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Increased methylation of RNA in SV40-infected interferon-treated cells
VIROLOGY
112, 109-118
Increased
(1981)
Methylation C. KAHANA, Department
of RNA
in SV40-infected
E. YAKOBSON,
of Virology,
The
M. REVEL,
Weizmann
Accepted
interferon-Treated
Institute
December
30,
AND
of Science,
Cells
Y. GRONERl Rehovot,
Israel
1980
Interferon treatment, late in the SV40 lytic cycle, results in a block of viral protein synthesis. Nevertheless, the quantity of the corresponding viral mRNAs is undiminished and the polyadenylation is normal. Under the same conditions, interferon treatment caused a threeto fivefold increase in internal methylation and a threefold increase in Cap II formation of SV40 mRNAs. Methylation of host mRNA and poly(A) minus RNA was also increased after interferon treatment, but only by a factor of l-2. No dimethylated adenosines (ma6A) were found in the overmethylated SV40 mRNAs and there was no change in the relative proportions of the various m6A sequences. Although the overmethylated SV40 mRNA was extracted from interferon-treated cells at a stage in which its in viva translation was inhibited by 80%, it was translated in vitro as efficiently as normally methylated control mRNA. INTRODUCTION
Interferon treatment, late in the SV40 lytic cycle, after early functions have been expressed and viral DNA replication was initiated, resulted in a marked reduction of viral protein synthesis. However, in the treated cells there was an undiminished quantity of what appeared to be normal 16 S and 19 S SV40 mRNA (Yakobson et al, 1977). Like the majority of mRNAs from eukaryotic cells and the viruses infecting them, SV40 mRNAs are blocked at their 5’ termini by a methylated cap structure (Shatkin, 1976; Groner et al., 1977; Canaani et al., 1979). The 5’-methylated cap structures are important for ribosome binding, mRNA translation, and stability (Revel and Groner, 1978). In addition, SV40 mRNAs contain N6-methyl-adenosine residues (m6A) at internal positions of the mRNA chain (Canaani et al., 1979b). These methylated m6A residues are found in two sequences: Gpm’ApC and (Ap),m’ApC, where n varies from 1 to 4 (Canaani et al., 1979b). These same sequences are also methylated in Hela cells, L cells, BSC-1 cells, and B77 avian sarcoma virus RNA (Wei and Moss, 1977; Wei et al., 1976; Schibler et al., 1977; Canaani et al., 1 To whom
reprint
requests
should
be addressed. 109
1979; Dimock and Stoltzfus, 1977). This remarkable degree of sequence specificity in evolutionary diversed systems, plus the fact that these methylated sequences are conserved during processing of hnRNA (Schibler et al., 1977; Lavi et al., 1977) argues for an important biological function. Recently, we have identified and mapped three m6A-containing sequences present in late SV40 16 S and 19 S mRNAs by hybridization of methyZ-3H-labeled mRNA to specific restriction fragments (Canaani et al., 1979b). These results, which have provided the first example of precise localization of internal methylations in specific mRNA species reveal that while all three m6As are present in the coding region, two appear to be clustered at the 5’ end. Since the latter two m6As are located near the region of the late SV40 mRNA in which an intervening sequence has been removed, these observations provide supportive indication for their potential role(s) in mRNA processing. This observation has led to speculation that these internal modifications may serve as specific cleavage sites for processing nucleases, most notably splicing and/or ligating enzymes. In this report a possible connection between the interferon-mediated block of late SV40 protein synthesis and the state of mRNA methylation was investigated. Detailed 0042~6822/81/090109-10$02,00/O Copyright All rights
B 1981 by Academic Press, Inc. of reproduction in any form reserved.
110
KAHANA
analysis of late SV40 mRNA methylations following interferon treatment revealed a three- to fivefold increase in internal m6A and about twofold increase in 2’-O-methylation of the nucleotide, adjacent to the cap (i.e., formation of Cap II). Further experiments showed that this increased methylation is not the direct cause for the interferon-induced impairment of viral mRNA translation. It is well established that various viruses are being affected differently by interferons. In this respect mRNA methylation is not an exception. In two other previously studied systems, vaccinia virusinfected chick embryo fibroblasts (Kroath et al., 19’78; Kroath et al., 1979) and reovirus-infected L929 cells (Desrosiers and Lengyel, 1977) interferon causes a decrease in cap methylation. In fact, these two systems also respond qualitatively differently to interferon treatment: the 50% reduction in vaccinia virus Cap I is due to a decrease in ribose methylation of the penultimate nucleotide in the cap (Kroath et al., 1979), whereas in reovirusinfected interferon-treated cells viral Cap I is not reduced, but the percentage of Cap II termini is lower from control cells (Desrosiers and Lengyel, 1977). MATERIALS
AND
METHODS
Preparation of monkey interferon. Monkey interferon was prepared from NDVinfected BSC-1 cells according to Desmyter et al. (1968) and Yakobson et al. (1977). The titer was measured by plaque reduction of vesicular stomatitis virus on BSC1 cells. Infection, interferon treatment, and labeling of RNA. BSC-1 cells were cultured in minimal Eagle’s medium with 10% calf serum. Two days after reaching confluency monolayers were infected at a multiplicity of about 25 plaque-forming units (PFU) per cell of SV40 strain 777 in serumfree medium. After 2 hr, unadsorbed virus was removed and incubation continued with medium containing 2% calf serum. Twenty-two hours postinfection, interferon was added to the culture at a final concentration of 100 U/ml, 22 hr later,
ET AL.
cultures were labeled for 7 hr with [methyl3H]methionine (75 ci/mmol) (New England Nuclear) or [32P]orthophosphate (Amersham, England). Isolation of SV40 mRNA. After labeling, cells were washed with cold phosphatebuffered saline (PBS) and monolayers were treated with hypotonic buffer (10 mM Tris-HCI, pH 7.6, 10 mM NaCl, 3 mM MgC12) containing 0.5% NP-40. Nuclei were collected by centrifugation (2000 rpm, 10 min 4”), and excluded. The cytoplasmic lysate was extracted twice with 1 vol phenol, 0.5 vol chloroform, 2% isoamylalcohol; and twice with chloroform 2% isoamylalcohol. Poly(A)-containing RNA was isolated via chromatography through oligo(dT)-cellulose, and SV40specific RNA was selected by hybridization to and elution from SV40 DNA covalently bound to Sepharose as outlined by (Groner et al., 1977). Enzymatic digestion. Combined digestion with Pi nuclease (Yamasa Shoyu Co.,) and alkaline phosphatase (Worthington Biochemicals), and with RNase T2 (Calbiochem) and alkaline phosphatase were carried out, as described by Groner and Hurwitz (1975). Digestion with RNases A and Ti (Worthington Biochemicals) was carried out as described by Canaani et al. (197913).
Chromatography
and
electrophoresis.
Paper electrophoresis in pyridine acetate (pH 3.5) was carried out as before (Groner and Hurwitz, 1975; Groner et al., 1978). Chromatography on acetylated dihydroxyboryl-cellulose (Rosenberg and Gilham, 1971) was essentially as previously described (Groner et al., 1976), but was carried out at 4” (Gelians and Roberts, 1977). Descending paper chromatography on Whatman 3MM was performed with acetonitrile:ethyl acetate:n-butanol:isopropanol:GN-NH3 (7:2:1:1:2.7) solvent A and with isopropanol:NH,:H,O (70:1:30) solvent B. Measurement of protein synthesis. Fortyfour hours postinfection cultures were labeled with [3H]leucine for 2 hr. Cells were lysed with electrophoresis sample buffer (100 mM Tris-HCl, pH 6.8, 1% SDS, 1%
INTERFERON-INDUCED
OVERMETHYLATION
2-mercaptoethanol, and 10% glycerol) and boiled for 5 min. Equal volume aliquots were fractionated on 12% polyacrylamide gel. In vitro translation in reticulocyte lysates. Reticulocyte lysates from rabbits were prepared by the method of Pelham and Jackson (1976). Micrococcal nuclease treatment of the lysate was carried out at a concentration of 20 pg/ml at 20” for 20 min. Protein synthesis assays contained 0.015 M creatin phosphate, 90 mM potassium chloride, 125 mM each amino acid except methionine, 0.5 mM magnesium acetate, and 0.1 mg of creatine kinase per milliliter. A 13.5 &i amount of [35S]methionine (900 Ci/mmol) and varying quantities of mRNA were added per 25 ~1 of reaction mixture. Incubation at 30” for 60 min was terminated by freezing at -20”. RESULTS
Interferon-Mediated Increase of Internal Methylatiwn of SV40 Late mRNAs SV40-infected BSC-1 cells were treated by interferon and methyl-3H-labeled viral RNA isolated as described under Methods. methyl-3H-Labeled SV40 RNA was extensively digested with Pi nuclease and bacterial alkaline phosphatase. Cap structures were separated from internal m6A by high-voltage electrophoresis and their relative amount determined (Fig. 1, A, B). Table 1 shows that the ratio of m6A to caps was 3.5- to 5-fold higher in viral RNA from interferon-treated cells than in SV40 RNA extracted from control cells. This difference may result from either a decrease in SV40 cap methylations or an increase in internal m6A methylation. To distinguish between these two possibilities, a doublelabel experiment was performed. RNA was uniformly labeled with [14C]uridine and the methyl groups were labeled as before with methyl-3H. SV40 RNA was isolated. from control and interferontreated cells, digested with nuclease P1 plus alk.aline phosphatase and analyzed by paper electrophoresis (Fig. 1, C, D). Quantitative comparison of the various radioactive components separated on the elec-
OF RNA
111
tropherogram in Fig. 1, C, D, is presented in Table 2. No significant differences were found between IF-treated and control cells in the ratio: methyl-3H-cap/[14C]uridine plus cytidine (?30% of the [14C]uridine was converted in vivo to cytidine). However, the ratio m6A/uridine plus cytidine was 5.5 times higher in SV40 mRNA from IF-treated cells. These data imply that the increased ratio of m6A to cap (Table 1) is a result of enhanced internal methylation rather than a reduction in cap methylation. This enhancement of m6A is not merely a result of interferon-induced inhibition of viral protein synthesis. Since SV40 mRNA extracted from cells in which protein synthesis was 20-fold inhibited by 25pg/ml cycloheximide displayed a normal pattern of methylation. The number of pm6A residues in SV40 mRNA was previously determined by analysis of in vivo 32P-labeled RNA (Canaani et al., 1979b). Similar measurements of [32P]m6A in SV40 mRNA from interferon-treated cells confirmed the observed IF-induced enhancement of internal methylation (data not shown). Interferon-Induced Overmethylation of Cellular mRNA and poly(A)-minus RNA methyl-3H-Labeled poly(A)-containing cellular RNA from SV40-infected interferon-treated cells was analyzed as described above. The nuclease P, and alkaline phosphatase digestion was followed by electrophoretic separation of the internal m6A and caps. Results are summarized in Table 1. While the ratio IF/control in SV40 mRNA varied between 2.9 and 5.4, the overmethylation of cellular poly(A) plus RNA was much lower (IF/control, 1.26-1.68). Double-labeled experiments, using [14C]uridine and [methyl-3H]methionine, showed also in this case an increase in the internal m6A residues. Overmethylation of poly(A)-minus RNA from IF-treated cells was also examined. methyl3H radioactivity per OD260 was twofold higher in RNA extracted from interferontreated cells (not shown). We concluded from these data that IF-induced over-
112
KAHANA
ET AL.
FIG. 1. Paper electrophoretic analysis of nuclease P, and alkaline phosphatase digestion of labeled late SV40 mRNAs. Labeled SV40 mRNAs were digested with nuclease PI, followed by alkaline phosphatase and analyzed by paper electrophoresis as described under Methods. (A, B) methyl-3HLabeled RNA from (A) control and (B) interferon-treated cells. (C, D) [‘%]Uridine and rneth~&~Hlabeled RNA from (C) control and (D) interferon-treated cells.
TABLE
1
RELATIVEPROPORTIONSOF~~AANDCAPCORESIN SV40 AND CELLULAR mRNAs FROM INTERFERONTREATED
AND
CONTROL
CELLS
Distribution of Interferon-Induced m6A among the Methylated Sequences
wm mRNA sv40 Control Exp 1 Exp 2 Exp 3 Interferon Enp 1 Exp 2 Exp 3 CdlUlU Control Erp 1 Exp 2 Interferon Exp 1 Exp 2
methylation is not restricted to the viral mRNA. Nevertheless, it is definitely more pronounced in late SV4Q mRNA than in the cellular message and therefore, interesting to characterize.
&An
cap
m6A/eap
3570 4350 2150
7070 7750 7930
0.50 0.56 0.27
9320 4850 6350
5480 2900 4300
1.70 1.67 1.47
3807 1971
2360 1643
1.61 1.19
2997 1729
1468 862
2.04 2.00
treated
treated
IF/control
Exp 1 Exp 2 Exp 3
3.40 2.98 5.44
Exp 1 Exp 2
1.26 1.68
” meth~1-3H-Labeled mRNAs were digested with nuclease P, and alkaline phosphatase and products separated by paper electrophoresis as in Fig. 1.
In SV40 late mRNAs, as well as in mRNAs from other cellular and viral systems, m6A occurs mainly in two sequences: Gpm’ApC and Apm’ApC (see Introduction). In late SV40 mRNA there are an additional three minor m6A-containing sequences: ApApm’ApC; ApApApm’ApC, and ApApApApm’ApC. We examined whether the interferon-induced m6As were confined to those sequences in the same relative abundance as in untreated viral RNA. methyl-3H-Labeled SV40 mRNA from IF-treated cells was isolated and digested, as outlined under Methods, with a mixture of RNases A and T1 (which resulted in cleavage of phosphodiester bonds adjacent to U, C, and G, but not A). This
INTERFERON-INDUCED
OVERMETHYLATION TABLE
MEASUREMENT
2
OF m6A AND CAPS IN UNIFORMLY LABELED SV40 AND CONTROL CELLS 14C cpm
sv40 mRNA
Uridine”
Control Interferon
3260 9891
a Double-labeled SV40 mRNA as described in Fig. 1, C, D.
with
n~thyl-~H
cpm
Cytidine
m6A
cap
1301 3163
503 8080
1564 4983
[‘%]uridine
plus
treatment generates [methyZ-3H]m6A-containing oligonucleotides plus methgb3Hcapped termini. To separate the m6A oligonucleotides from caps, digested RNA was chromatographed through a DBAEcellulose column. Caps containing free 2’3’-hydroxyl groups at the 5’-terminal m7G are retained by the DBAE-cellulose column due to the affinity of the cis-diols for the dihyd.roxyboryl groups (Rosenberg and Gilham, 19’71; Groner et al., 1976). The unbound material containing the internal m6A oligonucleotides was desalted, treated with alkaline phosphatase, and analyzed by paper electrophoresis. Figure 2 depicts the separation of SV4Q m6A-containing oligonucleotides derived from control (A) and interferon-treated cells (B). The five previously characterized (Canaani et al., 1979b) methylated sequences were resolved (numbered l-5); two major peaks, migrating with ApC and ApApC markers, plus three more minor peaks. The relative proportions of the m6A-containing oligonucleotid.es are summarized in Table 3. There was no significant difference between SV40 mRNA extracted from control or IF-treated cells, indicating that the interferon-induced m6As are evenly distributed among the normal five methylated sequences.
Interferon Treatment Does Not Induce Fomnation of Dimethyladenosine Interferon treatment of SV40-infected cells inhibits SV40 protein synthesis (see Introduction). Monomethylation at the N6 position of adenosine is known to desta-
113
OF RNA
cytidine
mRNA
FROM INTERFERON-TREATED
m’A/U
Cap/U
+ C
0.11 0.61
and methyl-sH
+ C
0.34 0.38
was digested
and fractionated
bilize Watson-Crick base pairing. A second methylation in the 6 position to form 6,6-dimethyladenosine will totally disrupt hydrogen bonding (Engle and Hippel, 1974; Giffen et al., 1964). Canaani et al. have shown that in late SV40 mRNA, m6A residues are present in the coding regions.
0
20
40
60
SO
100
FIG. 2. Paper electrophoresis analysis of m6A-containing oligonucleotides derived from SV40 mRNA. m6A oligonucleotides generated by digestion with RNases T1 and A were isolated by passage through DBAE-cellulose as described under Methods, treated with alkaline phosphatase, and subjected to paper electrophoresis in pyridinum acetate (pH 3.5). (A) Control; (B) interferon treated.
114
KAHANA TABLE
3
RELATIVE PROPORTIONS OF m6A OLIGONUCLEOTIDES IN SV40 mRNA FROM INTERFERON-TREATED AND CONTROLCELLS SV40
mRNA
Control m6A oligonucleotide# Gpm’ApC Apm’ApC ApApm’ApC ApApApm’ApC ApApApApm”ApC
analyzed Interferon
wm
%
wm
%
1752 458 108 234 117
65.6 17.1 4.0 8.8 4.4
1556 468 116 262 113
62.0 18.6 4.6 10.4 4.5
a methyl-3H-Labeled SV40 mRNA with RNases A and Ti. m6A-containing tides were isolated by passage through lulose column, treated with alkaline and analyzed by paper electrophoresis
was digested oligonucleoa DBAE-celphosphatase, as in Fig. 2.
Thus, the introduction of a second methyl group at the N6 position may create a nontranslatable mRNA. Since the interferoninduced overmethylation is confined to the normally found sequences, the possibility that interferon treatment results in the formation of dimethyladenosine was considered. methyl-3H-Labeled SV40 mRNA from interferon-treated cells was digested by nuclease P1 followed by treatment with alkaline phosphatase and separated by paper electrophoresis, as in Fig. 1B. Radioactivity comigrating with m6A marker was eluted and further analyzed by descending paper chromatography with solvent A, which separates m6A from mz6A. As shown in Fig. 3, all the methylated adenosine of interferon-treated SV40 mRNA was a monomethylated derivative comigrating with m6A marker.
ET AL.
cores. To analyze the IF effect on Cap II formation methyZ-3H-labeled SV40 mRNA from IF-treated and nontreated BSC-1 cells, was digested with RNase T2 and alkaline phosphatase. RNase Tz cleaves phosphodiester linkages in polynucleotides, except those containing 2’-O-methylated residues, to yield 3’-phosphate nucleotides. Therefore, RNase Tz digestion, followed by phosphatase treatment of methyZ-3H-labeled SV40 mRNA, should generate m6A as well as 5’ cap structures of the type m7GpppXmpY (Cap I) and m7GpppmXmpYmpZ (Cap II). Figure 4 depicts electrophoretic separations of the m6A and Cap I and II species of control (A) and interferon-treated (B) late SV40 mRNA. Peaks 1, 2, and 3 were eluted and analyzed by partial digestion with nucleotide pyrophosphatase as detailed before (Canaani et al., 1979). They have been identified as: (1) m7GpppmAmpC (2) m7GpppmAmpUmpU, and (3) m7GpppmAmpU. Quantitative comparison is summarized in Table 4. While the amount of Cap I species was similar in control and IF-treated cells, the proportion of Cap II m7GpppmAmpUmpU was 3.6 times higher in IF-treated cells. From these results we
0
Interferon
Enhances
Formatiwn
of SV40
Cap II Late SV40 mRNA contains heterogenous populations of 5’ capped termini. Ninety percent of the Cap I structures are m7GpppmAmpU and 65% of the Cap II are m7GpppmAmpUmpU (Canaani et al., 1979). As shown in Table 2, interferon treatment did not alter the amount of late SV40 cap
IO
20 Fraction
30
40
50
Number
FIG. 3. Paper chromatography separation of methylated adenosine residues derived from interferontreated methyl-3H-labeled SV40 mRNA. methyl-3HLabeled material comigrating with adenosine marker in paper electrophoresis at pH 3.5 was eluted and analyzed by descending paper chromatography in acetonitryle:ethyl acetate:n-butanol:isopropanol:GN-NH, (7:2:1:1:2.7) as described under Meth-
ads.
INTERFERON-INDUCED
OVERMETHYLATION
concluded that the enhanced methylation is not specific for the internal N6 adenosine, but also occurs at the ribose of the subpenultimate nucleotide. In Vitro Translation of mRNA from Interferon-Treated Cells In vitro translation of a given messenger RN.A in cell-free extracts is a useful measure for its biological activity. We have used this criterion to analyze and compare late SV40 mRNAs extracted from IF-treated and nontreated BSC-1 cells. RNA was labeled with [“Hluridine to a low specific activity, and poly(A)-containing RNA was isolated from control and IFtreated cells. The percentage of SV40-specific mRNA, as determined by hybridization to SV40 DNA, was approximately the same in both samples. mRNA, 0.01 to 0.5 pg, was added to reticulocyte cell-free systems. Radioactivity incorporated from I
I
I
I
1
I
1
1
1
’
‘I
OF RNA TABLE
115 4
RELATIVEPROPORTIONOF SV40 CAP STRUCTURES FROM INTERFERON-TREATED TROL CELLS Control (wm)
Interferon
cap structure" m'GpppmAmpC mrGpppmAmpU m7GpppmAmpUmpU
3730 7880 3190
3550 8410 11780
(cpm)
I ANDCAP II AND CON-
IF/control
0.95 1.07 3.69
'mrthyl-3H-Labeled SV40 mRNA was digested with RNase T2 and alkaline phospbatase.Caps were separated as described in Fig.4.
[35S]methionine was determined and aliquots containing the same amount of radioactivity were fractionated on 12% polyacrylamide gels. Figure 5 shows that overmethylated mRNA from IF-treated cells was as efficient as control mRNA in supporting [35S]methionine incorporation. The onthoradiogram shows that the relative intensity of VP-l bands was practically the same in the two RNA samples. These results imply that in reticulocyte cell-free systems the biological activity of interferon-induced overmethylated late SV40 mRNA is indistinguishable from control viral mRNA. DISCUSSION
3 1
aI 0
I 20
I 40
I
I 60
I
I 80
I 100
FIG. 4. Paper electrophoresis analysis of RNase TEresistant capped oligonucleotides derived from SV40 mRNA. ,methyl-%Labeled capped oligonucleotides and m6Ap generated by digestion with RNase Tz were treated with alkaline phosphatase and subjected to paper electrophoresis in pyridinim acetate (pH 3.5). (A) Control; (B) interferon treated.
In the present paper, we have shown that in response to interferon treatment late in SV40 lytic cycle viral-specific mRNA was three- to fivefold overmethylated at all the internal m6A-containing sequences. There was, however, no change in the type of m6A sequences and the relative proportion of the various species was maintained. The major late SV40 5’-terminal sequence m7GpppmAmpUpU is also two to three times overmethylated after interferon treatment and converted into Cap II m7GpppmAmpUmpU. A plausible explanation for the increased methylation is that in normal infection not all the potential sites are methylated but they become methylated in interferon-treated cells. The biological significance of the overmethylation is still unclear. Recently it was shown that specific cleavage of ribosomal RNA occurs in cytoplasmic extracts of BSC-1 infected with SV40 and
116
KAHANA
57 s
I
I
0.1
0.2
ET AL.
I
0.3 p-s
I 0.4
I
0.5
RNA
FIG. 5. Comparison of mRNAs activity in reticulocyte cell-free system. Amino acid incorporation was directed by increasing concentrations of mRNA extracted from SV40-infected control (0) interferon-treated (0) cells. Three-microliter aliquots were removed and the incorporation [%]methionine into material precipitated by hot trichloroacetie acid was determined. Autoradiogram depicts the analysis by SDS-polyacrylamide gel electrophoresis of 10 ~1 [%]methioninelabeled cell-free products. SV40 capsid polypeptide marker was derived from [%]methionine-labeled purified SV40 virus.
treated by interferon (Revel et al., 1979). This degradation may reflect the turning on of the (2’-5’)oligo(A)-dependent RNase via an activation of the interferon-induced (2’-5’)oligo(A) synthetase by some overmethylated RNA present in these cells (Revel et al., 1979). As shown here, mRNA extracted from SV40-infected interferontreated cells can be translated in reticulocyte cell-free systems. The activity of these SV40 mRNAs was the same as that of SV40 mRNAs from control cells, despite the fact that the in vivo rate of VP-l synthesis at the time the mRNA was extracted, was only 20% of that in the control cells. The increased methylation was seen at the time when SV40 mRNAs were no longer translated in the interferon-treated cells, but the arrest of translation by cycloheximide did not cause such an overmethylation. Thus, the increased methylation is not merely the result of viral protein synthesis inhibition in the interferon-treated cells. Methylation of host mRNA and poly(A)minus RNA was also increased after inter-
or of
feron treatment, but by a factor of 1.3-2, instead of the three- to fivefold increase seen for SV40 RNA. In various virus-host cell systems different steps in the virus growth cycle are influenced by interferons and multivarious cellular responses to interferon treatment were observed (Revel, 1979). In this respect, methylation of mRNA is not an exception: Rossi et al. (1977) have reported an increase in RNA-methylating activity in interferon-treated cells. An unidentified modification of mouse L cell mRNA after interferon treatment was also reported (Riley and Levy, 1972). It is not clear if these observations are related to the overmethylation of RNA reported here. The increased methylation of SV40 RNA contrasts with the observation that in extracts from interferon-treated mouse cells the methylation of the 7-methylguanosine in the 5’ cap of in vitro-synthesized reovirus RNA is impaired, as a result of the presence of an inhibitor of methylation (Sen et al., 1975, 1977). VSV cap methylation was not decreased in interferon-
INTERFERON-INDUCED
OVERMETHYLATION
treated chick cell extracts (Ball and White, 1978). In infected mouse cells, a reduction in reovirus mRNA ribose methylation, leading to a shift of Cap II to Cap I, was reported (Desrosiers and Lengyel, 1979). This reduction is qualitatively different from that observed. in interferon-treated chick cel.ls where a decreased methylation of the penultimate cap nucleotide was reported for both vaccinia and host mRNA (Kroath et al., 19’78, 1979). Since no decrease in the total methylated nucleotides per RNA was found it is possible that in fact methylation increased in other parts of the RNA chain. In the present study, the ratio of methyl label over uridine incorporation was used to obtain an absolute measure of the Cap and m6A per RNA and the increase in m6A was confirmed by [32P]phosphate labeling. The fact that vaccinia and reovirus RNA synthesis takes place in the cytoplasm, while that of SV40 occurs in the nucleus, could also explain the differences observed. ACKNOWLEDGMENTS We thank A. Mukamel for her excellent technical assistance. Support for this work was provided by funds from BSF and NCRD (Jerusalem, Israel) and GSF (Munich, West Germany). REFERENCES BALL, L. A., and WHITE, C. N. (1978). Effect of interferon pretreatment on coupled transcription and translation in cell-free extracts of primary chick embryo cells. Virology 84, 496-508. CANAANI, D., KAHANA, C., MUKAMEL, A., and GRONER, Y. (1979). Sequence heterogeneity at the 5’ termini of late Simian virus 40 19s and 16s mRNAs. Proc. Nut. Acad. Sci. USA 76, 3078-3082. CANAANI, D., KAHANA, C., LAVI, S., and GRONER, Y. (1979b). Identification and mapping of N’-methyladenosine sequences in Simian virus 40 RNA. Nucleic Acid Res. 6, 2879-2899. DESMYTER, J., MELNICK, J. L., and RAWIS, W. E. (1968). Defectiveness of interferon production and of rubella virus interference in a line of African green monkey cells (vero). J. Viral. 2, 955-961. DESROSIERS, R. C., and LENGYEL, P. (1977). Impairment of Reovirus mRNA cap methylation in interferon-treated L cells. Fed. Proc. 36, 812. DIMOCK, K., and STOLTZFUS, C. M. (1977). Sequence specificity of internal methylation in B77 Avian
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sarcoma virus RNA subunits. Biochemistry 16, 471-478. ENGLE, J. D., and VON HIPPEL, P. H. (1974). Effects of methylation on the stability of nucleic acid conformations: Studies at the monomer level. Biochemistry 13, 4143-4158. GELIANS, R. E., and ROBERTS, R. J. (1977). One predominant 5’-undecanucleotide in adenovirus 2 late messenger RNAs. Cell 11, 533-544. GRIFFIN, B. E., HASLAM, W. J., and REESE, C. B. (1964). Synthesis and properties of some methylated polyadenylic acids. J. Mol. Biol. 10, 353-356. GRONER, Y., and HURWITZ, J. (1975). Synthesis of RNA containing a methylated blocked 5’ terminus by HeLa nuclear homogenates. Proc. Nut. Acad. Sci. USA 72, 2930-2934. GRONER, Y., GROSFELD, H., and LITTAUER, U. Z. (1976). 5’-capping structures of Artemia salina mRNA and the translational inhibition by cap analogs. Eur. J. Biochem. 71,281-293. GRONER, Y., CARMI, P., and ALONI, Y. (1977). Capping structures of Simian virus 40 19s and 16s mRNAs. Nucleic Acid Res. 4, 3958-3968. GRONER, Y., GILBOA, E., and AVIV, H. (1978). Methylation and capping of RNA polymerase II primary transcripts by HeLa nuclear homogenates. Biochemistry 17, 977-982. KROATH, H., GROSS, H. J., JUNGWIRTH, C., and BODO, G. (1978). RNA methylation in vaccinia-infected chick embryo fibroblasts treated with homologous interferon. Nucleic Acid Res. 5, 2441-2454. KROATH, H., JANDA, H. G., HILLER, G., KUHN, E., JUNGWIRTH, C., GROSS, H. J., and BODO, G. (1979). Methylation of vaccinia virus-specific mRNA in the interferon-treated chick cells. Virology 92,572-577. LAVI, U., FERNANDEZ-MUNOS, R., and DARNELL, J. E. (1977). Content of N’-methyl-adenylic acid in heterogenous nuclear and messenger RNA of HeLa cells. Nucleic Acid Res. 4, 63-79. PELHAM, H. R. B., and JACKSON, R. J. (1976). An efficient mRNA-dependent translation system from reticulocyte lysates. Eur. J. Biochem. 67, 247-256. REVEL, M., and GRONER, Y. (1978). Post-transcriptional and translational controls of gene expression in eukaryotes. Annu. Rev. Biochem. 47,10791126. REVEL, M. (1979). Molecular mechanisms involved in the antiviral effects of interferon. In “Interferon” (I. Gresser, ed.), Vol. 1, pp. 102-157. Academic Press, New York. REVEL, M., KIMCHI, A., SCHMIDT, A., SHULMAN, L., CHERNAJOVSKY, Y., RAPOPORT, S., and LAPIDOT, Y. (1979). Studies on interferon action: Synthesis, degradation and biological activity of (2’-5’)oligoisoadenylate. In “Regulation of Macromolecular Synthesis by Low Weight Mediators” (G. Koch and R. Dietmar, eds.), pp. 341-359. Academic Press, New York.
118
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RILEY, F. L., and LEVY, H. B. (1977). Studies on the mechanism of interferon action: Characterization of polysome-associated cellular RNAs modified by interferon. Virology 76, 459-463. ROSENBERG, M., and GILHAM, P. T. (1971). The isolation of 3’-terminal polynucleotides from RNA molecules. Biochem. Biophys. Acta 246, 337-340. ROSSI, G. B., DOLEI, A., CIOE, L., BENEDETTO, A., MATARESE, G. P., and BELARDELLI, F. (1977). Inhibition of transcription and translation of globin messenger RNA in dimethyl sulfoxide-stimulated Friend erythroleukemic cells treated with interferon. Proc. Nat. Acad. Sci. USA 74, 2036-2040. SCHIBLER, U., KELLEY, D. E., and PERRY, R. P. (1977). Comparison of methylated sequences in messenger RNA and heterogeneous nuclease RNA from mouse L-cells. J. Mol. Biol. 115, 695-714. SEN, G. C., LEBLEU, B., BROWN, G. E., REBELLO, M. A., FURNICHI, Y., MORGAN, M., SHATKIN, A., and LENGYEL, P. (1975). Inhibition of reovirus messenger RNA methylation in extracts of interferontreated Ehrlich ascites tumor cells. Biochem. Biophys. Res. Commun. 65, 427-434. SEN, G. C., SHAILU, S., LEBLEU, B., BROWN, G. E.,
ET AL. DESROSIERS, R. C., and LENGYEL, P. (1977). Impairment of reovirus mRNA methylation in extracts of interferon-treated Ehrlich ascites tumor cells: Further characteristics of the phenomenon. J. Viral. 21, 69-83. SHATKIN, A. J. (1976). Capping of eukaryotic mRNAs. Cell 9, 645-653. WEI, C.-M., GUERSHOWITZ, A., and Moss, B. (1976). 5’-terminal and internal methylated nucleotide sequenees in HeLa cells mRNA. Biochemistry 15, 397-401. WEI, C.-M., and MOSS, B. (1977). Nucleotide sequences at the N’-methyladenosine sites of HeLa cells messenger ribonucleic acid. Biochemistry 16, 16721676. YAKOBSON, E., PRIVES, C., HARTMAN, J.R., WINOCOUR, E., and REVEL, M. (1977). Inhibition of viral protein synthesis in monkey cells treated with interferon late in Simian virus 40 lytic cycle. Cell 12, 73-81. YAKOBSON, E., REVEL, M., and WINOCOUR, E. (1977b). Inhibition of Simian virus 40 replication by interferon treatment late in the lytic cycle. Virology 80, 225-228.
112, 109-118
Increased
(1981)
Methylation C. KAHANA, Department
of RNA
in SV40-infected
E. YAKOBSON,
of Virology,
The
M. REVEL,
Weizmann
Accepted
interferon-Treated
Institute
December
30,
AND
of Science,
Cells
Y. GRONERl Rehovot,
Israel
1980
Interferon treatment, late in the SV40 lytic cycle, results in a block of viral protein synthesis. Nevertheless, the quantity of the corresponding viral mRNAs is undiminished and the polyadenylation is normal. Under the same conditions, interferon treatment caused a threeto fivefold increase in internal methylation and a threefold increase in Cap II formation of SV40 mRNAs. Methylation of host mRNA and poly(A) minus RNA was also increased after interferon treatment, but only by a factor of l-2. No dimethylated adenosines (ma6A) were found in the overmethylated SV40 mRNAs and there was no change in the relative proportions of the various m6A sequences. Although the overmethylated SV40 mRNA was extracted from interferon-treated cells at a stage in which its in viva translation was inhibited by 80%, it was translated in vitro as efficiently as normally methylated control mRNA. INTRODUCTION
Interferon treatment, late in the SV40 lytic cycle, after early functions have been expressed and viral DNA replication was initiated, resulted in a marked reduction of viral protein synthesis. However, in the treated cells there was an undiminished quantity of what appeared to be normal 16 S and 19 S SV40 mRNA (Yakobson et al, 1977). Like the majority of mRNAs from eukaryotic cells and the viruses infecting them, SV40 mRNAs are blocked at their 5’ termini by a methylated cap structure (Shatkin, 1976; Groner et al., 1977; Canaani et al., 1979). The 5’-methylated cap structures are important for ribosome binding, mRNA translation, and stability (Revel and Groner, 1978). In addition, SV40 mRNAs contain N6-methyl-adenosine residues (m6A) at internal positions of the mRNA chain (Canaani et al., 1979b). These methylated m6A residues are found in two sequences: Gpm’ApC and (Ap),m’ApC, where n varies from 1 to 4 (Canaani et al., 1979b). These same sequences are also methylated in Hela cells, L cells, BSC-1 cells, and B77 avian sarcoma virus RNA (Wei and Moss, 1977; Wei et al., 1976; Schibler et al., 1977; Canaani et al., 1 To whom
reprint
requests
should
be addressed. 109
1979; Dimock and Stoltzfus, 1977). This remarkable degree of sequence specificity in evolutionary diversed systems, plus the fact that these methylated sequences are conserved during processing of hnRNA (Schibler et al., 1977; Lavi et al., 1977) argues for an important biological function. Recently, we have identified and mapped three m6A-containing sequences present in late SV40 16 S and 19 S mRNAs by hybridization of methyZ-3H-labeled mRNA to specific restriction fragments (Canaani et al., 1979b). These results, which have provided the first example of precise localization of internal methylations in specific mRNA species reveal that while all three m6As are present in the coding region, two appear to be clustered at the 5’ end. Since the latter two m6As are located near the region of the late SV40 mRNA in which an intervening sequence has been removed, these observations provide supportive indication for their potential role(s) in mRNA processing. This observation has led to speculation that these internal modifications may serve as specific cleavage sites for processing nucleases, most notably splicing and/or ligating enzymes. In this report a possible connection between the interferon-mediated block of late SV40 protein synthesis and the state of mRNA methylation was investigated. Detailed 0042~6822/81/090109-10$02,00/O Copyright All rights
B 1981 by Academic Press, Inc. of reproduction in any form reserved.
110
KAHANA
analysis of late SV40 mRNA methylations following interferon treatment revealed a three- to fivefold increase in internal m6A and about twofold increase in 2’-O-methylation of the nucleotide, adjacent to the cap (i.e., formation of Cap II). Further experiments showed that this increased methylation is not the direct cause for the interferon-induced impairment of viral mRNA translation. It is well established that various viruses are being affected differently by interferons. In this respect mRNA methylation is not an exception. In two other previously studied systems, vaccinia virusinfected chick embryo fibroblasts (Kroath et al., 19’78; Kroath et al., 1979) and reovirus-infected L929 cells (Desrosiers and Lengyel, 1977) interferon causes a decrease in cap methylation. In fact, these two systems also respond qualitatively differently to interferon treatment: the 50% reduction in vaccinia virus Cap I is due to a decrease in ribose methylation of the penultimate nucleotide in the cap (Kroath et al., 1979), whereas in reovirusinfected interferon-treated cells viral Cap I is not reduced, but the percentage of Cap II termini is lower from control cells (Desrosiers and Lengyel, 1977). MATERIALS
AND
METHODS
Preparation of monkey interferon. Monkey interferon was prepared from NDVinfected BSC-1 cells according to Desmyter et al. (1968) and Yakobson et al. (1977). The titer was measured by plaque reduction of vesicular stomatitis virus on BSC1 cells. Infection, interferon treatment, and labeling of RNA. BSC-1 cells were cultured in minimal Eagle’s medium with 10% calf serum. Two days after reaching confluency monolayers were infected at a multiplicity of about 25 plaque-forming units (PFU) per cell of SV40 strain 777 in serumfree medium. After 2 hr, unadsorbed virus was removed and incubation continued with medium containing 2% calf serum. Twenty-two hours postinfection, interferon was added to the culture at a final concentration of 100 U/ml, 22 hr later,
ET AL.
cultures were labeled for 7 hr with [methyl3H]methionine (75 ci/mmol) (New England Nuclear) or [32P]orthophosphate (Amersham, England). Isolation of SV40 mRNA. After labeling, cells were washed with cold phosphatebuffered saline (PBS) and monolayers were treated with hypotonic buffer (10 mM Tris-HCI, pH 7.6, 10 mM NaCl, 3 mM MgC12) containing 0.5% NP-40. Nuclei were collected by centrifugation (2000 rpm, 10 min 4”), and excluded. The cytoplasmic lysate was extracted twice with 1 vol phenol, 0.5 vol chloroform, 2% isoamylalcohol; and twice with chloroform 2% isoamylalcohol. Poly(A)-containing RNA was isolated via chromatography through oligo(dT)-cellulose, and SV40specific RNA was selected by hybridization to and elution from SV40 DNA covalently bound to Sepharose as outlined by (Groner et al., 1977). Enzymatic digestion. Combined digestion with Pi nuclease (Yamasa Shoyu Co.,) and alkaline phosphatase (Worthington Biochemicals), and with RNase T2 (Calbiochem) and alkaline phosphatase were carried out, as described by Groner and Hurwitz (1975). Digestion with RNases A and Ti (Worthington Biochemicals) was carried out as described by Canaani et al. (197913).
Chromatography
and
electrophoresis.
Paper electrophoresis in pyridine acetate (pH 3.5) was carried out as before (Groner and Hurwitz, 1975; Groner et al., 1978). Chromatography on acetylated dihydroxyboryl-cellulose (Rosenberg and Gilham, 1971) was essentially as previously described (Groner et al., 1976), but was carried out at 4” (Gelians and Roberts, 1977). Descending paper chromatography on Whatman 3MM was performed with acetonitrile:ethyl acetate:n-butanol:isopropanol:GN-NH3 (7:2:1:1:2.7) solvent A and with isopropanol:NH,:H,O (70:1:30) solvent B. Measurement of protein synthesis. Fortyfour hours postinfection cultures were labeled with [3H]leucine for 2 hr. Cells were lysed with electrophoresis sample buffer (100 mM Tris-HCl, pH 6.8, 1% SDS, 1%
INTERFERON-INDUCED
OVERMETHYLATION
2-mercaptoethanol, and 10% glycerol) and boiled for 5 min. Equal volume aliquots were fractionated on 12% polyacrylamide gel. In vitro translation in reticulocyte lysates. Reticulocyte lysates from rabbits were prepared by the method of Pelham and Jackson (1976). Micrococcal nuclease treatment of the lysate was carried out at a concentration of 20 pg/ml at 20” for 20 min. Protein synthesis assays contained 0.015 M creatin phosphate, 90 mM potassium chloride, 125 mM each amino acid except methionine, 0.5 mM magnesium acetate, and 0.1 mg of creatine kinase per milliliter. A 13.5 &i amount of [35S]methionine (900 Ci/mmol) and varying quantities of mRNA were added per 25 ~1 of reaction mixture. Incubation at 30” for 60 min was terminated by freezing at -20”. RESULTS
Interferon-Mediated Increase of Internal Methylatiwn of SV40 Late mRNAs SV40-infected BSC-1 cells were treated by interferon and methyl-3H-labeled viral RNA isolated as described under Methods. methyl-3H-Labeled SV40 RNA was extensively digested with Pi nuclease and bacterial alkaline phosphatase. Cap structures were separated from internal m6A by high-voltage electrophoresis and their relative amount determined (Fig. 1, A, B). Table 1 shows that the ratio of m6A to caps was 3.5- to 5-fold higher in viral RNA from interferon-treated cells than in SV40 RNA extracted from control cells. This difference may result from either a decrease in SV40 cap methylations or an increase in internal m6A methylation. To distinguish between these two possibilities, a doublelabel experiment was performed. RNA was uniformly labeled with [14C]uridine and the methyl groups were labeled as before with methyl-3H. SV40 RNA was isolated. from control and interferontreated cells, digested with nuclease P1 plus alk.aline phosphatase and analyzed by paper electrophoresis (Fig. 1, C, D). Quantitative comparison of the various radioactive components separated on the elec-
OF RNA
111
tropherogram in Fig. 1, C, D, is presented in Table 2. No significant differences were found between IF-treated and control cells in the ratio: methyl-3H-cap/[14C]uridine plus cytidine (?30% of the [14C]uridine was converted in vivo to cytidine). However, the ratio m6A/uridine plus cytidine was 5.5 times higher in SV40 mRNA from IF-treated cells. These data imply that the increased ratio of m6A to cap (Table 1) is a result of enhanced internal methylation rather than a reduction in cap methylation. This enhancement of m6A is not merely a result of interferon-induced inhibition of viral protein synthesis. Since SV40 mRNA extracted from cells in which protein synthesis was 20-fold inhibited by 25pg/ml cycloheximide displayed a normal pattern of methylation. The number of pm6A residues in SV40 mRNA was previously determined by analysis of in vivo 32P-labeled RNA (Canaani et al., 1979b). Similar measurements of [32P]m6A in SV40 mRNA from interferon-treated cells confirmed the observed IF-induced enhancement of internal methylation (data not shown). Interferon-Induced Overmethylation of Cellular mRNA and poly(A)-minus RNA methyl-3H-Labeled poly(A)-containing cellular RNA from SV40-infected interferon-treated cells was analyzed as described above. The nuclease P, and alkaline phosphatase digestion was followed by electrophoretic separation of the internal m6A and caps. Results are summarized in Table 1. While the ratio IF/control in SV40 mRNA varied between 2.9 and 5.4, the overmethylation of cellular poly(A) plus RNA was much lower (IF/control, 1.26-1.68). Double-labeled experiments, using [14C]uridine and [methyl-3H]methionine, showed also in this case an increase in the internal m6A residues. Overmethylation of poly(A)-minus RNA from IF-treated cells was also examined. methyl3H radioactivity per OD260 was twofold higher in RNA extracted from interferontreated cells (not shown). We concluded from these data that IF-induced over-
112
KAHANA
ET AL.
FIG. 1. Paper electrophoretic analysis of nuclease P, and alkaline phosphatase digestion of labeled late SV40 mRNAs. Labeled SV40 mRNAs were digested with nuclease PI, followed by alkaline phosphatase and analyzed by paper electrophoresis as described under Methods. (A, B) methyl-3HLabeled RNA from (A) control and (B) interferon-treated cells. (C, D) [‘%]Uridine and rneth~&~Hlabeled RNA from (C) control and (D) interferon-treated cells.
TABLE
1
RELATIVEPROPORTIONSOF~~AANDCAPCORESIN SV40 AND CELLULAR mRNAs FROM INTERFERONTREATED
AND
CONTROL
CELLS
Distribution of Interferon-Induced m6A among the Methylated Sequences
wm mRNA sv40 Control Exp 1 Exp 2 Exp 3 Interferon Enp 1 Exp 2 Exp 3 CdlUlU Control Erp 1 Exp 2 Interferon Exp 1 Exp 2
methylation is not restricted to the viral mRNA. Nevertheless, it is definitely more pronounced in late SV4Q mRNA than in the cellular message and therefore, interesting to characterize.
&An
cap
m6A/eap
3570 4350 2150
7070 7750 7930
0.50 0.56 0.27
9320 4850 6350
5480 2900 4300
1.70 1.67 1.47
3807 1971
2360 1643
1.61 1.19
2997 1729
1468 862
2.04 2.00
treated
treated
IF/control
Exp 1 Exp 2 Exp 3
3.40 2.98 5.44
Exp 1 Exp 2
1.26 1.68
” meth~1-3H-Labeled mRNAs were digested with nuclease P, and alkaline phosphatase and products separated by paper electrophoresis as in Fig. 1.
In SV40 late mRNAs, as well as in mRNAs from other cellular and viral systems, m6A occurs mainly in two sequences: Gpm’ApC and Apm’ApC (see Introduction). In late SV40 mRNA there are an additional three minor m6A-containing sequences: ApApm’ApC; ApApApm’ApC, and ApApApApm’ApC. We examined whether the interferon-induced m6As were confined to those sequences in the same relative abundance as in untreated viral RNA. methyl-3H-Labeled SV40 mRNA from IF-treated cells was isolated and digested, as outlined under Methods, with a mixture of RNases A and T1 (which resulted in cleavage of phosphodiester bonds adjacent to U, C, and G, but not A). This
INTERFERON-INDUCED
OVERMETHYLATION TABLE
MEASUREMENT
2
OF m6A AND CAPS IN UNIFORMLY LABELED SV40 AND CONTROL CELLS 14C cpm
sv40 mRNA
Uridine”
Control Interferon
3260 9891
a Double-labeled SV40 mRNA as described in Fig. 1, C, D.
with
n~thyl-~H
cpm
Cytidine
m6A
cap
1301 3163
503 8080
1564 4983
[‘%]uridine
plus
treatment generates [methyZ-3H]m6A-containing oligonucleotides plus methgb3Hcapped termini. To separate the m6A oligonucleotides from caps, digested RNA was chromatographed through a DBAEcellulose column. Caps containing free 2’3’-hydroxyl groups at the 5’-terminal m7G are retained by the DBAE-cellulose column due to the affinity of the cis-diols for the dihyd.roxyboryl groups (Rosenberg and Gilham, 19’71; Groner et al., 1976). The unbound material containing the internal m6A oligonucleotides was desalted, treated with alkaline phosphatase, and analyzed by paper electrophoresis. Figure 2 depicts the separation of SV4Q m6A-containing oligonucleotides derived from control (A) and interferon-treated cells (B). The five previously characterized (Canaani et al., 1979b) methylated sequences were resolved (numbered l-5); two major peaks, migrating with ApC and ApApC markers, plus three more minor peaks. The relative proportions of the m6A-containing oligonucleotid.es are summarized in Table 3. There was no significant difference between SV40 mRNA extracted from control or IF-treated cells, indicating that the interferon-induced m6As are evenly distributed among the normal five methylated sequences.
Interferon Treatment Does Not Induce Fomnation of Dimethyladenosine Interferon treatment of SV40-infected cells inhibits SV40 protein synthesis (see Introduction). Monomethylation at the N6 position of adenosine is known to desta-
113
OF RNA
cytidine
mRNA
FROM INTERFERON-TREATED
m’A/U
Cap/U
+ C
0.11 0.61
and methyl-sH
+ C
0.34 0.38
was digested
and fractionated
bilize Watson-Crick base pairing. A second methylation in the 6 position to form 6,6-dimethyladenosine will totally disrupt hydrogen bonding (Engle and Hippel, 1974; Giffen et al., 1964). Canaani et al. have shown that in late SV40 mRNA, m6A residues are present in the coding regions.
0
20
40
60
SO
100
FIG. 2. Paper electrophoresis analysis of m6A-containing oligonucleotides derived from SV40 mRNA. m6A oligonucleotides generated by digestion with RNases T1 and A were isolated by passage through DBAE-cellulose as described under Methods, treated with alkaline phosphatase, and subjected to paper electrophoresis in pyridinum acetate (pH 3.5). (A) Control; (B) interferon treated.
114
KAHANA TABLE
3
RELATIVE PROPORTIONS OF m6A OLIGONUCLEOTIDES IN SV40 mRNA FROM INTERFERON-TREATED AND CONTROLCELLS SV40
mRNA
Control m6A oligonucleotide# Gpm’ApC Apm’ApC ApApm’ApC ApApApm’ApC ApApApApm”ApC
analyzed Interferon
wm
%
wm
%
1752 458 108 234 117
65.6 17.1 4.0 8.8 4.4
1556 468 116 262 113
62.0 18.6 4.6 10.4 4.5
a methyl-3H-Labeled SV40 mRNA with RNases A and Ti. m6A-containing tides were isolated by passage through lulose column, treated with alkaline and analyzed by paper electrophoresis
was digested oligonucleoa DBAE-celphosphatase, as in Fig. 2.
Thus, the introduction of a second methyl group at the N6 position may create a nontranslatable mRNA. Since the interferoninduced overmethylation is confined to the normally found sequences, the possibility that interferon treatment results in the formation of dimethyladenosine was considered. methyl-3H-Labeled SV40 mRNA from interferon-treated cells was digested by nuclease P1 followed by treatment with alkaline phosphatase and separated by paper electrophoresis, as in Fig. 1B. Radioactivity comigrating with m6A marker was eluted and further analyzed by descending paper chromatography with solvent A, which separates m6A from mz6A. As shown in Fig. 3, all the methylated adenosine of interferon-treated SV40 mRNA was a monomethylated derivative comigrating with m6A marker.
ET AL.
cores. To analyze the IF effect on Cap II formation methyZ-3H-labeled SV40 mRNA from IF-treated and nontreated BSC-1 cells, was digested with RNase T2 and alkaline phosphatase. RNase Tz cleaves phosphodiester linkages in polynucleotides, except those containing 2’-O-methylated residues, to yield 3’-phosphate nucleotides. Therefore, RNase Tz digestion, followed by phosphatase treatment of methyZ-3H-labeled SV40 mRNA, should generate m6A as well as 5’ cap structures of the type m7GpppXmpY (Cap I) and m7GpppmXmpYmpZ (Cap II). Figure 4 depicts electrophoretic separations of the m6A and Cap I and II species of control (A) and interferon-treated (B) late SV40 mRNA. Peaks 1, 2, and 3 were eluted and analyzed by partial digestion with nucleotide pyrophosphatase as detailed before (Canaani et al., 1979). They have been identified as: (1) m7GpppmAmpC (2) m7GpppmAmpUmpU, and (3) m7GpppmAmpU. Quantitative comparison is summarized in Table 4. While the amount of Cap I species was similar in control and IF-treated cells, the proportion of Cap II m7GpppmAmpUmpU was 3.6 times higher in IF-treated cells. From these results we
0
Interferon
Enhances
Formatiwn
of SV40
Cap II Late SV40 mRNA contains heterogenous populations of 5’ capped termini. Ninety percent of the Cap I structures are m7GpppmAmpU and 65% of the Cap II are m7GpppmAmpUmpU (Canaani et al., 1979). As shown in Table 2, interferon treatment did not alter the amount of late SV40 cap
IO
20 Fraction
30
40
50
Number
FIG. 3. Paper chromatography separation of methylated adenosine residues derived from interferontreated methyl-3H-labeled SV40 mRNA. methyl-3HLabeled material comigrating with adenosine marker in paper electrophoresis at pH 3.5 was eluted and analyzed by descending paper chromatography in acetonitryle:ethyl acetate:n-butanol:isopropanol:GN-NH, (7:2:1:1:2.7) as described under Meth-
ads.
INTERFERON-INDUCED
OVERMETHYLATION
concluded that the enhanced methylation is not specific for the internal N6 adenosine, but also occurs at the ribose of the subpenultimate nucleotide. In Vitro Translation of mRNA from Interferon-Treated Cells In vitro translation of a given messenger RN.A in cell-free extracts is a useful measure for its biological activity. We have used this criterion to analyze and compare late SV40 mRNAs extracted from IF-treated and nontreated BSC-1 cells. RNA was labeled with [“Hluridine to a low specific activity, and poly(A)-containing RNA was isolated from control and IFtreated cells. The percentage of SV40-specific mRNA, as determined by hybridization to SV40 DNA, was approximately the same in both samples. mRNA, 0.01 to 0.5 pg, was added to reticulocyte cell-free systems. Radioactivity incorporated from I
I
I
I
1
I
1
1
1
’
‘I
OF RNA TABLE
115 4
RELATIVEPROPORTIONOF SV40 CAP STRUCTURES FROM INTERFERON-TREATED TROL CELLS Control (wm)
Interferon
cap structure" m'GpppmAmpC mrGpppmAmpU m7GpppmAmpUmpU
3730 7880 3190
3550 8410 11780
(cpm)
I ANDCAP II AND CON-
IF/control
0.95 1.07 3.69
'mrthyl-3H-Labeled SV40 mRNA was digested with RNase T2 and alkaline phospbatase.Caps were separated as described in Fig.4.
[35S]methionine was determined and aliquots containing the same amount of radioactivity were fractionated on 12% polyacrylamide gels. Figure 5 shows that overmethylated mRNA from IF-treated cells was as efficient as control mRNA in supporting [35S]methionine incorporation. The onthoradiogram shows that the relative intensity of VP-l bands was practically the same in the two RNA samples. These results imply that in reticulocyte cell-free systems the biological activity of interferon-induced overmethylated late SV40 mRNA is indistinguishable from control viral mRNA. DISCUSSION
3 1
aI 0
I 20
I 40
I
I 60
I
I 80
I 100
FIG. 4. Paper electrophoresis analysis of RNase TEresistant capped oligonucleotides derived from SV40 mRNA. ,methyl-%Labeled capped oligonucleotides and m6Ap generated by digestion with RNase Tz were treated with alkaline phosphatase and subjected to paper electrophoresis in pyridinim acetate (pH 3.5). (A) Control; (B) interferon treated.
In the present paper, we have shown that in response to interferon treatment late in SV40 lytic cycle viral-specific mRNA was three- to fivefold overmethylated at all the internal m6A-containing sequences. There was, however, no change in the type of m6A sequences and the relative proportion of the various species was maintained. The major late SV40 5’-terminal sequence m7GpppmAmpUpU is also two to three times overmethylated after interferon treatment and converted into Cap II m7GpppmAmpUmpU. A plausible explanation for the increased methylation is that in normal infection not all the potential sites are methylated but they become methylated in interferon-treated cells. The biological significance of the overmethylation is still unclear. Recently it was shown that specific cleavage of ribosomal RNA occurs in cytoplasmic extracts of BSC-1 infected with SV40 and
116
KAHANA
57 s
I
I
0.1
0.2
ET AL.
I
0.3 p-s
I 0.4
I
0.5
RNA
FIG. 5. Comparison of mRNAs activity in reticulocyte cell-free system. Amino acid incorporation was directed by increasing concentrations of mRNA extracted from SV40-infected control (0) interferon-treated (0) cells. Three-microliter aliquots were removed and the incorporation [%]methionine into material precipitated by hot trichloroacetie acid was determined. Autoradiogram depicts the analysis by SDS-polyacrylamide gel electrophoresis of 10 ~1 [%]methioninelabeled cell-free products. SV40 capsid polypeptide marker was derived from [%]methionine-labeled purified SV40 virus.
treated by interferon (Revel et al., 1979). This degradation may reflect the turning on of the (2’-5’)oligo(A)-dependent RNase via an activation of the interferon-induced (2’-5’)oligo(A) synthetase by some overmethylated RNA present in these cells (Revel et al., 1979). As shown here, mRNA extracted from SV40-infected interferontreated cells can be translated in reticulocyte cell-free systems. The activity of these SV40 mRNAs was the same as that of SV40 mRNAs from control cells, despite the fact that the in vivo rate of VP-l synthesis at the time the mRNA was extracted, was only 20% of that in the control cells. The increased methylation was seen at the time when SV40 mRNAs were no longer translated in the interferon-treated cells, but the arrest of translation by cycloheximide did not cause such an overmethylation. Thus, the increased methylation is not merely the result of viral protein synthesis inhibition in the interferon-treated cells. Methylation of host mRNA and poly(A)minus RNA was also increased after inter-
or of
feron treatment, but by a factor of 1.3-2, instead of the three- to fivefold increase seen for SV40 RNA. In various virus-host cell systems different steps in the virus growth cycle are influenced by interferons and multivarious cellular responses to interferon treatment were observed (Revel, 1979). In this respect, methylation of mRNA is not an exception: Rossi et al. (1977) have reported an increase in RNA-methylating activity in interferon-treated cells. An unidentified modification of mouse L cell mRNA after interferon treatment was also reported (Riley and Levy, 1972). It is not clear if these observations are related to the overmethylation of RNA reported here. The increased methylation of SV40 RNA contrasts with the observation that in extracts from interferon-treated mouse cells the methylation of the 7-methylguanosine in the 5’ cap of in vitro-synthesized reovirus RNA is impaired, as a result of the presence of an inhibitor of methylation (Sen et al., 1975, 1977). VSV cap methylation was not decreased in interferon-
INTERFERON-INDUCED
OVERMETHYLATION
treated chick cell extracts (Ball and White, 1978). In infected mouse cells, a reduction in reovirus mRNA ribose methylation, leading to a shift of Cap II to Cap I, was reported (Desrosiers and Lengyel, 1979). This reduction is qualitatively different from that observed. in interferon-treated chick cel.ls where a decreased methylation of the penultimate cap nucleotide was reported for both vaccinia and host mRNA (Kroath et al., 19’78, 1979). Since no decrease in the total methylated nucleotides per RNA was found it is possible that in fact methylation increased in other parts of the RNA chain. In the present study, the ratio of methyl label over uridine incorporation was used to obtain an absolute measure of the Cap and m6A per RNA and the increase in m6A was confirmed by [32P]phosphate labeling. The fact that vaccinia and reovirus RNA synthesis takes place in the cytoplasm, while that of SV40 occurs in the nucleus, could also explain the differences observed. ACKNOWLEDGMENTS We thank A. Mukamel for her excellent technical assistance. Support for this work was provided by funds from BSF and NCRD (Jerusalem, Israel) and GSF (Munich, West Germany). REFERENCES BALL, L. A., and WHITE, C. N. (1978). Effect of interferon pretreatment on coupled transcription and translation in cell-free extracts of primary chick embryo cells. Virology 84, 496-508. CANAANI, D., KAHANA, C., MUKAMEL, A., and GRONER, Y. (1979). Sequence heterogeneity at the 5’ termini of late Simian virus 40 19s and 16s mRNAs. Proc. Nut. Acad. Sci. USA 76, 3078-3082. CANAANI, D., KAHANA, C., LAVI, S., and GRONER, Y. (1979b). Identification and mapping of N’-methyladenosine sequences in Simian virus 40 RNA. Nucleic Acid Res. 6, 2879-2899. DESMYTER, J., MELNICK, J. L., and RAWIS, W. E. (1968). Defectiveness of interferon production and of rubella virus interference in a line of African green monkey cells (vero). J. Viral. 2, 955-961. DESROSIERS, R. C., and LENGYEL, P. (1977). Impairment of Reovirus mRNA cap methylation in interferon-treated L cells. Fed. Proc. 36, 812. DIMOCK, K., and STOLTZFUS, C. M. (1977). Sequence specificity of internal methylation in B77 Avian
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