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Primary microRNA processing assay reconstituted using recombinant Drosha and DGCR8.

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.

SUBMITTER: Barr I

PROVIDER: S-EPMC4788497 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Primary microRNA processing assay reconstituted using recombinant Drosha and DGCR8.

Barr Ian I Guo Feng F

Methods in molecular biology (Clifton, N.J.) 20140101

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 prote ...[more]

PMID: 24166303

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