The RNase activity of endouc is involved in repressing the Human uORFchop- mediated Translational Inhibition during ER Stress
Date Issued
2016
Date
2016
Author(s)
Huang, Ting-Ying
Abstract
The C/EBP homologous protein (CHOP), which determines that cells undergo apoptosis or survival, starts to be translated when cells encounter endoplasmic reticulum (ER) stress. It has been reported that the 5’UTR of chop mRNA contains an inhibitory upstream open reading frame (uORFchop) which inhibits the translation of the downstream coding sequence (CDS) such as chop during normal condition. When cells encounter ER stress, this uORFchop–mediated translational inhibition (uORF-MTI) is repressed, resulting that the chop located at CDS in mRNA is translated. However, the molecular mechanism underlying uORFchop-MTI is still controversial. To understand the plausible mechanism involved in uORFchop-MTI, I employed the zebrafish transgenic line huORFZ, which harbors an exogenous DNA fragment that the CDS GFP cDNA fused with human uORFchop (huORFchop) and driven by a cytomegalovirus promoter. When huORFZ embryos were treated with heat-shock stress, GFP was exclusively expressed in the central nerve system. Employing laser-capture microdissection combined with microarray to compare the gene expression levels between GFP(+) brain cells and GFP(-) brain cells, we found that the endonuclease poly(U)-specific C gene (endouc) of zebrafish was significantly up-regulated in GFP(+) cells. Overexpression of endouc mRNA was able to repress uORF-MTI, resulting that GFP was expressed in the non-stressed huORFZ embryos. Moreover, the phosphorylation of eIF2α (p-eIF2α) and CHOP proteins were increased greatly in the endouc-overexpressive embryos (in vivo) and HEK293T cells (in vitro), indicating that endouc is involved in the repression of uORF-MTI. We proved that levels of many stress factors including Bip, p58IPK, and pPERK were not ectopic expression, suggesting that endouc overexpression per se did not induce ER stress intracellularly. To identify which domain of Endouc is involved in repression of uORF-MTI, I performed domain mapping and found a domain that contains amino acid residues between 137 and 299 is necessary for repression of uORF-MTI. Furthermore, I generated a single mutation of Endouc at H181 (EndoucH181A) and K242 (EndoucK242A), which disturb the endonuclease activity of Endouc, and found that overexpression of either endoucH181A or endoucK242A led to reduce the protein levels of p-eIF2α and CHOP. I also demonstrated that Endouc was able to enzymatically digest the huORF RNA fragment, while the mutant EndoucK242A was not, suggesting that the function of Endouc to disrupt uORF-MTI is depend on its endoribonuclease activity. Finally, using polysome profiling assay, I clearly demonstrated that, under stress condition, Endouc presented as a free protein and a 40S-80S associated complex. On contrast, the mutant EndoucK242A was always associated with polysomes, suggesting that Endouc was able to be released from polysomes under stress due to its RNase activity. Taken together, I hypothesize a model to explicit how Endouc plays role on the suppression of uORF-MTI: During ER stress, the RNase activity of Endouc might be triggered to digest the huORFchop motif located at 5''UTR of mRNA, resulting in a cap-independent mRNA, which in turn, the downstream CDS of CHOP cDNA is therefore reinitiated to translate through bypassing the hindrance of huORFchop structure. Furthermore, I found a positive feedback involved in suppression of uORF-MTI: the ectopic expression of Endouc enhances the eIF2α phosphorylation, which helps to disrupt the uORF-MTI, resulting in the increase of downstream CDS translation.
Subjects
uORF
RNase
endouc
Type
thesis