Gallia G L, Darbinian N, Jaffe N, Khalili K
Center for NeuroVirology and Cancer Biology, Laboratory of Molecular Neurovirology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania 19122, USA.
J Cell Biochem. 2001;83(3):355-63. doi: 10.1002/jcb.1247.
Pur alpha is a highly conserved, eukaryotic sequence-specific DNA- and RNA-binding protein involved in diverse cellular and viral functions including transcription, replication, and cell growth. Pur alpha exerts its activity in part by interacting with other viral and cellular proteins. One such protein is the human immunodeficiency virus (HIV) type I regulatory protein Tat. Earlier studies have demonstrated that this interaction is mediated by Pur alpha-associated RNA (PARNA) and that RNA immunopurified from mammalian expressed Pur alpha was capable of reconstituting the interaction between these two proteins. In the current study, we characterize four RNA species which were immunopurified with Pur alpha. Northern blot analysis with one of the PARNAs revealed a highly abundant signal of approximately 2.0 kilobases (kb) present in all cell lines tested. Sequence analysis of each of the four PARNA clones revealed a high homology to different regions of the human 18S ribosomal RNA sequence. Based on this homology, we investigated the influence of Pur alpha on translation. Luciferase assays were performed after coupled in vitro transcription/translation reactions with a vector containing a luciferase reporter construct and increasing concentrations of BSA, GST, and GST-Pur alpha. Inclusion of GST-Pur alpha in these reactions resulted in a dose-dependent inhibition of luciferase activity. Similar inhibition was observed with in vitro translation reactions performed with in vitro transcribed luciferase RNA and increasing concentrations of GST-Pur alpha. In control experiments, inclusion of increasing concentrations of GST-Pur alpha with luciferase protein resulted in no effect on luciferase activity. Taken together, these data demonstrate that Pur alpha inhibits translation reactions in vitro. Moreover, this Pur alpha-mediated inhibition of translation can be abrogated by HIV-1 Tat protein.
Purα是一种高度保守的真核生物序列特异性DNA和RNA结合蛋白，参与多种细胞和病毒功能，包括转录、复制和细胞生长。Purα部分通过与其他病毒和细胞蛋白相互作用发挥其活性。其中一种蛋白是人类免疫缺陷病毒（HIV）I型调节蛋白Tat。早期研究表明，这种相互作用由Purα相关RNA（PARNA）介导，并且从哺乳动物表达的Purα免疫纯化的RNA能够重建这两种蛋白之间的相互作用。在本研究中，我们鉴定了用Purα免疫纯化的四种RNA物种。用其中一种PARNA进行Northern印迹分析显示，在所有测试的细胞系中均存在约2.0千碱基（kb）的高度丰富信号。对四个PARNA克隆中的每一个进行序列分析，发现与人类18S核糖体RNA序列的不同区域具有高度同源性。基于这种同源性，我们研究了Purα对翻译的影响。在用含有荧光素酶报告构建体的载体以及增加浓度的牛血清白蛋白（BSA）、谷胱甘肽S-转移酶（GST）和GST-Purα进行体外转录/翻译偶联反应后，进行荧光素酶测定。在这些反应中加入GST-Purα导致荧光素酶活性呈剂量依赖性抑制。在用体外转录的荧光素酶RNA和增加浓度的GST-Purα进行的体外翻译反应中也观察到类似的抑制作用。在对照实验中，加入增加浓度的GST-Purα与荧光素酶蛋白一起对荧光素酶活性没有影响。综上所述，这些数据表明Purα在体外抑制翻译反应。此外，HIV-1 Tat蛋白可以消除这种由Purα介导的翻译抑制。
