Versatile applications of transcriptional pulsing to study mRNA turnover in mammalian cells
Versatile applications of transcriptional pulsing to study mRNA turnover in mammalian cells
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DOI：
10.1261/rna.663507
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发表时间：
2007-10-01
期刊：
影响因子：
4.5
通讯作者：
Shyu, Ann-Bin
中科院分区：
文献类型：
--
作者：
Chen, Chyi-Ying A.;Yamashita, Yukiko;Shyu, Ann-Bin
使用 c-fos 和 Tet-off 启动子系统的转录脉冲方法的开发极大地促进了哺乳动物细胞中 mRNA 周转的研究。然而，这些方法的最佳方案因不同的细胞类型和/或生理条件而异，限制了它们的广泛应用。在这项研究中，我们进一步优化了不同细胞系的转录脉冲系统，并开发了新的方案以促进 mRNA 周转各个方面的研究。我们应用 Tet-off 转录脉冲策略来研究药物抑制剂在细胞周期各个阶段停滞的人红白血病 K562 细胞中 ARE 介导的 mRNA 衰减。该应用有助于研究 mRNA 稳定性在控制细胞周期依赖性基因表达中的作用。为了推进对 mRNA 周转及其调控相关因素的研究，我们还将最近开发的转染和 siRNA 试剂纳入转录脉冲方法中。使用这些方案，siRNA和DNA质粒可以有效地共转染到小鼠NIH3T3细胞中以获得高敲低效率。此外，我们使用人支气管上皮 BEAS-2B 细胞建立了含有 tTA 的稳定细胞系，并应用转录脉冲方法监测该细胞系统中 mRNA 脱腺苷化和衰变动力学。这拓宽了转录脉冲系统在研究与过敏性炎症相关的 mRNA 周转调节方面的应用。对采用这些方法时需要考虑的关键因素进行了描述和讨论。
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.