Interactions of CCCH zinc finger proteins with mRNA - Non-binding tristetraprolin mutants exert an inhibitory effect on degradation of Au-rich element-containing mRNAs
Interactions of CCCH zinc finger proteins with mRNA - Non-binding tristetraprolin mutants exert an inhibitory effect on degradation of Au-rich element-containing mRNAs
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DOI：
10.1074/jbc.m110395200
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发表时间：
2002-03-15
影响因子：
4.8
通讯作者：
Blackshear, PJ
中科院分区：
文献类型：
--
作者：
Lai, WS;Kennington, EA;Blackshear, PJ
Tristetraprolin（TTP）是CCCH串联锌指（TZF）结构蛋白的一小部分的原型，它是编码肿瘤坏死因子A和粒细胞/巨噬细胞/巨噬细胞刺激某些细胞类型中mRNA的不稳定性的生理刺激剂。 TTP与两个mRNA的X-非翻译区域内的II类Au富元素（ARE）结合后，刺激了mRNA转换。反过来，这种结合取决于TZF域中的关键CCCH残基。为了评估这个新型的mRNA结合结构域的其他主要序列要求是结合的，我们突变了人类TTP的TZF结构域内的许多保守残基，并评估了这些突变对无细胞系统和TTP-的RNA结合的影响在细胞转染实验中诱导的mRNA不稳定。这些突变揭示了许多保守的氨基酸，这些氨基酸是结合所需的，并开始定义这种新型mRNA结合基序的主要蛋白质序列要求。出乎意料的是，在细胞转染实验中，所有阻止TTP结合RNA的点突变也导致稳态含有含量的mRNA。放线菌素D实验表明，这种作用是由于抑制mRNA更新。尽管TTP突变形式的表达也可以抑制野生型TTP对肿瘤坏死因子-a mRNA的破坏，但主要机制不涉及与野生型TTP的异二聚体化，因为这些研究中使用的293个细胞无表达可检测的内源性内源性的细胞TTP。这些数据表明，TTP至少可以部分地通过与酶活性或蛋白质复合物进行物理相互作用，并在功能上刺激其Deadenylate II类具有含有的mRNA的能力。
Tristetraprolin (TTP), the prototype of a small family of CCCH tandem zinc finger (TZF) domain proteins, is a physiological stimulator of instability of the mRNAs encoding tumor necrosis factor-a and granulocyte/macrophage colony-stimulating factor in certain cell types. TTP stimulates mRNA turnover after binding to class II AU-rich elements (AREs) within the X-untranslated regions of both mRNAs. In turn, this binding is dependent upon the key CCCH residues in the TZF domain. To evaluate other primary sequence requirements for ARE binding in this novel mRNA-binding domain, we mutated many of the conserved residues within the TZF domain of human TTP and evaluated the effects of these mutations on RNA binding in a cell-free system and TTP-induced mRNA instability in cell transfection experiments. These mutations revealed a number of conserved amino acids that were required for binding and begin to define the primary protein sequence requirements for this novel mRNA-binding motif. Unexpectedly, all of the point mutations that prevented TTP binding to RNA also caused an increase in steady-state levels of ARE-containing mRNAs in cell transfection experiments. Actinomycin D experiments suggested that this effect was due to inhibition of mRNA turnover. Although expression of the mutant form of TTP could also inhibit the destruction of tumor necrosis factor-a mRNA by wild-type TTP, the primary mechanism did not involve heterodimerization with wild-type TTP because the 293 cells used in these studies express no detectable endogenous TTP. These data suggest that TTP may act, at least in part, by physically interacting with an enzyme activity or protein complex and functionally stimulating its ability to deadenylate class II ARE-containing mRNAs.