An analysis of the functional significance of the 3'-untranslated region of CHOP/Gadd153 messenger RNA

Abstract

CHOP, or Gadd153, is a 29 kDa protein that plays a pivotal role in the mediation of cellular stress-induced cell death. The expression of the gene encoding CHOP/Gadd153 is regulated at both the transcriptional and post-transcriptional levels. Compared to transcription, the regulation of Chop gene expression at the post-transcriptional level is much less understood. In this study, the role played by the 3’-untranslated region of CHOP mRNA (3UTRChop) in mediating mRNA stability was examined. A reporter plasmid was constructed so that the mRNA expressed has 3UTRChop as its 3’-untranslated region. Partial 5’ deletions, or deletion of short internal (~30 nucleotides) sequences, or the deletion of a putative AU-rich element (ARE) within 3UTRChop, all resulted in the elevation of the steady state levels of mRNA and the encoded reporter protein, EGFP. Deletion of the ARE or sequences remote from ARE resulted in the reduced rate of mRNA degradation. Such data suggested that 3UTRChop is closely related to mRNA stability.

The effect of cellular stress on the functioning of 3UTRChop was studied by examining the change in mRNA level in cells treated with arsenic trioxide (ATO). The presence of arsenic stress stimulated a marked increase in the steady state level of not only the reporter mRNA, but also control mRNAs that did not have 3UTRChop as the 3’-untranslated region. A non-specific effect of arsenic stress on mRNA levels was therefore suggested. Consistent with the increase in the level of reporter mRNA, the expression of EGFP protein was also increased. Arsenic stress and partial deletion of 3UTRChop produce additive increase in mRNA level and EGFP protein level implying that the mRNA destabilizing function of 3UTRChop is unlikely to be stress-regulated.

The contribution of 3UTRChop in mRNA translation was then examined using reporter constructs that expressed EGFP mRNA having its original 5’ and 3’-untranslated regions replaced with 5UTRChop and 3UTRChop respectively. In the presence of wild-type 3UTRChop, the abolition of the translation repressor functions of 5UTRChop produced only mild increase in EGFP expression. However, the additional partial deletion of 3UTRChop resulted in massive increase in EGFP expression. In the presence of wild-type 5UTRChop, the partial deletion of 3UTRChop resulted in only a small increase in EGFP expression. Such data demonstrated a complementary relationship between 5UTRChop and 3UTRChop in the regulation of Chop expression in unstressed cells. EGFP mRNA having 5UTRChop and 3UTRChop as the 5’ and 3’-untranslated region respectively expressed significant EGFP protein only in the presence of ATO. The expression of EGFP was not significantly affected with swopping of 3UTRChop with another 3UTR. 3UTRChop is therefore not essential for the mediation of ATO-stimulated expression of EGFP.

The present study demonstrated that full length 3UTRChop may have constitutive mRNA destabilizing effect that is not alleviated by cellular stress. The evaluation of the relative contributions of 5UTRChop and 3UTRChop in mRNA translation suggested a model for Chop gene expression whereby the eventual protein level of Chop is determined by 5UTRChop-mediated translation as well as by 3UTRChop-mediated destabilization of mRNA template.