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

Abstract

CHOP is a stress-inducible protein that acts as a transcription co-regulatory factor for genes that are involved in cellular responses to stress. Previous studies demonstrated that the activation of promoter activity of the CHOP gene is a major mechanism underlying the increase in the mRNA level of the CHOP gene in stressed cells. It is however not certain if the cellular mRNA degradation mechanism for CHOP mRNA might also be stress-sensitive so that together with stress-stimulated transcription a highly precise control of CHOP mRNA level can be achieved. To examine such a possibility, a series of CHOP-expressing plasmids were constructed and used to express the full length CHOP mRNA with or without deletion mutations in its 3’untranslated region in cultured cells. Realtime PCR quantitation demonstrated that the level of the expressed full length CHOP mRNA was the same in unstressed and stressed cells. Deletion mutations of the 3UTR of CHOP mRNA resulted in changes of CHOP-mRNA levels relative to the wild type mRNA but otherwise such changes were not affected by cellular stress. To ascertain if deletion mutations of the 3’UTR of CHOP mRNA alone were sufficient to cause changes in mRNA levels without requiring the participation of the rest of the CHOP mRNA sequence, another series of expression plasmids were constructed that will express EGFP mRNA having its original 3’UTR replaced with either the 3’UTR of CHOP mRNA, or 3’UTR of CHOP mRNA with deletions mutations the same as those of the pCHOP series of plasmids. Analysis of real-time PCR data obtained for the quantitation of mRNA levels expressed from the E3 series of plasmids showed the same changes of the expressed mRNA levels caused by the deletion mutations in the 3’UTR. The changes in mRNA levels were again independent of cellular stress. To examine if the change in mRNA levels would result in proportionate changes of translation, the abundance of the reporter protein (GFP) expressed from plasmids of the E3-series was quantitated directly by Western blot and indirectly by measurement of FITC fluorescence by FACS analysis. The decrease in mRNA expression due to the deletion of the first 30 ribonucleotides in the 3’UTR CHOP is always met with more of less proportionate decrease in the reporter protein expressed from this mutant mRNA. However, the change in mRNA level expressed from other pCHOP and E3 deletion mutants may not always cause a corresponding change in level of translational product. Results of this study suggest that unlike transcriptional synthesis of CHOP mRNA that is tightly stress-regulated, the degradation mechanism of CHOP mRNA is stress-insensitive and it may only be passively involved in preventing excessive synthesis of the CHOP mRNA. The degradation of the CHOP is closely related to the nucleotide sequence of its 3’UTR. Additional function of the nucleotide sequence of the 3’UTR of CHOP mRNA is also suggested by results of the present study.