Regulation of Nucleolar RNA Metabolism by Hydrocortisone

Abstract

Purified rat liver nucleoli were examined for RNA polymerase activity in the presence of Mg2+ and of Mn2+ with added ammonium sulfate. Both types of activity were found to be present in this organelle.A single injection of hydrocortisone 21-phosphate into adrenalectomized rats resulted, after 3.5 hours, in a threefold increase in the Mg2+-dependent polymerase activity while the Mn2+-dependent polymerase activity was not significantly altered. When rats were injected with[14C]orotic acid hydrocortisone induced a 60°/, increase in the synthesis of 45 S RNA isolated from nucleoli. Pulse-chase experiments using low doses of actinomycin D suggested that the hormone enhances the turnover of newly synthesized 45 S RNA.Similar studies on extra-nucleolar RNA produced smaller changes in the synthesis of this RNA fraction following hydrocortisone treatment and the pattern of RNA turnover in the chase experiments was not affected by the hormone.Much attention has been focused on hormoneinduced changes in the synthesis of RNA [l-81. Studies on whole nuclei reveal a complex picture due to the presence of many species of RNA including polydisperse extra-nucleolar RNA, much of which turns over in the nucleus without reaching the cytoplasm [9]. I n order to simplify identification of some of the effects of hormonal stimulation on RNA metabolism, we have isolated nucleoli and have examined the effects of corticosteroid treatment on the synthesis of RNA by this organelle. The nucleolus is considered to be the principal site of formation of rRNA [lo-131 and possibly the site of ribosome biogenesis [14]. In the course of our investigations a Mn2+/(NH4).$O4-activated RNA polymerase, which presumably produces a non-ribosomal type of RNA, was detected in purified liver nucleoli as well as the Mg2+-dependent polymerase activity, which produces GC-rich rRNA. Preliminary notes on this observation have already appeared [15,16]. We have correlated changes in these two polymerase activities after hormone administration with alterations in the labelling patterns of nucleolar RNA following injection of [14C]orotic acid. MATERIALS AND METHODS Animals and TreatmentsNormal and adrenalectomized male albino rats, weighing 150-200g Chow, were obtained from the Charles River Breeding Laboratories (North Wilmington, Mass.). Adrenalectomized rats were normally used within 6 days after the operation.Labelling of Nucleolar RNA in vivo Animals, in groups of three, each received hydrocortisone 21-phosphate (Merck, Sharp, and Dohme) a t a dose of 1 mg/100 g body weight while control animals were given 0.9O/, NaC1. A dose of 2 pC/100 g body weight of [6-14C]orotic acid (Schwarz Bioresearch, 35 mC/mmole) was administered intravenously 3.5 hours after hormone treatment, and the animals were sacrificed 15 min later. For pulse-chase studies, actinomycinD (generously supplied by Merck, Sharp and Dohme) was injected intravenously (30 pg/ 100 g body weight) after the pulse labelling and 30 min prior to sacrifice of the animals. Isolation of Nucleoli and Extra-Nuc...