Multiple domains of EBER 1, an Epstein-Barr virus noncoding RNA, recruit human ribosomal protein L22
Multiple domains of EBER 1, an Epstein-Barr virus noncoding RNA, recruit human ribosomal protein L22
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DOI：
10.1261/rna.2339606
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发表时间：
2006-05-01
期刊：
影响因子：
4.5
通讯作者：
Steitz, JA
中科院分区：
文献类型：
--
作者：
Fok, V;Mitton-Fry, RM;Steitz, JA
EBER 1是一种在EB病毒转化的所有细胞中大量表达的非编码病毒小RNA，已被证明与人类核糖体蛋白L22有关。在这里，我们介绍了使用纯化的RNA和重组蛋白进行的体外结合研究。凝胶迁移率改变分析(EMSA)表明，重组L22(RL22)和麦芽糖结合蛋白(MBP)标记的L22蛋白在体外与EBER1结合，两者都形成三种依赖于蛋白质的迁移率改变。使用rL22和MBP-L22的混合物表明，这三个移位在每个EBER 1分子中包含一个、两个或三个L22蛋白。用EBER 1缺失构建体和EBER 1茎环插入非结合RNA HSUR 3进行的EMSA鉴定茎环I、III和IV为L22结合位点。体内紫外光交联实验证明EBER1在细胞内存在多个L22结合位点。我们的结果针对EBV感染的人B细胞中EBER1的功能进行了讨论。
EBER 1, a small noncoding viral RNA abundantly expressed in all cells transformed by Epstein-Barr virus (EBV), has been shown to associate with the human ribosomal protein L22. Here we present in vitro binding studies using purified RNAs and recombinant proteins. Electrophoretic mobility-shift assays (EMSAs) show that recombinant L22 (rL22) and maltose-binding protein (MBP)-tagged L22 protein bind EBER 1 in vitro, both forming three specific protein-dependent mobility shifts. Use of a mixture of rL22 and MBP-L22 indicates that these three shifts contain one, two, or three L22 proteins per EBER 1 molecule. EMSAs performed with EBER 1 deletion constructs and EBER 1 stem-loops inserted into a nonbinding RNA, HSUR 3, identify stem-loops I, III, and IV as L22 binding sites. The existence of multiple L22 binding sites on EBER 1 inside cells is demonstrated by in vivo UV cross-linking. Our results are discussed with respect to the function of EBER 1 in EBV-infected human B cells.