Estrogen regulation of peptidylglycine alpha-amidating monooxygenase messenger ribonucleic acid levels by a nuclear posttranscriptional event

Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a bifunctional protein containing two enzymes that act sequentially to catalyze the conversion of glycine-extended peptides into COOH-terminal amidated peptides. We have previously shown that PAM messenger RNA (mRNA) levels in the anterior pituitary of intact cycling adult female rats showed changes inversely related to the physiological variations of plasma estrogen levels during the estrous cycle. Chronic treatment of ovariectomized (OVX) rats with 17 beta-estradiol was accompanied by a 4.5 +/- 0.5-fold decrease in total PAM mRNA and a 2-fold decrease in PAM activity in the anterior pituitary gland. To investigate the cellular site at which 17 beta-estradiol acts to affect the PAM mRNA, we made parallel measurements of the relative levels of PAM mRNA and nuclear precursor RNA and the relative rate of gene transcription after treatments designed to alter the estrogen status. The transcription rate experiments indicated that these 17 beta-estradiol effects were not due to reduced PAM gene activity, suggesting that a posttranscriptional mechanism was involved. The most common mechanism of posttranscriptional regulation affects cytoplasmic mRNA stability. Primary rat pituitary cell cultures from OVX and OVX-17 beta-estradiol-treated rats in the presence of actinomycin D showed that 17 beta-estradiol treatment decreased the half-life of PAM mRNA from 15-16 h to 8-9 h. There was no effect of 17 beta-estradiol on PAM mRNA poly(A) tail length or site of polyadenylation. However, in this study the down-regulation of PAM, was identified as a nuclear event. Analysis of nuclear RNA with probes specific for PAM intron sequences shows that decreased PAM expression after 17 beta-estradiol treatment was largely due to intranuclear destabilization of the primary transcript. The levels of nuclear precursor RNA were decreased roughly 5- to 6-fold in OVX + 17 beta-estradiol compared with OVX rats. The decrease in PAM mRNA is blocked by cycloheximide, indicating that its requires new protein synthesis. Mechanisms that would generate such an effect include altered stability of unprocessed message in the nucleus. The proportional changes observed in the nuclear precursor and mRNA levels suggest that the site of control is at the level of stability of the nuclear precursor RNA for PAM mRNA.