一个组成型表达的草鱼ADAR1基因剪切异构体ADAR1a的鉴定与特征
发布时间：2019-02-19 18:33
【摘要】：作为ADAR家族中的一员,ADAR1(adenosine deaminase acting on RNA)能够将双链RNA的腺嘌呤(A)置换为次黄嘌呤(I)。在哺乳动物中,ADAR1拥有很多剪切异构体,包括研究较为普遍的能被干扰素诱导表达的150kD大小的剪切异构体ADAR1-p150和组成型表达的110kD大小的剪切异构体ADAR1-p110。ADAR1这种结构的多样性可能反应了其不同剪切异构体之间功能的多样及差异。ADAR1不同剪切异构体也被发现存在于鱼类之中。在我们之前的研究中,我们已经在草鱼中克隆得到了ADAR1两种不同剪切异构体Ci ADAR1和CiADAR1-like,它们都可以被干扰素诱导表达。在本文中,我们鉴定了一个新的草鱼ADAR1基因剪切异构体ADAR1a。ADAR1a基因包含15个外显子和14个内含子,它的全长cDNA序列包括359 bp的5'UTR,229 bp的3'UTR和一个2592 bp的最大ORF,编码983个氨基酸的多肽。ADAR1a蛋白具有1个Z-DNA结合域,3个dsRNA结合域和一个高度保守的水解脱氨酶激酶活性区。在草鱼CIK细胞中,通过western blot分析,草鱼ADAR1a蛋白表现出组成型表达特性并不受poly(I:C)刺激影响。在正常状况下,草鱼肾细胞中ADAR1a蛋白主要分布在细胞核,而通过poly(I:C)刺激,随着时间推移,ADAR1a蛋白由细胞核转移至细胞质,其具体分子机制尚不清楚。为了进一步研究草鱼ADAR1a基因的转录调控机制,通过5'full Race技术,我们确定了草鱼ADAR1a的转录起始位点,位于ADAR1基因的第二个外显子上。然后我们克隆了其包含部分5'UTR及其上游序列在内共1303 bp作为ADAR1a基因的启动子,通过软件分析,其中包含4个干扰素调节因子元件。通过构建草鱼IRF1、IRF3的真核表达载体pcDNA3.1-IRF1和pcDNA3.1-IRF3与pGL3-Ci ADAR1a启动子报告载体共转入草鱼肾细胞。双荧光素酶活性分析结果显示在草鱼肾细胞中无论是草鱼IRF1、IRF3蛋白还是Poly I:C都不能影响草鱼ADAR1a蛋白的表达。通过其分子与表达机制,细胞内亚定位结果及其转录调控机制,我们推测草鱼ADAR1a与哺乳动物ADAR1蛋白剪切异构体ADAR1-p110具有高度同源性。随后,通过序列比对推测其核输出信号(NES)序列,并构建pEGFP-C1-ADAR1a,pEGFP-C1-ADAR1N,pEGFP-C1-ADAR1NΔNES表达载体,瞬时转染进CIK细胞,通过荧光显微镜观察,发现转染pEGFP-C1-ADAR1a质粒的细胞荧光集中于细胞核,转染pEGFP-C1-ADAR1N(ADAR1 N端区域,1-505aa)质粒的细胞在细胞质与细胞核都有荧光,而转染pEGFP-C1-ADAR1NΔNES载体的细胞荧光主要在细胞核,证实了草鱼ADAR1 N端区域拥有一段NES序列。以上所有结果都表明,草鱼ADAR1a是一个组成型表达的ADAR1基因剪切异构体。
[Abstract]:As a member of the ADAR family, ADAR1 (adenosine deaminase acting on RNA) can replace the adenine (A) of double-stranded RNA with Hypoxanthine (I). In mammals, ADAR1 has many shearing isomers, Including the study of the more common shearing isomer ADAR1-p150, which can be induced to express 150kD by interferon, and the shearing isomer ADAR1-p110.ADAR1, which is composed of 110kD, the diversity of structures may reflect their different shearing. The diversity and difference of the function of the isomers. The different shear isomers of ADAR1 are also found in fish. In our previous studies, we have cloned two different shearing isomers of ADAR1, Ci ADAR1 and CiADAR1-like, from grass carp, both of which can be induced by interferon. In this paper, we have identified a new ADAR1 gene shearing isomer ADAR1a.ADAR1a gene containing 15 exons and 14 introns. Its full-length cDNA sequence consists of 5 UTR229 bp 3'UTR of 359 bp and a maximum ORF, of 2592 bp. ADAR1a protein has one Z-DNA binding domain, three dsRNA binding domains and a highly conserved active domain of water deaminase kinase. In grass carp CIK cells, western blot analysis showed that the constitutive expression of ADAR1a protein in grass carp was not affected by poly (I: C) stimulation. Under normal conditions, the ADAR1a protein in the kidney cells of grass carp is mainly distributed in the nucleus, but through the stimulation of poly (I: C), the ADAR1a protein is transferred from the nucleus to the cytoplasm with the passage of time, and its molecular mechanism is not clear. In order to further study the transcriptional regulation mechanism of grass carp ADAR1a gene, we identified the ADAR1a transcription initiation site of grass carp by 5'full Race technique, which is located on the second exon of ADAR1 gene. Then we cloned 1303 bp which contains part of 5'UTR and its upstream sequence as promoter of ADAR1a gene. By software analysis, it contains four interferon regulatory factors. The eukaryotic expression vectors pcDNA3.1-IRF1, pcDNA3.1-IRF3 and pGL3-Ci ADAR1a promoter of grass carp IRF1,IRF3 were co-transferred into the kidney cells of grass carp by constructing the eukaryotic expression vector of grass carp IRF1,IRF3. The results of double luciferase activity analysis showed that neither grass carp IRF1,IRF3 protein nor Poly I: C could affect the expression of ADAR1a protein in grass carp kidney cells. Based on its molecular and expression mechanism, intracellular sublocalization and transcriptional regulation, we speculate that ADAR1a has high homology with mammalian ADAR1 shearing isomer ADAR1-p110. Then, the (NES) sequence of nuclear output signal was deduced by sequence alignment, and pEGFP-C1-ADAR1a,pEGFP-C1-ADAR1N,pEGFP-C1-ADAR1N 螖 NES expression vector was constructed. The expression vector was transiently transfected into CIK cells and observed by fluorescence microscope. It was found that the fluorescence of the transfected pEGFP-C1-ADAR1a plasmid was concentrated in the nucleus, and that the cells transfected with the pEGFP-C1-ADAR1N (ADAR1 N-terminal region, 1-505aa) plasmid had fluorescence in both the cytoplasm and the nucleus. The fluorescence of the transfected pEGFP-C1-ADAR1N 螖 NES vector was mainly in the nucleus, which confirmed that there was a NES sequence in the N-terminal region of grass carp ADAR1. All the above results suggest that ADAR1a is a constitutive expression of ADAR1 gene shearing isomer.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78
本文编号：2426750
[Abstract]:As a member of the ADAR family, ADAR1 (adenosine deaminase acting on RNA) can replace the adenine (A) of double-stranded RNA with Hypoxanthine (I). In mammals, ADAR1 has many shearing isomers, Including the study of the more common shearing isomer ADAR1-p150, which can be induced to express 150kD by interferon, and the shearing isomer ADAR1-p110.ADAR1, which is composed of 110kD, the diversity of structures may reflect their different shearing. The diversity and difference of the function of the isomers. The different shear isomers of ADAR1 are also found in fish. In our previous studies, we have cloned two different shearing isomers of ADAR1, Ci ADAR1 and CiADAR1-like, from grass carp, both of which can be induced by interferon. In this paper, we have identified a new ADAR1 gene shearing isomer ADAR1a.ADAR1a gene containing 15 exons and 14 introns. Its full-length cDNA sequence consists of 5 UTR229 bp 3'UTR of 359 bp and a maximum ORF, of 2592 bp. ADAR1a protein has one Z-DNA binding domain, three dsRNA binding domains and a highly conserved active domain of water deaminase kinase. In grass carp CIK cells, western blot analysis showed that the constitutive expression of ADAR1a protein in grass carp was not affected by poly (I: C) stimulation. Under normal conditions, the ADAR1a protein in the kidney cells of grass carp is mainly distributed in the nucleus, but through the stimulation of poly (I: C), the ADAR1a protein is transferred from the nucleus to the cytoplasm with the passage of time, and its molecular mechanism is not clear. In order to further study the transcriptional regulation mechanism of grass carp ADAR1a gene, we identified the ADAR1a transcription initiation site of grass carp by 5'full Race technique, which is located on the second exon of ADAR1 gene. Then we cloned 1303 bp which contains part of 5'UTR and its upstream sequence as promoter of ADAR1a gene. By software analysis, it contains four interferon regulatory factors. The eukaryotic expression vectors pcDNA3.1-IRF1, pcDNA3.1-IRF3 and pGL3-Ci ADAR1a promoter of grass carp IRF1,IRF3 were co-transferred into the kidney cells of grass carp by constructing the eukaryotic expression vector of grass carp IRF1,IRF3. The results of double luciferase activity analysis showed that neither grass carp IRF1,IRF3 protein nor Poly I: C could affect the expression of ADAR1a protein in grass carp kidney cells. Based on its molecular and expression mechanism, intracellular sublocalization and transcriptional regulation, we speculate that ADAR1a has high homology with mammalian ADAR1 shearing isomer ADAR1-p110. Then, the (NES) sequence of nuclear output signal was deduced by sequence alignment, and pEGFP-C1-ADAR1a,pEGFP-C1-ADAR1N,pEGFP-C1-ADAR1N 螖 NES expression vector was constructed. The expression vector was transiently transfected into CIK cells and observed by fluorescence microscope. It was found that the fluorescence of the transfected pEGFP-C1-ADAR1a plasmid was concentrated in the nucleus, and that the cells transfected with the pEGFP-C1-ADAR1N (ADAR1 N-terminal region, 1-505aa) plasmid had fluorescence in both the cytoplasm and the nucleus. The fluorescence of the transfected pEGFP-C1-ADAR1N 螖 NES vector was mainly in the nucleus, which confirmed that there was a NES sequence in the N-terminal region of grass carp ADAR1. All the above results suggest that ADAR1a is a constitutive expression of ADAR1 gene shearing isomer.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78
【参考文献】
相关硕士学位论文 前1条
1 孙志成;草鱼ADAR1(adenosine deaminase acting on RNA 1)及其剪接异构体ADAR1-like的转录调控分析[D];南昌大学;2016年
，本文编号：2426750
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