A broader role for AU-rich element-mediated mRNA turnover revealed by a new transcriptional pulse strategy.

The widespread occurrence of AU-rich elements (AREs) in mRNAs encoding proteins with diversified functions and synthesized under a vast variety of physiological conditions suggests that AREs are involved in finely tuned and stringent control of gene expression. Thus it is important to investigate the regulation of ARE-mediated mRNA decay in a variety of mammalian cells in different physiological states. The tetracycline (Tet)-regulatory promoter system appears appropriate for these investigations. However, we found that efficient degradation of mRNAs bearing different AREs cannot be observed simply by blocking constitutive transcription from the Tet-regulated promoter with Tet, possibly due to saturation of the cellular decay machinery. In addition, deadenylation kinetics and their relationship to mRNA decay cannot be adequately measured under these conditions. To overcome these obstacles we have developed a new strategy that employs the Tet-regulated promoter system to achieve a transient burst of transcription that results in synthesis of a population of cytoplasmic mRNAs fairly homogeneous in size. Using this new system we show that ARE-destabilizing function, necessary for down-regulating mRNAs for cytokines, growth factors and transcription factors, is maintained in quiescent or growth-arrested cells as well as in saturation density-arrested NIH 3T3 cells. We also demonstrate that the ARE-mediated decay pathway is conserved between NIH 3T3 fibroblasts and K562 erythroblasts. These in vivo observations support a broader role for AREs in the control of cell growth and differentiation. In addition, we observed that there is a significant difference in deadenylation and decay rates for beta-globin mRNA expressed in these two cell lines. Deadenylation and decay of beta-globin mRNA in K562 cells is extraordinarily slow compared with NIH 3T3 cells, suggesting that the increased stability gained by beta-globin mRNA in K562 cells is mainly controlled at the deadenylation step. Our strategy for studying mammalian mRNA turnover now permits a more general application to different cell lines harboring the Tet-regulated system under various physiological conditions.富含AU元件（AU-rich elements，AREs）广泛存在于功能多样、且在多种生理条件下合成的蛋白质编码mRNA中，这提示AREs参与了基因表达的精细调控与严格质控。因此，在不同生理状态下的多种哺乳动物细胞中研究ARE介导的mRNA降解调控机制，具有重要学术意义。四环素（tetracycline，Tet）调控启动子系统似乎适合开展此类研究。但我们发现，仅通过用Tet阻断四环素调控启动子的组成型转录，无法有效观测到携带不同AREs的mRNA的降解过程，这可能是由于细胞降解机制饱和所致。此外，在此类条件下，亦无法充分测定脱腺苷酸化动力学及其与mRNA降解的关联。为克服这些障碍，我们开发了一种新策略：借助四环素调控启动子系统实现瞬时爆发式转录，从而获得一批大小均一的细胞质mRNA群体。利用这一新系统，我们证实：细胞因子、生长因子及转录因子编码mRNA的下调所必需的ARE去稳定功能，在静止或生长阻滞细胞，以及处于饱和密度阻滞状态的NIH 3T3细胞中均得以保留。我们还证明，ARE介导的降解通路在NIH 3T3成纤维细胞与K562红系母细胞中具有保守性。这些体内观测结果支持AREs在细胞生长与分化调控中发挥更广泛的作用。此外，我们发现，在这两种细胞系中表达的β-珠蛋白mRNA，其脱腺苷酸化速率与降解速率存在显著差异。相较于NIH 3T3细胞，K562细胞中β-珠蛋白mRNA的脱腺苷酸化与降解过程异常缓慢，这提示K562细胞中β-珠蛋白mRNA稳定性的提升主要受控于脱腺苷酸化步骤。我们用于研究哺乳动物mRNA周转的新策略，如今可更广泛地应用于携带四环素调控系统、且处于不同生理状态的各类细胞系中。