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.

Fig. 1

Domain structures and recombinant expression constructs of human Drosha and DGCR8. The heme-binding domain (HBD) of DGCR8 includes a dimerization (sub)domain (DD)

Fig. 2

Electronic absorbance spectrum of Fe(III) heme-bound NC1 dimer. The solid line, corresponding to the left y-axis, shows the relative absorbencies of the heme and protein peaks. The dashed line, corresponding to the right y-axis, shows the extinction coefficients of the heme, as determined using the pyridine hemochromagen assay as recently reported [30]

Fig. 3

Example of a pri-miRNA processing assay. Uniformly labeled pri-miR-30a was incubated with 4 nM His₆-Drosha^390–1374, either alone (lane 2) or with 50 nM Fe(III) heme-bound NC1 dimer (lane 3), at 37 °C for 45 min. The reactions were analyzed using a 7 M urea, 15 % polyacrylamide gel. LMWM: low molecular weight marker