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Primary microRNA processing assay reconstituted using recombinant drosha and DGCR8

  • Author(s): Barr, I
  • Guo, F
  • et al.

Published Web Location

https://doi.org/10.1007/978-1-62703-703-7-5
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Abstract

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.

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This item is under embargo until December 31, 2999.