Abstract
Estradiol up-regulates expression of the estrogen receptor alpha gene in the uterus by stabilizing estrogen receptor alpha mRNA. Previously, we defined two discrete minimal estradiol-modulated stability sequences (MEMSS) within the extensive 3'-untranslated region of estrogen receptor alpha mRNA with an in vitro stability assay using cytosolic extracts from sheep uterus. We report here that excess MEMSS RNA inhibited the enhanced stability of estrogen receptor alpha mRNA in extracts from estradiol-treated ewes compared with those from control ewes. Several estradiol-induced MEMSS-binding proteins were characterized by UV cross-linking in uterine extracts from ewes in a time course study (0, 8, 16, and 24 h after estradiol injection). The pattern of binding proteins changed at 16 h post-injection, concurrent with enhanced estrogen receptor alpha mRNA stability and the highest rate of accumulation of estrogen receptor alpha mRNA. The predominant MEMSS-binding protein induced by estradiol treatment was identified as AUF1 (A + U-rich RNA-binding factor 1) protein isoform p45 (a product of the heterogeneous nuclear ribonucleoprotein D gene). Immunoblot analysis indicated that only two of four AUF1 protein isoforms were present in the uterine cytosolic extracts and that estradiol treatment strongly increased the ratio of AUF1 isoforms p45 to p37. Nonphosphorylated recombinant AUF1p45 protected estrogen receptor alpha mRNA in vitro in a dose-dependent manner. These studies describe estrogenic induction of AUF1p45 binding to the estrogen receptor alpha mRNA as a molecular mechanism for post-transcriptional up-regulation of gene expression.
Highlights
Expression of the ER␣ gene is tightly regulated by steroid hormones from the ovary in responsive tissues
The data presented in this report provide the first characterization of the proteins that are induced by E2 treatment to bind stabilizing sequences of ER␣ mRNA
The predominant one was identified as AUF1p45, which associates with several distinct proteins in ribonucleoprotein complexes on mRNAs that are stabilized in response to cellular signals (30, 32, 35)
Summary
Expression of the ER␣ gene is tightly regulated by steroid hormones from the ovary in responsive tissues. These examples lead us to hypothesize that E2 treatment of ewes induces such uterine proteins to bind and stabilize ER␣ mRNA. The recombinant proteins were preincubated with uterine extract proteins from control ewes (1.8 g) in the stability assay buffer for 10 min at 37 °C prior to the addition of radiolabeled ER␣ RNA (10,000 cpm) and a 5-min incubation at room temperature.
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