Stability of α-fetoprotein messenger RNA in mouse yolk sac

DEVELOPMENTAL

BIOLOGY

83.111-116 (1382)

Stability of a-Fetoprotein GLEN Department

K. ANDREW&’

of Medical

Received

Biochemistry,

Messenger RICHARD

G. JANZEN,

University

of Calgary,

January 13, 1981; accepted

in revised

RNA in Mouse Yolk Sac AND TAIKI Calgary,

form

Alberta

August

TAMAOKI’ T2N

1N4, Can&a

4, 1981

Incubation of mouse yolk sac explants in the presence of ol-amanitin led to a 2.7-fold reduction in total protein synthesis and a 2-fold increase in the relative rate of cu-fetoprotein synthesis. This indicated that a-fetoprotein messenger RNA was more stable than other yolk sac messenger RNA. This was further documented by pulse-chase experiments in which decay of o-fetoprotein messenger RNA was monitored by hybridization to filter-bound o-fetoprotein complementary DNA. cu-Fetoprotein messenger RNA was found to be stable during a 30-hr-chase period whereas most other poly(A)+ RNA species decayed with a half-life of 6 hr. There appeared to be no changes in the stability of c*-fetoprotein messenger RNA from Day 11.5 to Day 17.5 of gestation. This supports the conclusion that the selective stabilization of a-fetoprotein messenger RNA is an important mechanism for maintaining the abundancy of this message in the developing yolk sac. INTRODUCTION

MATERIALS

a-Fetoprotein (AFP) is synthesized by the liver and yolk sac during mammalian fetal development (Abelev, 1971; Ruoslahti et al, 1974; Hirai, 1979; Sell et d, 1980). We have focused attention on AFP synthesis in the developing mouse yolk sac because the yolk sac is a relatively simple structure amenable to biochemical analysis. Our recent studies of AFP synthesis in the mouse yolk sac have shown that (i) the relative rate of AFP synthesis increases steadily from Day 9.5 to Day 15 of gestation reaching to 25% of total protein synthesis, then gradually decreases to 18% near term (Janzen et al., 1982), and (ii) there is one copy of the AFP gene per haploid genome in yolk sac cells (submitted for publication). Comparative studies of AFP gene organization in the yolk sac and other AFP-producing and nonproducing tissues indicate that amplification, deletion, or gross sequence rearrangements are not involved in regulation of AFP gene expression (submitted for publication; Sala-Trepat et aZ., 1979). As a part of our continued studies on regulation of AFP gene expression we examined the stability of AFP mRNA in the developing yolk sac. This paper describes experimental results which show that the half-life of AFP mRNA is at least five times greater than the majority of yolk sac mRNAs. This indicates an involvement of AFP mRNA stabilization in regulation of AFP gene expression during mouse yolk sac development. ’ National Institutes of Health Postdoctoral Fellow. ’ Research Associate of the National Cancer Institute

of Canada.

AND METHODS

Assay of protein synthesis. Yolk sac explants were incubated in Eagle’s minimum essential medium (MEM) supplemented with nonessential amino acids, penicillin (100 units/ml) and streptomycin (100 pg/ml) at 37°C with vigorous shaking. When used, a-amanitin was present throughout the incubation at 10 )cg/ml (Somers et aL, 1975). The medium was replaced at indicated times with prewarmed methionine-free medium containing 50 pCi/ml of L-[35S]-methionine (935 Ci/mmol, New England Nuclear) and incubation was continued for 1 hr. Phenylmethylsulfonyl fluoride, methionine, sodium deoxycholate (DOC), and Triton X-100 were added to the culture to final concentrations of 100 piV, 1 mM, 0.5%, and 0.5%) respectively, and the culture was then subjected to three cycles of freeze and thaw. The concentration of DOC and Triton X-100 was increased to 1% and the freeze-thaw procedure was repeated three more times. The lysate was centrifuged at 100,000~ for 60 min and the supernatant was assayed for total protein synthesis by measuring radioactivity in hot trichloroacetic acid (TCA)-precipitable material. Assay of AFP synthesis. AFP synthesis was assayed by measuring radioactivity in the precipitate formed by antibodies to mouse AFP as described previously (Miura et aL, 1979). Two-dimensional gel electrophoresis. Two-dimensional gel electrophoresis was performed according to O’Farrell(l975) with some modifications. Samples were heated at 70°C for 20 min in the presence of 0.5% SDS and electrophoresed in 4% gels (0.3 X 10.5 cm) containing 9 M urea, 2% Nonidet P-40, and 2% ampholines

111 0012-1606/82/010111-06$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

DEVELOPMENTAL BIOLOGY

VOLUME 89,1982

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FIG. 1. Effect of cY-amanitin of AFP synthesis in the yolk sac. Day-15.5 yolk sac was incubated with [%]methionine in the presence or absence of cr-amanitin as described under Materials and Methods. Protein synthesis was determined by hot tricbloroacetic acid precipitation and AFP synthesis by immunoprecipitation. Total protein in the yolk sac was determined according to Bradford (1976). (A) Time course of total protein synthesis (0) and AFP synthesis (0) in the presence of a-amanitin. (B) Changes in the relative rate of AFP synthesis in control (m) and a-amanitin-treated (0) cultures as a function of incubation time.

(Bio-Rad; pH 4-6, pH 4-8, pH 3-10; 2:2:1) at 350 V for 19 hr. The gels were then equilibrated with a buffer containing 3% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 62.5 mM Tris-HCl, pH 6.8, for 1 hr. The second-dimension electrophoresis was performed on 816% linear gradient slab gels. The gels were stained with Coomasie brilliant blue and radioactive proteins were detected by fluorography using Kodak XR-1 film according to Bonner and Laskey (1974). Pulse-chase expe&nents. Uridine pulse-chase experiments were conducted by incubating yolk sac explants in the media described above but also containing rH]uridine (250 &i/ml, 50 Ci/mmol, Amersham) for 4 hr followed by incubation for 1 to 30 hr in nonradioactive medium containing 5 mM uridine, 5 mM cytidine, and 5 mM glucosamine (Scholtissek, 1971; Levis and Penman, 19’77). Binding of AFP cumpkmentayl DNA (AFP cDNA) tofilters. Binding of AFP cDNA to nitrocellulose filters was done essentially according to Gillespie (1968). A chimeric plasmid containing a 900-base-pair AFP cDNA insert (Law et aL, 1980) was linearized by cleavage with the restriction endonuclease EcoRI and denatured in 0.4 M NaOH (25 pg DNA/ml). An equal volume of cold 2 M ammonium acetate, pH 7.0, was added and the solution was pumped through a 2.5-cm nitrocellulose filter

(BA-85, Schleicher and Schuell), at a flow rate of 500 pl/min. The filters were subsequently processed as described by Tsai et al (1980a), which involved baking at 70°C followed by treatment with Denhardt’s solution (Denhardt, 1966). Smaller disks (4 mm in diameter) were punched from the processed filter. Each of the small discs contained 7-10 pg of DNA. Control filters which contained an equivalent amount of chicken DNA or no DNA were also prepared as described above. Hybridization of labeled yolk sac RNA to~lter-bound AFP cDNA. Labeled total cellular RNA was isolated by SDS-phenol extraction (Andrews and Teng, 1979) and poly(A)+ RNA was fractionated by oligo d(T)-cellulose chromatography (Aviv and Leder, 1972). Hybridization of labeled RNA to filter-bound cDNA was carried out according to Tsai et al. (1980b). RNA (20-100 pg; 5 X 105-5 X lo6 cpm) was incubated for 18 hr at 66°C in 100 ~1 of hybridization buffer (0.6 M NaCl, 10 mM Hepes, pH 7.0, 3 mM EDTA, 0.1% SDS, 10 pg/ml poly(A)) in the presence of one cDNA-containing filter and one control filter. After hybridization the filters were washed in 2 ml of 2X SSC, 0.1% SDS (1X SSC = 150 mM NaCl, 15 mM sodium citrate, pH 6.8) for 15 min at 22”C, 1 ml of hybridization buffer for 1 hr at 66”C, 4 ml of 2~ SSC, 0.1% SDS, for 1 hr at 66”C, and 4 ml of 2~ SSC for 1 hr at 22°C. Filters were then

ANDREW& JANZEN, AND TAMAOKI

a-Fetqmotein

Mess~w

RNA Stability

25

25

FIG. 2. Two-dimensional gel electrophoretic analysis of proteins synthesized in control and a-amanitin-treated yolk sacs. Yolk sacs were incubated overnight in the absence (A) or presence (B) of a-amanitin. The incubation was continued for 1 hr with [%]methionine, and radioactive proteins (200,000 cpm) from control and treated yolk sacs were analyzed by two-dimensional gel electrophoresis. Tf is transferrin.

incubated in 500 pl of 2X SSC containing 20 pg/ml RNase A for 1 hr at 22’C, washed twice with 2 ml of 2~ SSC for 15 min at 22”C, and dried. Radioactivity

associated with washed filters was determined by liquid scintillation counting in toluene-based scintillation fluid at 25% efficiency of counting for tritium.

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VOLUME 89.1982

ence of cY-amanitin total protein synthesis decreased 2.‘7-fold in 24 hr, while AFP synthesis remained relatively constant (Fig. 1A). As a result, the relative rate of AFP synthesis increased 2-fold to nearly 40% of the total protein synthesis (Fig. 1B). These results indicated that AFP may be translated from an mRNA which is more stable than the average yolk sac mRNA. Figure 2 shows two-dimensional gel electrophoretic analysis of proteins synthesized in cY-amanitin-treated and control yolk sacs. A significant reduction of minor protein spots was observed on the gel of the treated yolk sac (Fig. 2B). In addition to AFP, transferrin and proteins X and Y became dominant protein species in the cY-amanitin-treated yolk sac. All these proteins were found to be secretory proteins (Janzen et al., 1982). Kinetics of Decay of Total Cellular Poly(A)+ RNA

0 0-e

RNA and

o--qAz

20 1 0

ii

ib

15 HOURS

20

i5

30

OF CHASE

FIG. 3. Kinetics of decay of total RNA and poly(A)+ RNA of yolk sacs at different stages of gestation. Yolk sacs were labeled with [3H]uridine for 4 hr and changes in specific activity of labeled total RNA (0) and poly(A)+ RNA (0) during a chase were determined. Decay is expressed as percentage of the zero time value. (A) Day-11.5 yolk sac RNA; (B) Day-15.5 yolk sac RNA; (C) Day-17.5 yolk sac RNA.

Determination of thermal melting pro$les of RNADNA hybrids. Filters containing AFP cDNA-RNA hybrids were placed in 200 ~1 of 2X SSC and incubated at temperatures ranging from ‘70 to 95°C for 2 min. The buffer was recovered and the filter was washed with 100 ~1 of cold buffer. The wash was combined with the original buffer. The total radioactivity released was determined by liquid scintillation counting in xylenebased scintillation fluid. The percentage of radioactivity released was plotted as a function of incubation temperature. The T,,,, the temperature at which 50% of the hybrid was denatured, was determined from the curve. RESULTS AND DISCUSSION

Synthesis of AFP in Yolk Sac Explants a-Amanitin

and Effects of

Yolk sac explants were incubated with [?S]methionine in the presence or absence of a-amanitin. In the pres-

We analyzed the stability of total cellular RNA and poly(A)+ RNA of the yolk sac at Days 11.5, 15.5, and 17.5 by pulse-labeling these RNAs with [3H]uridine and following their decay during the subsequent chase with cold nucleotides. The decay of total cellular RNA was slow at all three stages of gestation; 50% decay was not reached within the 30-hr-chase period (Fig. 3). By extending the decay curves we estimated the half-life of the total RNA preparations to be about 55 hr. This long half-life was likely due to ribosomal RNA in the samples (Emerson, 1971; Singer and Penman, 1973). A 4-hr pulse was used to label poly(A)+ RNA as well as total cellular RNA. This long pulse may have led to an overrepresentation of stable transcripts in the total labeled RNA and to an overestimation of the half-life of poly(A)+ RNA. However, this did not appear to be the case as the initial kinetics of decay of poly(A)+ RNA in Day-11.5 and -15.5 yolk sacs indicated a half-life of about 6 hr (Figs. 3A and B) which is similar to the initial kinetics of decay of poly(A)+ RNA in other cell types (Greenberg, 1975). In contrast, the poly(A)+ RNA in Day-17.5 yolk sac decayed with a half-life of about 15 hr. This indicated that stable mRNA species were more predominant in the yolk sac toward the end of gestation. Kinetics

of Decay of AFP mRNA

In order to determine the half-life of AFP mRNA directly, we analyzed the kinetics of decay of AFP mRNA by hybridization of pulse-labeled RNA to AFP cDNA immobilized on nitrocellulose filters. Similar experimental approaches have successfully been used to quantitate relative rates of transcription and decay of

ANDREW&

JANZEN,

a-Fetoprohin

AND TAMAOKI

TABLE CHARACTERIZATION

DNA bound to filter (A) None Chicken AFP cDNA AFP cDNA

Day Day Day Day

(B) None AFP cDNA (C) Chicken AFP cDNA

11.5 11.5 11.5 11.5

yolk yolk yolk yolk

1

OF THE FILTER

Input nucleic acid sac RNA sac RNA sac RNA sac RNA

115

Messenger RNA Stability

HYDRIDIZATION ASSAY Bound radioactivity (cpm)

Input radioactivity (cw) 0.9 1.0 0.9 1.0

x x x x

Percentage bound”

lo6 lo6 lo6 lo6

18 23 296 275

0.004 0.005 0.066 0.055

Chinese hamster cell RNA Chinese hamster cell RNA

1.5 x lo6 1.5 x lo6

21 29

0.003 0.004

rH]AFP cDNAb [3H]AFP cDNA

1.7 x lo4 1.7 x lo4

54 994

0.6 11.7

’ % bound = (bound cpm/input cpm) X 100 X 2 (the factor of two was employed to correct for efficiency of counting of 3H-labeled RNA bound to filters compared to counting this RNA free in liquid scintillation media). b rH]AFP cDNA was synthesized as described by Miura et al. (1979).

several other mRNA species (Guyette et al, 1979; McKnight and Palmiter, 1979; Mullinix et aL, 1979). Initially, we performed several control experiments to examine the validity of the assay procedure (Table 1). When labeled yolk sac RNA was incubated with a filter which contained no DNA or chicken DNA, only 0.004% of the input radioactivity was retained by the filter. At least lo-fold more radioactivity was retained by the AFP cDNA-containing filter (Table 1A). RNA from Chinese hamster ovary cells (non-AFP-producing cells) did not hybridize either to control or AFP cDNA filters (Table 1B). Annealing of picogram quantities of 3H-labeled AFP cDNA with filter-bound AFP cDNA resulted in retention of 12% of the radioactivity while control filters retained less than 0.6% of the label (Table 1C). Furthermore, the T, of hybrids retained by the AFP cDNA filter was 88’C which was similar to the T,,, value of 92°C previously reported for AFP mRNA-cDNA hybrids formed in solution (Miura et cd, 1979). These results indicated that the filter hybridization assay was highly specific and sensitive for AFP mRNA. The decay of AFP mRNA in total cellular RNA from Day-11.5, -15.5, and -17.5 yolk sacs is presented in Fig. 4. The results showed that AFP mRNA did not decay appreciably during the 30-hr-chase period, Longer chase periods were not attempted due to loss of yolk sac viability. However, by extrapolating results from several experiments the half-life of AFP mRNA was estimated to be between 60 and 120 hr. There appeared to be no significant changes in the stability of AFP mRNA from Day 11.5 to Day 17.5 of gestation. The decay of AFP mRNA in Day-11.5 yolk sac was also assayed using a poly(A)+ RNA preparation (Fig. 4A). The decay curve was identical with that obtained with the total RNA preparation. The results presented above indicated that the half-

life of AFP mRNA was at least five times greater than the half-life of the majority of yolk sac mRNA species at Days-11.5 and 15.5 of gestation. Rat AFP mRNA in cultured hepatoma cells has been shown to have a halflife of 40 hr which is five-fold greater than the average 1

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FIG. 4. Kinetics of decay of AFP mRNA of yolk sacs at different stages of gestation. Yolk sacs were incubated with rH]uridine for 4 hr and the decay of 3H-labeled AFP mRNA in total RNA (0) or poly(A)+ RNA (0) during a chase was determined by hybridization to filter-bound AFP cDNA as described under Materials and Methods. Decay is expressed as percentage of the highest value. (A) Day-11.5 yolk sac RNA, (B) Day-15.5 yolk sac RNA; (C) Day-17.5 yolk sac RNA.

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DEVELOPMENTAL BIOLOGY

hepatoma mRNA (Innis and Miller, 1979). Selective stabilization of mRNA has been shown to occur in several cell types which synthesize a large amount of specific proteins (Greenberg, 1975). Modulation of mRNA stability may also be involved in hormonal regulation of protein synthesis (Guyette et ak, 1979; Palmiter and Carey, 1974). Hybridization analysis using AFP cDNA has shown that the number of AFP mRNA molecules per cell increases during yolk sac development (Nishi et aZ., 1979; Janzen et al., 1982). At Day 15.5, AFP mRNA represents about 20% of the total mRNA population. It is conceivable that the high stability of AFP mRNA shown in this study plays an important role in ensuring the abundancy of this mRNA observed during yolk sac development. We thank Mr. John Stockton, Drs. K. Ohara, and L. Bryan for assistance in propagating plasmid DNA. We also thank Mrs. Joanne Moggert for technical assistance. This work was supported by the National Cancer Institute of Canada, the Medical Research Council of Canada, and the Alberta Heritage Savings Trust Fund. REFERENCES ABELEV, G. I. (1971). Alpha-fetoprotein in ontogenesis and its association with malignant tumors. Advan Cancer Res. 14.295-358. ANDREW& G. K., and TENG, C. S. (1979). Studies on sex-organ development: Prenatal effect of oestogenic hormone on tubular gland cell morphogenesis and ovalbumin gene expression in the chick Mullerian duct. B&hem, f. 182,271-286. AVIV, H., and LEDER, P. (1972). Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid cellulose. Proc. Nat. Acad Sci. USA 69,1408-1412. BONNER, W. M., and LASKEY, R. A. (1974). A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. B&hem. 45,83-88. BRADFORD,M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochcm, 72, 248-254. DENHARDT, D. T. (1966). A membrane-filter technique for the detection of complementary DNA. Biochxm Biophys. Res. Commun. 23, 641-646. EMERSON, C. P. (1971). Regulation of the synthesis and the stability of ribosomal RNA during contact inhibition of growth. Nature New BioL 232,101-106. GILLESPIE, D. (1968). The formation and detection of DNA-RNA hybrids. 1% “Methods in Enzymology” (K. Moldave and L. Grossman, eds.), Vol. 12B, pp. 641-668. Academic Press, New York. GREENBERG, J. R. (1975). Messenger RNA metabolism of animal cells: Possible involvement of untranslated sequences and mRNA-associated proteins. J. CeU BioL 64, 269-288. GUYETTE, N. A., MATUSIK, R. J., and ROSEN, J. M. (1979). Prolactinmediated transcriptional and post-transcriptional control of casein gene expression. CeU 17.1013-1023. HIRAI, H. (1979). Model systems of AFP and CEA expression. In “Methods in Cancer Research” (H. Busch, ed.), pp. 39-97. Academic Press, New York. INNIS, M. A., and MILLER, D. L. (1979). ol-Fetoprotein gene expression: Control of cY-fetoprotein mRNA levels in cultured rat hepatoma cells. J. BioL Chem. 254, 9148-9154. JANZEN, R. G., ANDREWS, G. K., and TAMAOKI, T. (1982). Synthesis

VOLUME 89, 1982

of secretory proteins in developing mouse yolk sac. Develop. Biol., in press. LAW, S., TAMAOKI, T., KREUZALER, F., and DUGAICZYK, A. (1980). Molecular cloning of DNA complementary to a mouse a-fetoprotein mRNA sequence. Gene 10,53-61. LEVIS, R., and PENMAN, S. (1977). The metabolism of poly(A)+ and poly(A)) hnRNA in cultured Drosophila cells studied with a rapid uridine pulse-chase. Cell 1.105-113. MCKNIGHT, S. G., and PALMITER, R. D. (1979). Transcriptional regulation of the ovalbumin and conalbumin genes by steroid hormones in chick oviduct. J. BioL Chem. 254,9050-9058. MIURA, K., LAW, S., NISHI, S., and TAMAOKI, T. (1979). Isolation of a-fetoprotein messenger RNA from mouse yolk sac. J. Biol Chem 254, 5515-5521. MULLINIX, K. P., MEYERS, M. B., CHRISTMANN, J. L., DEELEY, R. G., GORDON,J. I., and GOLDBERGER, R. F. (1979). Specific transcription in chicken liver chromatin hy endogenous RNA polymerase II: Comparison of an estrogen-inducible gene with a constitutively expressed gene. J. BioL Chem 254.9860-9866. NISHI, S., MIURA, K., and TAMAOKI, T. (1979). Mouse alpha-fetoprotein mRNA: Characterization and quantitation. In “Carcinoembryonic Proteins” (F. G. Lehman, ed.), Vol. 1, pp. 137-143. Elsevier, Amsterdam. O’FARRELL, P. H. (1975). High resolution two-dimensional phoresis of proteins. J BioL &em. 250,4007-4021.

electro-

PALMITER, R. D., and CAREY, N. H. (1974). Rapid interaction of ovalbumin messenger ribonucleic acid after acute withdrawal of estrogen. Proc Nat. Acad Sci. USA 71,2357-2361. RUOSLAHTI, E., PIHKO, H., and SEPPALA, M. (1974). Alpha-fetoprotein: Immunochemical purification and chemical properties: Expression in normal and in malignant and non-malignant liver disease. Transplant. Rev. 20,38-60. SALA-TREPAT, J. M., SARGENT, T. D., SELL, S., and BONNER, J. (1979). cY-Fetoprotein and albumin genes of rats: No evidence for amplification, deletion or rearrangement in rat liver carcinogenesis. Proc. Nat Acad Sci USA 76,695-699. 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. SELL, S., SALA-TREPAT, J. M., SARGENT, T. D., THOMAS, K., NUHON, J. L., GOODMAN, J. A., and BONNER, J. (1980). Molecular mechanisms of control of albumin and alpha-fetoprotein production: A system to study early effects of chemical hepatocarcinogenesis. CelE BioL Int Rep. 4, 235-254. SINGER, R. H., and PENMAN, S. (1973). Messenger RNA in Hela cells: Kinetics of formation and decay. J. Mel BioL 78, 321-334. SOMERS,D. G., PEARSON, M. L., and INGLES, C. J. (1975). Isolation and characterization of an cy-amanitin-resistant rat myoblast mutant cell line possessing a-amanitin-resistant RNA polymerase II. J. BioL Chum. 250,4825-4831. TSAI, M. -J., LAWSON, G. M., TOWLE, H. C., TSAI, S. Y., MCCLURE, M. E., and O’MALLEY, B. W. (198Oa). Gene transcription in nuclei and chromatin. In “Laboratory Methods Manual for Hormone Action and Molecular Endocrinology” (W. T. Schraeder and B. W. G’Malley, eds.), Chap. ‘7, pp. l-79. TSAI, S. Y., ROOP, D. R., STUMPH, W. E., TSAI, M. -J., and O’MALLEY, B. W. (198Ob). Evidence that deoxyribonucleic acid sequences flanking the ovalbumin gene are not transcribed. Biochemistry 19,17551761.