Multimerization of poly(rC) binding protein 2 is required for translation initiation mediated by a viral IRES
Multimerization of poly(rC) binding protein 2 is required for translation initiation mediated by a viral IRES
复制标题
DOI：
10.1261/rna.7070304
复制
发表时间：
2004-08-01
期刊：
影响因子：
4.5
通讯作者：
Semler, BL
中科院分区：
文献类型：
--
作者：
Bedard, KM;Walter, BL;Semler, BL
已知细胞蛋白多（rC）结合蛋白2 （PCBP2）在小核糖核酸帽无关翻译中起作用。我们进一步研究了翻译所需的PCBP2的RNA结合特性和蛋白-蛋白相互作用。我们利用酵母双杂交试验和体外生化方法，包括谷胱甘肽s -转移酶（GST）下拉试验和凝胶过滤，研究了其假定的多聚特性。通过遗传分析，该多聚结构域定位于蛋白质的第二个k -同源（KH） RNA结合结构域，位于氨基酸125和158之间。为了检验多聚体在脊髓灰质炎病毒翻译中的功能，我们利用了截断的蛋白DeltaKH1-PCBP2，它能够形成多聚体，但不结合脊髓灰质炎病毒茎环IV RNA（翻译所需的相互作用）。利用RNA结合和体外翻译实验，该蛋白显示为显性阴性，表明PCBP2多聚在脊髓灰质炎病毒翻译和RNA结合中起作用。此外，含有多聚域缺失的PCBP2 （DeltaKH2-PCBP2）不能结合脊髓灰质炎病毒茎环IV RNA，并且在缺乏内源性PCBP2的提取物中不能恢复翻译。这些实验结果表明，PCBP2的多聚是有效的RNA结合和脊髓灰质炎病毒RNA的帽无关翻译所必需的。通过研究细胞蛋白PCBP2的功能相互作用，我们发现了小核糖核酸帽无关翻译机制中的一个新的决定因素。
The cellular protein, poly(rC) binding protein 2 (PCBP2), is known to function in picornavirus cap-independent translation. We have further examined the RNA binding properties and protein-protein interactions of PCBP2 necessary for translation. We have studied its putative multimerization properties utilizing the yeast two-hybrid assay and in vitro biochemical methods, including glutathione S-transferase (GST) pull-down assays and gel filtration. Through genetic analysis, the multimerization domain has been localized to the second K-homologous (KH) RNA binding domain of the protein between amino acids 125 and 158. To examine the function of multimerization in poliovirus translation, we utilized the truncated protein, DeltaKH1-PCBP2, which is capable of multimer formation, but does not bind poliovirus stem-loop IV RNA (an interaction required for translation). Utilizing RNA binding and in vitro translation assays, this protein was shown to act as a dominant negative, suggesting that PCBP2 multimerization functions in poliovirus translation and RNA binding. Additionally, PCBP2 containing a deletion in the multimerization domain (DeltaKH2-PCBP2) was not able to bind poliovirus stem-loop IV RNA and could not rescue translation in extracts that were depleted of endogenous PCBP2. Results from these experiments suggest that the multimerization of PCBP2 is required for efficient RNA binding and cap-independent translation of poliovirus RNA. By examining the functional interactions of the cellular protein PCBP2, we have discovered a novel determinant in the mechanism of picornavirus cap-independent translation.