Barr Ian, Guo Feng
Department of Biological Chemistry, University of California Los Angeles, Los Angeles, CA, USA.
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。
