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Life Sciences Commons

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Biochemistry, Biophysics, and Structural Biology

Humans

2014

Dominican University of California

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Primary Microrna Processing Assay Reconstituted Using Recombinant Drosha And Dgcr8., Ian Barr, Feng Guo Jan 2014

Primary Microrna Processing Assay Reconstituted Using Recombinant Drosha And Dgcr8., Ian Barr, Feng Guo

Natural Sciences and Mathematics | Faculty Scholarship

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.