Intron retention may regulate expression of Epstein-Barr virus nuclear antigen 3 family genes
Intron retention may regulate expression of Epstein-Barr virus nuclear antigen 3 family genes
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DOI：
10.1128/jvi.73.2.1195-1204.1999
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发表时间：
1999-02-01
影响因子：
5.4
通讯作者：
Sculley, TB
中科院分区：
文献类型：
--
作者：
Kienzle, N;Young, DB;Sculley, TB
EB病毒核抗原3家族基因(EBNA-3、EBNA-4和EBNA-6)对EB病毒诱导的B淋巴细胞永生化和存活具有重要作用。然而，人们对这些基因的表达是如何调控的知之甚少。EBNA-3、EBNA-4和EBNA-6基因均由两个外显子组成，由一个小内含子隔开。逆转录酶聚合酶链式反应分析表明，绝大多数EBNA-3、EBNA-4和EBNA-6mRNA在转基因和EBV感染的B细胞中表达，保留内含子序列。Northern印迹和S1保护分析证实，EBNA-3mRNA大部分含有内含子。对EBNA-3缺失突变体的检测表明，EBNA-3蛋白不是内含子保留所必需的，并且在EBNA-3mRNA中不存在剪接沉默元件。细胞分裂和RNA梯度分析表明，未剪接的EBNA 3家族mRNAs被转运到细胞质中，并与多聚体结合。然而，标记了FLAG表位的EBNA-3的Western印迹分析没有表明EBNA-3存在剪接变异蛋白形式。相比之下，瞬时表达EBNA-3的细胞显示，基因组EBNA-3基因的EBNA-3蛋白表达是EBNA-3cDNA的6倍。这些数据表明，内含子序列可以影响EBNA-3蛋白的表达，并提示内含子保留可能为微调单个EBNA 3家族基因的表达提供了一种手段。
The nuclear antigen 3 family genes (EBNA-3, EBNA-4, and EBNA-6) of Epstein-Barr virus (EBV) are important for EBV-induced immortalization and survival of B lymphocytes. However, little is known about how the expression of these genes is regulated. Each of the EBNA-3, EBNA-4 and EBNA-6 genes consists of two exons separated by a small intron. Reverse transcriptase PCR assays revealed that the vast majority of the EBNA-3, EBNA-4, and EBNA-6 mRNA, expressed in transfected and EBV-infected B cells, retained intron sequences. Northern blot and S1 protection assays confirmed that most of the EBNA-3 mRNA contained intron. Examination of deletion mutants of EBNA-3 indicated that the EBNA-3 protein was not necessary for intron retention and that there was no splicing silencing element encoded in the EBNA-3 mRNA. Cell fractionation and RNA gradient analysis revealed that the unspliced EBNA 3 family mRNAs were transported into the cytoplasm and associated with the polysomes. However, Western blot analysis of FLAG-epitope tagged EBNA-3 gave no indication of the presence of splice variant protein forms of EBNA-3. In contrast, transiently transfected cells expressing EBNA-3 revealed a sixfold increase in EBNA-3 protein expression from the genomic EBNA-3 gene compared to EBNA-3 cDNA. These data show that the intronic sequences can influence EBNA-3 protein expression and suggest that intron retention may provide a means for the fine-tuning of expression of the individual EBNA 3 family genes.