Members of the poly (rC) binding protein family stimulate the activity of the c-myc internal ribosome entry segment in vitro and in vivo
Members of the poly (rC) binding protein family stimulate the activity of the c-myc internal ribosome entry segment in vitro and in vivo
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DOI：
10.1038/sj.onc.1206645
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发表时间：
2003-09-11
期刊：
影响因子：
8
通讯作者：
Willis, AE
中科院分区：
文献类型：
--
作者：
Evans, JR;Mitchell, SA;Willis, AE
原癌基因 c-myc 的 5' 非翻译区包含内部核糖体进入片段，c-Myc 翻译可以通过帽独立和帽依赖性机制启动。与帽依赖性启动过程相反，人们对进入细胞内部核糖体的反式作用因子的要求知之甚少。在这里，我们表明，聚（rC）结合蛋白家族的成员，聚（rC）结合蛋白1（PCBP1），聚（rC）结合蛋白2（PCBP2）和hnRNPK与N-ras和unr相互作用蛋白上游组合添加时，能够在体外将IRES激活高达三倍。通过紫外交联分析和电泳迁移率变化测定，PCBP1、PCBP2 和 hnRNPK 与 c-myc-IRES-RNA 的相互作用被证明是特异性的，而使用特异性抗体对这三种蛋白进行免疫沉淀，然后进行逆转录酶-聚合酶链反应表明它们能够结合 c-myc mRNA。通过编码 PCBP1、PCBP2 和 hnRNPK 的质粒的共转染来刺激 c-myc-IRES 介导的报告载体翻译。有趣的是，多发性骨髓瘤患者中普遍存在的c-myc IRES突变版本在体外更有效地结合hnRNPK，并且在体内更大程度地受到hnRNPK的刺激。
The 5' untranslated region of the proto-oncogene c-myc contains an internal ribosome entry segment and c-Myc translation can be initiated by cap-independent as well as cap-dependent mechanisms. In contrast to the process of cap-dependent initiation, the trans-acting factor requirements for cellular internal ribosome entry are poorly understood. Here, we show that members of the poly (rC) binding protein family, poly (rC) binding protein 1 (PCBP1), poly (rC) binding protein 2 (PCBP2) and hnRNPK were able to activate the IRES in vitro up to threefold when added in combination with upstream of N-ras and unr-interacting protein. The interactions of PCBP1, PCBP2 and hnRNPK with c-myc-IRES-RNA were shown to be specific by ultraviolet crosslinking analysis and electrophoretic mobility shift assays, while immunoprecipitation of the three proteins using specific antibodies followed by reverse transcriptase-polymerase chain reaction showed that they were able to bind c-myc mRNA. c-myc-IRES-mediated translation from the reporter vector was stimulated by cotransfection of plasmids encoding PCBP1, PCBP2 and hnRNPK. Interestingly, the mutated version of the c-myc IRES that is prevalent in patients with multiple myeloma bound hnRNPK more efficiently in vitro and was stimulated by hnRNPK to a greater extent in vivo.