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Differential regulation and polyadenylation of transferrin mRNA in Xenopus liver and oviduct
J. Steroid Biochem. Molec. Biol. Vol. 42, No. 7, pp. 649~57, 1992
I)960-0760/92 $5.00 + 0.00 Pergamon Press Ltd
Printed in Great Britain
DIFFERENTIAL OF
REGULATION
TRANSFERRIN
AND
mRNA AND
POLYADENYLATION
IN XENOPUS LIVER
OVIDUCT
RICARDO L. PASTORI, JOHN E. MOSKAITIS, SUSAN W. BUZEK a n d DANIEL R. SCHOENBERG* Department of Pharmacology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799, U.S.A. (Received 9 November 1991)
Summary--Estrogen destabilizes transferrin mRNA in male Xenopusliver in the same manner as observed for albumin and 7-fibrinogen. The present study examined estrogen regulation of transferrin gene expression in female Xenopus liver and oviduct. In female Xenopus liver estrogen causes the same enhanced degradation of transferrin mRNA from the cytoplasm as seen in males. In contrast, transferrin is induced 3- to 4-fold in both oviduct nuclear and cytoplasmic RNA. The similar increase in transferrin RNA in both preparations suggests a transcriptional mechanism is responsible for this stimulation. Therefore, transferrin expression is differentially regulated in these tissues by the same hormone. Previous experiments showed that Xenopus serum albumin mRNA has a very short (17 residue) poly(A) tail that may play a role in its hormone-regulated instability. Transferrin mRNA has a similarly short poly(A) tail in liver of both male and female Xenopus. Estrogen has no effect on transferrin polyadenylation in liver. Similarly short poly(A) is found on transferrin mRNA from estrogen-deprived oviducts in explant culture. However, addition of estradiol to the medium results in the appearance of a 50-200 nucleotide poly(A) concurrent with induction. Therefore, transferrin mRNA is differentially polyadenylated in Xenopus liver and oviduct. In the latter tissue polyadenylation is under hormonal control.
INTRODUCTION
The female sex steroid estradiol exerts a broad spectrum o f effects on gene expression in oviparous vertebrates. In chick liver estradiol causes the transcriptional induction of the genes encoding the yolk protein precursor vitellogenin [1], and apo-very low density lipoprotein II (apoV L D L II[2]). Apo-VLDL II mRNA is subsequently stabilized in the continued presence of estradiol, however abrupt withdrawal of hormone is accompanied by rapid degradation [3]. Also in avian liver estrogen causes a slight increase in both transferrin gene transcription and mRNA. In chick oviduct estrogen induces ovalbumin transcription and causes a significant stimulation in conalbumin transcription (reviewed in[4]). Several lines of evidence, including D N A sequence analysis, have shown conalbumin and transferrin to be the same protein [5]. Microinjection of SV40 T antigen reporter constructs bearing the promoter and 5' flanking regions of ovalbumin and transferrin *To whom correspondence should be addressed. 649
into primary cultures of a variety of chicken cells shows both promoters to be active only in hepatocytes and oviduct tubular gland cells [6]. In liver cells neither promoter is stimulated by estradiol, whereas both are stimulated by hormone in oviduct cells. Therefore, while avian liver and oviduct are both estrogen responsive, there is differential transcriptional regulation of the transferrin gene in each tissue. In liver of both birds and amphibia estrogen causes the transcriptional induction of the vitellogenin genes (reviewed in [7, 8]). In frogs estrogen also stabilizes vitellogenin m R N A [8] and destabilizes serum albumin [9] and ?-fibrinogen [10] mRNA. The changes in the stabilities of these mRNAs, like the induction of vitellogenin [11] are dependent on the action of the estrogen receptor[12]. A recent report from this laboratory demonstrated that transferrin mRNA is also destabilized by estrogen in Xenopus liver[13]. This is in contrast to the minimal increase in transferrin mRNA observed in avian liver [14]. In general, studies on estrogen-regulated gene expression in chicken and Xenopus liver
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RICARDO L. PASTORI et al.
have shown considerable similarity between these two species [7]. Therefore the differences in transferrin expression were striking. This led us to examine the regulation of transferrin expression in Xenopus oviduct. Tata and coworkers [15, 16] have identified a number of cDNAs whose cognate mRNAs are induced by estrogen in primary cultures of oviduct cells. None of these corresponds to known mRNAs, although FOSP-I (frog oviduct-specific protein-I) has been subjected to DNA sequence analysis[16]. FOSP-! mRNA is unique to oviduct and its mRNA is induced 5-fold by estrogen. In the present study we report that transferrin mRNA is expressed constitutively in the absence of estradiol in oviduct explant cultures. Upon addition of estradiol to the culture medium transferrin mRNA is induced 3- to 4-fold. The transcriptional induction of transferrin mRNA in oviduct contrasts with its posttranscriptional destabilization in liver. In addition, the estrogen-induced transferrin mRNA in oviduct has a significantly longer poly(A) than that found in liver or nonstimulated oviduct.
10-rM estradiol was added twice daily to the medium. Oviduct nuclear RNA was prepared by our previously described procedure[18] modified by an extraction procedure that removes the viscous jellycoat proteins[19]. Briefly, tissue fragments from oviduct explant cultures were washed for 5min (10ml/mg of tissue) in DB-Tris buffer, pH 8.9, followed by a wash in DB-Tris buffer, pH 7.8. DB-Tris consisted of 0.11 M NaCI, 1.34mM CaCI2, 1.32 mM KC1, 10 mM Tris-HCl. The tissue was next rinsed in 5 ml/gm 0.3 M sucrose, 10mM Tris-HCI, pH 8.0 followed by homogenization in the same buffer. The homogenate was diluted 2-fold in sucrose-Tris and filtered through nitex mesh as described previously for preparation of liver nuclear RNA [18]. This was then diluted 2-fold again with 2 M sucrose, 10mM Tris, pH 8.0 and layered over 15 ml of 2 M sucrose in a SW27 centrifuge tube. The nuclei were collected by centrifugation at 25,000 rpm in an SW27 rotor. RNA was subsequently extracted from the nuclear pellet. All of the preceding steps were performed at 4:C.
EXPERIMENTAL
The analysis of transferrin and vitellogenin polyadenylation was performed as described previously[18] by a modification of the procedure of Donis-Keller [20]. An oligonucleotide (5'-ATGTGCAGGCAGCCA-Y) complementary to the sequence -166 nucleotides 5' to the poly(A) addition site was used to direct cleavage of transferrin mRNA by RNase H. Cleavage of A2 vitellogenin mRNA was directed by the oligonucleotide 5'-CACATGAACAAGCGGTG-3' to generate a fragment containing the terminal 205 nucleotides plus poly(A). 10 #g of total liver or oviduct RNA was mixed with 3 #g of oligonucleotide + 3 #g oligo(dT)~2_~s in 19 #1 40 mM Tris-HCl, pH 7.9, 4 mM MgCI 2, 1 mM dithiothreitol, 30 ng/ml bovine serum albumin. This was heated at 85°C for 5 min 42°C for 10min and cooled to room temperature. Two units of RNase H was then added and the samples were incubated at 37°C for 30min. The reaction was terminated by the addition of 80/11 H20, followed by sequential extraction with HCCI3-isoamyl alcohol then phenol, and ethanol precipitation. The RNA obtained from this treatment was electrophoresed on a 1.5% agarose gel and blotted onto Nytran. The blots used for poly(A) length analysis of transferrin were hybridized to a 250bp 3' EcoRI fragment of pXtrans. Vitellogenin A2 poly(A) was
Experimental animals and oviduct explant culture Laboratory bred female Xenopus laer'is were obtained from Nasco, Inc. (Fort Atkinson, WI) and kept in Plexiglas aquaria at 20°C with a 12 h light-dark cycle. They were fed frog brittle twice weekly. Injections of l mg of estradiol were performed in 0.1 ml 5% (v/v) dimethyl sulfoxide in propylene glycol in the dorsal lymph sac. Control animals received injection vehicle 24 h prior to death. Before killing, animals were anesthetized by immersion in a solution of tricaine methanesulfonate (Finquel, Ayerst, Rouses Point, NY). Livers were perfused of blood with sterile 1 x SSC (1 x SCC = 0.15 M NaCI, 0.015 M sodium citrate), rinsed in cold 1 x SSC and used immediately for the isolation of RNA. Oviducts cut into 1 mm fragments were cultured in 0.65 x Coon's modified Ham's FI2 medium essentially as described for liver explant cultures by Brock and Shapiro [17]. They were maintained on a rocking platform at 25°C. A complete change of medium was done after 60 h, and the medium was changed each 24 h subsequently. The first 60 h was used to acclimate the fragments to culture prior to initiation of experimental procedures. Where indicated
Poly(A ) analysis
Transferrin mRNA in liver and oviduct
analyzed with a probe prepared by polymerase chain reaction of the 3' end of the A2 vitellogenin cDNA clone pXlvc 10 (a gift from Walter Wahli). The 5' PCR primer (adjacent to the cleavage site) was 5'-AGACATCTCCATGTGCA-3'. The 3' PCR primer consisted of the sequence immediately adjacent to the poly(A) addition site (5'-TTCTATTTGAATGCACAG-3'). Radiolabeled probe was prepared by 30 cycles of PCR using 100 pg of Hind IIIdigested pXlvc 10 and 150/a Ci of ct-[32P]dATP by the method of Schowalter and Summer [21]. All blotting and hybridization conditions were the same as described previously[18]. The Northern blot in Fig. 2 and slot blots were hybridized to a central 1164 bp EcoRl fragment of pXtrans [22]. Slot blot data were quantified by laser scanning densitometry of autoradiograms and by scintillation counting of the individual slots identified by autoradiography.
Enzymes, isotopes and reagents RNase H, restriction enzymes, ultrapure guanidine isothiocyanate, and CsCI were obtained from Bethesda Research Laboratories, Inc. (Gaithersburg, MD). Taq polymerase was obtained from Cetus. ~t-[32p]dCTP and dATP (4000 Ci/mmol) were obtained from New England Nuclear (Boston, MA). All other reagents and salts were the highest quality available from Sigma Chemical Co. (St Louis, MO). RESULTS
Estrogen regulates transferrin mRNA expression in female liver Data presented previously [13] demonstrated that liver transferrin mRNA, like albumin, Tronsferrin
651
is regulated by cytoplasmic destabilization in response to estrogen administration to male Xenopus laevis. The first experiments in this study examined whether the same response occurred in the liver of female animals. In the experiment shown in Fig. 1 two young adult female Xenopus were injected with vehicle control and two were injected with 1 mg of estradiol 24 h prior to death. Liver cytoplasmic RNA was prepared from each animal and analyzed separately by solid phase hybridization. Approx. 90% of the transferrin mRNA disappeared from the cytoplasm of each of the animals that received estradiol compared to the animals that received vehicle alone. There was relatively little vitellogenin mRNA present in livers from control females. However this was substantially induced following estrogen administration. Hybridization to /3-giobin (which is expressed constitutively [23]) was performed to control for variations in sample loading. This data indicate that transferrin mRNA is regulated by the same process of cytoplasmic destabilization in male and female liver. This response is distinct from that observed in chickens, where estrogen causes a slight increase in liver transferrin mRNA.
Estrogen induces transferrin mRNA oviduct
The induction of transferrin (conalbumin) mRNA is a characteristic response of the chicken oviduct to estrogen. Since transferrin mRNA is destabilized in Xenopus liver we asked whether estrogen had the same effect on its expression in oviduct. Transferrin mRNA was not inducible in oviduct following injection of estradiol in vivo. However, it became inducible
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Fig. !. Enhanced degradation of transferrin mRNA in liver from female Xenopusupon estrogen treatment. Two groups of 2 adult female frogs were injected with either vehicle (Ctrl) or 1 mg of estradiol (E) 24 h prior to death. Liver cytoplasmic RNA was isolated from each animal separately and 5 #g from each were applied to nylon membrane in triplicate. The filters were hybridized to transferrin, vitellogenin and /Lglobin cDNA clones and autoradiographed.
652
RICARDO L. PASTOR! et al.
if animals were first injected with 4-hydroxytamoxifen followed 7 days later by estradiol (data not shown). From these observations we c o n c l u d e d that transferrin mRNA was maintained above its basal state by estrogen present in vivo. To study transferrin regulation without background effects of endogenous estrogen the following experiments were performed with oviduct explant cultures. Oviducts were cut into 1 mm fragments as described previously for liver explant cultures[13]. These were maintained on a rocking platform in serum-free Coon's modified Ham's F I2 medium for 60 h prior to the beginning of the experiment (see Experimental). This period of time was used to acclimate the tissue to culture conditions and to remove as much endogenous estrogen as possible by diffusion and medium changes. At the beginning of the experiment the medium was changed to one containing either no steroid or 10-6M estradiol. The experiment shown in Fig. 2(A) is a Northern blot of oviduct RNA from cultures maintained for 24h in the presence of medium +estradiol. In contrast to the data obtained with liver, estrogen caused a significant increase in the amount of transferrin mRNA in the oviduct. A time course of transferrin induction is shown in Fig. 2(B). Cultures
exposed to estradiol showed maximal (3- to 4-fold) induction of transferrin mRNA within the first 24 h. There was no change in transferrin mRNA levels in control oviduct cultures maintained over the 3 day interval. These data indicate that estradiol exerts opposite effects on the expression of transferrin mRNA in liver and oviduct. The constancy of transferrin mRNA over 3 days in culture in the absence of hormone indicates that either estradiol is not required for basal expression or that transferrin mRNA is very stable in the absence of hormone. The very low transcription rate in poikilothermic vertebrates limits successful transcription run-on experiments to only the most abundantly transcribed RNAs, like vitellogenin and albumin [9, 23]. However, we have previously shown that a good approximation of the relative transcription rate of a given gene can be determined by analyzing changes in its nuclear RNA [10]. The jellycoat proteins produced by oviduct explant cultures were removed by short-term exposure to increased pH (see Experimental) and oviduct nuclear RNA was prepared essentially as described previously for liver[18]. This procedure has been shown to yield nuclear RNA free of cytoplasmic contamination. Duplicate cultures were then exposed to medium with or without estradiol for 48 h and
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Fig. 2. Induction of transferrin mRNA in Xenopus oviduct. (A) Oviduct explants acclimated to culture for 60 h were exposed to medium alone (Ctrl) or medium containing 10-4 M estradiol (E) for 24 h. Total RNA was isolated after 24 h and 10 #g samples of each were examined by Northern blot. (B) Oviduct explant cultures were exposed to medium (open bars) or medium + estradiol (shaded bars) as in A. Total RNA was isolated at the indicated times, applied in duplicate to nylon filters and hybridized to transferrin cDNA. After autoradiography the bands were excised from the filters and quantified by liquid scintillation spectrometry. The data are presented as the amount relative to transferrin mRNA content at time 0.
Transferrin mRNA in liver and oviduct nuclear transferrin R N A was assayed by solid phase hybridization. Since globin is not produced in this tissue, actin m R N A was used as an internal control. The data in Fig. 3 indicate that transferrin nuclear R N A was induced 3to 4-fold, much like the induction observed in total oviduct RNA. We conclude from these data that the most likely action of estrogen is to stimulate transferrin gene transcription in Xenopus oviduct.
Differential polyadenylation mRNA in liver and oriduct
of
Oviduct
75
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RNA
nucleor
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Tronsferrin
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transferrin Fig. 3. Induction of transferrin RNA in oviduct nuclei. Duplicate oviduct explant cultures were incubated for 48 h in medium + 10-6M estradiol. Nuclear RNA from each culture was immobilized onto nylon membrane and hybridized to transferrin or actin cDNA.
A previous study demonstrated that albumin m R N A has a remarkably short poly(A) tail consisting of a discrete length of 17 adenosine residues[18]. Furthermore, estrogen has no effect on the length of albumin poly(A) throughout the process of its cytoplasmic destabilization. We have postulated that because of this short poly(A) tail albumin m R N A is poised to be destabilized following estrogen administration. Since transferrin m R N A is co-regulated with albumin in liver it was of interest to determine whether it bears a similarly short poly(A).
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The experimental protocol used to measure transferrin poly(A) employed an oligonucleotide complementary to the sequence 166 nucleotides 5' to the poly(A) addition site to guide cleavage of transferrin m R N A by RNase H. Digestion of the transferrin m R N A - D N A hybrid with RNase H generates a fragment Control
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Fig. 4. Poly(A) analysis of liver transferrin mRNA. 10#g samples of liver RNA from male animals (panels l and 2) or female animals (panels 3 and 4) were analyzed for transferrin poly(A). In panels 1 and 3 animals received vehicle injection (control) 24 h prior to death. In panels 2 and 4 the animals received I mg of estradiol. In the first lane of each panel liver RNA was incubated with RNase H but with no added oligonucleotides. In lane 2 the - 166 oligo was present in the reaction. In lane 3 both - 166 oligo and oligo(dT) were present in the reaction. After termination of the reaction the extracted RNA samples were electrophoresed on a 1.5% agarose gel, blotted onto nylon membrane and hybridized to a probe made from the 252 bp 3' EcoRI fragment of transferrin eDNA. The band at approx. 1 kb is a nonenzymatic cleavage fragment generated by heating transferrin mRNA at 85°C in the presence of Mg + +.
654
RICARDOL. PASTORIet al.
containing 166 nucleotides of the Y end plus the poly(A) tail. Inclusion of oligo(dT) in the reaction results in the removal of poly(A). The difference in size between the fragments generated in the presence or absence of oligo(dT) gives the size of the poly(A) tail. In these experiments transferrin cleavage products were detected with a cDNA probe containing the terminal 252 nucleotides. The experiment in Fig. 4 examines transferrin polyadenylation in both male and female liver of animals that received either injection vehicle or estradiol 24 h prior to death. The gel in the first panel shows transferrin poly(A) in male animals that received only the injection vehicle. Cleavage with RNase H in the presence of the 166 oligonucleotide alone generated a discrete fragment approximately the size expected for the 3' fragment alone. There was no detectable change in the mobility of this fragment on the agarose gel upon removal of poly(A) with oligo(dT), indicating the presence of a very short poly(A) tail. We know from oligo(dT) cellulose selection and sequence data that transferrin mRNA contains at least 12 adenosine residues. Although the exact length of liver transferrin poly(A) was not determined by RNase A + T! analysis of hybrid-selected RNA (as was done previously for albumin [18]), we estimate the length of transferrin poly(A) to be very close to that of albumin. In the second panel poly(A) analysis was performed on RNA from male animals that had received estradiol. As previously shown for albumin m R N A [18], estrogen had no effect on transferrin polyadenylation in liver. The third and fourth panels show the same poly(A) analysis applied to transferrin m R N A of liver from female animals that received vehicle or estradiol, respectively. Transferrin mRNA has a short poly(A) tail in female animals and, as in males, estrogen has no effect on its length. The presence of such short poly(A) on albumin and transferrin mRNA led us to question whether short poly(A) is a feature characteristic of all liver mRNAs. Vitellogenin m R N A is one of the most well-characterized mRNAs in Xenopus liver. Nevertheless, the length of vitellogenin poly(A) has never been directly determined. The experiment in Fig. 5 shows that vitellogenin has poly(A) ranging from 50-200 residues in length, similar to that seen in most eucaryotic mRNAs. Therefore, short poly(A) is not a generalized characteristic of Xenopus liver mRNAs. -
Estrogen-regulated polyadenylation of oviduct transferrin mRNA The next experiment examined the polyadenylation of transferrin mRNA in oviduct explant cultures (Fig. 6). In this experiment, oviduct explant cultures were exposed to either medium or medium containing estradiol for 24h prior to isolation of RNA for poly(A) analysis. Transferrin mRNA from control oviducts has the same short poly(A) tail as found in liver. However estrogen treatment results in a striking increase in transferrin polyadenylation evidenced by the broad band observed in the middle lane of the second panel. The range of this poly(A) tail length varies from 50-200 nucteotides, resembling that found on vitellogenin mRNA in liver. DISCUSSION The data presented above and in our preceding paper[13] demonstrate that, while
7,5
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Fig. 5. Poly(A)analysis of vitellogeninmRNA. Liver cytoplasmic RNA was isolated 48 h after a single injection of estradiol and processed as in Fig. 4 for determination of vitellogenin poly(A) length. The probe for vitellogenin consisted of a PCR product specificfor the portion of the mRNA between the site of cleavagewith RNase H and the poly(A) addition site.
Transferrin mRNA in liver and oviduct
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Fig. 6. Poly(A) analysis o f oviduct transferrin m R N A . I 0 # g samples o f R N A from oviduct explant cultures exposed for 2 4 h to medium alone (control) or 1 0 - 6 M estradio] (estrogen) were treated as described in the legend to Fig. 4.
transferrin mRNA is regulated like albumin in both male and female liver, the regulation in oviduct is quite distinct. We had previously postulated that the short poly(A) found on albumin mRNA might serve to predispose it to enhanced degradation in response to estradiol. The presence of the same feature on an unrelated mRNA in liver (where it is degraded) but not oviduct (where it is induced) lends support to this concept. A brief high pH extraction procedure enabled us to remove jellycoat proteins and hence isolate oviduct nuclear RNA free from cytoplasmic contamination. However, we were less convinced that cytoplasmic RNA isolated from the same fractions was free of nuclear contamination. Therefore the experiments in this study were limited to preparations of total oviduct or nuclear RNA. Estrogen induces transferrin mRNA 3- to 4-fold in both total and nuclear RNA. We conclude that the increased amount of transferrin mRNA is due most likely to increased synthesis. The transcriptional induction of transferrin (conalbumin) mRNA in avian oviduct is well-
655
characterized [24]. The increased synthesis of transferrin mRNA in amphibian oviduct therefore provides another link between steroid hormone regulation in these oviparous vertebrates. The role played by poly(A) in transferrin gene expression in oviduct is unclear. Changes in poly(A) length correlate with the hormoneregulated stabilization of apo-VLDL II [2] and growth hormone mRNA[25]. It may be that transferrin mRNA is also stabilized in oviduct. We believe this to be unlikely, since estrogen increases nuclear and total oviduct transferrin mRNA to the same extent. Alternatively, long poly(A) may enhance the translatability of transferrin mRNA. Munroe and Jacobson [26] have shown that poly(A) length correlates with degree of translation initiation. They propose that poly(A) acts analogously to an enhancer in stimulating the formation of translation initiation complexes. Similarly, Sachs and Davis [27] have shown that the complex between poly(A) and poly(A)-binding protein plays an essential, if yet unknown, role in translation initiation. The short poly(A) found on transferrin mRNA in both liver and oviduct is sufficient to bind only a single molecule of poly(A)-binding protein[28]. Since poly(A)binding protein binds with an average density of one molecule per 20-25 residues [28], a 200 residue poly(A) tail could bind 8-10 molecules of this protein. The net effect would be to enhance translation initiation on these templates. One could therefore envision a scenario in which the large demands for transferrin production during ®®genesis could be met by both increased transcription and enhanced translatability of new mRNAs bearing long poly(A). Poly(A) is added to mRNAs in the nucleus in a two-step mechanism in which 10 residues are added slowly, followed by the rapid addition of 200 residues[29]. The size heterogeneity observed in the cytoplasm is caused by exonucleolytic degradation, perhaps related to the age of the mRNA. The short poly(A) found on mRNAs encoding secreted proteins in Xenopus liver is unusual. We have never observed a long poly(A) on albumin, even in nuclear RNA. The experiments shown in Fig. 5 demonstrate that vitellogenin mRNA has significantly longer poly(A) than albumin or transferrin. The long viteilogenin poly(A) determined here corresponds well to the length estimated previously by solution hybridization [30]. Therefore the presence of short poly(A) is not characteristic of all liver mRNAs.
656
RncAat,vo L. PASTORnet al.
If addition of short poly(A) represents a basal state for certain mRNAs, then there may be factors present in liver and estrogen-deprived oviduct which recognize the shared sequences to limit the action of poly(A) polymerase. In the oviduct this limitation may be overcome by estrogen to result in the observed increase in polyadenylation. Alternatively, all mRNAs may receive a long poly(A) that is rapidly cleaved to the short, discrete length observed for albumin in liver and transferrin in liver and estrogendeprived oviduct. The discrete size of albumin and transferrin poly(A) suggests a template molecule might protect this portion of the RNA. A nuclease responsible for poly(A) trimming may then be inactivated specifically under estrogen regulation in the oviduct. In either case, it is clear that estrogen is regulating polyadenylation of the same mRNA in a tissuespecific manner. Of note in this regard is the observation that only long poly(A) is found on oviduct transferrin mRNA isolated directly from female Xenopus, consistent with the constitutive estrogenic stimulation of this tissue (data not shown). Experiments are in progress to address the polyadenylation of these mRNAs in cell-free extracts to further characterize the processes regulating transferrin polyadenylation. Acknowledgements--This work was supported by Public Health Service Grant GM-38277 from the National Institute of General Medical Sciences and Grant CO7577 from the Uniformed Services University. The experiments reported herein were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals, Institute of Animal Resources. National Research Council, Department of Health and Human Services Pub. No. (NIH) 78-23. All recombinant organisms and molecules were handled under conditions of the NIH guidelines for recombinant DNA research. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as otficial or reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences.
REFERENCES I. Jost J.-P., Geiser M. and Seldran M.: Specific modulation of the transcription of cloned avian vitellogenin I1 gene by estradiol-receptor complex in vitro. Proc. Natn. Acad. Sci. U.S.A. $2 (1985) 988-991. 2. Cochrane A. W. and Deeley R. G.: Estrogen-dependent activation of the avian very low density apolipoprotein II and vitellogenin genes: Transient alterations in mRNA polyadenylation and stability early during induction. J. Molee. Biol. 203 (1988) 555-567. 3. Gordon D. A., Sheln¢~ G. S., Nicosia M. and Williams D. L.: Estrogen-induced destabilizaton of yolk precursor protein mRNAs in avian liver. J. Biol. Chem. 263 (1988) 2625-2631. 4. Chambon P., Dierich A., Gaub M.-P., Jakowlev S., Jonggtra J., Krust A,, l.xPennec J.-P., Oudet P. and Reudelhuber T.: Promoter elements of genes coding for
proteins and modulation of transcription by estrogens and progesterone. Recent Prog. Horm. Res. 40 (1984) I ..42.
5. Thibodeau S. N., Lee D. C. and Palmiter R. D.: Identical precursors for serum transferrin and egg white conalbumin. J. Biol. Chem. 253 (1978) 3771-3774. 6. Dierich A., Gaub M.-P., LePennec J.-P., Astinotti D. and Chambon P.: Cell-specificity of the chicken ovalbumin and conalbumin promoters. EMBO Jl 6 (1987) 2305-2312. 7. Wahli W.: Evolution and expression of vitellogenin genes. Trends Genet. 4 (1988) 227-232. 8. Shapiro D. J., Barton M. C., McKearin D. M., Chang T.-C., Lew D., Blume J.. Nielsen D. and Gould L.: Estrogen regulation of gene transcription and mRNA stability. Recent Prog. Horm. Res. 45 (1989) 29-64. 9. Riegel A. T., Martin M. B. and Schoenberg D, R.: Transcriptional and p~st-transcriptional inhibition of albumin gene expression by estrogen in Xenopus liver. Molec. Cell. Endocr. 44 (1986) 201-209. 10. Pastori R. L., Moskaitis J. E., Smith L. H. and Schoenberg D. R.: Estrogen regulation of Xenopus laet'is 7-fibrinogen gene expression. Biochemistry 29 (1990) 2599 2605. I 1. Riegel A. T., Jordan V. C., Bain R. R. and Schoenberg D. R.: Effects of antiestrogens on the induction of vitellogenin and its mRNA in Xenopus laevis. J. Steroid Biochem. 24 (1986) 1141-I 149. 12. Riegel A. T., Aitken S. C., Martin M. B. and Schoenberg D. R.: Posttranscriptional regulation of albumin gene expression in Xenopus liver: Evidence for an estrogen receptor-dependent mechanism. Molec. Endocr. 1 (1987) 160-167. 13. Pastori R. L., Moskaitis J. E., Buzek S. B. and Schoenberg D. R.: Coordinate estrogen-regulated instability of serum protein-coding messenger RNAs in Xenopus laevis. Molec. Endocr. 5 (1991) 461 468. 14. Lee D. C., McKnight G. S. and Palmiter R. D.: The action of estrogen and progesterone on the expression of the transferrin gene: A comparison of the response in chick liver and oviduct. J. Biol. Chem. 253 (1978) 2494-3503. 15. Marsh J. and Tara J. R.: Hormonal regulation of RNA synthesis and specific gene expression in Xenopus oviduct cells in primary culture. Molec. Cell. Endocr. 53 (1987) 141-148. 16. Lerivary H., Smith J. A. and Tata J. R.: FOSP-I (frog oviduct-specific protein- 1) gene: cloning of cDNA and induction by estrogen in primary cultures of Xenopus oviduct cells. Molec. Cell. Endocr. 59 (1988) 24t-248. 17. Brock M. L. and Shapiro D. J.: Estrogen stabilizes vitellogenin mRNA against cytoplasmic degradation. Cell 34 (1983) 207-214. 18. Schoenberg D. R., Moskaitis J. E., Smith L. H. and Pastori R. L.: Extranuclear estrogen-regulated destabilization of Xenopus laevis serum albumin mRNA. Molec. Endocr. 3 (1989) 805-814. 19. Gerton G. L. and Hedrick J. L.: The coelomic envelope to vitelline envelope conversion in eggs of Xenopus laet'is. J. Cell. Biochem. 30 (1986) 341-350. 20. Donis-Keller H.: Site specific enzymatic cleavage of RNA. Nucleic Acids Res. 7 (1979) 179-192. 21. Schowalter D. B. and Summer S. S.: The generation of radiolabeled DNA and RNA probes with polymerase chain reaction. Analyt. Biochem. 177 (1989) 90-94. 22. Moskaitis J. E., Pastori R. L. and Schoenberg D. R.: The nucleotide sequence of Xenopus laevis transferrin messenger RNA. Nucleic Acids Res. lg (1990) 6135. 23. Martin M. B., Riegel A. T. and Schoenberg D. R.: Differential induction of vitellogenin gene transcription and total transcriptional activity by estrogen in Xenopus laevis liver. J. Biol. Chem. 261 (1986) 2355-2361.
Transferrin mRNA in liver and oviduct 24. McKnight G. S., Lee D. C. and Palmiter R. D.: Transferrin gene expression: Regulation of mRNA transcription in chick liver by steroid hormones and iron deficiency. J. Biol. Chem. 255 (1980) 148-153. 25. Paek I. and Axel R.: Glucocorticoids enhance stability of human growth hormone mRNA. Molec. Cell. Biol. 7 (1987) 1496-1507. 26. Munroe D. and Jacobson, A.: mRNA poly(A) tail, a 3' enhancer oftranslational initiation. Molec. Cell. Biol. 10 (1990) 3441-3455. 27. Sachs A. B. and Davis R. W.: The poly(A) binding protein is required for poly(A) shortening and 60S
657
ribosomal subunit-depcndent translation initiation. Cell 59 (1989) 857-867. 28. Sachs A. B., Davis R. W. and Kornberg R. D.: A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability. Molec. Cell. Biol. 7 (1987) 3268-3276. 29. Sheets M. D. and Wickens M.: Two phases in the addition of a poly(A) tail. Genes De~. 3 (1990) 1401-1412. 30. Shapiro D. J. and Baker H. J.: Purification and characterization of Xenopus laevis vitellogenin messenger RNA. J. Biol, Chem. 252 (1977) 5244-5250.
I)960-0760/92 $5.00 + 0.00 Pergamon Press Ltd
Printed in Great Britain
DIFFERENTIAL OF
REGULATION
TRANSFERRIN
AND
mRNA AND
POLYADENYLATION
IN XENOPUS LIVER
OVIDUCT
RICARDO L. PASTORI, JOHN E. MOSKAITIS, SUSAN W. BUZEK a n d DANIEL R. SCHOENBERG* Department of Pharmacology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799, U.S.A. (Received 9 November 1991)
Summary--Estrogen destabilizes transferrin mRNA in male Xenopusliver in the same manner as observed for albumin and 7-fibrinogen. The present study examined estrogen regulation of transferrin gene expression in female Xenopus liver and oviduct. In female Xenopus liver estrogen causes the same enhanced degradation of transferrin mRNA from the cytoplasm as seen in males. In contrast, transferrin is induced 3- to 4-fold in both oviduct nuclear and cytoplasmic RNA. The similar increase in transferrin RNA in both preparations suggests a transcriptional mechanism is responsible for this stimulation. Therefore, transferrin expression is differentially regulated in these tissues by the same hormone. Previous experiments showed that Xenopus serum albumin mRNA has a very short (17 residue) poly(A) tail that may play a role in its hormone-regulated instability. Transferrin mRNA has a similarly short poly(A) tail in liver of both male and female Xenopus. Estrogen has no effect on transferrin polyadenylation in liver. Similarly short poly(A) is found on transferrin mRNA from estrogen-deprived oviducts in explant culture. However, addition of estradiol to the medium results in the appearance of a 50-200 nucleotide poly(A) concurrent with induction. Therefore, transferrin mRNA is differentially polyadenylated in Xenopus liver and oviduct. In the latter tissue polyadenylation is under hormonal control.
INTRODUCTION
The female sex steroid estradiol exerts a broad spectrum o f effects on gene expression in oviparous vertebrates. In chick liver estradiol causes the transcriptional induction of the genes encoding the yolk protein precursor vitellogenin [1], and apo-very low density lipoprotein II (apoV L D L II[2]). Apo-VLDL II mRNA is subsequently stabilized in the continued presence of estradiol, however abrupt withdrawal of hormone is accompanied by rapid degradation [3]. Also in avian liver estrogen causes a slight increase in both transferrin gene transcription and mRNA. In chick oviduct estrogen induces ovalbumin transcription and causes a significant stimulation in conalbumin transcription (reviewed in[4]). Several lines of evidence, including D N A sequence analysis, have shown conalbumin and transferrin to be the same protein [5]. Microinjection of SV40 T antigen reporter constructs bearing the promoter and 5' flanking regions of ovalbumin and transferrin *To whom correspondence should be addressed. 649
into primary cultures of a variety of chicken cells shows both promoters to be active only in hepatocytes and oviduct tubular gland cells [6]. In liver cells neither promoter is stimulated by estradiol, whereas both are stimulated by hormone in oviduct cells. Therefore, while avian liver and oviduct are both estrogen responsive, there is differential transcriptional regulation of the transferrin gene in each tissue. In liver of both birds and amphibia estrogen causes the transcriptional induction of the vitellogenin genes (reviewed in [7, 8]). In frogs estrogen also stabilizes vitellogenin m R N A [8] and destabilizes serum albumin [9] and ?-fibrinogen [10] mRNA. The changes in the stabilities of these mRNAs, like the induction of vitellogenin [11] are dependent on the action of the estrogen receptor[12]. A recent report from this laboratory demonstrated that transferrin mRNA is also destabilized by estrogen in Xenopus liver[13]. This is in contrast to the minimal increase in transferrin mRNA observed in avian liver [14]. In general, studies on estrogen-regulated gene expression in chicken and Xenopus liver
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RICARDO L. PASTORI et al.
have shown considerable similarity between these two species [7]. Therefore the differences in transferrin expression were striking. This led us to examine the regulation of transferrin expression in Xenopus oviduct. Tata and coworkers [15, 16] have identified a number of cDNAs whose cognate mRNAs are induced by estrogen in primary cultures of oviduct cells. None of these corresponds to known mRNAs, although FOSP-I (frog oviduct-specific protein-I) has been subjected to DNA sequence analysis[16]. FOSP-! mRNA is unique to oviduct and its mRNA is induced 5-fold by estrogen. In the present study we report that transferrin mRNA is expressed constitutively in the absence of estradiol in oviduct explant cultures. Upon addition of estradiol to the culture medium transferrin mRNA is induced 3- to 4-fold. The transcriptional induction of transferrin mRNA in oviduct contrasts with its posttranscriptional destabilization in liver. In addition, the estrogen-induced transferrin mRNA in oviduct has a significantly longer poly(A) than that found in liver or nonstimulated oviduct.
10-rM estradiol was added twice daily to the medium. Oviduct nuclear RNA was prepared by our previously described procedure[18] modified by an extraction procedure that removes the viscous jellycoat proteins[19]. Briefly, tissue fragments from oviduct explant cultures were washed for 5min (10ml/mg of tissue) in DB-Tris buffer, pH 8.9, followed by a wash in DB-Tris buffer, pH 7.8. DB-Tris consisted of 0.11 M NaCI, 1.34mM CaCI2, 1.32 mM KC1, 10 mM Tris-HCl. The tissue was next rinsed in 5 ml/gm 0.3 M sucrose, 10mM Tris-HCI, pH 8.0 followed by homogenization in the same buffer. The homogenate was diluted 2-fold in sucrose-Tris and filtered through nitex mesh as described previously for preparation of liver nuclear RNA [18]. This was then diluted 2-fold again with 2 M sucrose, 10mM Tris, pH 8.0 and layered over 15 ml of 2 M sucrose in a SW27 centrifuge tube. The nuclei were collected by centrifugation at 25,000 rpm in an SW27 rotor. RNA was subsequently extracted from the nuclear pellet. All of the preceding steps were performed at 4:C.
EXPERIMENTAL
The analysis of transferrin and vitellogenin polyadenylation was performed as described previously[18] by a modification of the procedure of Donis-Keller [20]. An oligonucleotide (5'-ATGTGCAGGCAGCCA-Y) complementary to the sequence -166 nucleotides 5' to the poly(A) addition site was used to direct cleavage of transferrin mRNA by RNase H. Cleavage of A2 vitellogenin mRNA was directed by the oligonucleotide 5'-CACATGAACAAGCGGTG-3' to generate a fragment containing the terminal 205 nucleotides plus poly(A). 10 #g of total liver or oviduct RNA was mixed with 3 #g of oligonucleotide + 3 #g oligo(dT)~2_~s in 19 #1 40 mM Tris-HCl, pH 7.9, 4 mM MgCI 2, 1 mM dithiothreitol, 30 ng/ml bovine serum albumin. This was heated at 85°C for 5 min 42°C for 10min and cooled to room temperature. Two units of RNase H was then added and the samples were incubated at 37°C for 30min. The reaction was terminated by the addition of 80/11 H20, followed by sequential extraction with HCCI3-isoamyl alcohol then phenol, and ethanol precipitation. The RNA obtained from this treatment was electrophoresed on a 1.5% agarose gel and blotted onto Nytran. The blots used for poly(A) length analysis of transferrin were hybridized to a 250bp 3' EcoRI fragment of pXtrans. Vitellogenin A2 poly(A) was
Experimental animals and oviduct explant culture Laboratory bred female Xenopus laer'is were obtained from Nasco, Inc. (Fort Atkinson, WI) and kept in Plexiglas aquaria at 20°C with a 12 h light-dark cycle. They were fed frog brittle twice weekly. Injections of l mg of estradiol were performed in 0.1 ml 5% (v/v) dimethyl sulfoxide in propylene glycol in the dorsal lymph sac. Control animals received injection vehicle 24 h prior to death. Before killing, animals were anesthetized by immersion in a solution of tricaine methanesulfonate (Finquel, Ayerst, Rouses Point, NY). Livers were perfused of blood with sterile 1 x SSC (1 x SCC = 0.15 M NaCI, 0.015 M sodium citrate), rinsed in cold 1 x SSC and used immediately for the isolation of RNA. Oviducts cut into 1 mm fragments were cultured in 0.65 x Coon's modified Ham's FI2 medium essentially as described for liver explant cultures by Brock and Shapiro [17]. They were maintained on a rocking platform at 25°C. A complete change of medium was done after 60 h, and the medium was changed each 24 h subsequently. The first 60 h was used to acclimate the fragments to culture prior to initiation of experimental procedures. Where indicated
Poly(A ) analysis
Transferrin mRNA in liver and oviduct
analyzed with a probe prepared by polymerase chain reaction of the 3' end of the A2 vitellogenin cDNA clone pXlvc 10 (a gift from Walter Wahli). The 5' PCR primer (adjacent to the cleavage site) was 5'-AGACATCTCCATGTGCA-3'. The 3' PCR primer consisted of the sequence immediately adjacent to the poly(A) addition site (5'-TTCTATTTGAATGCACAG-3'). Radiolabeled probe was prepared by 30 cycles of PCR using 100 pg of Hind IIIdigested pXlvc 10 and 150/a Ci of ct-[32P]dATP by the method of Schowalter and Summer [21]. All blotting and hybridization conditions were the same as described previously[18]. The Northern blot in Fig. 2 and slot blots were hybridized to a central 1164 bp EcoRl fragment of pXtrans [22]. Slot blot data were quantified by laser scanning densitometry of autoradiograms and by scintillation counting of the individual slots identified by autoradiography.
Enzymes, isotopes and reagents RNase H, restriction enzymes, ultrapure guanidine isothiocyanate, and CsCI were obtained from Bethesda Research Laboratories, Inc. (Gaithersburg, MD). Taq polymerase was obtained from Cetus. ~t-[32p]dCTP and dATP (4000 Ci/mmol) were obtained from New England Nuclear (Boston, MA). All other reagents and salts were the highest quality available from Sigma Chemical Co. (St Louis, MO). RESULTS
Estrogen regulates transferrin mRNA expression in female liver Data presented previously [13] demonstrated that liver transferrin mRNA, like albumin, Tronsferrin
651
is regulated by cytoplasmic destabilization in response to estrogen administration to male Xenopus laevis. The first experiments in this study examined whether the same response occurred in the liver of female animals. In the experiment shown in Fig. 1 two young adult female Xenopus were injected with vehicle control and two were injected with 1 mg of estradiol 24 h prior to death. Liver cytoplasmic RNA was prepared from each animal and analyzed separately by solid phase hybridization. Approx. 90% of the transferrin mRNA disappeared from the cytoplasm of each of the animals that received estradiol compared to the animals that received vehicle alone. There was relatively little vitellogenin mRNA present in livers from control females. However this was substantially induced following estrogen administration. Hybridization to /3-giobin (which is expressed constitutively [23]) was performed to control for variations in sample loading. This data indicate that transferrin mRNA is regulated by the same process of cytoplasmic destabilization in male and female liver. This response is distinct from that observed in chickens, where estrogen causes a slight increase in liver transferrin mRNA.
Estrogen induces transferrin mRNA oviduct
The induction of transferrin (conalbumin) mRNA is a characteristic response of the chicken oviduct to estrogen. Since transferrin mRNA is destabilized in Xenopus liver we asked whether estrogen had the same effect on its expression in oviduct. Transferrin mRNA was not inducible in oviduct following injection of estradiol in vivo. However, it became inducible
Vitellogemn
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Fig. !. Enhanced degradation of transferrin mRNA in liver from female Xenopusupon estrogen treatment. Two groups of 2 adult female frogs were injected with either vehicle (Ctrl) or 1 mg of estradiol (E) 24 h prior to death. Liver cytoplasmic RNA was isolated from each animal separately and 5 #g from each were applied to nylon membrane in triplicate. The filters were hybridized to transferrin, vitellogenin and /Lglobin cDNA clones and autoradiographed.
652
RICARDO L. PASTOR! et al.
if animals were first injected with 4-hydroxytamoxifen followed 7 days later by estradiol (data not shown). From these observations we c o n c l u d e d that transferrin mRNA was maintained above its basal state by estrogen present in vivo. To study transferrin regulation without background effects of endogenous estrogen the following experiments were performed with oviduct explant cultures. Oviducts were cut into 1 mm fragments as described previously for liver explant cultures[13]. These were maintained on a rocking platform in serum-free Coon's modified Ham's F I2 medium for 60 h prior to the beginning of the experiment (see Experimental). This period of time was used to acclimate the tissue to culture conditions and to remove as much endogenous estrogen as possible by diffusion and medium changes. At the beginning of the experiment the medium was changed to one containing either no steroid or 10-6M estradiol. The experiment shown in Fig. 2(A) is a Northern blot of oviduct RNA from cultures maintained for 24h in the presence of medium +estradiol. In contrast to the data obtained with liver, estrogen caused a significant increase in the amount of transferrin mRNA in the oviduct. A time course of transferrin induction is shown in Fig. 2(B). Cultures
exposed to estradiol showed maximal (3- to 4-fold) induction of transferrin mRNA within the first 24 h. There was no change in transferrin mRNA levels in control oviduct cultures maintained over the 3 day interval. These data indicate that estradiol exerts opposite effects on the expression of transferrin mRNA in liver and oviduct. The constancy of transferrin mRNA over 3 days in culture in the absence of hormone indicates that either estradiol is not required for basal expression or that transferrin mRNA is very stable in the absence of hormone. The very low transcription rate in poikilothermic vertebrates limits successful transcription run-on experiments to only the most abundantly transcribed RNAs, like vitellogenin and albumin [9, 23]. However, we have previously shown that a good approximation of the relative transcription rate of a given gene can be determined by analyzing changes in its nuclear RNA [10]. The jellycoat proteins produced by oviduct explant cultures were removed by short-term exposure to increased pH (see Experimental) and oviduct nuclear RNA was prepared essentially as described previously for liver[18]. This procedure has been shown to yield nuclear RNA free of cytoplasmic contamination. Duplicate cultures were then exposed to medium with or without estradiol for 48 h and
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Fig. 2. Induction of transferrin mRNA in Xenopus oviduct. (A) Oviduct explants acclimated to culture for 60 h were exposed to medium alone (Ctrl) or medium containing 10-4 M estradiol (E) for 24 h. Total RNA was isolated after 24 h and 10 #g samples of each were examined by Northern blot. (B) Oviduct explant cultures were exposed to medium (open bars) or medium + estradiol (shaded bars) as in A. Total RNA was isolated at the indicated times, applied in duplicate to nylon filters and hybridized to transferrin cDNA. After autoradiography the bands were excised from the filters and quantified by liquid scintillation spectrometry. The data are presented as the amount relative to transferrin mRNA content at time 0.
Transferrin mRNA in liver and oviduct nuclear transferrin R N A was assayed by solid phase hybridization. Since globin is not produced in this tissue, actin m R N A was used as an internal control. The data in Fig. 3 indicate that transferrin nuclear R N A was induced 3to 4-fold, much like the induction observed in total oviduct RNA. We conclude from these data that the most likely action of estrogen is to stimulate transferrin gene transcription in Xenopus oviduct.
Differential polyadenylation mRNA in liver and oriduct
of
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nucleor
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transferrin Fig. 3. Induction of transferrin RNA in oviduct nuclei. Duplicate oviduct explant cultures were incubated for 48 h in medium + 10-6M estradiol. Nuclear RNA from each culture was immobilized onto nylon membrane and hybridized to transferrin or actin cDNA.
A previous study demonstrated that albumin m R N A has a remarkably short poly(A) tail consisting of a discrete length of 17 adenosine residues[18]. Furthermore, estrogen has no effect on the length of albumin poly(A) throughout the process of its cytoplasmic destabilization. We have postulated that because of this short poly(A) tail albumin m R N A is poised to be destabilized following estrogen administration. Since transferrin m R N A is co-regulated with albumin in liver it was of interest to determine whether it bears a similarly short poly(A).
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The experimental protocol used to measure transferrin poly(A) employed an oligonucleotide complementary to the sequence 166 nucleotides 5' to the poly(A) addition site to guide cleavage of transferrin m R N A by RNase H. Digestion of the transferrin m R N A - D N A hybrid with RNase H generates a fragment Control
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Fig. 4. Poly(A) analysis of liver transferrin mRNA. 10#g samples of liver RNA from male animals (panels l and 2) or female animals (panels 3 and 4) were analyzed for transferrin poly(A). In panels 1 and 3 animals received vehicle injection (control) 24 h prior to death. In panels 2 and 4 the animals received I mg of estradiol. In the first lane of each panel liver RNA was incubated with RNase H but with no added oligonucleotides. In lane 2 the - 166 oligo was present in the reaction. In lane 3 both - 166 oligo and oligo(dT) were present in the reaction. After termination of the reaction the extracted RNA samples were electrophoresed on a 1.5% agarose gel, blotted onto nylon membrane and hybridized to a probe made from the 252 bp 3' EcoRI fragment of transferrin eDNA. The band at approx. 1 kb is a nonenzymatic cleavage fragment generated by heating transferrin mRNA at 85°C in the presence of Mg + +.
654
RICARDOL. PASTORIet al.
containing 166 nucleotides of the Y end plus the poly(A) tail. Inclusion of oligo(dT) in the reaction results in the removal of poly(A). The difference in size between the fragments generated in the presence or absence of oligo(dT) gives the size of the poly(A) tail. In these experiments transferrin cleavage products were detected with a cDNA probe containing the terminal 252 nucleotides. The experiment in Fig. 4 examines transferrin polyadenylation in both male and female liver of animals that received either injection vehicle or estradiol 24 h prior to death. The gel in the first panel shows transferrin poly(A) in male animals that received only the injection vehicle. Cleavage with RNase H in the presence of the 166 oligonucleotide alone generated a discrete fragment approximately the size expected for the 3' fragment alone. There was no detectable change in the mobility of this fragment on the agarose gel upon removal of poly(A) with oligo(dT), indicating the presence of a very short poly(A) tail. We know from oligo(dT) cellulose selection and sequence data that transferrin mRNA contains at least 12 adenosine residues. Although the exact length of liver transferrin poly(A) was not determined by RNase A + T! analysis of hybrid-selected RNA (as was done previously for albumin [18]), we estimate the length of transferrin poly(A) to be very close to that of albumin. In the second panel poly(A) analysis was performed on RNA from male animals that had received estradiol. As previously shown for albumin m R N A [18], estrogen had no effect on transferrin polyadenylation in liver. The third and fourth panels show the same poly(A) analysis applied to transferrin m R N A of liver from female animals that received vehicle or estradiol, respectively. Transferrin mRNA has a short poly(A) tail in female animals and, as in males, estrogen has no effect on its length. The presence of such short poly(A) on albumin and transferrin mRNA led us to question whether short poly(A) is a feature characteristic of all liver mRNAs. Vitellogenin m R N A is one of the most well-characterized mRNAs in Xenopus liver. Nevertheless, the length of vitellogenin poly(A) has never been directly determined. The experiment in Fig. 5 shows that vitellogenin has poly(A) ranging from 50-200 residues in length, similar to that seen in most eucaryotic mRNAs. Therefore, short poly(A) is not a generalized characteristic of Xenopus liver mRNAs. -
Estrogen-regulated polyadenylation of oviduct transferrin mRNA The next experiment examined the polyadenylation of transferrin mRNA in oviduct explant cultures (Fig. 6). In this experiment, oviduct explant cultures were exposed to either medium or medium containing estradiol for 24h prior to isolation of RNA for poly(A) analysis. Transferrin mRNA from control oviducts has the same short poly(A) tail as found in liver. However estrogen treatment results in a striking increase in transferrin polyadenylation evidenced by the broad band observed in the middle lane of the second panel. The range of this poly(A) tail length varies from 50-200 nucteotides, resembling that found on vitellogenin mRNA in liver. DISCUSSION The data presented above and in our preceding paper[13] demonstrate that, while
7,5
kb
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Fig. 5. Poly(A)analysis of vitellogeninmRNA. Liver cytoplasmic RNA was isolated 48 h after a single injection of estradiol and processed as in Fig. 4 for determination of vitellogenin poly(A) length. The probe for vitellogenin consisted of a PCR product specificfor the portion of the mRNA between the site of cleavagewith RNase H and the poly(A) addition site.
Transferrin mRNA in liver and oviduct
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Fig. 6. Poly(A) analysis o f oviduct transferrin m R N A . I 0 # g samples o f R N A from oviduct explant cultures exposed for 2 4 h to medium alone (control) or 1 0 - 6 M estradio] (estrogen) were treated as described in the legend to Fig. 4.
transferrin mRNA is regulated like albumin in both male and female liver, the regulation in oviduct is quite distinct. We had previously postulated that the short poly(A) found on albumin mRNA might serve to predispose it to enhanced degradation in response to estradiol. The presence of the same feature on an unrelated mRNA in liver (where it is degraded) but not oviduct (where it is induced) lends support to this concept. A brief high pH extraction procedure enabled us to remove jellycoat proteins and hence isolate oviduct nuclear RNA free from cytoplasmic contamination. However, we were less convinced that cytoplasmic RNA isolated from the same fractions was free of nuclear contamination. Therefore the experiments in this study were limited to preparations of total oviduct or nuclear RNA. Estrogen induces transferrin mRNA 3- to 4-fold in both total and nuclear RNA. We conclude that the increased amount of transferrin mRNA is due most likely to increased synthesis. The transcriptional induction of transferrin (conalbumin) mRNA in avian oviduct is well-
655
characterized [24]. The increased synthesis of transferrin mRNA in amphibian oviduct therefore provides another link between steroid hormone regulation in these oviparous vertebrates. The role played by poly(A) in transferrin gene expression in oviduct is unclear. Changes in poly(A) length correlate with the hormoneregulated stabilization of apo-VLDL II [2] and growth hormone mRNA[25]. It may be that transferrin mRNA is also stabilized in oviduct. We believe this to be unlikely, since estrogen increases nuclear and total oviduct transferrin mRNA to the same extent. Alternatively, long poly(A) may enhance the translatability of transferrin mRNA. Munroe and Jacobson [26] have shown that poly(A) length correlates with degree of translation initiation. They propose that poly(A) acts analogously to an enhancer in stimulating the formation of translation initiation complexes. Similarly, Sachs and Davis [27] have shown that the complex between poly(A) and poly(A)-binding protein plays an essential, if yet unknown, role in translation initiation. The short poly(A) found on transferrin mRNA in both liver and oviduct is sufficient to bind only a single molecule of poly(A)-binding protein[28]. Since poly(A)binding protein binds with an average density of one molecule per 20-25 residues [28], a 200 residue poly(A) tail could bind 8-10 molecules of this protein. The net effect would be to enhance translation initiation on these templates. One could therefore envision a scenario in which the large demands for transferrin production during ®®genesis could be met by both increased transcription and enhanced translatability of new mRNAs bearing long poly(A). Poly(A) is added to mRNAs in the nucleus in a two-step mechanism in which 10 residues are added slowly, followed by the rapid addition of 200 residues[29]. The size heterogeneity observed in the cytoplasm is caused by exonucleolytic degradation, perhaps related to the age of the mRNA. The short poly(A) found on mRNAs encoding secreted proteins in Xenopus liver is unusual. We have never observed a long poly(A) on albumin, even in nuclear RNA. The experiments shown in Fig. 5 demonstrate that vitellogenin mRNA has significantly longer poly(A) than albumin or transferrin. The long viteilogenin poly(A) determined here corresponds well to the length estimated previously by solution hybridization [30]. Therefore the presence of short poly(A) is not characteristic of all liver mRNAs.
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RncAat,vo L. PASTORnet al.
If addition of short poly(A) represents a basal state for certain mRNAs, then there may be factors present in liver and estrogen-deprived oviduct which recognize the shared sequences to limit the action of poly(A) polymerase. In the oviduct this limitation may be overcome by estrogen to result in the observed increase in polyadenylation. Alternatively, all mRNAs may receive a long poly(A) that is rapidly cleaved to the short, discrete length observed for albumin in liver and transferrin in liver and estrogendeprived oviduct. The discrete size of albumin and transferrin poly(A) suggests a template molecule might protect this portion of the RNA. A nuclease responsible for poly(A) trimming may then be inactivated specifically under estrogen regulation in the oviduct. In either case, it is clear that estrogen is regulating polyadenylation of the same mRNA in a tissuespecific manner. Of note in this regard is the observation that only long poly(A) is found on oviduct transferrin mRNA isolated directly from female Xenopus, consistent with the constitutive estrogenic stimulation of this tissue (data not shown). Experiments are in progress to address the polyadenylation of these mRNAs in cell-free extracts to further characterize the processes regulating transferrin polyadenylation. Acknowledgements--This work was supported by Public Health Service Grant GM-38277 from the National Institute of General Medical Sciences and Grant CO7577 from the Uniformed Services University. The experiments reported herein were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals, Institute of Animal Resources. National Research Council, Department of Health and Human Services Pub. No. (NIH) 78-23. All recombinant organisms and molecules were handled under conditions of the NIH guidelines for recombinant DNA research. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as otficial or reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences.
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