Upstream regions of the estrogen receptor alpha proximal promoter transcript regulate ER protein expression through a translational mechanism

• Published in: Molecular and cellular endocrinology Vol. 229; no. 1; pp. 83 - 94
• Main Authors: Pentecost, B.T., Song, R., Luo, M., DePasquale, J.A., Fasco, M.J.
• Format: Journal Article
• Language: English
• Published: Ireland Elsevier Ireland Ltd 14.01.2005
• Subjects: 5' Untranslated Regions; Alternate promoters; Alternate splicing; Estrogen receptor; Estrogen Receptor alpha - genetics; Estrogen Receptor alpha - metabolism; Gene Expression Regulation, Neoplastic; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Humans; Open Reading Frames - physiology; Promoter Regions, Genetic - genetics; Protein Biosynthesis; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; RNA, Messenger - genetics; Transfection; Translational control; Tumor Cells, Cultured; Upstream open reading frame; Estrogen receptor; Alternate promoters; DCS: Donor calf serum; GFP: Green fluorescent protein; ER: Estrogen receptor alpha; Translational control; DMEM: Dulbecco's modified Eagle's medium; PCR: Polymerase chain reaction; HRP: Horse radish peroxidase; aa: Amino acid; nt: Nucleotide; ORF: Open reading frame; Alternate splicing; uORF: Upstream ORF; parental GFP from pEGFP-N1; Upstream open reading frame; DC10: 10% DCS in DMEM; RT: Reverse transcriptase; wt-GFP: Wild type
• ISSN: 0303-7207; 1872-8057
• DOI: 10.1016/j.mce.2004.09.002

Estrogen receptor alpha (ER) mRNA is primarily transcribed from two promoters, the two transcripts share identical sequence encoding the same ER protein but differ in upstream regions. The 5′ region of the two transcripts contain upstream open reading frames (uORFs) encoding potential peptides of 20 and 18 amino acids. The peptides have five C-terminal residues in common. These studies were undertaken to determine if the uORFs and encoded peptides differentially affected expression of ER. Expression of green fluorescent protein (GFP) reporter constructs containing upstream proximal promoter transcript sequences with the first 18 codons of ER fused to GFP was tested in HeLa cells. The cells expressed reduced levels of GFP as compared to the pEGFP-N1 parent vector; the effect was dependent on the presence of an intact proximal ER transcript uORF. Similar regulation by the uORF was seen in transfected MCF-7, MDA MB-231 and Ishikawa cells. Only protein expression was affected by eliminating the uORF; RNA levels were unchanged. This indicates the mechanism is translational rather than being an effect of the introduced point mutations on either mRNA stability or transcription. Eliminating the uORF did not significantly increase expression from similar distal promoter transcript ER-GFP constructs. However, study of in-frame fusions of GFP to the entire proximal and distal uORFs and to their translational start motifs showed that the translational start region of the distal uORF was inherently better at initiating translation than the AUG environment of the proximal promoter transcript uORF. The data indicate there are regulatory properties suppressing expression from the ER translation start which are specific to the unique regions of the ER proximal promoter transcript and these are likely associated with the proximal transcript uORF peptide product.