Ctenopharyngodon idella IRF2 and ATF4 down-regulate the transcriptional level of PRKRA
Ctenopharyngodon idella IRF2 and ATF4 down-regulate the transcriptional level of PRKRA
复制标题
草鱼IRF2和ATF4下调PRKRA转录水平
DOI：
10.1016/j.fsi.2017.03.002
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发表时间：
2017
影响因子：
4.7
通讯作者：
Chengyu Hu
中科院分区：
文献类型：
--
作者：
Keyi Huang;Guoqin Qi;Zhicheng Sun;Xiancheng Liu;Xiaowen Xu;Haizhou Wang;Zhen Wu;Yiqi Wan;Chengyu Hu
PRKRA（interferon-inducible double-stranded RNA-dependent protein kinase activator A，干扰素诱导的双链RNA依赖性蛋白激酶激活因子A）是一种保护性蛋白，通过激活PKR调节细胞对内质网应激和病毒刺激信号通路的适应。本研究克隆并鉴定了草鱼（Ctenopharyngodon idella）PRKRA全长cDNA（命名为CiPRKRA，KT 891991）。该基因全长由36 bp的5′ UTR、350 bp的3′ UTR和882 bp的开放阅读框组成，编码293个氨基酸。推导的CiPRKRA的氨基酸序列包含三个典型的dsRNA结合基序（dsRBM）。系统进化树分析表明，CiPRKRA与其它鱼类PRKRA，尤其是斑马鱼PRKRA的进化关系较近。qRT-PCR结果显示，经衣霉素（Tm）和Poly I：C刺激后，CIK细胞中CiPRKRA表达显著上调。为了进一步研究其转录调控功能，我们利用Tail-PCR技术克隆了CiPRKRA基因的部分启动子序列（1463 bp），其中包含一个ISRE和一个CARE。随后，在大肠杆菌BL 21中表达了草鱼IRF 2（CiIRF 2）和ATF 4（CiATF 4），经Ni-NTA His-Bind Resin亲和层析纯化后，凝胶迁移率变动分析表明，两者均能与CiPRKRA启动子高亲和力结合，表明IRF 2和ATF 4可能是CiPRKRA的潜在转录调控因子。应用双荧光素酶报告基因分析进一步研究CiPRKRA在体内的转录调控。构建pGL 3-PRKRAPro重组质粒，分别与pcDNA3.1-CiIRF 2和pcDNA3.1-CiATF 4共转染CIK细胞。结果表明，CiIRF 2和CiATF 4均能显著降低pGL 3-PRKRAPro的荧光素酶活性，提示它们对CiPRKRAPro的转录起负作用。
PRKRA (interferon-inducible double-stranded RNA-dependent protein kinase activator A) is a protective protein which regulates the adaptation of cells to ER stress and virus-stimulated signaling pathways by activating PKR. In the present study, a grass carp (Ctenopharyngodon idella) PRKRA full-length cDNA (namedCiPRKRA, KT891991) was cloned and identified. The full-length cDNA is comprised of a 5′ UTR (36 bp), a 3′ UTR (350 bp) and the longest ORF (882 bp) encoding a polypeptide of 293 amino acids. The deduced amino acid sequence ofCiPRKRA contains three typical dsRNA binding motifs (dsRBM). Phylogenetic tree analysis revealed a closer evolutionary relationship ofCiPRKRA with other fish PRKRA, especially withDanio rerioPRKRA. qRT-PCR showed thatCiPRKRAwas significantly up-regulated after stimulation with tunicamycin (Tm) and Poly I:C inC. idellakidney (CIK) cells. To further study its transcriptional regulation, the partial promoter sequence ofCiPRKRA(1463 bp) containing one ISRE and one CARE was cloned by Tail-PCR. Subsequently, grass carp IRF2 (CiIRF2) and ATF4 (CiATF4) were expressed inEscherichia coliBL21 and purified by affinity chromatography with the Ni-NTA His-Bind Resin.In vitro, bothCiIRF2 andCiATF4 bound toCiPRKRApromoter with high affinity by gel mobility shift assays, revealing that IRF2 and ATF4 might be potential transcriptional regulatory factors forCiPRKRA. Dual-luciferase reporter assays were applied to further investigate the transcriptional regulation ofCiPRKRA in vivo. Recombinant plasmid of pGL3-PRKRAPro was constructed and transiently co-transfected into CIK cells with pcDNA3.1-CiIRF2 and pcDNA3.1-CiATF4, respectively. The results showed that bothCiIRF2 andCiATF4 significantly decreased the luciferase activity of pGL3-PRKRAPro, suggesting that they play a negative role inCiPRKRAtranscription.