Biology Reference
In-Depth Information
Chapter 5
Primary MicroRNA Processing Assay Reconstituted
Using Recombinant Drosha and DGCR8
Ian Barr and Feng Guo
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.
Key words RNA processing, DiGeorge syndrome, Heme, RNA-binding protein, Nucleic acid-
binding protein, Pasha, Ribonuclease III
1
Introduction
The Microprocessor complex minimally contains the proteins
Drosha [ 1 ], an RNase III family member, and DGCR8 [ 2 , 3 ], an
RNA-binding protein that also contains the cofactor heme [ 4 - 6 ].
Drosha and DGCR8 are essential for processing of all canonical
microRNAs (miRNAs) in animals [ 7 - 9 ]. They are also sufficient to
reconstitute pri-miRNA processing activity in vitro [ 3 , 10 ].
Furthermore, quite a few proteins have been shown to regulate the
Drosha/DGCR8-mediated cleavage of pri-miRNAs [ 11 - 18 ].
The domain structures of Drosha and DGCR8 have been dis-
sected in several studies. Drosha contains two RNase III domains
and a double-stranded RNA-binding domain (dsRBD) in the
C-terminal region (Fig. 1a ). The central region of Drosha, includ-
ing residues 390-900, is highly conserved and required for pri-
miRNA processing but contains no recognizable sequence motifs
[ 10 , 19 ]. DGCR8 contains a heme-binding domain, two dsRBDs,
and a C-terminal tail (CTT) (Fig. 1b ). The dsRBDs contribute to
pri-miRNA binding [ 4 , 20 , 21 ]. The C-terminal tail has been
1.1 The
Microprocessor
Complex
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