Transgenic zebrafish model to study translational control mediated by upstream open reading frame of human chop gene

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 (huORFchop) is added to the leader of GFP and is driven by a cytomegalovirus promoter. The translation of transgenic huORFchop-gfp mRNA was absolutely inhibited by the huORFchop 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 huORFchop-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 huORFchop-mediated translational control under heat-shock stress. Therefore, using the huORFZ embryos allows us to study the regulatory network involved in human uORFchop-mediated translational inhibition.

Multiple Mechanisms of Unfolded Protein Response–Induced Cell Death

“…49e51 A second mechanism by which ATF4 promotes cell death is via transcription of the proapoptotic Chop gene (official name DDIT3), whose translation is also enhanced by eIF2aP (Figure 3). 16,43,44,52,53 By dual transcriptional and translational up-regulation, CHOP is highly enriched when PERK is strongly activated. CHOP's role as a proapoptotic transcription factor has been clearly shown in vitro where CHOPnull cells are resistant to cell death induced by the chemical Figure 3 Consequences of acute and chronic PKR-like endoplasmic reticulum (ER) kinase (PERK) activation.…”

The role of endoplasmic reticulum stress in maintaining and targeting multiple myeloma: a double-edged sword of adaptation and apoptosis

“…The CHOP promoter includes binding sites for both ATF4 and ATF6, which appear to synergize (Okada et al, 2002). In addition, the CHOP mRNA includes an upstream inhibitory ORF that is preferentially translated during ER stress (Jousse et al, 2001; Lee et al, 2011). Expression of this protein is very tightly regulated and eventual convergence on CHOP activation signals a likely shift into macroautophagy and/or apoptosis (Gomez-Santos et al, 2005; Kim et al, 2006; Emdad et al, 2011).…”

Subtypes of hypoxia‐responsive cells differentiate into neurons in spinal cord of zebrafish embryos after hypoxic stress

“…Lee et al. () generated huORFZ , a zebrafish transgenic line harbouring an inhibitory uORF of human chop (huORF chop ) fused with GFP reporter. It is not expressed in unstressed huORZF embryos, but it is exclusively expressed in both brain and spinal cord of hypoxia‐exposed huORFZ embryos.…”