Human TOB, an antiproliferative transcription factor, is a poly(A)-binding protein-dependent positive regulator of cytoplasmic mRNA deadenylation
Human TOB, an antiproliferative transcription factor, is a poly(A)-binding protein-dependent positive regulator of cytoplasmic mRNA deadenylation
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DOI：
10.1128/mcb.01254-07
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发表时间：
2007-11-01
影响因子：
5.3
通讯作者：
Shyu, Ann-Bin
中科院分区：
文献类型：
--
作者：
Ezzeddine, Nader;Chang, Tsung-Cheng;Shyu, Ann-Bin
在哺乳动物细胞中，mRNA的衰变始于去腺苷化，这涉及两个连续的阶段，分别由PAN 2-PAN 3和CCR 4-CAF 1复合物介导。关键的去腺苷酸化步骤的调控及其与RNA加工体（P体）的关系，这被认为是一个网站，其中聚（A）缩短的mRNA得到降解，知之甚少。使用Tet-Off转录脉冲方法来研究小鼠NIH 3 T3成纤维细胞中的mRNA衰变，我们发现TOB，一种抗增殖转录因子，在体内增强mRNA去腺苷化。谷胱甘肽S-转移酶下拉和免疫共沉淀实验的结果表明，TOB可以同时与多聚（A）核酸酶复合物CCR 4-CAF 1和细胞质多聚（A）结合蛋白PABPC 1相互作用。结合这些发现与那些从诱变研究，我们进一步确定了蛋白基序的TOB和PABPC 1是必要的，它们的相互作用，并发现与PABPC 1的相互作用是必要的TOB的deadenylation增强效果。此外，我们的免疫荧光显微镜结果显示，TOB与P-体共定位，表明TOB在连接去腺苷化的P-体的作用。我们的发现揭示了一种新的机制，通过这种机制，哺乳动物mRNA的命运在去腺苷酸化步骤中被一种蛋白质调节，该蛋白质将多聚（A）核酸酶募集到3 '多聚（A）尾-PABP复合物中。
In mammalian cells, mRNA decay begins with deadenylation, which involves two consecutive phases mediated by the PAN2-PAN3 and the CCR4-CAF1 complexes, respectively. The regulation of the critical deadenylation step and its relationship with RNA-processing bodies (P-bodies), which are thought to be a site where poly(A)-shortened mRNAs get degraded, are poorly understood. Using the Tet-Off transcriptional pulsing approach to investigate mRNA decay in mouse NIH 3T3 fibroblasts, we found that TOB, an antiproliferative transcription factor, enhances mRNA deadenylation in vivo. Results from glutathione S-transferase pull-down and coimmunoprecipitation experiments indicate that TOB can simultaneously interact with the poly(A) nuclease complex CCR4-CAF1 and the cytoplasmic poly(A)-binding protein, PABPC1. Combining these findings with those from mutagenesis studies, we further identified the protein motifs on TOB and PABPC1 that are necessary for their interaction and found that interaction with PABPC1 is necessary for TOB's deadenylation-enhancing effect. Moreover, our immunofluorescence microscopy results revealed that TOB colocalizes with P-bodies, suggesting a role of TOB in linking deadenylation to the P-bodies. Our findings reveal a new mechanism by which the fate of mammalian mRNA is modulated at the deadenylation step by a protein that recruits poly(A) nuclease(s) to the 3 ' poly(A) tail-PABP complex.