Versatile applications of transcriptional pulsing to study mRNA turnover in mammalian cells

Chyi Ying A Chen, Yukiko Yamashita, Tsung Cheng Chang, Akio Yamashita, Wenmiao Zhu, Zhenping Zhong, Ann Bin Shyu

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

Development of transcriptional pulsing approaches using the c-fos and Tet-off promoter systems greatly facilitated studies of mRNA turnover in mammalian cells. However, optimal protocols for these approaches vary for different cell types and/or physiological conditions, limiting their widespread application. In this study, we have further optimized transcriptional pulsing systems for different cell lines and developed new protocols to facilitate investigation of various aspects of mRNA turnover. We apply the Tet-off transcriptional pulsing strategy to investigate ARE-mediated mRNA decay in human erythroleukemic K562 cells arrested at various phases of the cell cycle by pharmacological inhibitors. This application facilitates studies of the role of mRNA stability in control of cell-cycle dependent gene expression. To advance the investigation of factors involved in mRNA turnover and its regulation, we have also incorporated recently developed transfection and siRNA reagents into the transcriptional pulsing approach. Using these protocols, siRNA and DNA plasmids can be effectively cotransfected into mouse NIH3T3 cells to obtain high knockdown efficiency. Moreover, we have established a tTA-harboring stable line using human bronchial epithelial BEAS-2B cells and applied the transcriptional pulsing approach to monitor mRNA deadenylation and decay kinetics in this cell system. This broadens the application of the transcriptional pulsing system to investigate the regulation of mRNA turnover related to allergic inflammation. Critical factors that need to be considered when employing these approaches are characterized and discussed. Published by Cold Spring Harbor Laboratory Press.

Original languageEnglish (US)
Pages (from-to)1775-1786
Number of pages12
JournalRNA
Volume13
Issue number10
DOIs
Publication statusPublished - Oct 2007

    Fingerprint

Keywords

  • Cell cycle
  • Deadenylation
  • mRNA turnover
  • siRNA knockdown
  • Transcriptional pulsing

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology

Cite this

Chen, C. Y. A., Yamashita, Y., Chang, T. C., Yamashita, A., Zhu, W., Zhong, Z., & Shyu, A. B. (2007). Versatile applications of transcriptional pulsing to study mRNA turnover in mammalian cells. RNA, 13(10), 1775-1786. https://doi.org/10.1261/rna.663507