| Summary: | 碩士 === 國立臺灣大學 === 分子與細胞生物學研究所 === 103 === In response of endoplasmic reticulum (ER) stress, C/EBP homologous protein (CHOP) is critically involved in either cell survival or apoptosis. It has been reported that the 5’UTR of chop mRNA contains an upstream open reading frame (uORFchop) which inhibits its translation. During ER stress, the uORFchop–mediated translational inhibition is abolished, resulting in generating CHOP protein. However, underlying molecular mechanisms of uORFchop -mediated translational inhibition is not fully understood. To answer this issue, we employed the zebrafish transgenic line huORFZ, which harbors the GFP reporter fused with the human uORFchop (huORFchop) and driven by a cytomegalovirus promoter. Interestingly, GFP was expressed only when huORFZ embryos were treated with ER stresses. The number of GFP(+) cells in the brain of huORFZ embryos was dependent on the duration of heat-treatment. Taking advantage of Laser Microdissection, we collected the neuronal cells expressing GFP from the brain of heat-shocked huORFZ embryos to perform microarray analysis using GFP-negative neuronal cells as a background. Among the putative genes, dkey was selected for further study because it was one of the most up-regulated genes presented in GFP(+) cells, whose A value was 7.2 (A value greater than 7 indicates high reliability) and M value was 2.4 (M value greater than 0 indicates high intensity). Whole mount in situ hybridization demonstrated that dkey transcripts were expressed in the brain and spinal cord of embryos at 96 hpf. The expressional level of dkey was greatly increased when embryos were treated with 40℃,which was corresponding with the results obtained from microarray. To confirm whether dkey plays role on the huORFchop-mediated translational inhibition, we injected a DNA construct of huORFchop-luciferase and a DNA fragment of dkey into zebrafish embryos. Results showed that the luciferase activity of the injected embryos was increased, suggesting that overexpression of dkey did suppress the huORFchop –translational inhibition in vivo. Furthermore, Western blot analysis revealed that overexpressive dkey in embryos increased the protein levels of phosphorylated eIF2α (p-eIF2α) and CHOP. Results obtained from in vitro studies of luciferase assay and Western blot analysis were correspondent with those of in vivo study. We also demonstrated that increase of the non-phosphorylated mutant of eIF2α (eIF2α/S51A) in cells reduced the suppressive capability of dkey on the huORFchop - translational inhibition in vitro. In contrast, increase of the phosphorylated eIF2α (eIF2α/S51D) enhanced the suppressive capability of dkey on the huORFchop -mediated translational inhibition. Furthermore, co-immunoprecipitation revealed that Dkey protein was able to interact with eIF2α. Thus, we concluded that eIF2α and Dkey interact to control the huORFchop translational inhibition. Interestingly, Northern blot exhibited that Dkey could digest the huORFchop -tagged mRNA, suggesting that the endoribonucleases activity of Dkey may function in huORFchop -mediated translational inhibition. Collectively, we suggested that (1) Dkey is effectively involved in the huORFchop -mediated translational inhibition through the increase of p-eIF2α; and (2) Dkey may combine with eIF2α to locate the binding site of huORFchop, resulting in altering the huORFchop structure through its endoribonucleases ability, which in turn, the huORFchop –mediated translational inhibition is abolished, and CHOP is translated during ER stress.
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