Identification of a novel AU-Rich element in the 3' untranslated region of epidermal growth factor receptor mRNA that is the target for regulated RNA-binding proteins.
The epidermal growth factor receptor (EGF-R) plays an important role in the growth and progression of estrogen receptor-negative human breast cancers. EGF binds with high affinity to the EGF-R and activates a variety of second messenger pathways that affect cellular proliferation. However, the underlying mechanisms involved in the regulation of EGF-R expression in breast cancer cells are yet to be described. Here we show that the EGF-induced upregulation of EGF-R mRNA in two human breast cancer cell lines that overexpress EGF-R (MDA-MB-468 and BT-20) is accompanied by stabilization (>2-fold) of EGF-R mRNA. Transient transfections using a luciferase reporter identified a novel EGF-regulated approximately 260-nucleotide (nt) cis-acting element in the 3' untranslated region (3'-UTR) of EGF-R mRNA. This cis element contains two distinct AU-rich sequences (~75 nt), EGF-R1A with two AUUUA pentamers and EGF-R2A with two AUUUUUA extended pentamers. Each independently regulated the mRNA stability of the heterologous reporter. Analysis of mutants of the EGF-R2A AU-rich sequence demonstrated a role for the 3' extended pentamer in regulating basal turnover. RNA gel shift analysis identified cytoplasmic proteins (~55 to 80 kDa) from breast cancer cells that bound specifically to the EGF-R1A and EGF-R2A cis-acting elements and whose binding activity was rapidly downregulated by EGF and phorbol esters. RNA gel shift analysis of EGF-R2A mutants identified a role for the 3' extended AU pentamer, but not the 5' extended pentamer, in binding proteins. These EGF-R mRNA-binding proteins were present in multiple human breast and prostate cancer cell lines. In summary, these data demonstrate a central role for mRNA stabilization in the control of EGF-R gene expression in breast cancer cells. EGF-R mRNA contains a novel complex AU-rich 260-nt cis-acting destabilizing element in the 3'-UTR that is bound by specific and EGF-regulated trans-acting factors. Furthermore, the 3' extended AU pentamer of EGF-R2A plays a central role in regulating EGF-R mRNA stability and the binding of specific RNA-binding proteins. These findings suggest that regulated RNA-protein interactions involving this novel cis-acting element will be a major determinant of EGF-R mRNA stability.
Laboratory for Cancer Medicine, Royal Perth Hospital, University of Western Australia, Perth, Western Australia, Australia 6000.
L A
LA
Balmer
D J
DJ
Beveridge
J A
JA
Jazayeri
A M
AM
Thomson
C E
CE
Walker
P J
PJ
Leedman
D020413:3' Untranslated Regions; D001483:Base Sequence; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004252:DNA Mutational Analysis; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000494:pharmacology; D066246:ErbB Receptors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D020871:RNA Stability; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured
D020413:3' Untranslated Regions; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D013755:Tetradecanoylphorbol Acetate