Abstract
Upstream open reading frame (uORF)-mediated translational inhibition is important in controlling key regulatory genes expression. However, understanding the underlying molecular mechanism of such uORF-mediated control system in vivo is challenging in the absence of an animal model. Therefore, we generated a zebrafish transgenic line, termed huORFZ, harboring a construct in which the uORF sequence from human CCAAT/enhancer-binding protein homologous protein gene (huORF(chop)) is added to the leader of GFP and is driven by a cytomegalovirus promoter. The translation of transgenic huORF(chop)-gfp mRNA was absolutely inhibited by the huORF(chop) cassette in huORFZ embryos during normal conditions, but the downstream GFP was only apparent when the huORFZ embryos were treated with endoplasmic reticulum (ER) stresses. Interestingly, the number and location of GFP-responsive embryonic cells were dependent on the developmental stage and type of ER stresses encountered. These results indicate that the translation of the huORF(chop)-tag downstream reporter gene is controlled in the huORFZ line. Moreover, using cell sorting and microarray analysis of huORFZ embryos, we identified such putative factors as Nrg/ErbB, PI3K and hsp90, which are involved in huORF(chop)-mediated translational control under heat-shock stress. Therefore, using the huORFZ embryos allows us to study the regulatory network involved in human uORF(chop)-mediated translational inhibition.