使用重组Drosha和DGCR8重建的初级微小RNA加工检测法。
Primary microRNA processing assay reconstituted using recombinant Drosha and DGCR8.
作者信息
Barr Ian, Guo Feng
机构信息
Department of Biological Chemistry, University of California Los Angeles, Los Angeles, CA, USA.
出版信息
Methods Mol Biol. 2014;1095:73-86. doi: 10.1007/978-1-62703-703-7_5.
In animals, the Microprocessor complex cleaves primary transcripts of microRNAs (pri-miRNAs) to produce precursor microRNAs in the nucleus. The core components of Microprocessor include the Drosha ribonuclease and its RNA-binding partner protein DiGeorge critical region 8 (DGCR8). DGCR8 has been shown to tightly bind an Fe(III) heme cofactor, which activates its pri-miRNA processing activity. Here we describe how to reconstitute pri-miRNA processing using recombinant human Drosha and DGCR8 proteins. In particular, we present the procedures for expressing and purifying DGCR8 as an Fe(III) heme-bound dimer, the most active form of this protein, and for estimating its heme content.
在动物中，微处理器复合物在细胞核内切割微小RNA（pri-miRNA）的初级转录本以产生前体微小RNA。微处理器的核心组件包括Drosha核糖核酸酶及其RNA结合伴侣蛋白迪乔治关键区域8（DGCR8）。DGCR8已被证明能紧密结合Fe(III)血红素辅因子，从而激活其pri-miRNA加工活性。在这里，我们描述了如何使用重组人Drosha和DGCR8蛋白重建pri-miRNA加工过程。特别是，我们介绍了将DGCR8表达并纯化成为结合Fe(III)血红素的二聚体（该蛋白最具活性的形式）的步骤，以及估算其血红素含量的方法。
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