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