Regulation of GADD34 and CReP mRNA Expression in the Unfolded Protein Response
Format
Poster Presentation
Faculty Mentor Name
Doug Weiser
Faculty Mentor Department
Biological Sciences
Additional Faculty Mentor Name
Lisa Wrischnik
Additional Faculty Mentor Department
Biological Sciences
Abstract/Artist Statement
The failure to balance protein synthesis, folding, and degradation in the endoplasmic reticulum (ER) leads to the accumulation of unfolded proteins, causing ER stress. Cells respond this increase in stress by activating a signaling pathway known as the Unfolded Protein Response (UPR). The UPR induces phosphorylation of eIF2α (Eukaryotic Initiation Factor 2) to attenuate global protein synthesis, allowing to clear misfolded proteins. This function is opposed by eIF2α phosphatases, which contain a catalytic subunit, Protein Phosphatase 1, and a scaffolding protein, either GADD34 or CReP. Inhibition of eIF2α phosphatases has shown to promote survival in cell types by prolonging the effects of the UPR. Understanding the gene regulation of GADD34 and CReP can be done by observing the changes in gene expression in zebrafish embryos after the induction of ER stress. Primers for the genes of interest (BIP, CHOP, GADD34, and CReP) were designed to test for changes in levels of gene expression. 24hpf (hours post fertilization) zebrafish embryos were treated with tunicamycin, thapsigargin, and salubrinal to induce ER stress and DMSO for a negative control for 1, 2, 3, 4, 5, 6, 8, 10, 12, 24, and 48 hours. The RNA was purified from the treated embryos to perform quantitative PCR (qPCR) to look at changes in gene levels to understand when eIF2α phosphatases are active. It is shown that the expression of GADD34 increases rapidly after the induction of stress and remains upregulated whereas the expression of CReP increases rapidly after the induction of stress and declines at the later time points. The expression of CHOP is seen to upregulate at the later time points, indicating that cells are beginning to undergo apoptosis due to not being able to clear the ER stress and recover.
Location
Virtual
Start Date
25-4-2020 1:00 PM
End Date
25-4-2020 3:00 PM
Regulation of GADD34 and CReP mRNA Expression in the Unfolded Protein Response
Virtual
The failure to balance protein synthesis, folding, and degradation in the endoplasmic reticulum (ER) leads to the accumulation of unfolded proteins, causing ER stress. Cells respond this increase in stress by activating a signaling pathway known as the Unfolded Protein Response (UPR). The UPR induces phosphorylation of eIF2α (Eukaryotic Initiation Factor 2) to attenuate global protein synthesis, allowing to clear misfolded proteins. This function is opposed by eIF2α phosphatases, which contain a catalytic subunit, Protein Phosphatase 1, and a scaffolding protein, either GADD34 or CReP. Inhibition of eIF2α phosphatases has shown to promote survival in cell types by prolonging the effects of the UPR. Understanding the gene regulation of GADD34 and CReP can be done by observing the changes in gene expression in zebrafish embryos after the induction of ER stress. Primers for the genes of interest (BIP, CHOP, GADD34, and CReP) were designed to test for changes in levels of gene expression. 24hpf (hours post fertilization) zebrafish embryos were treated with tunicamycin, thapsigargin, and salubrinal to induce ER stress and DMSO for a negative control for 1, 2, 3, 4, 5, 6, 8, 10, 12, 24, and 48 hours. The RNA was purified from the treated embryos to perform quantitative PCR (qPCR) to look at changes in gene levels to understand when eIF2α phosphatases are active. It is shown that the expression of GADD34 increases rapidly after the induction of stress and remains upregulated whereas the expression of CReP increases rapidly after the induction of stress and declines at the later time points. The expression of CHOP is seen to upregulate at the later time points, indicating that cells are beginning to undergo apoptosis due to not being able to clear the ER stress and recover.