Translational Regulation of Zebrafish GADD34 and CReP during the Unfolded Protein Response
Faculty Mentor Name
Dr. Lisa Wrischnik, Dr. Doug Weiser
Research or Creativity Area
Natural Sciences
Abstract
The biological activity of many proteins in eukaryotic cells relies on their ability to attain proper conformational structure in the endoplasmic reticulum (ER) during synthesis. Proteins may accumulate in the ER if they are misfolded or fail to fold efficiently, which induces a vital stress response called the Unfolded Protein Response (UPR). The UPR leads to the phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α), which suppresses cellular global protein synthesis while allowing selective translation of stress response genes. Two genes involved in cellular recovery during UPR are GADD34 (PPP1R15A) and CReP (PPP1R15B). GADD34 and CReP bind as subunits to Protein Phosphatase 1 (PP1), which rephosphorylates eIF2α, resuming global protein synthesis during UPR once the stress has been resolved. GADD34 is upregulated during stress and rapidly degraded upon its resolution, while CReP is considered constitutively expressed and maintains basal eIF2α dephosphorylation, but levels of both are very important for the proper timing of cell recovery. In human and mammalian models, one form of regulation of GADD34 and CReP expression is through translational regulation via upstream open reading frames (uORFs), though it is unclear whether these regulatory mechanisms are shared among other model systems. Zebrafish are widely used as a model organism due to their affordability and efficiency. Zebrafish GADD34 contains two uORFs in the 5’ UTR region likely involved in translational regulation. These uORFs were altered via site-directed mutagenesis and analyzed, using luciferase reporter assays, to determine if their effects on translation during ER stress mirrored the patterns of uORF regulation seen in their mammalian counterparts.
Translational Regulation of Zebrafish GADD34 and CReP during the Unfolded Protein Response
The biological activity of many proteins in eukaryotic cells relies on their ability to attain proper conformational structure in the endoplasmic reticulum (ER) during synthesis. Proteins may accumulate in the ER if they are misfolded or fail to fold efficiently, which induces a vital stress response called the Unfolded Protein Response (UPR). The UPR leads to the phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α), which suppresses cellular global protein synthesis while allowing selective translation of stress response genes. Two genes involved in cellular recovery during UPR are GADD34 (PPP1R15A) and CReP (PPP1R15B). GADD34 and CReP bind as subunits to Protein Phosphatase 1 (PP1), which rephosphorylates eIF2α, resuming global protein synthesis during UPR once the stress has been resolved. GADD34 is upregulated during stress and rapidly degraded upon its resolution, while CReP is considered constitutively expressed and maintains basal eIF2α dephosphorylation, but levels of both are very important for the proper timing of cell recovery. In human and mammalian models, one form of regulation of GADD34 and CReP expression is through translational regulation via upstream open reading frames (uORFs), though it is unclear whether these regulatory mechanisms are shared among other model systems. Zebrafish are widely used as a model organism due to their affordability and efficiency. Zebrafish GADD34 contains two uORFs in the 5’ UTR region likely involved in translational regulation. These uORFs were altered via site-directed mutagenesis and analyzed, using luciferase reporter assays, to determine if their effects on translation during ER stress mirrored the patterns of uORF regulation seen in their mammalian counterparts.
