P07 – Unusual hyperactivity of a glucocorticoid receptor fragment is automatized by specific deletion of the 3’UTR

Author(s):
David Greenhalgh, Tajia Green, Debora Lim, Stacey Leventhal, Kelly Tung, Kiho Cho, UC Davis Medical Center

Background: The glucocorticoid receptor (GR) is essential to the response to stress. We have previously reported that a 118 amino acid fragment of the N-terminus of GR [hGR-S1(-349A)] or “F1”, which lacks ligand and DNA binding domains has a markedly enhanced response to steroids compared to the full-length GR (hGRa). To determine the mechanism of the augmentation we dissected the 3’ untranslated region (3’UTR) of the mRNA and retested its activity in response to steroids.

Hypothesis: Elimination of the 3’UTR from F1 will eliminate its hyperactivity.

Methods: The 3’UTR of F1 had 300 base pair (bp) segments removed and its activity tested for response to hydrocortisone (Hyd) using our standard luciferase reporter assay. Values are expressed as luciferase + ¬SEM.

Results: As usual, there was a dose response to adding hydrocortisone to the control hGRa. The F1 fragment had an increase in activity 5 times that of hGRa after treatment with hydrocortisone. When removing 300 bp segments from the F1 3’UTR, all activity was lost until 1200 bps were removed. After eliminating these 1200 bps, which is near a polyA site, there was a doubling in activity beyond the highest F1 that did not require steroids.

Conclusions: The 3’UTR of F1 plays a major role in its hyperactivity. When 1200 bps are removed from the 3’UTR there is an increase in activity that is independent of steroids. The role of the poly A site needs to be determined.