KEPI plays a negative role in the repression that accompanies translational inhibition guided by the uORF element of human CHOP transcript during stress response

Abstract

Translation of the downstream coding sequence of some mRNAs may be repressed by the upstream open reading frame (uORF) at their 5′-end. The mechanism underlying this uORF-mediated translational inhibition (uORF-MTI) is not fully understood in vivo. Recently, it was found that zebrafish Endouc or its human orthologue ENDOU (Endouc/ENDOU) plays a positive role in repressing the uORF-MTI of human CHOP (uORFchop-MTI) during stress by blocking its activity However, the repression of uORFchop-MTI assisted by an as-yet unidentified negative effector remains to be elucidated. Compared to the upregulated CHOP transcript, we herein report that the kepi (kinase-enhanced PP1 inhibitor) transcript was downregulated in the zebrafish embryos treated with both heat shock and hypoxia. Quantitative RT-PCR also revealed that the level of kepi mRNA was noticeably decreased in both heat-shock-treated and hypoxia-exposed embryos. When kepi mRNA was microinjected into the one-celled embryos from transgenic line huORFZ, the translation of downstream GFP reporter controlled by the uORFchop-MTI was reduced in the hypoxia-exposed embryos. In contrast, when kepi was knocked down by injection of antisense Morpholino oligonucleotide, the translation of downstream GFP reporter was induced and expressed in the brain and spinal cord of injected embryos in the absence of stress. During normal condition, overexpression of KEPI increased eIF2α phosphorylation, resulting in inducing the translation of uORF-tag mRNA, such as ATF4 and CHOP mRNAs. However, during stress condition, overexpression of KEPI decreased eIF2α phosphorylation, resulting in reducing the GFP reporter and CHOP proteins. This is the first report to demonstrate that KEPI plays a negative role in uORFchop – mediated translation during ER stress.