Purchase this article with an account.
Lei Gu, Linda Ye, Jacky Man Kwong Kwong, Joseph Caprioli, Natik Piri; Transcriptional factor Sp1 stimulates expression of the RNA binding protein Rbpms. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4989.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The aim of the present study is to analyze the minimal promoter region of Rbpms, a specific marker for retinal ganglion cells (RGCs), and to identify transcriptional factors (TFs) involved in the regulation of its expression.
Progressive deletion constructs containing 5’-flanking (5’-FR) and 5’-untranslated (5’-UTR) regions of Rbpms, as well as constructs with predicted promoter regions and mutated promoter constructs were analyzed in human embryonic kidney 293T and RGC-5 cells. A pCMV-β-galactosidase plasmid was used as an internal normalization control for variations in transfection efficiency. The electrophoretic mobility shift assay (EMSA) was used to identify protein-DNA interactions.
Expression levels from construct containing both 5’-FR (-1616/-1) and 5’-UTR (+1/+665) was approximately 5 fold (in RGC-5) and 10 fold (in 293T) higher than that obtained from a promoter-less pGL3-Basic vector used as a negative control. Deletion of 5’-FR increased the expression by approximately 35 and 50 fold in RGC-5 and 293 cells, respectively indicating the critical role of 5’-UTR in activation of gene expression. Expression from constructs with 5’-FR only (-2000/-1) and with predicted promoter regions identified in the 5’-FR (-1603/-1353 and -259/-9) was reduced to the baseline levels. In the 5’-UTR, two potential transcription regulatory regions, +2 /+249 and +344/+597, were identified. Site-directed mutational analysis was used to evaluate the contribution of two GC-boxes (for Sp1) and one site for AP1 in +2/+249 region, as well as three GC-boxes and two sites for NFkB in the +344/+597 region. Mutations in the first three Sp1 sites decrease the expression level by about 2.5 fold, whereas mutations in the Ap1, NFkB and two downstream Sp1sites had no effect on gene expression compared to the non-mutated construct. EMSA showed the presence of DNA-protein interaction at the first GC-box (+20/+30). No protein binding to the downstream four Sp1 sites was observed.
The 5’-UTR of Rbpms is critical for the induction of gene expression and at least one Sp1 site within the 5’-UTR is involved in stimulation of transcription. The two predicted promoter regions in the 5’-FR have an inhibitory effect on gene expression in both RGC-5 and 293T cell lines.
This PDF is available to Subscribers Only