Rothenburg Stefan, Deigendesch Nikolaus, Dey Madhusudan, Dever Thomas E, Tazi Loubna
Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
BMC Biol. 2008 Mar 3;6:12. doi: 10.1186/1741-7007-6-12.
Double-stranded (ds) RNA, generated during viral infection, binds and activates the mammalian anti-viral protein kinase PKR, which phosphorylates the translation initiation factor eIF2alpha leading to the general inhibition of protein synthesis. Although PKR-like activity has been described in fish cells, the responsible enzymes eluded molecular characterization until the recent discovery of goldfish and zebrafish PKZ, which contain Z-DNA-binding domains instead of dsRNA-binding domains (dsRBDs). Fish and amphibian PKR genes have not been described so far.
Here we report the cloning and identification of 13 PKR genes from 8 teleost fish and amphibian species, including zebrafish, demonstrating the coexistence of PKR and PKZ in this latter species. Analyses of their genomic organization revealed up to three tandemly arrayed PKR genes, which are arranged in head-to-tail orientation. At least five duplications occurred independently in fish and amphibian lineages. Phylogenetic analyses reveal that the kinase domains of fish PKR genes are more closely related to those of fish PKZ than to the PKR kinase domains of other vertebrate species. The duplication leading to fish PKR and PKZ genes occurred early during teleost fish evolution after the divergence of the tetrapod lineage. While two dsRBDs are found in mammalian and amphibian PKR, one, two or three dsRBDs are present in fish PKR. In zebrafish, both PKR and PKZ were strongly upregulated after immunostimulation with some tissue-specific expression differences. Using genetic and biochemical assays we demonstrate that both zebrafish PKR and PKZ can phosphorylate eIF2alpha in yeast.
Considering the important role for PKR in host defense against viruses, the independent duplication and fixation of PKR genes in different lineages probably provided selective advantages by leading to the recognition of an extended spectrum of viral nucleic acid structures, including both dsRNA and Z-DNA/RNA, and perhaps by altering sensitivity to viral PKR inhibitors. Further implications of our findings for the evolution of the PKR family and for studying PKR/PKZ interactions with viral gene products and their roles in viral infections are discussed.
病毒感染过程中产生的双链（ds）RNA结合并激活哺乳动物抗病毒蛋白激酶PKR，PKR使翻译起始因子eIF2α磷酸化，从而导致蛋白质合成的全面抑制。尽管在鱼类细胞中已描述了类似PKR的活性，但直到最近发现金鱼和斑马鱼的PKZ（其含有Z-DNA结合结构域而非dsRNA结合结构域（dsRBDs）），负责的酶才得以进行分子表征。到目前为止，尚未描述鱼类和两栖类的PKR基因。
在此，我们报告了从包括斑马鱼在内的8种硬骨鱼和两栖类物种中克隆和鉴定出13个PKR基因，这表明PKR和PKZ在斑马鱼中共存。对它们基因组结构的分析显示，存在多达三个串联排列的PKR基因，它们以头对头的方向排列。在鱼类和两栖类谱系中至少独立发生了五次重复。系统发育分析表明，鱼类PKR基因的激酶结构域与鱼类PKZ的激酶结构域比与其他脊椎动物物种的PKR激酶结构域关系更密切。导致鱼类PKR和PKZ基因的重复发生在硬骨鱼进化早期，即在四足动物谱系分化之后。哺乳动物和两栖类的PKR中有两个dsRBD，而鱼类PKR中有一个、两个或三个dsRBD。在斑马鱼中，经免疫刺激后PKR和PKZ均强烈上调，且存在一些组织特异性表达差异。使用遗传和生化分析，我们证明斑马鱼的PKR和PKZ均可在酵母中使eIF2α磷酸化。
考虑到PKR在宿主抗病毒防御中的重要作用，PKR基因在不同谱系中的独立重复和固定可能通过导致识别更广泛的病毒核酸结构（包括dsRNA和Z-DNA/RNA）以及可能通过改变对病毒PKR抑制剂的敏感性而提供了选择优势。我们的发现对PKR家族进化以及研究PKR/PKZ与病毒基因产物的相互作用及其在病毒感染中的作用的进一步意义也进行了讨论。
