|dc.description.abstract||Administration of estradiol (E) to mature ovariectomized rats results in an early (4h) increase in nucleolar transcriptional activity above controls, this increase is totally due to an increased elongation rate of nascent transcripts. Salt extraction (150 mM NaCl) of isolated nucleoli eliminates the early stimulatory effect of E on nucleolar transcriptional activity. Addition of salt extracts obtained from uterine nucleoli of animals treated with E (4h) to nucleoli isolated from uteri of control animals resulted in increased transcriptional activity. The stimulation was due to an increased rate of elongation of nascent chains in uterine nucleoli. Treatment of animals with cycloheximide (eye) or actinomycin D (AD) eliminates the E induced stimulation and the stimulatory activity of salt extract from nucleoli isolated from E treated animals on control uterine nucleoli. Alkaline phosphatase treatment of nucleoli in vitro also is able to eliminate the E induced increase in transcriptional activity. Isolated uterine nucleoli and the salt extracts (E treated and control) contain active protein kinase(s), which are capable of in vitro phosphorylating a number of proteins in nucleoli isolated from control and E treated animals using ATP or GTP as substrate. In vivo E treatment results in the phosphorylation of a single protein with an apparent molecular weight of 105 kDa. Treatment of animals with AD or eye blocks the in vivo phosphorylation of a 105 kDa protein but not the in vitro phosphorylation, suggesting that the protein substrates and at least one of the endogenous nuclear kinase(s) are not the targets of the metabolic inhibitors. Decreased in vitro phosphorylation of a number of other proteins is also observed due to AD or eye treatment in vivo.
In vitro treatment of nucleoli with camptothecin, a specific inhibitor of topoisomerase I, was capable of inhibiting the E induced increase in nucleolar RNA synthesis. Results showed that topoisomerase was necessary but not sufficient for the E induced increase in nucleolar RNA synthesis. Leupeptin, an inhibitor of the processing of nucleolin, a 105 kDa phosphoprotein, was also capable of inhibiting E induced increase in nucleolar RNA synthesis. These results point to a role of phosphorylation of the 105 kDa protein in the early E induced stimulation of nucleolar RNA transcriptional activity. Nucleolin is a possible candidate for the 105 kDa protein that is phosphorylated in vivo||