Biology Reference
In-Depth Information
Chapter 6
In Vivo Processing Assay Based on a Dual-Luciferase
Reporter System to Evaluate DROSHA Enzymatic Activity
Vera Bilan, Danilo Allegra, Florian Kuchenbauer, and Daniel Mertens
Abstract
Luciferase reporter assays are widely used to study promoter activity, transcription factors, intracellular
signaling, protein interactions (Jia et al., PloS One 6:e26414), miRNA processing (Allegra and Mertens,
Biochem Biophys Res Commun 406:501-505), and target recognition (Jin et al., Methods Mol Biol
936:117-127). Here we describe the use of a dual-luciferase reporter system to evaluate the enzymatic
activity of a key enzyme involved in RNA maturation—DROSHA. This dual system is a simple and fast
method for the quantification of the DROSHA processing activity in live cells.
Key words
MicroRNA, Luciferase, miRNA processing assay, DROSHA, Ribonuclease III
1
Introduction
Luciferase reporter assays are widely used to study promoter activity,
transcription factors, intracellular signaling, protein interactions [
1
],
miRNA processing [
2
], and target recognition [
3
]. A dual-reporter
assay using firefly (
Photinus pyralis
) and
Renilla
(
Renilla reniformis
,
also known as sea pansy) luciferases improves experimental accuracy
by normalizing the results and reducing technical variance. Firefly
(FF) and Renilla luciferases do not require posttranslational modifi-
cations and function as an enzymatic reporter immediately after
translation. Additionally, these two luciferases have dissimilar sub-
strate requirements making it possible to measure them subsequently
from a single sample [
4
]. In dual-reporter assays Renilla luciferase is
normally used as an internal control to reduce experimental variabil-
ity [
5
]. We adapted a protocol that applies a dual-luciferase assay to
measure enzymatic activity of the DROSHA enzyme.
DROSHA is a member of the ribonuclease III family of pro-
teins and catalyzes cleavage of the primary long miRNA transcript
(pri-miRNA) to its precursor miRNA form. The process is critical
for miRNA maturation although a small subgroup of miRNA mol-
ecules are processed without this step. DROSHA has two RNase
