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. © 2014 Springer Science+Business Media, New York.
CITATION STYLE
Barr, I., & Guo, F. (2014). Primary microRNA processing assay reconstituted using recombinant drosha and DGCR8. Methods in Molecular Biology, 1095, 73–86. https://doi.org/10.1007/978-1-62703-703-7_5
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