DEVELOPMENTAL

67, 11-22

BIOLOGY

(1978)

RNA Synthesis in Avian Erythroid Cells ALLAN J. TOBIN,%SAN Department

of Biology

Received

E. SELVIG,' AND LARRYLASKY"

and Molecular Los Angeles,

November

Biology Institute, California 90024

17, 1977; accepted

in revised

University

form

May

of California,

24,1978

In nucleated erythroid cells, as in other vertebrate cells, less than 2% of the newly made RNA enters the cytoplasm. Posttranscriptional selection is therefore likely to play an important role in differential gene expression. The work reported here defines the relative roles of transcription, nuclear-cytoplasmic flow, and mRNA turnover in determining the distance of polysomal mRNAs in avian erythroid cells. Cells were preincubated with glucosamine to reduce endogenous pools of UTP and were then labeled with [:‘H]uridine. The specific activity of the UTP pools during the incubation was measured after high-pressure liquid chromatography, and the absolute rates of synthesis of nuclear and cytoplasmic RNA were determined. Under the labeling conditions described, the UTP pool reached a specific activity of 600 dpm/pmole within 5 min and remained between 600 and 800 dpm/pmole for 60 min. The total rate RNA synthesis was 0.9 fg/min/cell. The rate of synthesis of cytoplasmic RNA was 1.7% of the total. Comparison of these figures with published data for L cells indicates that these terminally differentiated, postmitotic cells are much more restricted in transcription than L cells, but similar in the efficiency of converting nuclear transcripts to cytoplasmic RNA. The rate of appearance and size distribution of polysomal RNAs were also determined. About 45% of the newly synthesized poiy(A)+ polysomal RNA has a sedimentation coefficient of approximately 9 S and was identified as globin mRNA by hybridization to recombinant plasmid DNA containing chicken P-globin mRNA sequence, Each species of globin mRNA is calculated to arrive on polysomes at about 1 molecule per minute per cell and each species of nonglobulin mRNA at about 0.02 molecules per minute per cell. These differences in the rates of appearance do not suffice to explain the 200-fold difference in the concentrations of individual globin and nonglobin poly(A)’ mRNA species determined in hybridization experiments. The nonglobin poly(A)’ mRNAs in these cells must therefore either be less stable than the globin mRNA or have accumulated for a shorter time than the globin mRNA.

of globin mRNA relative to other mRNAs could result from (i) a more rapid flow to the polysomes of globin mRNA than of other mRNAs, (ii) a greater stability of globin mRNA relative to other mRNAs, (iii) a longer period of accumulation of globin mRNA relative to other mRNAs, or (iv) a combination of these. In this paper we present data on the kinetics of appearance on erythroid cell polysomes of the 9 S globin mRNAs and of other poly(A)-containing mRNAs. We address the question of whether the differences in the rates of flow of globin and other mRNAs to poly-

INTRODUCTION

The concentration of hemoglobin in mature erythrocytes approaches that in hemoglobin crystals. It is thus not altogether surprising that about 90% of the polysomal mRNA of nearly mature erythroid cells is globin mRNA (Humphries, et al., 1976; Lasky et al., 1978). 3 The high concentration i Present address: Department of Biology, Immaculate Heart College, Los Angeles, California, 96027. ‘Present address: Division of Biology, California Institute of Technology, Pasadena, California 91125. 3 Abbreviations used: mRNA, messenger RNA; hnRNA, heterogeneous nuclear RNA; Hepes, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid; TCA, trichloroacetic acid; PPO, 2,5diphenyloxazole; EDTA, ethylenediaminetetraacetic acid; SDS, sodium dodecyl sulfate; DMSO, dirnethylsulfoxide; Tes, N-tris (hy-

droxymethyl) SSC, 0.15M pH 7.0; NT,

methyl-2-aminoethane sodium chloride, 0.015 nucleotide.

sulfonic M sodium

acid; citrate,

11 0012-1606/78/0671-0011$02.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

12

DEVELOPMENTAL

BIOLOGY

somes suffice to explain the observed differences in the concentrations of individual mRNA species. The circulating erythroid cells of anemic chickens are postmitotic and synthesize no detectable ribosomal RNA (Williamson and Tobin, 1977). Both their transcriptional and their translational repertoires are highly restricted. Their poly(A)’ nuclear RNA consists of only 4000 species, while their poly(A)+ mRNA contains fewer than 100 species (Lasky et al., 1978). These erythroid cells thus differ from all other cells that have been studied: even mouse erythroleukemic cells and the differentiated cells of the estrogen-stimulated chick oviduct contain more than 10,000 different mRNA species (Kleiman et al., 1977; Hynes et al., 1977). The question arises therefore whether avian erythroid cells are somehow uniquely specialized for the transcription of globin and a few other mRNAs. If the small number of proteins made by these cells results principally from a restriction of transcription, one might expect (i) a lower rate of RNA synthesis and (ii) a higher fraction of newly made RNA appearing in the cytoplasm than in other less specialized cells. In order to determine if this is the case, we have measured the absolute rates of synthesis of nuclear, cytoplasmic, and polysomal RNAs. Our measurements indicate that these cells synthesize RNA at about 1% the rate of L cells, but, as is the case for L cells, less than 2% of the newly synthesized RNA appears in the cytoplasm (Brandhorst and McConkey, 1974). The importance of differing mRNA stabilities in the accumulation of particular mRNAs during cytodifferentiation has been discussed in detail in several systems (Kafatos, 1972; Marks et al., 1962; Stewart and Papaconstantinou, 1967). Aviv and his colleagues have recently suggested, however, that the differing stabilities of mRNAs in mouse erythroid cells cannot explain the accumulation of globin mRNA if the stabilities are constant during erythropoiesis (Bastos and Aviv, 1977b). On the other

VOLUME

67,1978

hand, the data presented in this paper suggest that differences in the kinetics of appearance of different mRNAs on polysomes cannot wholly account for observed differences in polysomal concentrations of different mRNA species. It therefore seems likely that differences in the flows of individual mRNAs to the polysomes, as well as in the stabilities of these mRNAs or in the periods during which they appear, contribute to the striking accumulation of globin mRNAs during erythropoiesis. MATERIALS

AND

METHODS

Labeling of erythroid cells. White Leghorn hens were made anemic and bled as described by Lasky et al. (1978). The blood cells were separated from plasma by centrifugation for 5 min in a clinical centrifuge. The cells were then washed twice in cold isotonic saline containing 0.14 M sodium chloride, 0.05 M potassium chloride, 0.001 M magnesium chloride (NKM). The “buffy coat,” containing the nonerythroid cells, was carefully removed after the second NKM wash. The cells were then washed in labeling medium, which consisted of Dulbecco’s modified Eagle medium supplemented with 25 mM Hepes, 10% dialyzed fetal calf serum (GIBCO), 2 mit4 glutamine, and antibiotics (Penicillin-StreptomycinFungizone, GIBCO). After this final wash, cells were resuspended in 9 vol of labeling medium to give approximately 7 x 10” cells/ ml. Glucosamine (1 M), pH 7.4, was then added to a final concentration of 20 n-&f, and cells were incubated for 30 min at 37°C (Scholtissek, 1971). Cells were collected by centrifugation, washed once in labeling medium, and resuspended in 8.5 vol of warmed labeling medium. [5-3H]Uridine (25-30 Ci/ mmole, Amersham or New England Nuclear) was added in 0.5~cell volumes to give a final concentration of 50 @i/ml, or about 2 fl. Labeling was done at 37°C in a New Brunswick water-bath shaker. The flask was rotated at 175 rpm during the incubation. Analysis of nucleotide pools. After the indicated incubation times, the cells from 1

TOBIN,

SELVIG,

AND

LASKY

RNA

ml of suspension were harvested by centrifugation for 20 set in a Beckman microfuge, resuspended in 0.2 ml of NKM, and brought to 5% trichloroacetic acid (TCA). After incubation for 10 min at O”C, the precipitate was removed by centrifugation and the supernatant was removed and extracted three times with water-saturated ether (Brown, 1971). Nucleotide separation was done by high-pressure liquid chromatography on a Whatman Partisil SAX column connected to Waters Associates pumps and solvent programmer in the laboratory of Dr. J. D. O’Connor in this department. Resolution of ribonucleotides was accomplished by elution with a 45-ml concave gradient (number 8 on the solvent programmer) followed by 45 ml of final buffer. The initial elution buffer consisted of 0.007 M potassium phosphate, pH 4.0, and the final buffer of 0.25 M potassium phosphate, 0.5 M potassium chloride, pH 4.5. The column was eluted at 1.5 ml/min, and l-min fractions were collected. The resolution of all 12-ribonucleotide mono-, di-, and triphosphates required 60 min (Fig. 1A). The resolution of the four triphosphates alone can be accomplished in 30 min, eluting with the final buffer only. The radioactivity of each sample was measured by scintillation counting in a scintillation cocktail consisting of 2 parts toluene, 1 part Triton X-100, 2.7 g/l PPO (“Tritontoluene”). The efficiency of counting each sample was determined from the external standards ratio, calibrated from a quench curve using “H-toluene as a standard. Measurement of incorporated radioactivity in cells, nuclei, and cytoplasm. At each indicated incubation time, six O.l-ml aliquots of suspension were delivered into 1 ml of cold NKM in 1.5-ml tubes for the Beckman microfuge. Cells were harvested by centrifugation for 20 set and washed twice in cold NKM. The cells of three of the samples at each time point were then lysed in 1 ml of 10 n-&f calcium chloride, the nuclei were collected by centrifugation, and 0.4 ml of the cytoplasm was removed to separate tubes. The nuclei were then washed once with 10 mM calcium chloride.

Synthesis

in Avinn

Erythroid

13

Cells

The three sets of samples, containing washed whole cells, washed nuclei, and cytoplasm, were then brought to 5% TCA and allowed to stand for 10 min at 0°C. The resulting precipitates were collected by centrifugation and washed twice with 5% TCA, 0.01 M sodium pyrophosphate. The RNA was hydrolyzed in 1 ml of 0.3 N sodium hydroxide for 90 min at 37°C and the DNA and protein were reprecipitated by the addition of 0.25 ml of 50% TCA. The precipitate was removed by centrifugation, and the alkali-labile radioactivity was determined by scintillation counting in Triton-toluene. The efficiency of counting was approximately 15%. In the experiment shown in Fig. 3, the number of cells in each sample was determined by measurement of the DNA in the final TCA precipitate, using the diphenylamine procedure of Burton (1956). Isolation of polysomes. After the indiGMP

060-

~.

045c UDP

A i-4

G2P ADP ,

030

?

UMP

I

CDI

0

t (min)

FIG. 1. High-pressure liquid chromatography of ribonucleotides. Chromatography was performed as described in Materials and Methods. (A) Mixture of 20 nmoles of each of 12 ribonucleotides. Elution times of individual nucleotides were determined in separate experiments. (B) TCA extract of erythroid cells labeled for 30 min with [“H]uridine. (-) Absorbance; (-----) counts per minute in I-min (1.5.ml) fractions,

14

DEVELOPMENTAL BIOLOGY

cated incubation times, a lo-ml aliquot of the cell suspension was pipetted into 15 ml of cold NKM. Cells were harvested by centrifugation and lysed in 3 ml of lysis buffer containing 2% Triton X-100, 1 mg/ml of heparin, 25 mM sodium chloride, 5 mM magnesium chloride, 25 n&f Tris-HCl, pH 7.5 (Palmiter, 1974). Nuclei and cell membranes were removed by centrifugation at 4°C for 10 min at 27,000g. The cytoplasmic supernatant was removed and again centrifuged at 4°C for 10 min at 27,000g. Magnesium-precipitated polysomes and ribonucleoprotein particles were prepared by the method of Palmiter (1974). To the cytoplasmic supernatant was added an equal volume of a buffer containing 0.2 M magnesium chloride, 1.6% Triton X-100,0.8 mg/ml of heparin, 20 m&f sodium chloride, 20 n-J4 Tris-HCl, pH 7.5. The polysomes were then collected by centrifugation at 27,000g for 10 min at 4°C and incorporated radioactivity was determined by precipitation with TCA. EDTA-released polysomal RNA was prepared from polysomes that were isolated by differential centrifugation. Cells were lysed and the nuclei and cell membranes were removed as above. The cytoplasmic supernatant was then layered over 3 ml of 40% sucrose in 25 mM sodium chloride, 5 mM magnesium chloride, 25 r&f Tris-HCl, pH 7.5 (polysome buffer). Polysomes were collected by centrifugation at 4°C for 150 min at 40,000 rpm in a Beckman SW41 rotor. The polysomal pellet was resuspended in 20 mM Hepes, pH 7.4, and centrifuged at 4°C through a 10 to 40% sucrose gradient in polysome buffer for 105 min at 26,000 rpm in a Beckman SW27 rotor. Material sedimenting faster than monosomes was pooled, brought to 0.2 M sodium acetate, pH 6.0, and precipitated with 2 vol of ethanol. The precipitated polysomes were collected by centrifugation at 20,OOOgfor 30 min at 0°C and redissolved at lo-15 Azr;o/ ml in 5 miI4 EDTA, 25 miI4 sodium chloride, 25 n&f Tris-HCl, pH 7.5. The polysomes were then dissociated in 25 mM EDTA for 10 min at 0°C. The dissociated polysomes

VOLUME 67,1978

were layered over a 5 to 20% sucrose gradient in 5 mM EDTA, 25 mM sodium chloride, 25 mM Tris-HCl, pH 7.5, and centrifuged for 180 min at 26,000 r-pm in a Beckman SW27 rotor. Material sedimenting more slowly than 80s was pooled and precipitated with ethanol as above. Fractionation of RNA. EDTA-released polysomes were dissolved in buffer containing 0.1 M sodium chloride, 0.5% sodium dodecyl sulfate (SDS) (BDH), 0.001 M EDTA, and 10 n&f Tris-HCl, pH 7.4 (SDS buffer). Poly(A)’ mRNA was then prepared by chromatography on oligo(dT) cellulose (Krystosek et al., 1975). The RNA in SDS buffer was then heated for 1 min at 60°C and brought to 0.5 M sodium chloride, 0.5% SDS, 0.01 M Tris-HCl, pH 7.4 (binding buffer), for chromatography on oligo(dT) cellulose (T-3, Collaborative Research). A sample containing up to 10 Azm units of polyosmal RNA/ ml was applied to 50 mg of oligo(dT) cellulose, allowed to run through, and then reapplied. Poly(A)- RNA was washed from the column with 10 ml of binding buffer, and poly(A)’ RNA was eluted in 3 ml of elution buffer, which contained 0.5% SDS and 0.01 M Tris-HCl 7.4. The poly(A)’ RNA was brought to 0.2 M sodium acetate, pH 6.0, and precipitated with ethanol. The poly(A)+ polysomal RNA was then fractionated by centrifugation under denaturing conditions. The RNA was dissolved in 50% dimethylsulfoxide (DMSO), 0.1 M lithium chloride, 0.005 M EDTA, 0.2% SDS, 0.01 M Tris-HCl, pH 7.5 (DMSO buffer), heated at 65°C for 1 min, and then layered on a 5 to 20% sucrose gradient in DMSO buffer. The gradients were centrifuged at 29°C for 20 hr at 35,000 rpm in a Beckman SW41 rotor (Bantle and Hahn, 1976). Gradients were fractionated, and aliquots of each fraction were precipitated with 10% trichloroacetic acid and counted. The amount of poly(A) in each fraction was determined by hybridization to [‘HIpoly(U) (Miles) (Bishop et al., 1974). Detection of P-globin mRNA sequences. Labeled /I-globin sequences were detected

TOBIN,

SELVIG,

AND

LASKY

RNA Synthesis in Avian Erythroid

by hybridization to the recombinant plasmid DNA, pHb1001, whose inserted cDNA nucleotide sequence corresponds to the amino acid sequence of the adult chicken P-globin (Padayatty et al., in preparation). pHblOO1 was obtained from Dr. Winston Salser of this department. It was grown under P2,EKl conditions, as permitted by a reduction in containment requirements under the terms of a petition by Dr. Salser approved by the NIH Recombinant DNA Committee in January 1977. pHblOO1 DNA was sonicated, denatured, and bound to nitrocellulose filters (Gillespie and Spiegelman, 1965). Each filter was loaded with approximately 10 pg of pHblOO1 DNA, corresponding to 0.5 pg of DNA complementary to ,&globin mRNA. Nitrocellulose filters containing 10 pg of nonrecombinant pMB 9 DNA were similarly prepared. Aliquots of fractionated labeled RNA were incubated with filter-bound DNA for 24 hr at 65°C in 0.3 M sodium chloride, 0.01 M EDTA, 0.2% SDS, 0.01 M Tes, pH 7.4, which contained 10 pg/rnl of poly(A) and 10 pg/ml of tRNA. After incubation, filters were rinsed with 2~ SSC at 65”C, treated with 10 pg/ml of ribonuclease A and 5 units/ml of ribonuclease Tl for 1 hr at 37”C, rinsed with 2~ SSC at 37°C dried, and counted in toluene-Omnifluor. RESULTS

RNA precursor pools. To calculate the absolute rate of synthesis of RNA from measurements of incorporation of radioactivity, it is necessary to know the specific activity of the labeled precursor pool. The analysis of labeling kinetics is simplest if the specific activity of the precursor pool is constant during the incubation period, but this is not usually possible (Brandhorst and McConkey, 1974). The rate of equilibrium of an exogenous labeled nucleoside with the RNA precursor pool depends upon the size of the relevant pool. It is therefore advantageous to label with the precursor that has the smallest endogenous pool, often gua-

Cells

15

nosine (Puckett et al., 1975; Galau et al., 1976). In experiments reported here, the relative sizes of the nucleotide pools and the specific activities of the labeled pools were determined by high-pressure liquid chromatography. The solid line in Fig. 1B shows the absorbance of ribonucleotides in TCA extracts of erythroid cells that were preincubated with glucosamine. As expected, the predominant nucleotide is ATP. Since there is less UTP than ATP or GTP in these cells, we chose [‘lH]uridine as a label. Only CTP is present in lower amounts, but the analysis of cytidine labeling may be complicated by the CCA turnover of transfer RNA. Since these cells are postmitotic, there is no incorporation of label into DNA. The rate of entry of [‘Hluridine into the UTP pool is increased by preincubation with glucosamine, which converts much of the endogenous UTP to UDP-glucosamine (Scholtissek, 1971). Figure 1B shows the distribution of label among uridine nucleotides after a 30-min incubation with [‘HIuridine. Four radioactive species are present, whose elution times and probable identities (based on comparison with standards) are as follows: (1) 4 min, unphosphorylated uridine; (2) 26 min, UDP; (3) 30 min, UDP-glucosamine; (4) 39 min, UTP. The identification of the UTP peak was confirmed by two-dimensional thin-layer chromatography as described by Randerath and Randerath (1967). In a typical experiment, cells were preincubated with glucosamine, washed, and then labeled with [“Hluridine, as described in Materials and Methods. After the indicated times of incubation, aliquots of the cell suspension were extracted with TCA and the specific activity of individual nucleotides was determined after high-pressure liquid chromatography, as shown in Fig. 1B. Only a small fraction of the exogenous uridine was taken up by the cells, and the concentration of [‘Hluridine in the medium did not change during a 90-min incubation. The time course of the specific activity

16

DEVELOPMENTAL

BIOLOGY

of the UTP pool during the incubation period is shown in Fig. 2A. The specific activity rose rapidly, and at about 20 min reached a maximum value of 0.36 Ci/ mmole, about 1.5% that of the added uridine. After 30 min of incubation, the specific activity of the UTP began to decline, so that by 75 min, the specific activity had fallen to about 60% of the maximum. The curve shown in Fig. 2A is a best fit to Eq. (l), which decribes the specific activity of the UTP pool as a function of (i) the rate of entry of the exogenous uridine, (ii) the size of the endogenous UTP pool, and (iii) the rate of addition of salvaged uridine from the continuous turnover of nuclear RNA (Tobin, unpublished).

ds z

1 = ;

(kc

+ bry

-

Jrs),

(1)

where s is the specific activity of UTP at time t; u is the amount of UTP per cell; k, is the flow of exogenous uridine into the UTP pool; kDT is the first-order rate constant for the degradation of nuclear RNA;

VOLUME

67,1978

y is the amount of radioactivity in nuclear RNA at time t; and Jr is the total rate of RNA synthesis. To convert from the radioactivity found in RNA at a given time to the amount of newly made RNA, we used the average values of the specific activity of the UTP pool between the beginning of labeling and the time at which the radioactivity was measured. These calculated values for the average specific activity of the UTP pool are shown in Fig. 2B. Rates of appearance of RNA in the nucleus and cytoplasm. Using the specific activity data of Fig. 2B, one can convert the radiaoctivity incorporated into total, nuclear, and cytoplasmic RNAs to attomoles ( lo-l8 moles) of UTP incorporated into newly made RNA per cell. High-pressure liquid chromatography of an alkaline digest of labeled RNA showed that all of the acid-precipitable, alkali-labile radiaoctivity could be recovered as UMP, therefore, permitting a straightforward conversion of the measured radioactivity to amounts of newly made RNA. These data are shown in Fig.

3.

“&mJ d

/ 0

, 20

/ , 1 , 1 40 t (mln)

60

80

FIG. 2. Specific activity of UTP pools. Cells were labeled for the indicated times and extracted with TCA. The specific activity of the UTP pool was determined after high-pressure liquid chromatography. (A) Specific activity of UTP after the indicated times of labeling. (B) Calculated average specific activity between the beginning of incubation and the indicated times.

As can be seen from Fig. 3, the accumulation of newly made RNA in whole cells and nuclei approaches a plateau, while that in the cytoplasm continues to increase linearly during a 75-min incubation period. These data suggest (i) that the half-life of the nuclear RNA is approximately 15 min; (ii) that the half-life of most of the newly made cytoplasmic RNA is significantly longer than 75 min; and (iii) that nuclear RNA leakage does not significantly interfere with the measurements of cytoplasmic RNA labeling. The accumulation of newly made RNA in the nucleus of a nondividing cell can be described as an approach to the steady state governed by the following equation: (T)

= -

JT

(1 - epk”“) (2) b-r where (T) is the concentration of newly transcribed nuclear RNA, JT the rate of synthesis of nuclear RNA, and knr the first-

TOBIN,

SELVIG,

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RNA

Synthesis

in Auian

Erythroid

Cells

17

kDM is the first-order rate constant for the degradation of mRNA. For times short compared to the half-life of mRNA (i.e., for t CK l/k&, the exponential term is approximated as (1 - knMt), so Eq. (3) becomes: (M) = c&It.

0 0

30

60 t (min)

FIG. 3. Molar accumulation of newly made RNA. Cells were labeled for the indicated times and the amount of UMP incorporated into newly made RNA in each cell fraction was calculated as described in Materials and Methods. (A) Accumulation of newly made RNA in whole cells and in nuclei prepared by lysis in 10 m&f calcium chloride. (0) Whole cells; (0) nuclei. Curves are the best fits to Eq. (1). (B) Accumulation of newly made RNA in the cytoplasm. (C) Accumulation of newly made RNA in polysomes and ribonucleoproteins precipitated by 100 r& magnesium chloride.

order rate constant for the degradation of nuclear RNA. The parameters giving the best fit of the data shown in Fig. 3A to Eq. (2) were calculated using a nonlinear leastsquares fitting program in the UCLA Computer Center library, and are given in Table 1. The accumulation of newly made mRNA in a nondividing cell can also be described as an approach to steady state: (M) = 2

(1 - p’q(

(3)

where (M) is the concentration of newly made messenger RNA, JM is the rate of appearance of mRNA in the cytoplasm, and

(4)

The flow of RNA from nucleus to cytoplasm is thus given by the slope of the line shown in Fig. 3B, calculated from a linear least-squares fitting program. As summarized in Table 1, this flow is approximately 0.01 amoles of UTP/cell/min. Thus only about 1.7% of the newly made RNA flows from the nucleus to the cytoplasm. Analysis of polysomal RNA. In addition to analyzing the total nuclear and cytoplasmic RNA, we also examined the degree of utilization of newly made mRNA, that is, the rate of synthesis of polysomal RNAs. The rate of accumulation of labeled uridine in EDTA-released polysomal RNA is 1.7 x 10p2’ moles/cell/min, corresponding to 15% the rate of appearance of total cytoplasmic RNA (Table 1). Eighty percent of the newly made RNA binds to oligo(dT) cellulose, and is identified as poly(A)+ mRNA. In a separate experiment, newly made poly(A)’ mRNA was analyzed by sedimentation in denaturing sucrose gradients (Fig. 4). Approximately 45% of the radioactivity sedimented in the 9 S region of the gradient. The remaining poly(A)+ mRNA had a mean sedimentation coefficient of 14 S, corresponding to a mean length of approximately 1300 NT (Fig. 4A). The size distribution of unlabeled poly(A)+ mRNA was determined by hybridization to [“HIpoly(U). As expected, about 90% of the poly(A)* mRNA had a sedimentation coefficient of about 9 S. The size distribution of labeled P-globin mRNA sequences was determined by hybridization to recombinant DNA containing an adult ,8-globin cDNA insert (Fig. 4B). On the basis of these results, we identify the labeled 9 S poly(A)+ mRNA as newly made globin mRNA and the labeled 14 S poly(A)+ mRNA as newly

18

DEVELOPMENTAL

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VOISJME

67, 1978

TABLE 1 SUMMARYOFKINETICPARAMETERSOFCHICKENERYTHROIDCELLS UTP Total RNA synthesis Nuclear RNA synthesis Cytoplasmic RNA synthesis Magnesium-precipitated polysomes and ribonucleoprotein particles EDTA-released polysomal RNA Poly(A)’ polysomal RNA 9 S Poly(A)’ polysomal RNA Degradation

rate

constant

for nuclear

RNA

tamoles/min/celll

RNA

(fa/min/celll

0.688 0.617 0.0115 0.00414

+ + -+ +

0.015 0.012 0.0010 0.00025

0.908 0.814 0.0152 0.00546

-t + k +

0.0017 0.0014 0.00062

+ 0.0002 -t 0.9002 + 0.09008

0.0022 0.0018 o.ooo79

+ 0.0003 f 0.0002 f o.oooo9

0.0478

-t 0.0013

min

-

0.020 0.016 0.0013 0.00033

’

made nonglobin mRNA. As can be seen in Fig. 4B, the ratio of incorporated uridine to the amount of poly(A) in nonglobin poly(A)+ mRNA is approximately six times that in globin mRNA. DISCUSSION

From the specific activity of the pools and the accumulation kinetics of radioactivity into total RNA, we have determined the absolute synthetic rates of nuclear and cytoplasmic RNAs. These data indicate that the total rate of RNA synthesis in chicken erythroid cells is only 1.5% that in L cells and 25% that in erythroid cells from rabbit marrow. The fraction of the newly made RNA that exits to the cytoplasm, however, is about the same as that in L cells, rabbit erythroid cells, and Aedes cells in culture, viz., about 2% (Brandhorst and McConkey, 1974; Hunt, 1976; Lengyel and Penman, 1975). These cells thus seem to be highly restricted with respect to the total rate of transcription. Posttranscriptional selections of newly made RNA, however, seem to be quantitatively similar to those in other cells. Appropriateness of measured specific actiuity. Determination of absolute synthetic rates of RNA depends upon knowledge of the specific activity of the RNA precursor pool. It is possible that the specific activity of the total cell UTP measured in these experiments could differ from that of the UTP that serves as precursor to

Froctlon

Number

FIG. 4. Size distribution of labeled poly(A)’ polysomal RNA. Cells were labeled for 60 min and poly(A)’ RNA was isolated from EDTA-released polysomes. The poly(A)’ polysomal RNA was fractionated by centrifugation in 50% DMSO and analyzed as described in Materials and Methods. (A) (M) TCAprecipitable counts per minute; (A---A) ng poly(A). (B) (Ck-0) Counts per minute binding to P-globin recombinant DNA, pHb1001; (W---U) counts per minute binding to nonrecombinant plasmid DNA, pMBS; (A---A) TCA-precipitable counts per minute per nanogram of poly(A).

RNA. For example, a cytoplasmic UTP pool used in carbohydrate metabolism might not equilibrate rapidly with the nuclear pool used to make RNA. In such a case, the calculated rate of RNA synthesis in these experiments would not be correct. On the other hand, if the measured specific

TOBIN,

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RNA

activity was that of the RNA precursor pool, the steady-state level of nuclear RNA calculated from Eq. (2) (JT/~DT) should equal the amount of hnRNA in the nucleus. The kinetic parameters for nuclear RNA shown in Table 1 give a steady-state level of nuclear RNA of 13 amoles of UTP per cell, or approximately 17 fg of RNA. This is very close to the measured amount of nuclear RNA in these cells (Williamson, 1974; Blackman and Tobin, unpublished results). It is about 1% of the amount of nuclear RNA in L cells (Brandhorst and McConkey, 1974). The correspondence of the measured amounts of hnRNA to the steady-state amount calculated from the kinetic data indicates that the measured specific activity of the cellular UTP was the same as that of the RNA precursor pool. Polysomal Poly(A)+ mRNAs. In order to investigate the utilization of newly made RNA in protein synthesis, we have also analyzed the rate of synthesis and size distribution of labeled polysomal RNA. Poly(A)’ mRNA was prepared by oligo(dT) cellulose chromatography of EDTA-released polysomal RNA. About 90% of the poly(A)+ mRNA cosediments with globin mRNA. In contrast, only 45% of the poly(A)+ polysomal RNA synthesized during a 60-min pulse cosediments with globin mRNA, the remaining 55% of the newly made poly(A)’ polysomal RNA having a mean sedimentation coefficient of 14 S. We believe that all of the newly made 9 S poly(A)’ polysomal RNA is globin mRNA. Immature chicken erythroid cells contain two hemoglobins with a common /3 chain (Brown and Ingram, 1974; Vandecasserie et al., 1975). When 9 S poly(A)+ polysomal RNA from these cells is translated in a wheat germ cell-free protein synthesizing system, the three peptides produced are indistinguishable from adult chicken globins in two electrophoretic systems (Colot and Tobin, unpublished results). The sequence complexity of 9 S poly(A)+ polysomal RNA from these cells

Synthesis

in Avian

Erythroid

Cells

19

is about 1700 NT, just enough to code for the three adult globin chains (Lasky et al., 1978). When this globin mRNA is labeled in vitro with 12’1 and hybridized to cellulose-linked recombinant DNA containing a P-globin sequence, about 40% binds (Hansen and Tobin, unpublished results). We conclude from these data that virtually all of the accumulated 9 S poly(A)+ polysomal RNA in immature erythroid cell from adult chickens is globin mRNA and that at least 40% of it is P-globin mRNA. When 12”1labeled accumulated globin mRNA is hybridized to cellulose nitrate filters containing excess recombinant DNA, a maximum of about 11% is bound (Burch-Jaffe and Tobin, unpublished results). Similarly, when newly made 9 S poly(A)’ polysomal RNA (fractions 20-22 in the sucrose gradient shown in Fig. 4) is hybridized to filters containing j?-globin recombinant DNA, 13% is bound. This suggests that, as in the case of the accumulated 9 S mRNA, all of the newly made 9 S poly(A)’ polysomal RNA is globin mRNA. The 14 S labeled poly(A)’ polysomal RNA does not hybridize to a P-globin recombinant DNA and is therefore identified as nonglobin poly(A)* mRNA. The total rates of appearance of globin and nonglobin poly(A)’ mRNA are about equal in these cells, although the globin mRNA accounts for 90% of the accumulated mRNA. Specific activities of globin and nonglobin mRNAs. The specific activity of globin mRNA in the experiment shown in Fig. 4 can be calculated from the ratio of incorporated radioactivity to the amount of poly(A) (Fig. 4B). The number average length of poly(A) in the mRNA from these cells in 40 NT (Lasky et al., 1978), and the counting efficiency was 14%. The calculated specific activity of the UMP in globin mRNA after a 60-min label was thus approximately 62 dpm/pmole, 5.9% of the average specific activity of the UTP in this experiment. A similar calculation can be made for the specific activity of the UMP

20

DEVELOPMENTAL

BIOLOGY

in nonglobin poly(A)’ mRNA, but is subject to the uncertainty of the length of poly(A) in the nonglobin mRNA. Assuming that the poly(A) in nonglobin mRNA has the same average length as the poly(A) in globin mRNA, we calculate that the specific activity of nonglobin poly(A)’ mRNA in these cells is about 130 dpm/pmole, 12% that of the UTP pool in this experiment. If a given mRNA is in the steady state, its half-life may be estimated from the ratio of the specific activity of UMP in the mRNA to the specific activity of the UTP precursor pool, according to Eq. (5): s,(t) -=SUTP(t)

M(t) Mm

= 1 _ e-hv.f’ = 1 _ 2-w/2,

(5)

where SM(t) is the specific activity of the mRNA at time t, &TP(t) is the average specific activity of the UTP pool between the beginning of the incubation and time t, M(t) is the amount of newly made mRNA made between the beginning of the incubation and time t, M, is the steady-state amount of the mRNA, huM is the first-order rate constant for the degradation of the mRNA, and tI/2 is its half-life. We calculate from Eq. (5) that, if globin and nonglobin poly(A)+ mRNAs are in the steady state in these cells, the half-life of the globin mRNA is about 12 hr and that of the nonglobin mRNAs is about 5 hr. These calculations indicate that nonglobin poly(A)+ mRNA in these cells may be degraded approximately twice as rapidly as globin mRNA. Our estimates of mRNA half-lifes are indirect and depend upon the assumption that both globin and nonglobin mRNAs are in the steady state. A direct determination of mRNA half-lives would depend upon achieving relatively long culture times, which we have not yet been able to do with these cells, and a rapid dilution of the RNA precursor pool. The numerical values of the degradation rates derived in our calculations, moreover, depend upon the assumed length of poly(A), since we have not directly

VOLUME

67,1978

measured the specific activity of the UMP in the isolated mRNAs. We have directly determined the number average length of poly(A) in chicken globin mRNA in these cells to be 40 NT, but we have no data concerning the length of poly(A) in nonglobin mRNA (Lasky et al., 1978). In fact, the length of poly(A) in nonglobin mRNA is likely to be longer than that in globin mRNA, since the average age of the accumulated nonglobin mRNA is less than that of the globin mRNA (Mendecki et al., 1972). If the number average length of poly(A) in nonglobin mRNA is greater than 40 NT, the calculated specific activity of nonglobin mRNA would be higher and the estimated half-life would be shorter. If both globin and nonglobin mRNAs are in the steady state, we therefore conclude that nonglobin mRNA is degraded at least 2.4 times more rapidly than globin mRNA. Globin and nonglobin mRNA may not both be in the steady state. The lower specific activity of globin mRNA vis-a-vis nonglobin mRNAs could also reflect a longer period of accumulation of globin mRNA. Indeed, differences in the periods of accumulation of different mRNAs are likely to be important in terminally differentiating cells (Roberts et al., 1972). These differences, rather than changes in mRNA stability during differentiation, could explain the paradoxical accumulation of globin mRNA with respect to demonstrably more stable poly(A)+ mRNA in murine erythroid cells (Bastos and Aviv, 1977). Rates of appearance of individual mRNAs on polysomes. The total rate of appearance of RNA in EDTA-released polysomes is about 2.2 ag/min/cell, about 15% of the rate of appearance of total cytoplasmic RNA (Table 1). About 80% of the newly made polysomal RNA contains poly(A) and binds to oligo(dT) cellulose. From the size distribution of newly made poly(A)+ mRNA, we estimate that globin mRNA appears on polysomes at 0.8 ag/ min/cell and nonglobin poly(A)+ mRNA at 1 ag/min/cell. Since these cells contain

TORJN, SELVJG, AND LASKY

RNA

three species of globin mRNA, we calculate that each species of globin mRNA arrives on polysomes at about 1 molecule/min/cell. The complexity of the nonglobin poly(A)+ mRNA in these cells is 104,000 NT (Lasky et al., 1978). Taking the average length of nonglobin mRNA as 1300 NT, we calculate that each species of nonglobin poly(A)+ mRNA appears on polyosmes at about 0.02 molecule/min/cell. The rate of synthesis of individual species of poly(A)+ mRNA may also be calculated, either (i) from the total rate of synthesis of nuclear RNA, the fraction of newly made nuclear RNA that contains poly(A) (lo%), and the complexity of poly(A)+ hnRNA, or (ii) from the steady-state concentration of individual poly(A)* hnRNA species and the degradation rate constant for nuclear RNA determined from the approach to steady state. For both calculations, we have assumed that rare and abundant poly(A)’ hnRNA species are degraded at the same rate (Lasky, et al., 1978). Using either calculation, we estimate that abundant poly(A)+ hnRNA species are made at about 3 molecules/min/cell and that rare poly(A)’ hnRNA species are made at about 0.02 molecule/min. Nonglobin poly(A)+ mRNAs thus appear on polysomes at about the same rate that rare poly(A)+ hnRNA molecules are synthesized. Each globin mRNA species appears on polysomes at 50 times the rate of each nonglobin mRNA species. The total concentrations of globin and nonglobin poly(A)+ mRNAs in these cells, however, differ by a factor of about 200 (Lasky et al., 1978). Taken together, these data suggest either (i) that the nonglobin mRNAs are degraded more rapidly than the globin mRNAs, or (ii) that the relative rates of appearance of the globin and nonglobin mRNAs change during erythropoiesis. Most of the 200-fold difference in the final concentrations of individual mRNA species can be attributed to the different rates of appearance of individual mRNAs on polysomes. These differences can in turn

Synthesis

in Ackn

Erythroid

Cells

21

be due either to differences in rates of transcription, differences in the efficiencies of posttranscriptional processing, or differences in the efficiencies of initiation of translation. The relative roles of transcriptional and posttranscriptional regulation in differential gene expression are discussed in the accompanying paper (Tobin, 1978). We are grateful to Dr. Phyllis Brown for her advice on high-pressure liquid chromatography and Dr. J. D. O’Connor for the use of his chromatography apparatus. We thank Ms. Elizabeth Burch-Jaffe for preparing the filter-bound plasmid DNA; Drs. Tom Humphreys, Hans Lehrach, and Judith Lengyel for helpful discussions; and Ms. Ellen Thompson, Ms. Chai-Chi Kung, and Mr. Donald Clark for technical assistance. This work was supported by the UCLA University Research Committee, NIH Biomedical Research Support Grant to UCLA, National Science Foundation Grant PCM 7602859, and a Basil O’Connor Starter Research Grant from the National Foundation-March of Dimes. LL was supported in part by USPHS Institutional National Research Service Award GM-07104. REFERENCES BANTJX, J. A., AND HAHN, W. E. (1976). Complexity and characterization of polyadenylated RNA in the mouse brain. Cell 8, 139-150. BASTOS, R. N., VOLLOCH, Z., AND AVIV, H. (1977a). Messenger RNA population analysis during erythroid differentiation: A kinetical approach. J. Mol. Biol. 110,191-203. BASTOS, R. N., AND AVIV, H. (197713). Theoretical analysis of a model for globin messenger RNA accumulation during erythropoiesis. J. Mol. Biol. 110, 205-218. BISHOP, J. O., ROSBASH, M., AND EVANS, D. (1974). Polynucleotide sequences in eukaryotic DNA and RNA that form ribonuclease resistant complexes with polyuridylic acid. J. Mol. Biol. 85, 75-86. BRANDHORST, B. P., AND MCCONKRY, E. H. (1974). Stability of nuclear RNA in mammalian cells. J. Mol. Biol. 85, 451-463. BROWN, J. L., AND INGRAM, V. M. (1974). Structural studies on chick embryonic hemoglobins. J. Biol. Chem. 249,3960-3972. BURTON, K. (1956). Study of conditions and mechanism of diphenylamine reaction for calorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315-323. BROWN, P. (1971) Stability of nucleotide solutions on storage as determined by high-pressure liquid chromatography. Anal. Biochem. 43, 305-306. DRRMAN, E., GOLDBERG, S., AND DARNEI,I., J. E. (1976). HnRNA in HeLa cells: Distribution of transcript sizes estimated from nascent molecule profile.

22

DF,VEI.OPMF.NTAI,

Bror.ocu

Cell 9,465-472. GAI,AU, G. A., LIPSON, E. D., BRITTF,N, R. J., ANI) DAVIDSON, E. H. (1977). Synthesis and turnover of polysomal mRNAs in sea urchin embryos. Cell 10, 415-432. GILLESPIF., D., AND SPIEGELMAN, S. (1965). A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J. Mol. Biol. 12, 829-842. HUMPHRIES, S., WINDASS, J., AND WILLIAMSON, R. (1976). Mouse globin gene expression in erythroid and non-erythroid tissues. Cell 7, 267-277. HUNT, J. A. (1976). Ribonucleic acid synthesis in rabbit erythroid cells. Analysis of rates of synthesis of nuclear and cytoplasmic ribonucleic acid. Biochem. J. 160, 727-744. HYNF.S, N. E., GRONER, B., SIPPEL, A. E., NGUYENHuu, M. C., AND SCHULTZ, G. (1977). mRNA complexity and egg white protein mRNA content in mature and hormone-withdrawn oviduct. Cell 11, 923-932. KAFATOS, F. C. (1972). The cocoonase zymogen cells of silk moths: A model of terminal cell differentiation for specific protein synthesis. Curr. Top. Develop. Biol. 7, 125-191. KLEIMAN, L., BIRNIE, G. D., YOUNG, B. D., AND PAUL, J. (1977). Comparison of base sequence complexities of polysomal and nuclear RNAs in growing Friend erythroleukemia cells. Biochemistry 16, 1218-1228. KRYSTOSEK, A., CAWTHON, M. L., AND KABAT, D. (1975). Improved methods for purification and assay of eukaryotic messenger ribonucleic acids and ribosomes. J. Biol. Chem. 250,6077-6084. LASKY, L., NOZICK, N. D., AND TOBIN, A. J. (1978). Few transcribed RNAs are translated in avian erythroid cells. Deuelop. Biol. 67, 23-39. LF.NGYF,I,, J. A., AND PENMAN, S. (1975). HnRNA size and processing as related to different DNA content in two dipterans Drosophila and Aedes. Cell 5, 281-290. MARKS, P. A., BURKA, E. R., AND SCHLESSINCER, D. (1962). Protein synthesis in erythroid cells. I. retic-

VOUJMI?

67, 1978

ulocyte ribosomes active in stimulating amino acid incorporation. Proc. Nat. Acad. Sci. USA 48, 2183-2171. MENDIXKI, J., LF,F,, S. Y., AND BRAWERMAN, G. (1972). Characteristics of the polyadenylic acid segment associated with messenger ribonucleic acid in mouse sarcoma 180 ascites cells. Biochemistry 11, 792-798. PAI,MITER, R. D. (1974). Magnesium precipitation of ribonucleoprotein complexes. Expedient techniques for the isolation of undegraded polysomes and messenger ribonucleic acid. Biochemistry 13,3606-3615. PUCKETT, L., CHAMBERS, S., AND DARNELL, J. E. (1975). Short-lived messenger RNA in HeLa cells and its impact on the kinetics of accumulation of cytoplasmic polyadenylate. Proc. Nat. Acad. Sci. (USA) 72, 389-393. RANDERATH, K., AND RANDERATH, E. (1967). Thin layer separation methods for nucleic acid derivatives. In “Methods in Enzymology” (L. Grossman and K. Moldave, eds.), Vol. 12A, pp. 323-347. Academic Press, New York. ROBERTS, A. V., WEATHERALL, D. J., AND CLEGG, J. B. (1972). The synthesis of human hemoglobin AZ during erythroid maturation. Biochem. Biophys. Res. Commun. 47,81-87. SCHOLTISSEK, C. (1971). Detection of an unstable RNA in chick fibroblasts after reduction of the UTP pool by glucosamine. Eur. J. Biochem. 24,358-365. STEWART, J. A., AND PAPACONSTANTINOU, J. (1967). Stabilization of mRNA templates in bovine lens epithelial cells. J. Mol. Biol. 29, 357-370. TOBIN, A. J. (1978). Evaluating the contribution of posttranscriptional processing to differential gene expression. Develop. Biol. 67, in press. VANDECASSERIE, C., PAUL, C., SCHNER, A. G., AND L~ONIS, J. (1975). Probable identity of the /l chains from the two chicken hemoglobin components. Biochemie 57,843-844. WILLIAMSON, P. L., AND TOBIN, A. J. (1977). The heterogenous nuclear RNA of chicken erythroblasts. Biochim. Biophys. Acta 475, 366-382.