A broader role for AU-rich element-mediated mRNA turnover revealed by a new transcriptional pulse strategy.
AUTOR(ES)
Xu, N
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=147286Documentos Relacionados
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