RNA-dependent Release of Androgen and Other Steroid Receptor Complexes from DNA*

Certain poly- and oligonucleotides, at low concentra- tions, promoted the release of androgen- and other steroid-receptor complexes that were bound to DNA. DNA-cellulose and gradient centrifugation, were used to demonstrate that release of receptor was selective with respect to the base composition of the polymer. Among the homopolyribonucleotides studied, poly(U), poly(G), poly(X), poly(I), and others having bases with an oxygen or a sulfur atom at C-6 of the purines or C-4 of the pyrimidines were active, whereas poly(C) and poly(A) were inactive in promoting the release of the 5a-dihydr0[~H]testosterone* receptor complex of rat ventral prostate from DNA. Base pairing of the active nucleotide appeared to reduce this activity. Poly(U,G) with uracil/guanine ratios of 1 to 5 were more active than poly(G), poly(U) or equivalent mixtures of poly(G) and poly(U), indicating that the activity was dependent on the nucleotide sequence. The minimum length of the oligonucleotide needed to show activity appeared to be dependent on the type of nucleotide in the oligomer. Since various polyanions were significantly less active than poly(U1,G1), the release of receptor by polynucle- otides, was not due merely to a nonspecific polyionic interaction. Ethidium

1 Visiting scientist from Istituto di Chimica Biologica, University then interact with chromatin and presumably enhance the synthesis of certain RNA (1-5). It is generally believed that such an interaction involves binding of the receptor complex to DNA.
There are also indications that the steroid. receptor complex can bind to certain RNA or RNP' particles in the cell nuclei and cytoplasm of target tissues (7-12). We report here that polyribonucleotides with certain types of bases can compete effectively with DNA for binding to a steroid-receptor complex and promote the release of the receptor complex from DNA. These observations may be important since steroid. receptor complexes in target cells may participate in the regulation of the synthesis of certain RNA and by binding to RNA may also be involved in post-transcriptional control.  (40 Ci/mmol) were obtained from New England Nuclear. Pure enzymes were obtained from Worthington and Sigma. Poly-and oligonucleotides were obtained from P-L Biochemicals, Miles Laboratories, and Collaborative Research, Inc., or prepared in this laboratory with polynucleotide phosphorylase (13). The base composition and the size of the polymers were determined by a Waters Associate high pressure liquid chromatography system (14) equipped with an absorbance detector and a solvent programmer, ion exchange chromatography (15), and gradient centrifugation (16). Unless otherwise specified, the polyribonucleotides used have sedimentation coefficients of 5 2 1 S and, for heteropolymers, have equal amounts of individual bases. Sprague-Dawley rats (250 to 300 g) were purchased from Sasco Co., Omaha, Neb. Soluble RNA was extracted from the cytosol fraction of rat liver and chromatographed on oligo(dT)-cellulose as described by Miller and McCarthy (17). RNA that was not bound to oligo(dT)-cellulose was used as transfer RNA. Analysis of this RNA by sucrose gradient centrifugation (18) revealed only 4 S RNA. For the preparation of Potter-Elvehjem homogenizer in 4 volumes of 40 rnM Tris/HCI, pH other RNA, rat ventral prostate was homogenized with an all glass 7.5, and centrifuged a t 16,000 x g for 15 min. The supernatant was centrifuged again a t 130,000 x g for 1 h and the pellet used to prepare polysomal RNA. The pellets were resuspended in 10 mM Tris/HCl, pH 7.5, containing 3 m~ MgCI,, 250 m~ sucrose, 150 mM NaCI, and 0.5% SDS. RNA was extracted and then fractionated on oligo(dT)cellulose as previously described (17). RNA retained by oligo(dT)cellulose was used as poly(A)-RNA. RNA not retained was used as ribosomal RNA. Ribosomal RNA was fractionated into 5 S , 18 S , and 28 S ribosomal RNA by sucrose gradient centrifugation (18). The cytosol 1.5 S RNA was prepared as described elsewhere (19).

RNA-dependent Release of
Steroid. Receptor Complexes from DNA prostate (dihydrotestosterone), uterus (estradiol and progesterone), or liver (dexamethasone) of rats castrated or adrenalectomized. The radioactive steroid. receptor complexes thus formed were precipitated by the addition of ammonium sulfate to 40% saturation and then desalted by passing through a Sephadex G-25 gel column (20). The specific radioactivities of the steroid.receptor complexes used in the experiments were generally within the range of 20,000 to 100,000 cpm/mg of protein. DNA-cellulose was prepared as described by Alberts and Herrick (21) using calf thymus DNA (29% hyperchromicity at 260 nm) and Whatman CF-11 cellulose powder. The adduct contained about 1 mg of DNA/ml (packed volume) of DNA-cellulose.
DNA-Cellulose Column Assay-For binding studies, DNA-cellulose was equilibrated with Medium E T (20 mM Tris/HCI, pH 7.5, containing 1.5 mM EDTA) and packed into a glass column.
The volume of the packed DNA-cellulose was 0.5 ml (0.5 mg of DNA)/ column. "H-Labeled steroid. receptor complex, normally 10,000 cpm in 0.1 to 0.2 ml of Medium ET, was applied to the column. The column was washed with seven aliquots (0.5 ml each) of Medium E T to remove free steroid or the steroid.protein complex that did not bind to DNA-cellulose. The washed column was then eluted with seven aliquots of Medium E T (0.5 ml each) containing a polyribonucleotide or other test compounds (Fraction E). Finally, the steroid. receptor complex that remained attached to the DNA-cellulose was eluted from the column with seven aliquots of Medium E T (0.5 ml each) containing 0.6 M KC1 (Fractions R).
For the convenience of comparing the abilities of various test compounds to release the receptor complex, we determined the radioactivity in Fraction E ( e ) and in Fraction R (r) and calculated the percentage of the receptor complex that could be eluted from DNAcellulose by the test compound at the specified concentration according to the equation: The concentration of the individual test compound needed for 50% elution is termed ECw.
DNA-Cellulose Centrifugation Assay-In some experiments, we mixed DNA-cellulose (20 to 100 pg of DNA) and the radioactive complex (2,000 to 10, OOO cpm) in 0.5 ml of Medium E T and then added polynucleotides to study receptor binding by nucleic acids. The tubes containing all the components were incubated at 20°C for 5 min and then centrifuged at top speed in a clinical centrifuge or a Beckman microfuge. The DNA-cellulose pellet was washed three times with 1 ml of Medium ET. The radioactivity retained in the washed pellet was determined and compared. This method was convenient for an assay involving many tubes, and required less (-1Opg) polymer than the DNA-cellulose column assay. In the centrifugation assay, DNAcellulose must be washed extensively before assay to remove loosely associated DNA. DNA released from cellulose during the assay may carry the steroid.receptor complex into the elution medium. This results in lower binding of the receptor complex to DNA cellulose.
Gradient Centrifugation Assay-We also used gradient centrifugation to compare the relative abilities of various polynucleotides to compete with DNA for binding to the steroid.receptor complex. For this purpose, the radioactive steroid.receptor preparations were treated briefly with a small quantity of dextran-coated charcoal to minimize the amount of free steroid present. The receptor preparation (5,000 cpm) was mixed with 1 to 5 pg of DNA in 0.15 ml of Medium ET. The test polymer was then added to the tube and the mixture was incubated at 0°C for 10 min.
with an SW 60 rotor. The sucrose gradient (10 to 307r sucrose) Gradient centrifugation was performed in a Spinco ultracentrifuge contained 1.5 mM EDTA and 20 mM Tris/HCI at pH 7.5. The incubated sample was layered on top of the sucrose gradient and centrifuged for the length of time specified in the individual experiments. After centrifugation, fractions (0.2 ml each) were collected by an Isco fractionator and numbered from the top of the centrifuge tubes.
Under the conditions of our assay, the radioactive steroid.receptor complex bound to DNA (>20 S ) sedimented at the bottom of the tube whereas the receptor complex, free or bound to polyribonucleic acid, stayed in the upper portion of the sucrose gradient. The gradient centrifugation assay, although more tedious, is useful when only limited quantities (1 to 5 pg) of DNA or the test polymers are available.
The amount of polynucleotide was measured spectrophotometrically; the polymer concentration that, at pH 7, gave an absorbance of 1.0 at 260 nm (light path, 1 cm) was assumed to be 40.0 pg/ml for natural RNA, 35.4 pg/ml for poly(A), 32.5 pg/ml for poly(U), 58.7 pg/ ml for poly(C), 39.2 pg/rnl for poly(G), 35.8 pg/ml for Poly(U,,GI), and 50.0 pg/ml for DNA. The amount of polymer used in experiments was also expressed in monomer concentrations. DNA was also measured by the diphenylamine test, with calf thymus DNA as the standard (22). Protein was determined by the method of Lowry et al. (23) with bovine serum albumin as standard.

Retention of 5a-DihydrofH]testosterone-Receptor Com-
plex by DNA-Cellulose-The quantity of DNA-cellulose used in all the experiments reported here had the capacity for binding at least 10 times the radioactive steroid .receptor complex employed. Cellulose, free of DNA, did not retain the radioactive receptor complex to any significant extent. When the radioactive androgen. receptor complex was prepared in the manner described under "Experimental Procedures" and applied to the DNA-cellulose column under our assay conditions, about 50 to 70% of the radioactivity was retained and could not be washed out from the column by Medium E T ( Fig. 1). If the KC1 concentration of the medium was brought to 0.4 M or higher, all the radioactivity could be removed from the column. The initial flow-through fraction (Fractions 0 to 7 ) contained free steroid or other steroid-binding proteins that, unlike the androgen a receptor complex, were not retained by DNA-cellulose or by prostate cell nuclei (24,25). The major prostate cytosol protein (a protein) that binds sex steroids but not glucocorticoids (25)(26)(27) was also found in this flow-through fraction. When the radioactive androgen-receptor complex was inactivated by heating at 50°C for 30 min no radioactivity was retained by DNA-cellulose. The radio- active complex that was retained by DNA-cellulose and that was eluted from the column by 0.6 M KC1 (Fractions 16 to 20) sedimented as a 3 to 4 S entity after sucrose gradient centrifugation, supporting our contention that the retained radioactivity was associated with the Sa-dihydrotestosterone-receptor complex (25).
Effect of Homopolynucleotides on the Release of Androgen. Receptor Complex from DNA-Cellulose-The capability of poly(U) to promote the release of the radioactive 5a-dihydrotestosterone -receptor complex from DNA-cellulose is shown in Fig. 1. In this experiment the radioactive receptor complex was loaded onto DNA-cellulose columns and, after the initial washing, the columns were eluted with Medium ET alone (control) or with the Medium ET containing poly(U). The radioactivity that was not eluted was then removed from the column by Medium ET containing 0.6 M KCl.
Elution of the radioactive receptor complex after the addition of poly(U) proceeded rapidly. Most of the receptor complex that could be eluted a t a set concentration of the polymer emerged from the column within five fractions, taking only about 5 min. The difference in the amounts of the receptor complex that could be eluted from duplicate columns a t a set concentration of the polymer was generally within 10%. The effectiveness of poly(U) was not mimicked by high concentrations (1 to 5 mM) of inorganic phosphate, inorganic pyrophosphate, UMP, UDP, UTP, or other mononucleotides tested (see below).
When the radioactive complex eluted by poly(U) (Fractions 10 to 16 in Fig. 2 4 ) was treated with pancreatic RNase to destroy poly(U) and then reapplied to a DNA-cellulose column, practically all the radioactivity was retained on the column. The retained radioactivity could again be eluted by poly(U) (Fig. 2B). The radioactive complex eluted by poly(U) and treated with RNase also sedimented as a 3 to 4 S entity in sucrose gradients containing 0.6 M KC1 (Fig. 2C). These observations indicated that poly(U) eluted the receptor complex from DNA-cellulose without significantly altering the steroid-and DNA-binding activities and the sedimentation property of the receptor complex.
When the abilities of various synthetic polyribonucleotides to promote the release of the receptor complex from DNAcellulose were compared, we found a striking base specificity. As shown in Table I and Fig. 3, poly(G) and poly(U) were active, whereas poly(A) and poly(C) were essentially inactive at monomer concentrations up to 150 pM (about 50 p g / d ) .
Since the activity of poly(G) could be suppressed by the addition of poly(C) but not poly(A), whereas the activity of poly(U) could be reduced by poly(A) but not poly(C) (Table  I), the activity appeared to be dependent on an unpaired base structure.
Besides poly(G) and poly(U), other homopolymers, such as poly(X), poly(I), poly(4-thio-U), and poly(7-methyl-G), were very active, whereas poly(dU), poly(dT), and poly(dG) were much less active than the corresponding ribopolymers. Poly(dC) was inactive. The radioactive androgen. receptor complex could also be retained by columns packed with various oligodeoxyribonucleotide-celluloses. The relative effectiveness of the four major homopolyribonucleotides in promoting the release of the receptor complex from these columns (Table 11) was similar to those observed in the experiments using calf thymus DNA-cellulose.
Effect of Heteropolyribonucleotides on the Release of Androgen. Receptor Complex from DNA-Cellulose-Since poly(G) and poly(U) were effective in promoting the release of the androgen. receptor complex from DNA-cellulose, we also studied poly(U1,G1). For comparison, we fractionated the polymers by gradient centrifugation into groups with different sedimentation coeffkients (2 to 4 S, 4 to 6 S, 6 to 8 S). We The experiment was performed by the DNA-cellulose column assay. The monomer concentration of the polymers used in the elution of the radioactive complex was 150 p~. At polymer concentrations below 50 p~, poly(dT) was less than 50% as active as poly(U). receptor preparation (R) were also subjected to gradient centrifugation. oligodeoxyribonucleotide-cellulose by homopolynucleotides The experiment was performed using the DNA-cellulose column assay except that oligodeoxyribonucleotide-cellulose was used instead of DNA-cellulose. The amount of oligodeoxyribonucleotide in the cellulose adduct packed on the column was about 1 mg. The monomer concentration of the polyribonucleotide was 60 ELM (about 20 yglml). Since 3.5 ml of the polymer were used in the elution, the total amount of the individual polymer employed in the assay was 70 pg. found that poly(UI,GI) was much more active than poly(G) or poly(U) regardless of the size. As shown in Fig. 3, this difference was more clearly seen when the polymer concentration in the elution media was 3 to 15 p~ (about 1 to 5 pg/ml) rather than at higher concentrations (-150 p~) .

RNA-dependent Release
The receptor complex was retained more readily by DNA-cellulose at a pH below 7.0 than at a higher pH; however, the bound receptor complex could be released by poly(U1,G1) more effectively at a pH between 7.0 and 8.5 than at a more acidic pH.
Since mixtures of equivalent amounts of poly(G) and poly(U) were not as active as poly(U1,G1) at all concentrations tested (Fig. 3), the high activity of poly(U1,GI) appeared to be dependent on the presence of the two bases on the same polynucleotide chain. When poly(U,G) with different U/G ratios were compared, differences in the activities of polymers with U/G ratios ranging from 1 to 5 were small, but the activity decreased as the U/G ratio increased from 10 to 25 (Table I11 and Fig. 4).
Various RNA fractions isolated from rat ventral prostate were not as active as poly(U1,G1) but were moderately active at 30 p~ (Table IV).
Effect of Oligonucleotides on the Release of Androgen. Receptor Complex from DNA-Cellulose--In an attempt to study the minimum length of polyribonucleotides needed to promote the release of the receptor complex from DNA-cellulose, we tested various oligoribonucleotides listed in Table  V. ApUpU, ApUpG, and other oligomers that contained uracil and had a nucleotide chain length of six or less were inactive   testosterone. receptor complex from DNA-cellulose The experiments were performed by the DNA-celluose column assay. The activities of oligonucleotides were also determined by DNA-cellulose centrifugation assay. a t 150 PM nucleotide concentrations. Surprisingly, oligo(I)1o-2o was moderately active a t 30 p , but homo-oligomers with either adenine, cytosine, or uracil and with nucleotide chain lengths of 10 to 20 were inactive at this concentration. The effectiveness of the oligo(I)lo-n, was also confumed by the DNA-cellulose centrifugation assay. By the centrifugation assay, oligo(A)lo-2n, oligo(C)lo-m, and oligo(U)la-2o were not only inactive in promoting the release of the receptor but also slightly increased the amount of the receptor complex that could bind to DNA-cellulose.
As described above (Fig. 4), uracil-and guanine-containing polymers with high U/G ratios were less active than those with low U/G ratios. When various poly(U,G) with different U/G ratios were treated with T1-RNase, which could cleave the nucleotide chains at the site next to guanine, we found that the activities of the polymers were essentially abolished if the U/G ratio was below 10. Nuclease treatment, however, did not affect the activity of the poylmers with U/G ratios of 20 or above. These results suggested that the effective minimum chain length needed for Up(Up),G to exhibit activity was about 15 to 20 nucleotides. Use of Gradient Centrifugation to Demonstrate Release of Androgen. Receptor Complex from DNA by Polyribonucleotides-Since the receptor complex is not bound to cellulose in the absence of DNA, the phenomena described above were apparentIy due to binding of the receptor compIex by the DNA moiety of the DNA-cellulose adduct. To show that cellulose was not a necessary participant in the polyribonucleotide-dependent release of the receptor complex from DNA, we used the gradient centrifugation assay method. As shown in Fig. 5, the radioactive androgen.receptor complex stayed near the top of the tube after gradient centrifugation if no nucleic acid was present. If 4, y DNA was added to the tube, a large quantity of the radioactivity was found to associate with DNA that sedimented at the bottom of the tube. When poly(U1,GI) (5 S) was added to the receptor complex and +X DNA before centrifugation, the radioactivity was not found with DNA in the bottom of the tube, but was found associated with poly(U1,GI). Poly(A,C) was able to bind to the receptor complex if no DNA was present; however, it could not release the receptor complex from phage DNA. A similar result was obtained when SV-40 DNA was employed.
Effect of Nonnucleotide Compounds on the Release of Androgen. Receptor Complex from DNA-Cellulose- Table V summarizes the effect of various compounds on the release of 5a-dihydr0[~H]testosterone. receptor complex from DNA-cellulose columns. Group A includes compounds that can promote elution of 50% of the DNA-bound receptor complex at monomer concentrations lower than 150 p~ (ie. ECs < 150 p ) . In addition to various nucleotides described above, aurintricarboxylic acid which can dissociate nucleic acid.protein complexes (28) was as active as poly(UI,G1). Poly(L-aspartic acids) with molecular weights of 5,400 and 27,000 were active but required much higher concentrations. In contrast, pOly(Dglutamic) (Mr = 27,000) or poly@-glutamic acid) (M, = 66,000) and polyvinylsulfate were only weakly active (Group B) even at 150 PM. Poly@-lysine), poly(L-proline), a number of dipeptides, L-aspartic acid, and L-glutamic acid were not active at 150 FM (Group C). Ethidium bromide was weakly active, whereas actinomycin D and chloroquine were inactive. Rifamycin AF/05 and rifamycin AF/013 which inhibit eukaryotic RNA polymerase were significantly active but no activity was observed with rifampicin which inhibits bacterial but not eukaryotic RNA polymerase. Androgen. receptor (29) and estrogenreceptor (30) complexes have high affinities for heparin. At 1 mg/ml, heparin prevented binding of the radioactive androgen. receptor complex to DNA-cellulose. Spermine at 100 PM showed weak activity (20% elution) but prevented,   (Table   V).
It is not very clear why only a certain proportion of the receptor complex that is bound to DNA could be eluted at a set nucleotide concentration. This could be due to involvement of other DNA-binding proteins in the receptor interaction with DNA (5,12). In fact, we have found that poly(U1,Gd can release from DNA-cellulose a large number of prostate proteins that can not be released by poly(A). Differences in the local DNA sequence and structure may also contribute to the creation of multiple receptor binding sites. In addition, a change in the local DNA bihelical structure, including partial chain separation may occur during the binding and release of the receptor from DNA, creating binding sites with different affinities. It is conceivable that certain RNA having appropriate nucleotide sequences may be more effective than poly(U,G) and may show high specifkities toward different DNA-binding proteins and the steroid. receptor complexes.
The present study suggests that various steroid. receptor complexes may have higher affinities for certain types of RNA than for DNA. Since the concentration of RNA needed (1 to 5 pg/ml) to show this may be well within the range expected in the intact cell nuclei (35), preferential RNA binding of the steroid. receptor complexes in the nuclei is not inconceivable. Such a process may be important in the recycling of the receptor protein from nuclei to cytoplasm (8, 9, 36). The removal of RNA from DNA may also make the genetic template available for further transcription while receptor binding of RNA may be involved in the post-transcriptional control as we hypothesized before (7-9). In this scheme, different RNA molecules may contain, for example, identical or similar nucleotide sequences so that more than one RNA species can be selected, although with some preference, by the same steroid. receptor complex. These diversified specificities together with other cellular factors may provide the selectivity and multiplicity observed in the induction of different proteins by steroid hormones (7).
The interaction of the steroid. receptor complexes with RNA should be studied further since there are indications that steroid hormones may be involved in the stabilization of mRNA for proteins being induced by the hormones (37, 38). It is also plausible to speculate that the specific splicing of certain mRNA and removal of introns (39-42) may be controlled by a mechanism involving RNA binding by a steroid. receptor complex. Although we have not studied binding of the steroid. receptor complex to polydeoxyribonucleotide in detail, the receptor complex appears to have higher binding affinity toward the single-stranded deoxypolymers than to the double-stranded DNA. Whether such a preferential interaction may play a role in the local unwinding of DNA during the replication or transcription of DNA is worthy of further exploration.