An in Vitro
Nuclear Run-on Assay for Nuclear Function of Angiogenin
LI Ling-Yun1,2,
ZHANG Yao-Zhou2, ZHENG Yi-Fan1, HU Guo-Fu3, XU
Zheng-Ping1*
( 1 Department of Public Health, Zhejiang
University School of Medicine, Hangzhou 310031, China;
2 Institute of Biochemistry, College of Life Science, Zhejiang
University, Hangzhou 310029, China;
3 Center for Biochemical and Biophysical Sciences and Medicine,
Harvard Medical School, Cambridge, MA 02139, USA )
Abstract A sensitive and quantitative in vitro
analysis method was established to detect the nascent RNAs stimulated by
angiogenin using the nuclei isolated from human umbilical vein endothelial cells
(HUVE). Angiogenin was mixed with nuclei in the reaction buffer, then
transcription was initiated by adding the NTPs mixture. The RNA products were
measured quantitatively by [a–32P] CTP incorporation with a
liquid scintillation counter, either after removing free isotope by using spin
column, or cutting the electrophoresis lane and counting after autoradiography.
It was found that the optimum reacting temperature was 30 ºC,
the most suitable reaction time was 30 min for this system, and the
transcription enhancement activity of angiogenin was dose-dependent with the
feasible concentration being 1 mg/L. Higher concentration of angiogenin
degraded the RNA products in the system, suggesting that there is a mechanism
to control the entry and accumulation of angiogenin in the target cells, which
ensured angiogenin to play its role properly in the cells. Based on the
evidence that angiogenin bound to DNA in nucleolus and enhanced RNA
transcription, it was proposed that angiogenin might act as a trans-acting factor
in nucleus to regulate RNA transcription, and inhibition of
angiogenin-stimulated RNA transcription might be a promising target for
screening anti-angiogenesis inhibitor.
Key
words angiogenin; angiogenesis; RNA
transcription; molecular target
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