Raineri Ines, Wegmueller Daniel, Gross Brigitte, Certa Ulrich, Moroni Christoph
Institute for Medical Microbiology, University of Basel, Petersplatz 10, 4003 Basel, Switzerland.
Nucleic Acids Res. 2004 Feb 19;32(4):1279-88. doi: 10.1093/nar/gkh282. Print 2004.
HT1080 cells stably expressing green fluorescent protein (GFP) linked to a 3' terminal AU-rich element (ARE) proved to be a convenient system to study the dynamics of mRNA stability, as changes in mRNA levels are reflected in increased or decreased fluorescence intensity. This study examined whether mRNA stability can be regulated by small interfering RNAs (siRNAs) targeted to AU-binding proteins (AUBPs), which in turn should reveal their intrinsic role as stabilizers or destabilizers of ARE-mRNAs. Indeed, siRNAs targeting HuR or BRF1 decreased or increased fluorescence, respectively. This effect was abolished if cells were treated with both siRNAs, thus indicating antagonistic control of ARE-mRNA stability. Unexpectedly, downregulation of all four AUF1 isoforms by targeting common exons did not affect fluorescence whereas selective downregulation of p40AUF1/p45AUF1 strongly increased fluorescence by stabilizing the GFP-ARE reporter mRNA. This observation was fully confirmed by the finding that only selective reduction of p40AUF1/p45AUF1 induced the production of GM-CSF, an endogenous target of AUF1. These data suggest that the relative levels of individual isoforms, rather than the absolute amount of AUF1, determine the net mRNA stability of ARE-containing transcripts, consistent with the differing ARE-binding capacities of the isoforms.
稳定表达与3'末端富含AU元件（ARE）相连的绿色荧光蛋白（GFP）的HT1080细胞，被证明是研究mRNA稳定性动态变化的便捷系统，因为mRNA水平的变化会反映为荧光强度的增加或降低。本研究检测了mRNA稳定性是否可由靶向AU结合蛋白（AUBP）的小干扰RNA（siRNA）调控，而这反过来应能揭示它们作为ARE-mRNA稳定剂或去稳定剂的内在作用。实际上，靶向HuR或BRF1的siRNA分别降低或增加了荧光。如果用这两种siRNA处理细胞，这种效应就会消除，从而表明对ARE-mRNA稳定性存在拮抗调控。出乎意料的是，通过靶向共同外显子下调所有四种AUF1异构体并不影响荧光，而选择性下调p40AUF1/p45AUF1通过稳定GFP-ARE报告基因mRNA而强烈增加荧光。这一观察结果通过以下发现得到了充分证实：只有选择性降低p40AUF1/p45AUF1会诱导AUF1的内源性靶标GM-CSF的产生。这些数据表明，各个异构体的相对水平而非AUF1的绝对量决定了含ARE转录本的净mRNA稳定性，这与异构体不同的ARE结合能力一致。
