Identification and characterization of a constitutively expressed Ctenopharyngodon idella ADAR1 splicing isoform (CiADAR1a)
Identification and characterization of a constitutively expressed Ctenopharyngodon idella ADAR1 splicing isoform (CiADAR1a)
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组成型表达的草鱼 ADAR1 剪接亚型 (CiADAR1a) 的鉴定和表征
DOI：
10.1016/j.dci.2016.05.008
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发表时间：
2016-10-01
影响因子：
2.9
通讯作者：
Hu, Chengyu
中科院分区：
文献类型：
--
作者：
Liu, Xiancheng;Huang, Keyi;Hu, Chengyu
作为ADAR家族的一员，ADAR1(作用于RNA1的腺苷脱氨酶)可以在dsRNA中将腺苷转化为肌苷。哺乳动物中存在多种ADAR1剪接异构体，包括类似于150kD蛋白的诱导型干扰素(ADAR1-P150)和类似于110kD蛋白的结构性表达(ADAR1-p110)。ADAR1剪接异构体的结构多样性可能反映了它们的多重功能。在鱼类中也发现了ADAR1剪接异构体。在我们之前的研究中，我们已经克隆并鉴定了两种不同的草鱼ADAR1剪接异构体，即CiADAR1和CiADAR1-like，它们都是干扰素诱导的蛋白。在本论文中，我们发现了一个新的CiADAR1剪接异构体基因(命名为CiADAR1a)。CiADAR1a基因由15个外显子和14个内含子组成。其全长由3‘端非编码区(359 Bp)、3’端非编码区(229 Bp)和编码区(2952 Bp)组成，编码983个氨基酸的多肽，具有1个Z-DNA结合域、3个dsRNA结合基序和一个高度保守的水解脱氨基结构域。Western印迹分析表明，无论Poly I：C刺激与否，CiADAR1a均在草鱼肾细胞(CIK)中有结构性表达。在正常情况下，Ciadarla主要存在于细胞核中。经Poly I：C处理后，逐渐向胞浆转移。为了进一步研究CiADARla的转录调控机制，我们克隆并鉴定了其启动子序列。CiADARla的转录起始点位于截短的外显子2内。CiADARla启动子全长1303bp，含有4个IRF-ES。在本研究中，我们构建了带有IRF1和IRF3的真核表达载体pcDNA3.1，并将其与pGL3-CiADARla启动子共转染CIK细胞。结果表明，IRF1或IRF3过表达或Poly I：C刺激对CIK细胞CiADARla启动子活性均无明显影响。综上所述，根据分子和表达特征、亚细胞定位和转录调控机制，我们推测CiADAR1a与哺乳动物的ADAR1-p110有很高的同源性。(C)2016爱思唯尔有限公司。保留所有权利。
As one member of ADAR family, ADAR1 (adenosine deaminase acting on RNA 1) can convert adenosine to inosine within dsRNA. There are many ADAR1 splicing isoforms in mammals, including an interferon (IFN) inducible similar to 150 kD protein (ADAR1-p150) and a constitutively expressed similar to 110 kD protein (ADAR1-p110). The structural diversity of ADAR1 splicing isoforms may reflect their multiple functions. ADAR1 splicing isoforms were also found in fish. In our previous study, we have cloned and identified two different grass carp ADAR1 splicing isoforms, i.e. CiADAR1 and CiADAR1-like, both of them are IFN-inducible proteins. In this paper, we identified a novel CiADAR1 splicing isoform gene (named CiADAR1a). CiADAR1a gene contains 15 exons and 14 introns. Its full-length cDNA is comprised of a 5' UTR (359 bp), a 3' UTR (229 bp) and a 2952 bp ORF encoding a polypeptide of 983 amino acids with one Z-DNA binding domain, three dsRNA binding motifs and a highly conserved hydrolytic deamination domain. CiADAR1a was constitutively expressed in Ctenopharyngodon idella kidney (CIK) cells regardless of Poly I:C stimulation by Western blot assay. In normal condition, CiADARla was found to be present mainly in the nucleus. After treatment with Poly I:C, it gradually shifted to cytoplasm. To further investigate the mechanism of transcriptional regulation of CiADARla, we cloned and identified its promoter sequence. The transcriptional start site of CiADARla is mapped within the truncated exon 2. CiADARla promoter is 1303 bp in length containing 4 IRF-Es. In the present study, we constructed pcDNA3.1 eukaryotic expression vectors with IRF1 and IRF3 and co-transfected them with pGL3-CiADARla promoter into CIK cells. The results showed that neither the over-expression of IRF1 or IRF3 nor Poly I:C stimulation significantly impacted CiADARla promoter activity in CIK cells. Together, according to the molecular and expression characteristics, subcellular localization and transcriptional regulatory mechanism, we deduced that CiADARla shared a high degree of homology with mammalian ADAR1-p110. (C) 2016 Elsevier Ltd. All rights reserved.