and their mA-modified targets would provide further insight into the mechanisms and functions of mA in the cell.
真核信使 RNA（mRNA）的可逆 N6-甲基腺苷（m6A）修饰是一种广泛存在的调控机制，影响 mRNA 生命周期的每一个步骤。m6A 对 mRNA 命运的影响取决于“m6A 阅读蛋白”的结合——即特异性结合含有 m6A 的 RNA 的 RNA 结合蛋白。在这里，我们描述了一种 RNA 下拉方法，该方法可以从细胞或病毒 RNA 中的已知 m6A 修饰位点开始，用于鉴定新的 m6A 阅读蛋白。我们进一步描述了如何结合基于免疫沉淀的测序方法，在转录组水平上鉴定 m6A 阅读蛋白结合的 m6A 修饰位点。新的 m6A 阅读蛋白及其 m6A 修饰靶标的发现将进一步深入了解 m6A 在细胞中的作用机制和功能。
