The TAR RNA-binding protein, TRBP, stimulates the expression of TAR-containing RNAs in vitro and in vivo independently of its ability to inhibit the dsRNA-dependent kinase PKR
The TAR RNA-binding protein, TRBP, stimulates the expression of TAR-containing RNAs in vitro and in vivo independently of its ability to inhibit the dsRNA-dependent kinase PKR
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DOI：
10.1074/jbc.m208954200
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发表时间：
2003-02-14
影响因子：
4.8
通讯作者：
Vaquero, C
中科院分区：
文献类型：
--
作者：
Dorin, D;Bonnet, MC;Vaquero, C
TRBP（HIV-1反式激活反应（TAR）RNA结合蛋白）和PKR（干扰素诱导的dsRNA调节的蛋白激酶）含有两个dsRNA结合结构域。它们都通过不同的位点与HIV-1 TAR RNA结合。与dsRNA结合激活PKR，PKR磷酸化真核起始因子eIF-2 α，导致蛋白质合成抑制。TRBP和PKR可以异源二聚化，抑制PKR的激酶功能，对HIV-1的表达有积极影响。在这项研究中，体外网织红细胞测定显示，与CAT RNA相比，含有CAT RNA的TAR表达较差。TRBP的加入恢复了TAR-CAT RNA的翻译效率，降低了eIF-2 α的磷酸化状态，证实了其作为PKR抑制剂的作用。出乎意料的是，eIF-2 α在TAR-CAT以及缺乏TAR结构的CAT RNA存在下被磷酸化。在这两种情况下，TRBP抑制eIF-2 α磷酸化，这表明它独立地恢复了TAR-CAT RNA的翻译，并具有抑制PKR的能力。然后在无PKR的环境中使用PKR缺陷的鼠胚胎成纤维细胞分析TRBP对基因表达的活性。在瞬时报告基因测定中，TRBP刺激含TAR的荧光素酶的表达3.8倍，而具有突变的TAR结构或缺乏TAR的报告基因被刺激1.5至2.4倍。总的来说，当mRNA的5 '端被结构化并且由TRBP中的每个dsRBD独立介导时，TRBP 2的活性更高。增加浓度的TRBP显示荧光素酶RNA水平没有显著改变，表明TRBP刺激含TAR的RNA的翻译。因此，TRBP是有效翻译含dsRNA的转录物的重要细胞因子，通过抑制PKR和在PKR非依赖性途径中。
TRBP (HIV-1 transactivating response (TAR) RNA-binding protein) and PKR, the interferon-induced dsRNA-regulated protein kinase, contain two dsRNA binding domains. They both bind to HIV-1 TAR RNAs through different sites. Binding to dsRNA activates PKR that phosphorylates the eukaryotic initiation factor eIF-2alpha leading to protein synthesis inhibition. TRBP and PKR can heterodimerize, which inhibits the kinase function of PKR and has a positive effect on HIV-1 expression. In this study, an in vitro reticulocyte assay revealed the poor expression of TAR containing CAT RNAs compared with CAT RNAs. Addition of TRBP restored translation efficiency of TAR-CAT RNA and decreased the phosphorylation status of eIF-2alpha, confirm ing its role as a PKR inhibitor. Unexpectedly, eIF-2alpha was phosphorylated in the presence of TAR-CAT as well as CAT RNA devoid of the TAR structure. TRBP inhibited eIF-2alpha phosphorylation in both cases, suggesting that it restores the translation of TAR-CAT RNA independently and in addition to its ability to inhibit PKR. TRBP activity on gene expression was then analyzed in a PKR-free environment using PKR-deficient murine embryo fibroblasts. In a transient reporter gene assay, TRBP stimulated the expression of a TAR-containing luciferase 3.8-fold whereas the reporter gene with mutated TAR structures or devoid of TAR was stimulated 1.5- to 2.4-fold. Overall, the activity of TRBP2 was higher when the 5'-end of the mRNA was structured and was mediated independently by each dsRBD in TRBP. Increasing concentrations of TRBP showed no significant modification of the luciferase RNA levels, suggesting that TRBP stimulates translation of TAR-containing RNAs Therefore, TRBP is an important cellular factor for efficient translation of dsRNA containing transcripts, both by inhibiting PKR and in a PKR-independent pathway.