Estradiol and estrogen receptor-dependent stabilization of a minivitellogenin mRNA lacking 5,100 nucleotides of coding sequence.

Abstract

We have developed a transfection assay to investigate the estrogen-mediated stabilization of cytoplasmic vitellogenin mRNA. A miniviteliogenin (MV5) gene containing the 5' and 3' untranslated and coding regions but lacking 5,075 nucleotides of internal coding sequence was constructed. Cotransfection of the MV5 plasmid and a Xenopus estrogen receptor expression plasmid into Xenopus liver tissue culture cells yielded a 529-nucleotide MV5 mRNA, which was specifically stabilized by estrogen. MV5 mRNA exhibited the increased stability indicative of positive regulation when the estradiol-estrogen receptor complex was present and was not destabilized by unliganded estrogen receptor. Transfected estrogen receptor, estradiol, and 529 nucleotides of the 5,604-nucleotide vitellogenin Bl mRNA were sufficient for stabilization.Our quantitative studies indicate that regulation of vitellogenin gene transcription and control of the cytoplasmic stability of vitellogenin mRNA, not changes in the efficiency of nuclear vitellogenin RNA processing, are responsible for the massive estrogen induction of the hepatic mRNA coding for the Xenopus laevis egg yolk precursor protein vitellogenin (2, 5, 6). In Xenopus liver fragment cultures, vitellogenin mRNA is essentially stable, with a half-life of 500 h when estrogen is present and 16 h at 25°C or 30 h at 20°C after removal of estrogen from the culture medium (6; J. E. Blume and D. J. Shapiro, Nucleic Acids Res., in press). Vitellogenin mRNA undergoing cytoplasmic degradation can be specifically restabilized by addition of estrogen to the culture medium (6), indicating that vitellogenin mRNA stabilization is a reversible cytoplasmic effect of estrogen. The broad spectrum of estrogen effects on mRNA stability in Xenopus liver provides a striking example of the specificity and flexibility of this type of control. In hepatocytes, estrogen elicits a 30-fold increase in the stability of vitellogenin mRNA (6) and decreases the stability of albumin mRNA by 3-fold (30, 41) without affecting the 16-h half-life (at 25°C) of total poly(A) mRNA (6).The mechanisms governing the degradation of the more than 20 eucaryotic mRNAs whose stability is known to be regulated are currently attracting considerable attention (reviewed in references 3, 29, 31, and 36). Although hormones have been shown to regulate the stability of several mRNAs, the absence of functional assays for steroid hormone control of mRNA degradation has greatly hindered studies, and the mechanisms controlling mRNA degradation in these systems remain largely unknown. In this work, we demonstrate that the estrogen-mediated stabilization of vitellogenin mRNA can be reproduced in a transfection system. Only the estrogen receptor, estradiol, and 529 nucleotides of the 5,604- A. M. Nardulli, T.-C. Chang, and D. J. Shapiro, submitted for publication), were grown at 20°C in 0.6x phenol red-free Higuchi medium containing 10% charcoal-dextran-treated (12) fetal bovine serum. DNA was transfected into cells by a modification of the calcium phosphate...

Vigilin binding selectively inhibits cleavage of the vitellogenin mRNA 3′-untranslated region by the mRNA endonuclease polysomal ribonuclease 1

“…Whereas the complete 6.2-kb sequence of the B1 vitellogenin mRNA is not available, only two APyrUGA are elements present in the related A2 vitellogenin mRNA, and they are positioned similarly to the elements in the 3Ј-UTR of B1 vitellogenin mRNA. This segment of vitellogenin mRNA is implicated in estrogen regulation of vitellogenin mRNA stability (17). In contrast to vitellogenin mRNA, APyrUGA PMR-1 cleavage sites are scattered throughout the sequences of the serum protein mRNAs.…”

“…This portion of vitellogenin mRNA contains two consensus PMR-1 cleavage sites (boxed). Earlier work suggested that this region is important in the estrogen-induced stabilization of vitellogenin mRNA (17), and the presence of PMR-1 cleavage sites raised the possibility that mRNA stabilization might result from inhibition of PMR-1 endonuclease cleavage by vigilin binding over and occluding these sites. The feasibility of this concept was tested in the experiment shown in Fig.…”

“…PMR-1 preferentially cuts (14) RNA within a singlestranded consensus pentamer APyrUGA (16), and at a number of nonconsensus sites. Intriguingly, two copies of the consensus APyrUGA pentamer were found in a region of the vitellogenin mRNA 3Ј-UTR, which has been implicated in the control of vitellogenin mRNA stability (17). This region of vitellogenin mRNA is bound by an estrogen-induced polysome-associated protein (18), which was identified as vigilin͞scp160͞DDP1, a 155-kDa RNA-binding protein bearing 15 hnRNP K homology domains (19).…”

Expression of the Estrogen and Progesterone Receptors and Insulin-like Growth Factor-I Genes in the Porcine Myometrium during Early Pregnancy.

“…Ligand-activated steroid hormone receptors may affect the steady state of mRNA levels by other mechanisms than alterations in transcriptional activity, for example stabilization and degradation of mRNA [33][34][35][36][37]. Steroid receptors may also be activated by ligand-independent phosphorylation [30,[38][39][40][41][42].…”

Hormonal regulation of mRNA stability and RNA-protein interactions in the pituitary

“…The mRNA half-life of hepatic vitellogenin increases from 16 h to 500 h after the administration of estrogen (Brock & Shapiro 1983). Further analysis revealed that an estrogen-regulated cis-acting element was situated in the 3 UTR of vitellogenin mRNA (Nielsen & Shapiro 1990b), and required association of the mRNA with ribosomes for stabilization (Blume & Shapiro 1989).…”