Poly(rC) binding protein 2 binds to stem-loop IV of the poliovirus RNA 5' noncoding region: Identification by automated liquid chromatography tandem mass spectrometry
Poly(rC) binding protein 2 binds to stem-loop IV of the poliovirus RNA 5' noncoding region: Identification by automated liquid chromatography tandem mass spectrometry
复制标题
DOI：
10.1073/pnas.93.20.11115
复制
发表时间：
1996-10-01
影响因子：
11.1
通讯作者：
Ehrenfeld, E
中科院分区：
文献类型：
--
作者：
Blyn, LB;Swiderek, KM;Ehrenfeld, E
脊髓灰质炎病毒RNA的5'非编码区包含一个内部核糖体进入位点（IRES），用于帽独立的翻译起始。IRES的利用需要一个或多个介导翻译起始反应事件的细胞蛋白的参与，但其生化作用尚未确定。在本报告中，我们从未感染的HeLa细胞的核糖体盐洗中分离出一种细胞RNA结合蛋白，该蛋白特异性结合茎环IV。该蛋白通过特异性RNA亲和层析分离得到，并通过自动液相色谱-串联质谱法测定其55%的序列。该序列与先前鉴定的人细胞RNA结合蛋白PCBP2及其相关蛋白PCBP1在大肠杆菌中过表达后克隆到表达质粒中产生组氨酸标记的融合蛋白相匹配，通过RNA迁移位移分析证实重组PCBP2与脊髓灰质炎病毒茎环IV之间存在特异性相互作用。密切相关的PCBP1与RNA没有稳定的相互作用，含有3个核苷酸插入的茎环IV RNA无法结合PCBP2，从而破坏了翻译活性和病毒活力。
The 5' noncoding region of poliovirus RNA contains an internal ribosome entry site (IRES) for cap-independent initiation of translation, Utilization of the IRES requires the participation of one or more cellular proteins that mediate events in the translation initiation reaction, but whose biochemical roles have not been defined, In this report, we identify a cellular RNA binding protein isolated from the ribosomal salt wash of uninfected HeLa cells that specifically binds to stem-loop IV, a domain located in the central part of the poliovirus IRES, The protein was isolated by specific RNA affinity chromatography, and 55% of its sequence was determined by automated liquid chromatography-tandem mass spectrometry. The sequence obtained matched that of poly(rC) binding protein 2 (PCBP2), previously identified as an RNA binding protein from human cells, PCBP2, as well as a related protein, PCBP1, was over-expressed in Escherichia coli after cloning the cDNAs into an expression plasmid to produce a histidine-tagged fusion protein, Specific interaction between recombinant PCBP2 and poliovirus stem-loop IV was demonstrated by RNA mobility shift analysis, The closely related PCBP1 showed no stable interaction with the RNA, Stem-loop IV RNA containing a three nucleotide insertion that abrogates translation activity and virus viability was unable to bind PCBP2.