Figure 5: Semi-quantitative PCR analysis of CHOP splicing variants in plasmids and liver tissues, and CHOP exon arrangements. (A) CHOP1 mRNA contains four exons, whereas CHOP2 mRNA lacks the second exon of CHOP1. The 5 '-leader of CHOP is encoded within the first and second exons, while the coding sequence is encoded within the third and fourth exons of CHOP. (B) As expected, CHOP1 primers amplified CHOP1 transcripts at the size of 214 bp in the CHOP1 plasmid template containing the full sequence of CHOP1. While, CHOP2 isoforms were detected at the size of 207 bp in the CHOP2 plasmid expressing the whole sequence of CHOP2. The presence of a single band amplified by the corresponding primers in each plasmid indicates the specificity of CHOP
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(A) The CHOP1-luc plasmid is constructed to have an uORF preceding the luc ORF to determine the effect of CHOP1 uORF on luciferase activity. The CHOP2-luc plasmid is constructed to have CHOP2 uORF that overlaps out-of-frame with the luciferase CDS. (B) In comparison to non-treated (NT) MEF cells, CHOP1 and CHOP2 showed nearly a 2.5-fold induction of luciferase activity in MEF cells treated with thapsigargin (Tg), an ER stress agent, for 12 hours (12 hr). These findings indicate that CHOP1 and CHOP2 are preferentially translated during ER …show more content…
As a result of eIF2α phosphorylation in stressed cells, the cells produce ATF4 proteins in an attempt to restore cellular homeostasis; alternatively, ATF4 protein promotes CHOP expression in prolonged stressed conditions. Consequent to increased eIF2α phosphorylation, accumulation of CHOP mRNA, encoding a proapoptotic transcription factor, induces an apoptotic pathway leading to cellular death for the purpose of protection of neighboring cells.
CHOP mRNA is alternatively spliced at the second exon into CHOP1 and CHOP2 variants. Both CHOP transcripts encode the full-length protein, but differ in their 5 '-portions that carry out a regulatory role in CHOP translation. CHOP1 transcript appears to be more highly expressed compared to CHOP2 in tunicamycin-treated liver tissues (Figure 5C). This finding may indicate that there is differential expression of CHOP transcripts among mouse
(A) The CHOP1-luc plasmid is constructed to have an uORF preceding the luc ORF to determine the effect of CHOP1 uORF on luciferase activity. The CHOP2-luc plasmid is constructed to have CHOP2 uORF that overlaps out-of-frame with the luciferase CDS. (B) In comparison to non-treated (NT) MEF cells, CHOP1 and CHOP2 showed nearly a 2.5-fold induction of luciferase activity in MEF cells treated with thapsigargin (Tg), an ER stress agent, for 12 hours (12 hr). These findings indicate that CHOP1 and CHOP2 are preferentially translated during ER …show more content…
As a result of eIF2α phosphorylation in stressed cells, the cells produce ATF4 proteins in an attempt to restore cellular homeostasis; alternatively, ATF4 protein promotes CHOP expression in prolonged stressed conditions. Consequent to increased eIF2α phosphorylation, accumulation of CHOP mRNA, encoding a proapoptotic transcription factor, induces an apoptotic pathway leading to cellular death for the purpose of protection of neighboring cells.
CHOP mRNA is alternatively spliced at the second exon into CHOP1 and CHOP2 variants. Both CHOP transcripts encode the full-length protein, but differ in their 5 '-portions that carry out a regulatory role in CHOP translation. CHOP1 transcript appears to be more highly expressed compared to CHOP2 in tunicamycin-treated liver tissues (Figure 5C). This finding may indicate that there is differential expression of CHOP transcripts among mouse