mRNA in oocytes of Xenopus laevis?

Proc. Natl. Acad. Sci. USA Vol. 75, No. 10, pp. 5030-5033, October 1978

Cell Biology

Does 3'-terminal poly(A) stabilize human fibroblast interferon mRNA in oocytes of Xenopus laevis? [FS-4 cells/poly(I)poly(C)/polynucleotide phosphorylase (polyribonucleotide:orthophosphate nucleotidyltransferase, EC 2.7.7.8)]

PRAVINKUMAR B. SEHGAL, HERMONA SOREQ, AND IGOR TAMM The Rockefeller University, New York, New York 10021

Contributed by Igor Tamm, July 27, 1978

ABSTRACT Polynucleotide phosphorylase (polyribonucleotide:orthophosphate nucleotidyltransferase, EC 2.7.7.8) purified from Escherichia coli was used enzymatically to deadenylate polyadenylated human fibroblast interferon mRNA preparations obtained from human diploid fibroblasts (FS-4 strain) induced by poly(I)poly(C) (20 ttg/ml) in the presence of cycloheximide (50 ;g/ml, 4 hr). Both the polyadenylated and the deadenylated interferon mRNA preparations were translated into biologically active human interferon when injected into oocytes of Xenopus laevis. In the oocytes the functional stability of deadenylated interferon mRNA was indistinguishable from that of polyadenylated interferon mRNA. Polyadenylated human fibroblast interferon mRNA is translated into biologically active human interferon for 24-48 hr after microinjection into oocytes of Xenopus laevis (1, 2). This highly sensitive translation assay has been used by us earlier in an attempt to detect poly(A)-lacking interferon mRNA in poly(I)-poly(C)-induced diploid human fibroblasts (FS-4 strain) (2, 3). No translatable poly(A)-lacking interferon mRNA was detected in induced FS-4 cells (3). We have now evaluated the ability of X. laevis oocytes to translate poly(A)-lacking interferon mRNA produced by removal of the 3'-poly(A) tail enzymatically by using the enzyme polynucleotide phosphorylase (PNPase; polyribonucleotide:orthophosphate nucleotidyltransferase, EC 2.7.7.8) purified from Escherichta coli (4-8). Our results show that deadenylated interferon mRNA injected into oocytes is translated into biologically active human interferon. We have compared the functional stability of poly(A)containing interferon mRNA with that of poly(A)-lacking interferon mRNA molecules in an attempt to test the hypothesis that poly(A)-containing mRNA is more stable than poly(A)lacking mRNA in oocytes of X. laevis (9). We find that the functional stability of deadenylated interferon mRNA injected into oocytes is indistinguishable from that of polyadenylated interferon mRNA molecules.

are expressed in terms of the 69/19 reference standard for human interferon. Procedures used for the analysis of RNA by polyacrylamide gel electrophoresis have also been described (15-18). [3H]Uridine (26 Ci/mmol) was obtained from New England Nuclear. Removal of 3'-Poly(A) Tails. PNPase, purified from E. coli by the procedure of Soreq and Littauer (19), was used to deadenylate interferon mRNA essentially as described (4-8, 20). Briefly, mRNA (5-10 ,ug) was incubated at 0° for 20 min in a 100-Ad reaction mixture containing 80 mM Tris-HCI (pH 8.0), 10 mM MgCl2, 20 mM sodium phosphate (pH 8.0), 1 mM EDTA, and a molar excess of purified PNPase (10 ,Ag). Then, 2 ,ul of 20% sodium dodecyl sulfate was added and the mixture was extracted with phenol/chloroform/isoamyl alcohol. The RNA was then precipitated with ethanol.

RESULTS Effect of PNPase on Interferon mRNA Activity. We determined the effect of PNPase on interferon mRNA activity by subjecting an aliquot of a polyadenylated interferon mRNA preparation to digestion by an excess of PNPase for 20 min on ice followed by phenol extraction and ethanol precipitation of RNA from the reaction mixture. The RNA was then dissolved in 5 A1 of sterile distilled water and injected into oocytes. An equal aliquot of polyadenylated mRNA was handled in parallel except for the omission of PNPase from the digestion mixture. The injected oocytes were incubated at room temperature (21-23o) for approximately 40 hr and then homogenized in the incubation medium [modified Barth's medium (21), 0.2 ml/10 oocytes per assay]; the homogenate was assayed for interferon (2, 3). Table 1 summarizes the result of one such experiment. It is clear that PNPase digestion did not alter the translational activity of interferon mRNA. Evidence that PNPase Removes Poly(A) from Interferon mRNA Molecules. We determined the effect of PNPase on the electrophoretic profile of 3H-labeled mRNA preparations from FS-4 cells in polyacrylamide gels. Fig. 1 A and C shows the profiles of labeled mRNA from uninduced and induced cultures. Fig. 1 B and D illustrates the electrophoretic profiles of the two mRNA preparations after digestion with PNPase. All gel profiles show that the bulk of 3H-labeled mRNA is distributed between 18 and 28 S. Thus, overall, there was little or no degradation of RNA during digestion. A consistent reduction in size of mRNA due to phosphorolysis with PNPase was not observed. Because total cellular mRNA is displayed in these profiles, it would be difficult to discern a reduction in size of the order of 100-200 nucleotides. However, in parallel experiments, 32P-labeled late adenovirus 2-specific mRNA species were digested with PNPase and a reduction in size of the magnitude of 100-200 nucleotides was observed (H. Soreq and N. W. Fraser, not shown).

MATERIALS AND METHODS The procedures used for growing FS-4 cells in 150-mm Falcon petri dishes, for induction of interferon with poly(I)-poly(C) (P.-L. Biochemicals), for harvesting cells by trypsinization, for extraction of RNA, for poly(U)-Sepharose chromatography, and for the assay of interferon mRNA by translation of microinjected mRNA in X. Iaevts oocytes (10 oocytes per assay) have been reported (2, 3, 10-12). Polyadenylated' interferon mRNA was prepared from FS-4 cells induced with poly(I)-poly(C) (20 ,ug/ml) in the presence of cycloheximide (Polysciences, Inc.; 50 ,ug/ml, 4 hr) as described (2, 3). Interferon was assayed on FS-4 cells by a modified semimicro method with vesicular stomatitis virus as the challenge virus (13, 14). Interferon titers The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Abbreviation: PNPase, polynucleotide phosphorylase. 5030

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Proc. Natl. Acad. Sci. USA 75 (1978)

Table 1. Effect of PNPase on interferon mRNA activity

Sample

PNPase

Interferon titer in oocyte homogenate, ref. units/ml

1 2

+

32 64

mRNA (1-2 ,ug) from poly(I)-poly(C)-induced FS-4 cells was treated with PNPase, phenol extracted, ethanol precipitated, and dissolved in 5 ,l of sterile distilled water. An undigested sample was used as control. Approximately 2 Ml of each sample was assayed by injection into 10-12 oocytes that were then incubated in 0.2 ml of modified Barth's medium for approximately 40 hr at room temperature.

That the 3'-terminal poly(A) tails of interferon mRNA molecules had been largely removed is documented by data presented in Table 2. In this experiment the binding of polyadenylated interferon mRNA molecules to poly(U)-Sepharose in 0.4 M NaCl was assayed after digestion with PNPase. Because poly(U)-Sepharose can bind mRNA molecules with poly(A) tails approximately 30 residues long or longer (refs. 23, 24; M. Wilson and S. Sawicki, personal communication), the data in Table 2 show that, after PNPase digestion, >90% have 3'-poly(A) tails 100 residues (3), was injected into oocytes and the kinetics of accumulation of the synthesized interferon were monitored. In addition, deadenylated interferon mRNA which did not bind to poly(U)-Sepharose at 0.4 M NaCl, was prepared and injected into oocytes and the kinetics of interferon accumulation were determined. Fig. 2 presents the pooled results of such experiments. From these results we conclude that the kinetics of accumulation of interferon in oocytes injected with either poly(A)-containing or poly(A)lacking interferon mRNA are indistinguishable (see legend to Fig. 2 for summary of statistical analysis). It should also be noted C %d

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Gel slice nu FIG. 1. Electrophoretic profiles of [3H]uridine-labeled mRNA from uninduced and poly(I)-poly(C)-induced FS-4 cells before and after digestion with PNPase. Confluent 13-day-old cultures of FS-4 cells in 150-mm Falcon dishes (two per group) were induced and labeled with 10 ml of Eagle's medium (22) containing poly(I)-poly(C) (20 Mg/ml), cycloheximide (50 ,g/ml), and [3H]uridine (100 MCi/ml) for 4 hr at 37°. The cultures were then washed and incubated for another 1.5 hr in 20 ml of Eagle's medium supplemented with 20% heat-inactivated fetal calf serum (GIBCO) and cycloheximide (50 Mg/ml). Polyadenylated cellular RNA was then prepared by poly(U)-Sepharose chromatography from both the induced and the uninduced [poly(I)-poly(C) omitted] cultures. Half of each polyadenylated RNA preparation (-1,ug) was subjected to digestion with PNPase and the other half was handled in parallel with the omission of PNPase from the reaction mixture. A 2-Ml aliquot of each of the four 100-M1l reaction mixtures was precipitated with trichloroacetic acid (5%) and the 3H radioactivity was determined before the incubation. There were 22,113,38,189, 23,148, and 21,710 3H cpm in 2-Ml aliquots of the uninduced untreated, uninduced enzyme-treated, induced untreated, and induced enzyme-treated reaction mixtures, respectively, prior to incubation at 00 for 20 min. After the 20-min incubation period, the corresponding 3H cpm values were 24,448, 40,956, 17,993, and 20,279, respectively. The variation among the four samples probably results from differences in RNA recovery. The reextracted RNA was ethanol precipitated, and 10% of each sample was mixed with 14C-labeled HeLa cell ribosomal marker RNA and analyzed in 1.5% acrylamide/1% agarose tube gels as described (15-18). (A) Uninduced undigested; (B) uninduced digested; (C) induced undigested; (D) induced digested.

5032

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Proc. Natl. Acad. Sci. USA 75 (1978)

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Time, hr FIG. 2. Functional stability in oocytes of poly(A)-containing (0) and deadenylated (0) human fibroblast interferon mRNA. Appropriate amounts (1-2 ,wg) of each of poly(A)-containing interferon mRNA, which bound to and eluted from poly(U)-Sepharose [10-5096 (vol/vol) formamide step elution] and poly(A)-free interferon mRNA, which did not bind to poly(U)-Sepharose in 0.4 M NaCl (see Table 2), was injected into oocytes. Groups of 10 oocytes were homogenized in 0.2 ml of incubation medium at various times after injection and the interferon titer (Y) in the homogenates was determined. Pooled results of four experiments with polyadenylated mRNA (@) and three experiments with deadenylated mRNA (0) are presented. The two sets of data were initially fitted (least squares method) to a curve of the form Y = a exp (-bt) + c, in which Y represents cumulative yield, t represents time, and a, b, and c are constants, and Ymax = c was estimated. This value of Yma, was then used to plot ln (Y,,, - Y) = kt and a linear least squares fit was performed (linear correlation coefficient, 0.79 for * and 0.75 for 0). The difference between the slopes and the intercepts of the two lines was not statistically significant (Student's t test, 0.1 < P < 0.2). The half-life of poly(A)-containing interferon mRNA was estimated to be 7.9 I 2.5 (+SD) hr and that of poly(A)-lacking mRNA was 6 + 2.3 hr.

that the ability of 48-hr-old oocytes to synthesize biologically active interferon is identical to that of fresh oocytes (not shown). The cessation of the synthesis of interferon by 24-48 hr after injection reflects the functional decay of interferon mRNA and not that of the oocytes. The estimated half-life of both poly(A)-containing and poly(A)-lacking interferon mRNA was in the range of 6-8 hr (Fig. 2). At present we cannot exclude the possibility that the oocyte may have added poly(A) to the poly(A)-lacking interferon mRNA molecules injected into the oocyte cytoplasm. DISCUSSION The data presented in this paper strengthen the conclusion that poly(A)-lacking interferon mRNA is not present in detectable amounts in human fibroblasts (FS-4) induced with poly(I)poly(C) (3). Interferon mRNA in induced FS-4 cells appears exclusively in the polyadenylated mRNA fraction. However, our results suggest that the poly(A) sequence in interferon mRNA does not contribute to the functional stability of this mRNA. The demonstration that in the oocyte the functional stability of interferon mRNA containing poly(A) is indistinguishable from that lacking poly(A) is in contrast to the results reported for globin mRNA and for histone mRNA (4-9). Recent observations show that the translational stability, in oocytes, of poly(A)-containing mengovirus mRNA is also indistinguishable from that which is poly(A)-free (P. Cornelis, H. Soreq, and U. Z. Littauer, unpublished data). It therefore appears that the increased translational stability conferred upon globin and histone mRNA by the presence of a 3'-poly(A) tail (4-9) is not a general phenomenon. There is also evidence that the length of poly(A) may be unrelated to the 14-fold increase in half-life of human fibroblast (FS-4) interferon mRNA that is seen when poly(I)-poly(C)-

induced FS-4 cells are superinduced with 5,6-dichloro-1-,BD-ribofuranosylbenzimidazole, an inhibitor of hnRNA and mRNA synthesis (3, 25, 26). However, an inverse correlation between the rate of poly(A) shortening and half-life of mRNA in adenovirus-transformed cells has been reported recently (24). We thank Dr. James E. Darnell for helpful comments and for the poly(U)-Sepharose, Dr. Douglas S. Lyles for statistical analysis, Miss Toyoko Kikuchi for excellent technical assistance, Drs. S. Koide and L. Burzio for the X. laevis, and Mrs. Erna Olbert for their upkeep. P.B.S. is a postdoctoral fellow of the National Cancer Institute (5F32-CA05900-02) and H.S. is a Fogarty International Research Fellow (FO5TW2479-01) of the U.S. Public Health Service. This investigation was supported by Research Grant CA-18608 and Program Project Grant CA-18213 awarded by the National Cancer Institute.

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