Biology Reference
In-Depth Information
1.3 Preparation
of Recombinant
Drosha Protein
Full-length Drosha expressed in insect cells has been used in several
studies [
3
-
5
,
22
,
29
]. However, in our experience the full-length
His
6
-Drosha is poorly soluble and cannot be purified using either Ni
affinity or ion-exchange chromatography; and only a small amount
of partially purified active His
6
-Drosha may be obtained using size
exclusion chromatography [
4
]. The N-terminal 390 amino acid resi-
dues of Drosha are dispensable for in vitro activity [
10
]. Recently, we
found that truncation of this region greatly improves the solubility
of Drosha without compromising the activity [
6
]. The procedure for
purifying His
6
-Drosha
390-1374
is described below.
miRNAs may reside in introns or exons, messenger RNAs or inde-
pendent transcripts [
31
]. Processing of pri-miRNAs by Micro-
processor occurs co-transcriptionally [
32
]. Intronic pri-miRNAs
may be processed by Drosha before splicing catalysis [
33
]. The
exact 5′ and 3′ ends of pri-miRNAs at the time of processing are
often not known. Biochemical studies show that pri-miRNA frag-
ments containing the precursor miRNA (pre-miRNA) and certain
lengths of the immediate flanking regions can be processed by
Drosha and DGCR8 [
1
,
24
]. The minimal lengths of the flanking
regions for efficient processing by affinity-purified Microprocessor
complexes may be as short as 10-20 nt [
10
,
19
]. For reconstituted
pri-miRNA processing assays, we typically include 30-60 nt on
both sides of the pre-miRNA region.
The pri-miRNA fragments are typically prepared using T7 or
SP6 RNA polymerase. The protocol for how to use T7 RNA poly-
merase is provided here. The transcription template should contain
the T7 promoter, followed by the pri-miRNA coding sequence.
For high transcription yields, the first two nucleotides of the tran-
script should be guanosines [
34
]. Either a PCR product or a linear-
ized plasmid may serve as the template for the run-off transcription,
in which the 3′-end of the RNA is roughly defined by the end of
the template where the RNA polymerase simply falls off. The T7
RNA polymerase is known to add 0-3 non-templated residues at
the 3′-end of the transcripts [
34
]. In the case where a plasmid tem-
plate is used, a cleavage site for a restriction endonuclease such as
PstI
or
EarI
is engineered for linearization.
1.4 Design
of pri-miRNA
Constructs
for Reconstituted
pri-miRNA
Processing Assay
2
Materials
All solutions should be, to the greatest extent possible, free from
RNase contamination. This applies especially to the reagents and
buffers involved in transcription and pri-miRNA processing reac-
tions and in the storage of RNAs.
1. The NC1 expression plasmid contains the coding sequence of
amino acid residues 276-751 (NCBI accession no. of full-
length DGCR8 cDNA: BC037564) inserted between
NdeI
2.1 Expression and
Purification of DGCR8
