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 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.
Department of Orthopedic Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Sowa-ku, Nagoya, Aichi 466-8550, Japan.
Motomu
M
Tsutsui
Hitoki
H
Hasegawa
Koichi
K
Adachi
Maiko
M
Miyata
Peng
P
Huang
Naoki
N
Ishiguro
Michinari
M
Hamaguchi
Takashi
T
Iwamoto
D000818:Animals; D001483:Base Sequence; D053487:DEAD-box RNA Helicases / Q000235:genetics; D004722:Endoribonucleases / Q000235:genetics; D050939:Gene Fusion; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D051379:Mice; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins; D043244:Ribonuclease III / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
C474043:DGCR8 protein, human; D035683:MicroRNAs; D011506:Proteins; D016601:RNA-Binding Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004722:Endoribonucleases; C408087:DICER1 protein, human; C517918:DROSHA protein, human; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases