The polypyrimidine tract binding protein (PTB) represses splicing of exon 6B from the β-tropomyosin pre-mRNA by directly interfering with the binding of the U2AF65 subunit
The polypyrimidine tract binding protein (PTB) represses splicing of exon 6B from the β-tropomyosin pre-mRNA by directly interfering with the binding of the U2AF65 subunit
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DOI：
10.1128/mcb.00893-06
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发表时间：
2006-12-01
影响因子：
5.3
通讯作者：
Marie, Joelle
中科院分区：
文献类型：
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作者：
Sauliere, Jerome;Sureau, Alain;Marie, Joelle
剪接的外显子613从P-原肌球蛋白前mRNA的抑制在非肌肉细胞和成肌细胞的复杂阵列的内含子元件周围的外显子。在这项研究中，我们分析了通过结合上游元件介导外显子613剪接抑制的蛋白质。我们确定了多聚嘧啶道结合蛋白（PTB）作为从成肌细胞分离的复合物的组成部分，该复合物组装到分支点区域和嘧啶道上。体外剪接实验和PTB RNA干扰敲低实验表明，PTB作为外显子6 B剪接的阻遏物。使用peptien实验，我们表明PTB通过阻止U2 snRNA在分支点上的结合而在剪接体组装的早期阶段起作用。使用紫外线交联和免疫沉淀实验与位点特异性标记的RNA在PTB耗尽核提取物，我们发现，减少PTB与U2 AF 65的增加。此外，竞争实验表明，PTB是能够取代U2 AF 65上的聚嘧啶道的结合。我们的研究结果强烈支持一个模型，即PTB与U2 AF 65竞争结合到聚嘧啶道。
Splicing of exon 613 from the P-tropomyosin pre-mRNA is repressed in nonmuscle cells and myoblasts by a complex array of intronic elements surrounding the exon. In this study, we analyzed the proteins that mediate splicing repression of exon 613 through binding to the upstream element. We identified the polypyrimidine tract binding protein (PTB) as a component of complexes isolated from myoblasts that assemble onto the branch point region and the pyrimidine tract. In vitro splicing assays and PTB knockdown experiments by RNA interference demonstrated that PTB acts as a repressor of splicing of exon 6B. Using psoralen experiments, we showed that PTB acts at an early stage of spliceosome assembly by preventing the binding of U2 snRNA on the branch point. Using UV cross-linking and immunoprecipitation experiments with site-specific labeled RNA in PTB-depleted nuclear extracts, we found that the decrease in PTB was correlated with an increase in U2AF65. In addition, competition experiments showed that PTB is able to displace the binding of U2AF65 on the polypyrimidine tract. Our results strongly support a model whereby PTB competes with U2AF65 for binding to the polypyrimidine tract.