title: 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.
authors.affiliation: Department of Biological Chemistry, University of California Los Angeles, Los Angeles, CA, USA.
authors.forename: Ian
authors.initials: I
authors.lastname: Barr
authors.forename: Feng
authors.initials: F
authors.lastname: Guo
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D006418:Heme / Q000378:metabolism; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D059367:RNA Cleavage; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D043244:Ribonuclease III / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: C474043:DGCR8 protein, human; D035683:MicroRNAs; D011506:Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D006418:Heme; C517918:DROSHA protein, human; D043244:Ribonuclease III