The 3’-untranslated region of CHOP mRNA encodes information that determines the expression level of the CHOP gene

CHOP is a stress-inducible protein that functions as a transcription co-regulatory factor in cellular responses to stress. Amongst the various biochemical determinants that might control the ultimate level of CHOP in a stressed cell, the importance of the abundance of CHOP-mRNA template that is available for translation has received little attention. In the present study, we examined if the 3’-untranslated region of CHOP-mRNA might encode information that determines the intracellular level (abundance) of CHOP-mRNA. A reporter plasmid (E3) that expresses a Green Fluorescent Protein (EGFP) mRNA having its original 3’UTR replaced with the 3’UTR of CHOP mRNA was constructed. Reporter mRNA level in cells transfected with E3 was quantitated by real-time PCR (after reverse transcription) and compared with those of mutant reporter mRNA that had 30-nucleotide deletions in the 3UTR and expressed from the corresponding E3 mutants. Whereas the absence of the first 30 nucleotides in the 5’-end of CHOP 3’UTR resulted in approximately 4 fold decrease of the reporter mRNA level, E3 mutants bearing deletion of nucleotides 61-90, and 91-120 in the 3UTR displayed 6 to 7 fold increase in reporter mRNA levels. Such effects of 30-nucleotide deletion on mRNA levels were again observed using another construct in which the EGFP coding sequence was replaced with the CHOP coding sequence. The mutant mRNA levels relative to the wildtype mRNA was not affected by the presence of cellular stress. To examine if the change in mRNA levels would result in a corresponding change of translation, the abundance of the reporter protein (GFP) was quantitated directly by Western Blot and indirectly by measurement of FITC fluorescence by FACS analysis