Establishment of cells to monitor Microprocessor through fusion genes of microRNA and GFP

Microprocessor, the complex of Drosha and DGCR8, promotes the processing of primary microRNA to precursor microRNA, which is a crucial step for microRNA maturation. So far, no convenient assay systems have been developed for observing this step in vivo. Here we report the establishment of highly sen...

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Bibliographic Details
Published in:Biochemical and biophysical research communications 2008-08, Vol.372 (4), p.856-861
Main Authors: Tsutsui, Motomu, Hasegawa, Hitoki, Adachi, Koichi, Miyata, Maiko, Huang, Peng, Ishiguro, Naoki, Hamaguchi, Michinari, Iwamoto, Takashi
Format: Article
Language:English
Subjects:
60 APPLIED LIFE SCIENCES
Animals
Base Sequence
DEAD-box RNA Helicases - genetics
DGCR8
Dicer
Drosha
Endoribonucleases - genetics
Gene Fusion
GENES
Green fluorescent protein
Green Fluorescent Proteins - analysis
Green Fluorescent Proteins - genetics
Humans
IN VIVO
MATURATION
Mice
Microprocessor
MICROPROCESSORS
microRNA
MicroRNAs - genetics
MicroRNAs - metabolism
Molecular Sequence Data
MONITORING
NIH 3T3 Cells
PROTEINS
Proteins - genetics
Proteins - metabolism
Ribonuclease III - genetics
Ribonuclease III - metabolism
RNA
RNA interference
RNA-Binding Proteins
SCREENING
Transfection
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Summary:Microprocessor, the complex of Drosha and DGCR8, promotes the processing of primary microRNA to precursor microRNA, which is a crucial step for microRNA maturation. So far, no convenient assay systems have been developed for observing this step in vivo. Here we report the establishment of highly sensitive cellular systems where we can visually monitor the function of Microprocessor. During a series of screening of transfectants with fusion genes of the EGFP cDNA and primary microRNA genes, we have obtained certain cell lines where introduction of siRNA against DGCR8 or Drosha strikingly augments GFP signals. In contrast, these cells have not responded to Dicer siRNA; thus they have a unique character that GFP signals should be negatively and specifically correlated to the action of Microprocessor among biogenesis of microRNA. These cell lines can be useful tools for real-time analysis of Microprocessor action in vivo and identifying its novel modulators.
ISSN:0006-291X
1090-2104
DOI:10.1016/j.bbrc.2008.05.141