title: Ctenopharyngodon idella IRF2 and ATF4 down-regulate the transcriptional level of PRKRA.
abstract: 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 (named CiPRKRA, 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 of CiPRKRA contains three typical dsRNA binding motifs (dsRBM). Phylogenetic tree analysis revealed a closer evolutionary relationship of CiPRKRA with other fish PRKRA, especially with Danio rerio PRKRA. qRT-PCR showed that CiPRKRA was significantly up-regulated after stimulation with tunicamycin (Tm) and Poly I:C in C. idella kidney (CIK) cells. To further study its transcriptional regulation, the partial promoter sequence of CiPRKRA (1463 bp) containing one ISRE and one CARE was cloned by Tail-PCR. Subsequently, grass carp IRF2 (CiIRF2) and ATF4 (CiATF4) were expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind Resin. In vitro, both CiIRF2 and CiATF4 bound to CiPRKRA promoter with high affinity by gel mobility shift assays, revealing that IRF2 and ATF4 might be potential transcriptional regulatory factors for CiPRKRA. Dual-luciferase reporter assays were applied to further investigate the transcriptional regulation of CiPRKRA 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 both CiIRF2 and CiATF4 significantly decreased the luciferase activity of pGL3-PRKRAPro, suggesting that they play a negative role in CiPRKRA transcription.
authors.affiliation: College of Life Science, Key Lab of Aquatic Resources and Utilization of Jiangxi Province, Nanchang University, Nanchang 330031, China.
authors.forename: Keyi
authors.initials: K
authors.lastname: Huang
authors.affiliation: College of Life Science, Key Lab of Aquatic Resources and Utilization of Jiangxi Province, Nanchang University, Nanchang 330031, China.
authors.forename: Guoqin
authors.initials: G
authors.lastname: Qi
authors.affiliation: College of Life Science, Key Lab of Aquatic Resources and Utilization of Jiangxi Province, Nanchang University, Nanchang 330031, China.
authors.forename: Zhicheng
authors.initials: Z
authors.lastname: Sun
authors.affiliation: College of Life Science, Key Lab of Aquatic Resources and Utilization of Jiangxi Province, Nanchang University, Nanchang 330031, China.
authors.forename: Xiancheng
authors.initials: X
authors.lastname: Liu
authors.affiliation: College of Life Science, Key Lab of Aquatic Resources and Utilization of Jiangxi Province, Nanchang University, Nanchang 330031, China.
authors.forename: Xiaowen
authors.initials: X
authors.lastname: Xu
authors.affiliation: College of Life Science, Key Lab of Aquatic Resources and Utilization of Jiangxi Province, Nanchang University, Nanchang 330031, China.
authors.forename: Haizhou
authors.initials: H
authors.lastname: Wang
authors.affiliation: College of Life Science, Key Lab of Aquatic Resources and Utilization of Jiangxi Province, Nanchang University, Nanchang 330031, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Wu
authors.affiliation: College of Life Science, Key Lab of Aquatic Resources and Utilization of Jiangxi Province, Nanchang University, Nanchang 330031, China.
authors.forename: Yiqi
authors.initials: Y
authors.lastname: Wan
authors.affiliation: College of Life Science, Key Lab of Aquatic Resources and Utilization of Jiangxi Province, Nanchang University, Nanchang 330031, China. Electronic address: hucy2008@163.com.
authors.forename: Chengyu
authors.initials: C
authors.lastname: Hu
mesh_terms: D051701:Activating Transcription Factor 4 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D001483:Base Sequence; D002347:Carps / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics*; D050836:Interferon Regulatory Factor-2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D011070:Poly I-C / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D000998:Antiviral Agents; D018076:DNA, Complementary; D029941:Fish Proteins; D050836:Interferon Regulatory Factor-2; D012333:RNA, Messenger; D014415:Tunicamycin; D051701:Activating Transcription Factor 4; D011070:Poly I-C
keywords: Grass carp; IRF2 and ATF4; PKR; PRKRA; Transcriptional regulation