20-Hydroxyecdysone stimulates RNA polymerase I activity in silkmoth wing epidermis by increased synthesis and phosphorylation

MO C

lecular

and

ellular

E ndocrlnology

and Cellular

Molecular

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20-Hydroxyecdysone stimulates RNA polymerase I activity in silkmoth wing epidermis by increased synthesis and phosphorylation S. Sridhara*, Carol E. Mattes Department

of Biochemistry

and Molecular

Biology,

Received

Texas Tech University

29 December

1994; accepted

Health

Sciences

15 March

Center,

Lubbock.

TX 79430,

USA

1995

Abstract

The activities of RNA polymerases I and II in the wing epidermis of diapausing silkmoth pupae increased about tenfold during the first day after administration of either 20-hydroxyecdysone (20E) or 20E plus juvenile hormone (Katula et al., 1981a). The aim of these studies was to correlate these increases in RNA polymerase I and II activities to their amounts in hormone stimulated wing epidermis. The enzyme activities were measured by standard procedures while their amounts were determined by the application of a modified ELISA with subunit-specific monoclonal antibodies. Results showed that the increase in the amount of RNA polymerase I during the first 24 h accounted for only about 60% of the increase in activity. Alkaline phosphatase decreased the activity of the newly synthesized enzyme by 40-50%. These results indicate that hormone-stimulation of RNA polymerase I activity is due to a combination of synthesis of the enzyme and phosphorylation of the enzyme and/or tightly associated factors. RNA polymerases II and III determined by differential ELISA using a monoclonal antibody specific to a common subunit followed developmental changes similar to those of RNA polymerase I. The amounts and activity of the enzymes during the first 48 h were similar in wing tissue that followed the second pupa1 development (20E + juvenile hormone) compared to tissue that developed into adult wings (20E). Keywords:

20-Hydroxyecdysone;

Juvenile hormone; RNA polymerases; Silk moth (wing epidermis)

1. Introduction Transcriptional control in eucaryotes appears to be central to the determination and alteration of the cell phenotype with three distinct DNA-dependent RNA po-

lymerases (RNA ~01s) as the primary enzymes responsible for the production of RNAs. RNA polymerase I (EC 2.7.7.6) is responsible for transcription of ribosomal RNA which constitutes about half of the transcriptional capacity of cells (Sollner-Webb and Tower, 1986) and whose synthesis is intimately linked to cell cycle, growth and differentiation. The activity of RNA polymerase I (RNA pol I) varies in response to growth conditions and the presence of growth factors or hormones (Jacob et al., 1983; Sentenac, 1985; Sollner-Webb and Tower, 1986). We have shown that the wing epidermal cells of diapausing silkmoth pupae respond to the steroid hormone, 20hydroxyecdysone (20E) with enhanced RNA and protein synthesis (Katula et al., 198la,b). Concomitant with these * Corresponding

author.

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changes, about a tenfold increase in the total RNA pol I activity was observed during the first 24 h of hormonal stimulation which was dependent upon RNA and protein synthesis. Similar increases in RNA polymerase I activity were observed when juvenile hormone was administered along with 20E, a hormone regimen that prevents adult development and leads to a second pupal development. The increase in enzyme activity could be due to (i) an increase in the number of enzyme molecules, (ii) covalent modification of the pre-existing and/or new molecules for enhanced rates of initiation and elongation and (iii) an increase in the amount of other factor(s) that associate with the enzyme to modulate its activity. Some of these possibilities have been implicated in other systems of enhanced rRNA synthesis and some support found for each in different systems (Bateman and Paule, 1985; Liu and Rose, 1986; Sollner-Webb and Tower, 1986; Schnapp et al., 1992). To determine as to which of the possibilities is/are utilized in the wing epidermis, it was necessary to develop techniques to follow the quantitative changes of

40

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and Cellular

RNA pol I during pupal and adult development. Hence, monoclonal antibodies (MABs) against three individual subunits of the enzyme purified from the silk glands of Bombyx mori were isolated and characterized (Gowda and Sridhara, 1983; Mattes et al., 1991). A modification of the protein-avidin-biotin ELISA capture system (PABCELISA) was developed and shown to accurately measure RNA pol I present in tissue extracts with a linear response between 0.5 and 10 ng (Mattes and Sridhara, 1993). This procedure was applied to follow changes in wing epidermis enzyme levels for 48 h after hormonal stimulation. The results presented in this report demonstrate that increase in amount and phosphorylation of the enzyme or associated factors can account for the hormonally enhanced activity. 2. Experimental

procedures

2.1. Preparation of tissue extracts Commercially obtained Actias luna silkmoth pupae were stored at 4°C for at least 6 weeks prior to hormone stimulation. 20E and the natural juvenile hormone (JH I) obtained from Calbiochem were dissolved in water and olive oil, respectively. Development was elicited by removing the animals from the cold, placing at room temperature for 2 h and then injecting 20E (2pglg) alone or both 20E and JH (2pg/g) each. Three pupae in duplicate were used for each time point for quantitation of RNA pols. Wing epidermis was dissected, washed and homogenized in 2 ml of buffer (Katula et al., 1981a) with a tight fitting Dounce homogenizer and centrifuged at 20 000 X g. The supernatant was saved. The pellet was suspended in the buffer containing 0.4 M ammonium sulfate and sonicated while being cooled to 4°C. The sonicate was diluted to reduce the ammonium sulfate concentration to 100 mM and centrifuged at 20 000 X g. The supernatants from the first centrifugation and that from the sonicate were centrifuged at 150 000 X g for 60 min prior to concentration (Prodicon, Beaverton, OR) against buffer with 10% glycerol at 4°C. Extracts for enzyme activity measurements were prepared by the same procedure as above; however, wing epidermis from 40 pupae were used for the O-time point, 30 for the 8 h and 16 h time points, and 20 for the 24 h and 48 h samples, respectively. The amounts of buffer used for homogenization and sonication were proportionately increased (I 5-20 ml). The supernatant from the first homogenization was used to dilute the sonicate prior to reducing the ammonium sulfate concentration, and the combined extract was subjected to ultracentrifugation. The supernatant was then diluted as needed and loaded on DEAE-cellulose. 2.2. In vitro studies on hormone effects and phosphorylation Pupae injected with 20E for different periods were

Endocrinology

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cleaned and sterilized externally with 70% ethanol. Dissected wing epidermis was rinsed several times in tissue culture media containing saturated phenylthiourea (a phenol oxidase inhibitor) and the antibiotic-antimycotic supplement (GIBCO-BRL). Two pairs of wings in duplicate (one pair from each of two pupae) were placed in 300~1 of sterile Ex-cell 401 phosphate free media (JRH Bioscience, Lenexa, KS) containing the antibiotic-antifungal supplement. The media also contained either 1 PM 20E or 1FM 20E +2 PM JH. 20E was added after filter sterilization through a 0.22 pm filter; JH was dissolved in alcohol and added to the culture medium (alcohol was
S. Sridhara,

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100~1 of antibody present in PBST (phosphate buffered saline containing 1% bovine serum albumin (BSA) and 0.1% Tween 20). A fresh SO-p1aliquot of Protein-A agarose was added and the mixture gently mixed for an additional 2 h at 4°C. The beads were pelleted and washed three times with immunoprecipitation buffer (25 mM Tris, pH 7.4; 150 mM NaCl; 0.01% SDS; 0.05% Triton X-100; 0.01% deoxycholate and 0.01% BSA) followed by three washes with phosphate buffered saline (PBS). Bound material was solubilized with sample buffer and separated by SDS-PAGE. The gels were soaked in 10% trichloroacetic acid at 90°C for 1 h prior to Coomassie blue staining, destaining and drying. Radioactivity was visualized by autoradiography. Some of the gels were also treated with glutaraldehyde and potassium hydroxide to identify phosphotyrosine (Bourassa et al., 1988). 2.6. Alkaline phosphatase treatment and immunoprecipitation Prior to immunoprecipitation, aliquots of the same samples were treated with either 25 units of alkaline phosphatase (AP) conjugated agarose beads or 100 units of soluble calf intestine AP (both from Sigma) for 30 min. at room temperature. Both solutions were centrifuged in the cold and the supernatants were subjected to immunoprecipitation as described above. 2.7. Alkaline phosphatase treatment and enzyme assay The effect of AP treatment on enzyme activity was determined as follows. AP (Sigma P-8647, 4000 units) was suspended in 1 ml of 100 mM sodium bicarbonate and biotinylated with biotin-XXNHS (Calbiochem) in dimethylformamide (Mattes and Sridhara, 1993). The biotinylated AP (BAP) was dialyzed and concentrated to 0.5 ml against PBS containing 20% glycerol. Duplicate 200~~1 aliquots of the wing tissue fraction containing RNA pol I were mixed with 12~1 of BAP for 30 min at room temperature and transferred to a tube contain ing streptavidin magnetic particles (Promega). The particles were mixed with the solution in the cold for 10 min, removed with a magnet and the procedure repeated with a fresh aliquot of the magnetic particles. Fifty microliters of the supernatant were used to measure enzyme activity. Control enzyme measurements were done by incubating other aliquots with biotinylated BSA instead of BAP. 2.8. RNA polymerase assays RNA polymerase I was assayed at 35 mM ammonium sulfate, 4 mM Mn2+, 2Opg of native calf thymus DNA and 1 pg/ml a-amanitin. RNA pol II activity was determined with 20 pg denatured calf thymus DNA, 100 mM ammonium sulfate and 4 mM Mn2+. Incorporation of label from r3H]UTP was measured by precipitating the product with trichloroacetic acid and collecting the material on a glass filter (Katula et al., 1981a; Mattes and

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41

Sridhara, 1993). Enzyme activities were determined in triplicate. 2.9. RNA polymerase amounts The amounts of RNA pols in various extracts were measured by the modified PABC-ELISA developed in this laboratory using monoclonal antibodies (Mattes and Sridhara, 1993) The three MABs used in this study were I-D2, I-8, and I-39 recognizing the 135 kDa second largest subunit, a 55 kDa subunit and a 22 kDa subunit, respectively. The 22 kDa is common to RNA polymerases I, II and III. Triplicate aliquots (1-50~1) of wing extracts were diluted in PBS containing 0.05% BSA to bring the amount of enzyme in 100~1 within the linear range of the assay (l-10 ng). Extracts in a total volume of 100~1 each were then incubated overnight at 4°C with monoclonal antibodies immobilized in microtiter plate wells via biotinylated BSA, streptavidin and biotinylated antimouse antibody. The amount of enzyme captured by the MAB was measured with a polyclonal antibody and alkaline phosphatase conjugated second antibody. 2.10. Other procedures Protein concentrations were determined by the Biorad dye binding assay system (Bradford, 1976). SDS-PAGE was carried out in slab gels with 4% stacking and 10% resolving gels. Immunodetection on Western blots with the MABs was done by the ECL system (Amersham). 3. Results 3.1. Stimulation of RNA polymerase activity in wing epidermis by 20-hydroxyecdysone and juvenile hormone Due to availability considerations, the studies in this report were conducted with the silkmoth Actias luna, instead of the oak silkmoth Anthearea polyphemus which was studied earlier. Therefore, RNA pol I and II activities in A. luna silkmoth wing epidermis were measured at four time points after stimulation with the hormones. Approximately 12-fold increase in enzyme activities was observed after 48 h of administration of either 20E or 20E + JH (Fig. 1). These increases were slightly higher but followed similar kinetics to those observed earlier with the Anthearea pernyii and A. polyphemus silkmoths (Katula et al., 1981a; Nowock et al., 1978a). The enzyme activities in water injected controls were within 10% of the 0 h values, similar to earlier measurements, and hence not included (see below, Fig. 4). 3.2. Specificity of the monoclonal antibodies The ability of the monoclonal antibodies, produced against RNA pol I of the silkworm Bombyx mori, to recognize the native enzyme from the A. luna silkmoth was shown by ELISA (Mattes and Sridhara, 1993). Their ability to react with specific subunits of A. luna silkmoth RNA pol I was demonstrated by immunodetection on

42

S. Sridhara,

,”

z

ZOE. RNA pal I

=I es

60

q

20E+JH.

0 Hours

RNA pal I

8 After

C.E. Mattes

q

ZOE. RNA pol II

q

20E+JH.

16 Hormone

/ Molecular

and Cellular

48

Fig. 1. RNA polymerase activity of A. luna wing epidermis during 48 h of hormone administration. Pupae were injected with 20E alone or with 20E + JH and wing epidermis processed through the DEAE-cellulose step as described in Section 2. RNA pol I activity was measured in the 0.1 M salt fraction and RNA pol II activity was measured in the 0.5 M salt fraction. Data are expressed as picomoles of UMP incorporated into acid precipitable material under standard conditions of assay per set of wings from one pupa ? SE (n = 2).

Western blots (Fig. 2). The three monoclonal antibodies (l-D2, l-8, l-39) recognized a single polypeptide each, whose sizes (135, 55 and 22 kDa, respectively) correI- 01 12345

l-8 t23t

l-39

3.3. Quantitative analysis of RNA polymerases in the developing wing epidermis Developmental changes in the amount of RNA pol I were determined and the data are presented as nglpupa in Fig. 3. RNA pol I content began to increase soon after hormone administration although the first major change was observed at the 8 h. A sixfold increase in RNA pol I per animal was observed during the first 24 h and it increased to about tenfold by 48 h. These changes in the enzyme amounts during the 48 h of development were the same when measured with either of the two monoclonal antibodies, l-D2 and 1-8, both specific to RNA pol I. The differences between the values at 0 time and 8 h and the subsequent time intervals were significant by the t-test (P < 0.01) and analysis of variance (ANOVA). Analysis

I2345 600

KDa

1

116, 97 -

66

-

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I I I (I 995) 3949

sponded to those of RNA pol I purified from B. mori silkglands (Mattes and Sridhara, 1993). The absence of any immunoreactive material in the flow through material (first lane for each MAB in Fig. 2) confirmed that the RNA pols present in tissue extracts bound completely to the ion exchange column. The absence of cross reacting material in the 0.5 M fractions with the first two MABs compared to the detection of cross reacting material by the MAB l-39 in both the fractions showed that RNA pols I and III were present in fraction 1 and RNA pol II in fraction 2, respectively. Elution of RNA pols I and III together from DEAE cellulose has been seen earlier with the silkmoths and other eucaryotes (Roeder, 1976; Katula et al., 1981a; Gowda and Sridhara, 1983). Non-immune antibodies did not show any reaction with duplicate blots.

RNA pal II

24 Administration

Endocrinology

Fig. 2. Immunodetection of RNA polymerase subunits in silkmoth wing epidermal extracts. About 1OOpg of protein from the DEAE cellulose fractions 1 and 2 prepared from wing epidermis at I2 and 24 h of hormone administration was precipitated with trichloroacetic acid. The precipitate was collected by centrifugation, washed successively with ethanol/ether (1: 1) and ether. The final pellet was solubilized in sample buffer and separated by SDS-PAGE. Gels were electroblotted on to PVDF membranes, and probed with the monoclonal antibodies. Detection was with the ECL system. Lane I of each blot contained DEAEcellulose flow through material. Lanes 2 and 3 contained fraction I from 12 and 24 h extracts. Lanes 4 and 5 contained the corresponding fraction 2.

q n q

RNA pol I (MAB l-02)

-T

RNA pol I (MAE l-8) RNA pals II + Ill

0 0 Hours

2 After

4

8 Hormone

16

24

48

Administration

Fig. 3. Amounts of RNA polymerases in the developing wing epidermis. The amount of RNA pols in wing epidermis extracts as a function of time after 20E administration was determined by the modified ELISA with three monoclonal antibodies. The values from the mea.urements with the two subunit specific antibodies (I-D2 and 1-8) were averaged to derive the amount of RNA pal 1. This was subtracted from the amount obtained with the antibody l-39 to derive the amount of RNA pols II + III. Data are presented as ng per pupa basis f SE (n = 4).

S. Sridhara, 16

pal II+

amount

*

RNA

--t

RNA pal I amount

01

' 0 Hours

1 IO

1 20 After

C.E. Mattes

Ill

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I Molecular

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' ' 1 40 50 Administration

I1 I (1995)

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points (+) in the same figure at 8-48 h represent values relative to those of 0 h for total protein, enzyme activity and enzyme amounts determined in the wing epidermis of water injected controls. Only one data point is included at each time period since the relative values for the three parameters remained close to 1 and were overlapping.

ACT

RNA Pol I Act

'

Endocrinology

' 60

Fig. 4. Relative changes in the amounts and activities of RNA polymerases and protein in the wing epidermis as a function of time after 20E administration. The amounts of protein. RNA pols and the RNA pol activities in the wing epidermis at different times were normalized to time zero. Average of four experiments. The points (+) indicate the normalized values of RNA pals and protein in wing epidermis from pupae injected with water.

of the data in this manner (activity or amount in a set of wings in one pupa) circumvents the variations inherent to presentation of data as ng enzyme/unit protein or RNA or DNA as these three macromolecules also show variable increases following hormone administration (Nowock et al., 1978a; Ruh et al., 1980; Katula et al., 1981a,b; Bryzski and Ruh, 1982). Quantitation of the enzyme(s) with the MAB specific to the subunit common to all the three RNA pols (l-39) also demonstrated significant increases during the 48 h of development. The total amount of enzyme measured with the common antibody was slightly more than twice that of RNA pol I derived with the specific antibody. The values obtained with MAB I-8 or I-D2 (RNA pol I amount) were subtracted from those obtained with MAB I-39 to derive the amounts of RNA pals II + III. Consequently the amounts of RNA pol II presented in the figures actually correspond to that of RNA pols II + III. Since RNA pol III is usually present at much lower levels than the other two enzymes (a tenth or less and see below) and because of the inability to measure RNA pol III independently, the results obtained with MAB I-39 by differential ELISA are taken to represent the amounts of RNA pol II. The relative changes in the enzyme activity, enzyme content and protein of the wing epidermis of pupae following 20E administration are summarized in Fig. 4. Clearly, while the RNA pol activities and amounts increased faster than the general protein, the extent of increase in the enzyme amount at 24 h could account for only about 50-60% of the increase in enzyme activity. By 48 h, however, the increase in the enzyme content could account for more than 85% of the enzyme activity. The

3.4. 20-Hydroxyecdysone stimulates RNA polymerase activity and amount in vitro Modifications of the enzyme and/or associated factors have been proposed to account for changes in the activity of RNA pol I following growth stimulation. Phosphorylation being a common mechanism for such regulation, the phosphorylation status of the wing epidermal RNA pol I after 20E stimulation was studied. We were unable to detect phosphorylation of the enzyme by injecting [32Pi] to pupae along with 20E (see Section 4) and thus labeling of proteins by culturing wing tissue in vitro with [32PJ was next attempted. Wing tissue from pupae given 20E for various periods of time was dissected and put in tissue culture for 24 h as described in Section 2. Tissue was processed through sonication, concentration and RNA pals were measured in the extracts with two MABs (l-8 and l-39). Results showed that the amounts of both the RNA pols I and II increased during culture in the presence of 20E (Fig. 5), although the extent of increase was lower than that observed in vivo. While there was little enhancement in the enzymes of cultured epidermis from 0 h pupae, the largest increases occurred in the wing epidermis taken from pupae after 4 h or 8 h of in vivo hor-

0 Hours

4 After

a Hormone

16

24

Administration

Fig. 5. RNA polymerase amounts in wing epidermis cultured in vitro. Extracts were prepared from wing epidermis cultured in vitro in the presence of water, 20E, and 20E + JH as described in Section 2. The amounts of RNA pals present were determined with the monoclonal antibodies 1-8 and l-39. Results with the first antibody were subtracted from those with the second to derive amounts of RNA pol II + III. The values obtained with epidermis cultured without hormones (water) were subtracted from those with the hormones. Results are presented as ng enzyme/pupa 2 SE (n = 3).

44

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A KDa 205 116 97

116 97 -

29

29 -

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24

1s

A

B

c

0

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Fig. 6. Phosphorylation status of RNA polymerase I in wing epidermis cultured in vitro, (A) Wing epidermis derived from pupae 4 h after administration of 20E was cultured in vitro for 24 h in the presence of [32Pi] and processed. Aliquots of the total extract and DEAE cellulose fraction 1 were separated by SDS-PAGE (lanes A and B). Duplicate aliquots were subjected to immunoprecipitation with l-D2 and l-8 monoclonal antibodies followed by SDS-PAGE of precipitated material (lanes C and D). Arrows indicate the expected location of the three subunits (135, 55 and 22 kDa size) recognized by the monoclonal antibodies (LD2, 1-8 and l-39, respectively). The slight differences seen between the lengths of the gels in lanes (A) and (B) compared to others are due to the autoradiograms being taken from different gels and exposed to different lengths to X-ray film. (B) Other aliquots of the fraction 1 were first treated with AP conjugated to agarose (lane E). soluble AP (lanes F-H, 50, 75 and 100 units, respectively)and -100 units of biotinylated AP (lane I) prior to electrophoresis.

mone stimulation followed by culture in vitro. Measurement of enzyme activities showed changes similar to those seen in Fig. 5. 3.5. Incorporation of radioactive phosphate into RNA polymerase I and dephosphorylation Incorporation of [32Pi] into total protein and RNA p01 I was followed by measuring acid precipitable counts in tissue extracts and immunoprecipitates. Maximal incorporation occurred in wing epidermis recovered from pupae after 4 h of 20E injection and cultured for 24 h. Aliquots of the DEAE cellulose fraction 1 derived from such wing epidermises were immunoprecipitated. The patterns of radioactivity seen after SDS-PAGE and autoradiography of the immunoprecipitated material are shown in Fig. 6A. While a large number of polypeptides in the extract and the RNA pol I fraction were phosphorylated (lanes A and B), eight polypeptides of 200-20 kDa size were prominent after immunoprecipitation (lanes C and D). Immunoprecipitation with non-immune mouse antibodies showed very little radioactivity (data not included, and

see below). Arrows correspond to the location of the three RNA pol I subunits (135, 55, and 22 kDa size, respectively) recognized by the three monoclonal antibodies. The apparent differences in mobilities of the major radioactive bands in the first two lanes compared to those in the immunoprecipitates are probably due to two reasons. First, salt in the first two samples slows down or slightly distorts the mobility of the dye compared to the other samples. Secondly, the first two lanes had to be exposed to X-ray film for different periods in order to obtain images that can be compared to those of immunoprecipitates. Consequently those lanes had to be cut and pasted. Other gels containing the same four samples were treated with alkali prior to drying and autoradiography. Except for two faint non-specific bands in the first two lanes, all the radioactivity was eliminated indicating that little tyrosine phosphorylation occurred on the RNA pols (data not included). Portions of labeled fraction 1 from the DEAE cellulose column were treated with either conjugated or soluble alkaline phosphatase (AP). Incubation with 25 units of

S. Sridhara,

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Biotinylated

BSA

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ii .-c .-m

C.E. Maiies

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60

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16 Hormone.

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1 II (1995)

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wing epidermis derived after 48 h of development was lesser.

6

Hours

Endocrinology

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Administration

Fig. 7. Effect of alkaline phosphatase on RNA polymerase I activity of wing epidermis extracts derived from pupae treated with 20E. Concentrated DEAE cellulose fraction 1 was prepared in duplicate from wing epidermis at different times after hormone administration. Each preparation was divided into three portions, one of which was kept in the cold to measure enzyme activity. The other two portions were incubated with biotinylated BSA and biotinylated AP, respectively, as described in Section 2. Three 50-~1 aliquots were used to determine RNA pal I activity. Values are presented as percent of control, 100% being the activity in the extract before incubation with biotinylated proteins, Average of two experiments.

AP conjugated to agarose beads at 37°C for up to 30 min failed to remove all the incorporated [32Pt] as measured by trichloroacetic acid precipitation and autoradiography of SDS-PAGE gels (lane E). A series of assays showed that at high levels (>75 units) soluble AP hydrolyzed more than 90% of trichloroacetic acid precipitable counts within 30 min at room temperature. Autoradiography of gels containing such dephosphorylated proteins showed a significant reduction of the label in all the polypeptides (lanes F-H). Biotinylated AP under the same conditions was also capable of causing extensive dephosphorylation (lane I). The biotinylated AP was removed by two successive treatments with magnetic streptavidin particles (Promega) before addition of MABs for immunoprecipitation. 3.6. Effect of dephosphorylation on enzyme activity DEAE cellulose fraction 1 containing RNA pols I + III was prepared from wing epidermis of pupae at different times after hormone administration. Aliquots were treated with biotinylated AP or biotinylated BSA which were removed prior to measurement of RNA pol I activity. AP had little effect on enzyme activity present in extracts of wing tissue derived from unstimulated pupae (which is normally very low). However, the enzyme activity of the extracts derived from wing tissue 8-24 h after 20E administration was reduced by 40-50% as a consequence of AP activity (Fig. 7). The effect of AP on the activity of

3.7. Phosphorylation and dephosphorylation effects are confined to RNA pol I and/or associated factors Earlier studies demonstrated that silkmoth RNA pols I and III, like the enzymes of other organisms, separate from each other clearly on a DEAE Sephadex A25 ion exchange column (Gowda and Sridhara, 1983). Furthermore, the contribution of RNA pol III was shown to be minimal to the enzyme activity measured on native DNA in the DEAE cellulose fraction 1 under conditions optimal for RNA pol I (Nowock et al., 1978a,b; Katula et al., 1981a). Therefore fraction 1 of a DEAE-cellulose column from in vitro labeled wing epidermis extract (similar to that of Fig 6) was fractionated on DEAE-Sephadex A25. About 70% of the radioactivity was present in the RNA pol I peak and about 20% in the RNA pol III peak (Fig. 8A). Fractions (3-10 and 25-33) were combined separately, concentrated and aliquots immunoprecipitated with two MABs. It was seen that while both the MABs precipitated a number of radioactive polypeptides from peak 1, no radioactive peptides were present in immunoprecipitates from peak 2 (Fig. 8B). 3.8. Effects of juvenile hormone on RNA polymerase I in vivo and in vitro JH administered along with 20E diverts the developmental program towards a second pupa instead of the adult (Sridhara et al., 1980). However, stimulation of RNA synthesis and nuclear RNA pol activities observed after administration of 20E + JH was approximately similar to that by 20E itself (Katula et al., 1981a). Similarly, JH neither decreased nor enhanced the effects of 20E on RNA pol activities in A. luna silkmoth (Fig. 1). The small differences in the level of RNA pol activities in the wing epidermis given JH along with 20E compared to those given 20E alone (Fig. 1) were not statistically significant. The enzyme amounts in the tissue at four time points (8-48 h) after administration of 20E and JH as determined by ELISA were within 6% of those presented in Fig. 3 and hence not presented. The effects of JH on enzyme activities and contents were also determined in the wing epidermis cultured in vitro. At the two time points that were tested (8 and 24 h), JH which was added to the culture medium had little effect on the stimulation of enzyme activity brought about by 20E. Increases in the enzyme content seen with 20E alone were not affected by JH either (Fig. 5). 4. Discussion Increased transcription of RNAs is a general characteristic of cells exposed to hormones or mitogens (Jacob et al., 1983; Sentenac, 1985). Attempts have been made to correlate increases in RNA pol I activity to the changes in

46

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A 1 .o

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Radioactivity Salt Concentration - 0.8

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20

30

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Number

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Fig. 8. DEAE-Sephadex fractionation and immunoprecipitation of extracts !abeled in vitro. Extracts were prepared from five sets of wings combined after culturing as described in Fig. 6 and chromatographed on DEAE cellulose. Concentrated and dialyzed fraction 1 was fractionated on a DEAE-Sephadex column as described in Section 2. A. Aliquots of the fractions were counted (0). (B) Fractions 3-10 (peak 1) and 25-33 (peak 2) were combined. concentrated and portions subjected to immunoprecipitation with the monoclonal antibodies (ID2 and I-39) and non-immune antibody (c). Precipitated material was subjected to SDS-PAGE and autoradiography.

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its amounts, but not very successfully. For example, enzyme activity which increased 15fold after stimulation of murine B lymphocytes with mitogen was correlated with the amount of the largest subunit measured by immunoblotting with a subunit specific MAB. Although a correlation was seen between the increasing enzyme activity and amount of this subunit, it was not the case when other subunits of the enzyme were measured (Liu and Rose, 1986). These results showing that a significant number of molecules of a specific subunit existed free, left open the possibility that some subunits or associated polypeptides may act as rate limiting factors in rRNA synthesis (Liu and Rose, 1986). It was not clear how association of a single subunit could account for changes in enzyme activity. Other studies have given results indicating that while the amount of enzyme remains constant, its activity is modulated by covalent modifications, by varying levels of associated transcriptional factors, or elimination of pre-existing inhibitors (Bateman and Paule, 1985; Tower and Sollner-Webb 1987; Webb et al., 1989; Schnapp et al., 1992; Kermekchiev and Muramatsu, 1993; Larson et al., 1993). The modified PABC-ELISA system was developed to avoid the problems in quantitation by immunoblotting techniques, and also be able to measure the amount of the native enzyme. Immunoblotting and ELISA assays demonstrated that there were no inactive pools of any of the three subunits in either the diapausing or developing pupal wing epidermis (data not included). The absence of inhibitor(s) in the wing epidermis of diapausing pupa which was either eliminated or inactivated following hormone administration was also shown by mixing experiments (Katula, 1979, and unpublished data). Since enhancement of RNA pol activities was dependent on both RNA and protein synthesis (Katula et al., 1981a), the increases presented above do reflect a true enhancement of the enzyme activities and amounts as a consequence of hormone action. The results included in Fig. 1 showed that 20E stimulated RNA pol activity in wing epidermis of the A. ha silkmoth to the same extent as it did in A. polyphemus. RNA pols of other insects were also shown to respond to 2OE, mostly by enhanced activity, in larvae, cells and even isolated nuclei (Nishiura and Fristrom, 1975; Ruh and Dwyer, 1976; Schenkel and Scheller, 1982). Probably stimulation of RNA pol activity and RNA synthesis by 20E is universal to all diapusing silkmoth pupae. In most studies, including that on A. polyphemus, stimulation of RNA pol II activity was higher than that of RNA pol I. In our studies with A. ha, both RNA pol I and II activities increased approximately to the same extent during the first 24 h (Fig. 1). These differences can be attributed to the improvements in preparation of extracts for enzyme activity measurements and more so, to the difficulty of measuring the very low enzyme activities in 0 h wing tissue extracts which are used as the basis to calculate the

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relative increases. The absence of procedures to measure the amounts of RNA pols I, II and III has so far prevented a determination of the relative changes of the three RNA pols in any tissue (a few attempts to measure RNA pol II with binding of labeled a-amanitin have been only partly successful). ELISA was developed and applied here to correlate the changes in activity to the amounts of the enzymes. It has to be emphasized that while it is possible to quantitate RNA pol I with a subunit-specific antibody, the amounts of RNA pols II + III are derived by differential ELISA with a MAB specific to a common subunit. This approach although valid for developmental studies to measure relative changes, a determination of the absolute values of each of the native RNA pols, even with the silkmoth, must await purification of RNA pols II and III from the same organism to use as ELISA standards. Subject to this reservation, comparisons of the changes in the amounts of the enzymes during hormone induced development are valid. Thus the relative accumulation rates of RNA pols are higher than those of general protein (Fig. 4). The increase in amounts (about 5-6-fold at 24 h) was less than that of the enhancement in enzyme activity (about lO-12-fold). By 48 h however, the accumulation of the enzymes and their activities appear to equal one another. This lack of correlation between the increase in enzyme activity and amount of RNA pol I led us to look for alternative explanations. Phosphorylation is a common mechanism for modulation of various enzymatic activities and both RNA pols I and II have been shown to be phosphorylated (Bell et al., 1977; Breant et al., 1983; Jacob et al., 1983). Earlier attempts to correlate phosphorylation to enzyme activities showed no differences between phosphorylated and dephosphorylated yeast enzymes (Bell et al., 1977; Breant et al., 1983). However, more recent experiments with mammalian systems have shown a definite correlation between rDNA transcription and phosphorylation of one or more components of the rDNA transcription apparatus (Bateman and Paule, 1985; Belenguer et al., 1989; Schnapp et al., 1990). This question of the role of phosphorylation in regulation of enzyme activity became very interesting with the demonstration of one of its subunits having kinase activity and which caused autophosphorylation (Duceman et al., 198 1; Rose et al., 198 1). The same or similar kinase (casein kinase II or NK II) is also present in the nucleus and its level is hypothesized to play a central role in the growth dependent regulation of rRNA synthesis (Belenguer et al., 1989). No information exists as to whether a kinase is a subunit of RNA pol I of other animals or insects. In order to determine whether RNA pol I in wing tissue was phosphorylated, up to 0.5 mCi of [32Pi] was injected into each pupa along with the hormone and wing tissue processed at 24 h. Only a very low level of incorporation was seen in the RNA pol I fraction, and less than 10 DPM in the immunoprecipitates. Dilution of the label

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in vivo by a large pool of endogenous phosphate and uptake by tissues other than the wing epidermis could account for this low level of incorporation. Incorporation of [32Pi] into proteins was quite good during in vitro culture of the wing epidermis. The increases of enzyme levels which were much less than in vivo (comparing Figs. 3 and 5) is partly due to the use of phosphate-free medium. The resistance of phosphate incorporated in vivo to removal by immobilized AP, but sensitivity to higher amounts of the soluble enzyme were shown by SDSPAGE of AP treated material (Fig. 6). Such recalcitrance of phosphate moieties in native RNA pols to hydrolysis by AP was encountered earlier (Breant et al., 1983; Cadena and Dahmus, 1987). For example, only 1 unit of the enzyme was sufficient to hydrolyze label incorporated into RNA pol II by casein kinase in vitro compared to >200 units required to achieve the same result with label incorporated in vivo (Cadena and Dahmus, 1987). Our results parallel some of these observations in that a high concentration of soluble AP is required to dephosphorylate the native RNA pol I. While testing for the effects of dephosphorylation on RNA pol activity, it is necessary to eliminate the AP. Several gel filtration media (Sephacryl-200 or 300 Ultrogel AcA34, and Bio-Gel 1.5M) were tested, but none gave adequate separation, as some AP eluted with RNA pol I. Dilution and time involved also made these procedures unsuitable. By comparison, biotinylated AP can be removed rapidly in the cold with no dilution of the RNA pol. Since BAP reduced RNA pol I activity, the increase in enzyme activity during the first 24 h of hormone stimulation can be assumed to be due to a combination of increased amount of the enzyme and phosphorylation. The absence of phosphorylated polypeptides in immunoprecipitates from the peak containing RNA pol III (Fig. 8), and the lack of any effect of alkaline phosphatase on RNA pol III activity (data not included), indicate that the AP effects (Fig. 7) are likely due to its action on RNA pol I itself or some very tightly associated factor(s) that modulates its activity. Since polypeptides other than the subunits containing radioactivity were immunoprecipitated by the antibodies, it is not possible to correlate phosphorylation of specific subunits of RNA pol I to changes in its activity in vivo. These conclusions have to be further qualified by two reservations. First, the in vitro work regarding phosphorylation was done to maximize incorporation of r3*Pi] into protein, to determine whether RNA pol I was phosphorylated, and to optimize procedures for dephosphorylation. Consequently, the results of Fig. 6A may or may not reflect the in vivo situation. Second, recent studies on the role of phosphorylation of RNA pol I and/or associated factors in rRNA transcription have been done with in vitro transcription of specific rDNA templates while our studies were done with non-specific templates. Hence the results detailed above have to be confirmed by in vitro transcrip-

48

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tion of silkmoth rDNA templates. Efforts to do so are underway. Why wing epidermis at 48 h after hormone administration shows neither as much enhancement in activity, nor is its enzyme affected as much by dephosphorylation compared to the enzyme during the first 24 h (Figs. 4 and 7) is not clear. Probably the rate of enzyme synthesis slows down to a level conforming to that of general protein syntheses and the enzyme subunits and/or the associated peptides are not phosphorylated to the same extent. Such a differential phosphorylation can account for the presence of two isoforms of the enzyme detected during purification (Nowock et al., 1978b). The presence of subpopulations of RNA pols due to differential phosphorylation has been noted in other instances also (Kolodziej et al., 1990; Schnapp et al., 1990). The inability of JH to affect the content of RNA pols stimulated by 20E in vivo is very interesting. Evidently, JH has remarkable effects on all cells within the pupa, including the wing epidermis, as its presence during the first 24 h of 20E action changes the genetic expression program towards a second pupa rather than towards the adult (Sridhara et al., 1980; Brzyski and Ruh, 1982). During the first 48 h of hormone action, however, RNA synthetic rates, RNA content, DNA synthesis and even mRNA populations appear to be similar in 20E + JH treated and 20E treated wing epidermis (Sridhara et al., 1980; Katula et al., 1981a,b; Sridhara, 1994). In addition, the present demonstration of similar levels of RNA pol activities and contents suggest that the changes brought about by this hormone are not at the transcriptional activities of the RNA ~01s. The absence of any JH effects on the activities and contents of RNA pols in wing epidermis cultured in vitro was unexpected as it was shown earlier that JH inhibited macromolecular synthesis, especially protein synthesis, in cultured Drosophila imaginal discs (Fristrom et al., 1976). Since RNA pol I increased during incubation with JH, while RNA synthesis was inhibited, it was hypothesized that the increased enzyme activity was due to the blocking of synthesis of a rapidly turning over inhibitor of RNA pol I (Nishiura and Fristrom, 1975). This explanation cannot be applicable to the wing epidermal culture system since JH does not prevent the increase in either RNA pol activities or their levels (Figs. 1 and 3). These seemingly contradictory results on the effects of JH on Drosophila imaginal discs and the silkmoth wing epidermis are not necessarily incompatible since the hormonal effects depend on the age of the larvae from which wing discs are isolated. Imaginal discs isolated from younger larvae respond to the hormones very differently compared to those isolated from older larvae (Oberlander and Silhacek, 1976). The silkmoth pupal wing epidermis having already synthesized and secreted a pupal cuticle, is comparable to the discs isolated from the older larvae. The developmental stage is appropriate for JH to modify gene expression and/or chromatin structure so that

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the cells can synthesize a pupal instead of an adult cuticle several days later. During the early period of rearranging the developmental pathway, however, JH does not interfere with the stimulation of RNA pols by 20E. Acknowledgements This work was partly supported by the seed grant program of ‘ITUHSC. References Bateman,E.

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