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Sindbis virus messenger RNA: the 5′-termini and methylated residues of 26 and 42 S RNA
VIROLOGY‘
77,
457-470
Sindbis
Virus Messenger RNA: The 5’-Termini and Methylated Residues of 26 and 42 S RNA
DONALD
Department
(1977)
T. DUBIN,’ KATHLEEN
of Microbiology,
College
VICTOR TIMKO,
STOLLAR, CHUEN-CHIN HSUCHEN, AND GREGORY M. GUILD2
of Medicine Piscataway,
and New
Dentistry of New Jersey 08854
Accepted
November
Jersey-Rutgers
Medical
School,
8,1976
We have examined the methylation status in general and the 5’-termini in particular of the main mRNA species, “26 S” and “42 S” RNA, that accumulate in Sindbis virusinfected vertebrate cells and have compared these intracellular species with virion 42 S RNA. The 5’-termini examined were all “capped” and of the structure mG(5’)pppAp. The methylated guanosine (mG) caps consisted of rn,‘,‘G and rnz2J.‘G as well as m7G in the case of the intracellular RNAs, in contrast to virion RNA, which contained only m7G. In addition, one fourth to one half of the methyl label incorporated into the intracellular RNA species occurred as “internal” m5C! residues; such residues were absent from virion RNA. In addition to differing from virion RNA, the Sindbis mRNAs differ from host cell mRNA and from the mRNA specified by most other viruses of vertebrate cells both in the structure of their 5’-termini and in the nature of their internal methylated residues. Possible implications of these differences are discussed. INTRODUCTION
Recent work has shown that messenger RNA from eukaryotic systems, both cellular and viral, is characteristically slightly, but significantly, methylated. The methylated residues tend to be clustered at 5’termini in novel, “capped” structures of the form m7G(5’)pppN,pN,p. . ., where N, and N, can be 2’-0-ribose-methylated nucleosides. In addition, small numbers of internal methylated residues, mainly m6A, are found in most cellular and some viral mRNAs [see Shatkin (1976) for a general review]. There is evidence that the m7G residue is important in mRNA-ribosome binding (Muthukrishnan et al., 1975; Shafritz et al., 1976); the other methylated residues may play roles in the processing of mRNA precursors [see, for example, Rottman et al. (1974)]. Sindbis virus (SV) is an alphavirus ’ Address reprint 2 Present address: Stanford University California 94305. Copyright All rights
requests to Dr. Dubin. Department of Biochemistry, School of Medicine, Stanford,
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
whose genome consists of a positive polarity single-stranded RNA molecule, “42 S” RNA, approximately 12,000 nucleotides long (Levin and Friedman, 1971; Simmons and Strauss, 1972a). The 42 S RNA serves a messenger function in infected cells (Mowshowitz, 1973; Simmons and Strauss, 1974a), perhaps coding for nonstructural proteins such as a viral RNA polymerase (cf. Clewley and Kennedy, 1976). Most of the intracellular 42 S RNA is packaged ultimately into nucleocapsids to serve as the genome of progeny virus (Simmons and Strauss, 1974a). The main mRNA that accumulates in SV-infected cells, however, is “26 s” RNA, a species approximately 5000 nucleotides long (Levin and Friedman, 1971; Simmons and Strauss, 1972a) that codes for the viral structural proteins (Simmons and Strauss, 197413; Cancedda et al., 1975). It contains sequences equivalent to approximately one third of 42 S RNA (Simmons and Strauss, 1972a), and evidence from experiments with the closely related alphavirus, Semliki Forest virus (SFV; Kennedy, 1976; Wengler and Wen457 ISSN
0042-6822
458
DUBIN
gler, 1976) indicates that this third is at the 3’-end of 42 S RNA. We have shown that 42 S RNA from Sindbis virions contains a single methylated residue present in the 5’-terminus m’G(5’)pppAp (Hefti et al., 1976) and have presented evidence that 26 S RNA contains both methylated caps and internal methylated residues (Dubin and Stollar, 1975). We now report detailed analyses of the 5’-terminus and methylated residues of 26 S RNA, together with parallel studies on intracellular 42 S RNA and on virion 42 S RNA. The 26 S and intracellular 42 S RNA species were found to resemble virion 42 S RNA in containing the terminus . ; however, the intracelmGW)pppAp . . lular species differed from virion RNA in two respects: (i) The caps, especially of 26 S RNA, contained di- and trimethylated congeners of m7G (as well, as m7G itself, and (ii) a substantial portion of the methyl groups of both intracellular species occurred as internal m5C residues. Some of this work was presented at the 1976 Meeting of the American Society for Microbiology (Atlantic City, N.J.). MATERIALS
AND
METHODS
Cells, medium, virus. Procedures for preparing and growing baby hamster kidney (BHK-21) and primary chick embryo fibroblast (CEF) cultures and for preparing SV stocks (SV,,,) from CEF cells have been described (Stollar et al., 1976). Infection and labeling. For “short-term” experiments, monolayer cultures in lo-cm petri dishes were infected at input multiplicities of approximately 20 PFU/cell and were labeled from 3 to 8 hr after infection in the presence of 4 pg/ml of actinomycin D (Dubin and Stollar, 1975). For “longterm” experiments, confluent monolayers in one or two roller bottles (surface area, 690 cm? were infected at input multiplicities of approximately 1 PFU/cell and were labeled from 2 to 20 hr after infection in the presence of 0.5 pg/ml of antinomycin (Hefti et al., 1976). In all cases, we added adenosine and guanosine to minimize “leakage” of 3H from [methyZ-3H]methionine into purine rings (Dubin, 1974). Purification of virus and preparation of
ET
AL.
RNA. Virus was concentrated and purified by (NH&SO, precipitation followed by centrifugation to equilibrium in sucrose-D,O gradients (Shenk and Stollar, 1973). We prepared single-stranded RNA from infected cells using a phenol-sodium dodecyl sulfate (SDS) extraction procedure followed by LiCl precipitation (Dubin and Stollar, 1975). Viral RNA was purified from pelleted virus using phenol and SDS without a lithium step (Dubin, 1974). Digestion of RNA and oligonucleotides and fractionation of digests. Digestion with 1 N HCl and electrophoretic separation of the products at pH 3.5 were as described before (Dubin and Taylor, 1975). Routine chromatographic analysis of label recovered from electrophoretic peaks (Dubin, 1974) was performed in an alkaline system (“4”) (cf. Dubin and Taylor, 1975) for adenine and methylated adenines and an acid system (Y”) (cf. Dubin and Taylor, 1975) for guanine and methylated guanines. Digestion with ribonuclease T2 (500 U/ ml) was as described before (Dubin and Taylor, 1975). DEAE-cellulase chromatography was performed using a linear gradient of NaCl (0.05 to 0.5 N) in Tris-HCl, pH 7.8, containing 7 M urea; samples were recovered from columns by readsorption to a second column and elution with triethylammonium carbonate (Davenport et al., 1976). Oligonucleotides were digested with Escherichia coli alkaline phosphatase (100 Kg/ml) in 25 ~1 of 50 mM Tris-HCl, pH 8.6, and 5 mM MgCl, for 2 hr, at 37” (Wei and Moss, 1975). Prior to electrophoresis, digests were brought to approximately pH 4 with 0.5 N HCI, and samples were mixed with equal volumes of ammonium formate buffer (0.05 N, pH 3.5) containing appropriate markers. Methylated markers were obtained from commercial sources as specified earlier (Dubin and Taylor, 1975). In addition, m7G(5’)pppA and m7G(5’)pppG were obtained from P-L Biochemicals, Inc.; m’Cyt and m5Cyt from Cycle Biochemical Corp.; and m”Cyt from Sigma Chemical Co. A sample of m32,a,7G was kindly provided by
5’-TERMINI
OF
SINDBIS
VIRUS
459
mRNA
Dr. H. Busch, and, subsequently, samples of m;2*“*7G and m,‘s7G were synthesized from commercial m2G and mZ2G (HsuChen and Dubin, 1976). The corresponding bases were obtained by 1 N HCl hydrolysis as described above. m4Cyt was obtained from a sample of m4Cm (generously provided by Dr. B. Lane), as described in the footnote to Table 2. [MethyZ-3Hlmethionine was obtained from New England Nuclear Corp. (14.1 Ci/ mmolj or Schwartz Bioresearch Corp. (4.7 or 13 Ci/mmolj. 32Pi (carrier free) and [214Cluridine (55 mCi/mmolj were obtained from New England Nuclear Corp. RESULTS
Overall Methylation S RNA
Patterns of 26 and 42
The experiment illustrated in Figs. 1 and 2 was designed to compare directly the methylation patterns of Sindbis virion RNA and of the Sindbis-specified intracellular RNA species. The latter were obtained from cells labeled from 3 to 8 hr after infection, a period corresponding to near-maximal viral mRNA synthesis (Pfefferkorn and Shapiro, 1974; Stollar, unpublished observations). Virion RNA was obtained after more prolonged incubation so as to maximize release of virus; in this and other such experiments, the corresponding (“long-term-labeled’j intracellular RNA was also examined. The density gradient pattern shown in Fig. 1A is typical of 3- to 8-hr-labeled RNA. [14Cluridine provided a general RNA label, and the 14C pattern displays the 26 and 42 S RNA peaks. The 3H from methyl-labeled methionine yielded a major 4 S peak, representing residual labeling of tRNA (which is almost 1000 times as heavily methylated as Sindbis RNA), and also a peak corresponding to 26 S RNA. Generally, no discrete methyl-labeled peak corresponded to 42 S RNA in such gradients; as shown below, this reflects the fact that 42 S RNA, although methylated, is substantially less so than 26 S RNA or than other, minor species sedimenting between 26 and 42 S RNA. There was regularly a suggestion of a
Fraction
from Tap
FIG. 1. Sedimentation analysis of [“Hlmethyl-labeled RNA from Sindbis virus-infected cells. For short-term-labeled RNA (A!, BHK cells were labeled from 3 to 8 hr after infection with [methyl3H1methionine, 250 &i/3 Kg/ml, and [‘Cluridine, 0.1 &i/ml, 10e4 M, 40 ml of medium, five dishes; for long-term-labeled RNA (Bl, a roller bottle of BHK cells was labeled from 2 to 20 hr after infection with [methyPH]methionine, 100 &i/4.5 Kg/ml, 100 ml of medium (both as described in Materials and Methods). The LiCl-precipitable fractions of the total RNA extracted from the cells were subjected to density gradient sedimentation (Dubin and Stollar, 1975). The positions of cellular RNA peaks (arrows) were determined by 260~nm absorbance. (-O-O-), 3H; (-x-x-). “C.
shoulder ahead of the 26 S RNA peak (fractions 17, 18, and 19 of Fig. 1). When RNA was recovered from this region and resedimented, most of it ran as bona fide 26 S RNA; in addition, this material resembled 26 S RNA in methylation properties. We presume that it corresponds to 33 S RNA, a species thought to be a conformational variant of 26 S RNA (Simmons and Strauss, 1974a; Kennedy, 1976). The intracellular RNA from long-term, [“HImethyl-labeled cells yielded an 3H pattern (Fig. 1Bj essentially indistinguishable from that of 3- to 8-hr-labeled RNA.
460
DUBIN
RNA was concentrated from the 42 S regions of primary gradients of intracellular RNA, as indicated in Figs. 1A and B, and resedimented in parallel with a sample of L3H]methyl-labeled virion RNA from the long-term-labeled cells. As shown in Fig. 2, peaks of [3H]methyl label corresponded in all cases to 42 S RNA. For subsequent analysis, appropriate fractions from primary gradients (as in Figs. 1 and 2C) were pooled to obtain 26 S and virion 42 S RNA and from secondary gradients (as in Figs. 2A and B) to obtain intracellular 42 S RNA. RNA samples were screened for methylated residue patterns using acid hydrolysis followed by electrophoresis at pH 3.5 coupled with chromatographic analysis of certain electrophoretic fractions (cf. Dubin and Taylor, 1975; Dubin and Stollar, 1975). As shown in Fig. 3, 26 S RNA from the short- and the long-term-labeled cells yielded similar patterns. Each contained four discrete peaks, labeled accordingly. Peak 1 contained approximately 10% of the “H (slightly more in the long-term- than the short-term-labeled samples). Some of this label was found to be in m6Ade when corresponding peaks from parallel runs were eluted and fractionated by paper chromatography. However, most was found to be in adenine, representing leakage of label into purine rings. The small amount of “H in the guanine region also appears to represent leakage, since most or all of this 3H ran with guanine on paper chromatography. Peak 2 ran between Ade and m7Gua and Peak 3 ran in the m7Gua region. The latter peak tended to be rather broad, probably due to the fact that it contained two compounds. On paper chromatography, 3H eluted from this region yielded a peak running with m7Gua and a second peak that corresponded to no conventional methylated guanine marker, as illustrated in Fig. 5. Concurrent studies have established that peak 2 is the unusual base m,ZsZ,7Gua and the minor component of peak 3 is the previously unknown base m,“,7Gua (HsuChen and Dubin, 1976). Electrophoretic peak 4 ran slightly to the cathode side of Cp, as expected for
ET
AL.
Fmcilon from Top
FIG. 2. Sedimentation
analysis of [3Hlmethyl-labeled Sindbis 42 S RNA. (A), 3- to 8-hr intracellular RNA and (B), long-term-labeled intracerlular RNA from the 42 S regions of Figs. 1A and B, respectively, pooled as indicated by brackets “b” of these figures. (Cl, RNA isolated from virus prepared from the culture described for Fig. 1B. In the absence of uridine label, we have plotted the 260~nm absorbance patterns for (B) and (C) as an indicator of overall RNA distribution (the absorbance pattern of (A) resembled that of(B)). These patterns were transcribed from ISCO monitor tracings (cf. Dubin and Shine, 1976). The rising 260-nm absorbance approaching 28 S RNA in (C) is the result of our having added carrier cellular cytoplasmic RNA (100 pg) to this preparation. Centrifugation was in “standard salt” gradients (Dubin, 19741, SW 41 rotor, 18,000 rpm, 17 hr. (-O-O-), “H; (-x-x-l, I%; c---j, 260nm absorbance.
m5Cp (Brownlee, 1972). Radioactivity from this peak was characterized by perchloric acid digestion to release the free base, followed by chromatographic analysis in several systems. As summarized in Table 2, the results show that the parent nucleotide is indeed m5Cp. Electrophoretic patterns for acid hydrolysates of 42 S RNA are shown in Fig. 4. The simplest of these is that of virion 42 S
5’-TERMINI
OF
SINDBIS
10
%2 a” z z k” I
20
IO 0 Cm from Origin
IO
FIG. 3. Electrophoretic analysis of acid hydrolysates of [:iHlmethyl-labeled 26 S RNA from short- and long-term-labeled, Sindbis-infected BHK cells. Portions (2.5%, containing approximately 1000 cpm in “H each) of the 26 S RNA from the gradients of Fig. 1 (cuts “a”) were precipitated with ethanol in the presence of 100 Fg of carrier RNA and were subjected to HCl hydrolysis and electrophoresis at pH 3.5 (3 hr, 3OOOV), as described in Materials and Methods. To facilitate comparison, results are normalized to the counts in peak 3 (m7Gua plus m,‘,‘Gua). Here and in subsequent figures, ovals represent markers run with the samples, and rectangles indicate positions of markers as determined from other runs. Only the SH has been plotted. No radioactivity ran beyond Ade, where m’Ade would appear (cf. Dubin and Taylor, 1975). (-O-O-), Short-term-labeled RNA; (-x-x-), longterm-labeled RNA.
RNA. In agreement with earlier studies (Hefti et al., 19761, 80% of the 3H ran with m’Gua, and this was found to be essentially all in m’Gua on subsequent paper chromatography (Fig. 5). Approximately 15% of the label represented leakage into purine rings, and the remainder ran in heterodisperse fashion and was not further characterized. The 3- to 8-hr-labeled, intracellular 42 S RNA bore a closer resemblance to 26 S RNA. As shown in Figs. 4 and 5 and summarized in Table 1, it contained substantial label in m5Cp and some label (albeit significantly less than 26 S RNA) in rnZZ~‘Gua and m;‘v’,‘Gua. The 42 S RNA from long-term-labeled cells, as exemplified by Fig. 4, was intermediate in its methylation complexity between the other
VIRUS
461
mRNA
two types of 42 S RNA sample, containing a small but significant portion of its methyl label in m5C. In general, no more than about 5% of the total 3H of acid hydrolysates of 26 and 42 S RNA samples ran beyond Cp towards the anode, where products arising from Nm residues appear in this system (Dubin and Taylor, 1975). The paucity of Nm residues was confirmed by DEAE analyses of T2 ribonuclease digests (see next section). Table 1 summarizes these results, together with those of two other experiments on 3- to 8-hr-labeled RNA. In one (experiment II), we compared methylated residue patterns of RNA from infected BHK cells on the one hand and CEF cells on the other. There was regularly greater leakage into purine rings with the CEF than with the BHK cells; however, the intracellular 26 and 42 S species from the two hosts proved to have very similar methylation patterns. The results of experiment III of Table 1 are presented to illustrate the range of methylation patterns we noted for the 3- to 8-hr-labeled RNA. In particular, the percentage of methyl label in m5C ranged
mr -1 20
IO Cm from Orighn
0
IO
FIG. 4. Electrophoretic analysis of acid hydrolysates of 13H]methyl-labeled 42 S RNA. Portions of the RNA recovered from the bracketed cuts of the gradients of Fig. 2 were processed as for Fig. 3. We ran 10% of each sample (approximately 500-1500 cpm). (-O-O-), Short-term-labeled intracellular 42 S RNA; (- x-x -), long-term-labeled intracellular 42 S RNA); (-0-O-1, virion 42 S RNA.
DUBIN
462
IO Cm from
2o
F
Origin
FIG. 5. Chromatographic analysis of m’Gua Region of 26 to 42 S hydrolysates. Samples were processed in parallel with each sample described in Figs. 3 and 4, and segments corresponding to the m’Gua regions of electropherograms (generally 7 to 12 cm from the origins) were eluted in 0.05 N HCI. After lyophilization, portions (one-fourth to one-half) were subjected to paper chromatography in an acid system useful for resolving methylated guanines (see Materials and Methods). The methylated guanine markers illustrated can be readily distinguished by differing fluorescences. Not illustrated are Gua and m,Gua, which run more slowly than m’Gua (cf. Dubin and Taylor, 1975), and m,*,‘Gua and rn,‘~‘~‘Gua, both of which run between m*Gua and Cp. To facilitate comparison, counts are normalized to the counts present in m’Gua. (Al, 42 S RNA; (B), 26 S RNA. (-O-O-), Short-term-labeled intracellular RNA; (-x - x -1, long-term-labeled intracellular RNA; (-0-O-1, virion RNA.
from approximately 25 to 50% for both 26 and 42 S RNA, and the ratio of methyl label in m7G congeners to that in m7G proper ranged from approximately 0.4 to 1 for 26 S RNA and 0.1 to 0.2 for 42 S RNA. It should be noted that the molar ratios of congeners to m7G are considerably lower than the above ratios, since the congeners contain two or three times the amount of methyl label per base as does m7G.
ET AL.
We have also indicated in Table 1 (last column) the ratios of 7-methylated G (i.e., m7G plus congeners) to 14C (from [14C]uridine) for 26 S RNA relative to 42 S RNA. These normalized ratios should equal the ratio of the molecular weights of 42 S:26 S RNA (ca 2.4), if there is a one to one correspondence between 7-methylated G and 5’-termini; and, in fact, they are reasonably close to this value. Another estimate for the stoichiometry of capping was obtained for the 42 S RNA from the long-term-labeled cells of Figs. 1 and 2 by comparing 3H in 7-methylated G (assuming that the specific radioactivity of RNA methyl groups is the same as that of medium methionine) to 260-nm absorbance. The resulting values were approximately 1 mG per molecule for virion 42 S RNA and 0.8 for intracellular 42 S RNA. These results, as well as those described above, are compatible with the idea that all 26 and 42 S RNA 5’-termini are capped by 7-methylated G. Structure
of the 5’-Termini
Treatment of 26 and 42 S RNA (both virion and intracellular) with periodate followed by aniline (“p-elimination”) released 85-90% of the methylated guanosine residues and only these residues from the RNA. These results (data not shown) were obtained by electrophoretic analysis (as in Figs. 3 and 4) of acid hydrolysates of the treated RNA. Since the 3’-termini are A,,, (Deborde and Liebowitz, 1976), we can conclude that the methylated guanines are at 5’-termini in “inverted” 5’-ribotide linkage. This conclusion is not trivial, especially with regard to 42 S RNA, for which a “weak” internal initiation site has been reported (Cancedda et al., 1975). (One could postulate an internal equivalent of 7methylated G caps if there were sequential “inversions”; e.g., . . . pNpN(3’?p(3’)m7G(5’)pppNpNp
. .. ;
however, the cap-equivalent illustrated lacks vicinal hydroxyls and thus would not be susceptible to p-elimination.) More direct examination of 5’-termini was accomplished by subjecting T2 ribonuclease-treated samples to DEAE-cellulose
5’-TERMINI
OF
SINDBIS TABLE
DISTRIBUTION
Experiment, labeling time, and RNA speciesb
OF 3H FROM
IN SINDBIS
Percentage
of total
26 AND 42 S RNA”
methyl
mG: 14C (42 S = 1)
label
m 1 2. 7 &a
m2’). 7 Gua
1.4 2.2
7.2 1.4
11.7 4.8
47. 55.
28. 33.
<4. <5.
2.3
4.3 4.0
6.1 2. <2.
11.3 4. <2.
45. 69.
<6. <4. <3.
-
>so.
25. 16. <4.
3. 3.
13. 3.
20. 5.
31. 52
31. 30.
<3. <4.
2.3
7. 11.
3. 2.
9. 5.
22. 6.
30. 47.
33. 39.
<3. <2.’
2.1
4. 5.
3. 4.
4. 2.
15. 4.
31. 34.
45. 53.
<2. <2.
2.1
Gua
mGAde
1.7 4.1
1. 2.
3.1 7.1 11.
3. 3. 4.
4. 7.
2. 4.
CEF, 3- to 8hr label 26 S RNA 42 S RNA
15. 25.
BHK, 3- to 8-hr label 26 S RNA 42 S RNA
7. 10.
BHK, 3- to 8hr label 26 S RNA 42 S RNA
463
mRNA
I
[Methrl-“HIMETHIONINE
Percentage of total 3H Ade
VIRUS
m’Gua
mWyt
Nm
Ia.
Ib.
BHK, 2- to 20hr label 26 S RNA 42 S RNA Virion 42 S RNA
BHK, 3- to 8 hr label 26 S RNA 42 S RNA
IIa.
IIb.
III.
” Experiment I was that described in Figs. 1 and 2. In experiment II, parallel cultures of BHK and CEF cells (five dishes each) were labeled from 3 to 8 hr after infection with [methyl-3Hlmethionine, 100 &i/l.6 kg/ml, and [‘Quridine, 0.1 &i/ml, 10m4 M, and RNA samples were processed as for experiment I. The protocol for experiment III was essentially as for experiment Ia. Percentages of 3H were estimated by electrophoresis and chromatography of acid hydrolysates, as in Figs. 3, 4, and 5. Replicate analyses of the same sample generally agreed to +lO%. The “mG:W” ratios (last column) were determined taking into account the double and triple complements of “H in the di-and trimethylated congeners of m7G; these ratios refer to intracellular RNA and are normalized to the ratio for 42 S RNA. b Unless noted otherwise, all RNA species are intracellular. (I Determined by analysis of DEAE-cellulose peaks; for reasons that are unclear, this sample yielded a falsely high level of 3H in the Nm region of electropherogram.
chromatography. An elution pattern for 26 S RNA from experiment IIa of Table 1 is shown in Fig. 6. Approximately 65% of the 3H appeared as a peak corresponding to the trinucleotide (“-4”) marker, in good agreement with expectation if all methylated guanosines occurred at 5’-termini with the structure mG(5’)pppNp. .. . (Hefti et al., 1976). Approximately 30% of the 3H eluted with the mononucleotide (“-2”) marker, and, also as expected, this was found to be mainly m5Cp. The small
amount (ca. 3%) of the 3H corresponding to the -3 isostich can account for the apparent 2’-0-ribose methyl label in acid hydrolysates of this RNA; the nature of this fraction is unknown. No more than 1% corresponded to -5, -6 region, where blocked termini of the forms mGpppNmpNp and mGpppNmpNmpNp would occur (Wei et aZ., 1975; Furuichi et al., 1975b). Figure 7 illustrates the enzymatic analysis of the 26 S -4 peak. When the oligo-
DUBIN
464 TABLE
2
CHROMATOGRAPHY AND ELECTROPHORESIS METHYLATED CYTOSINEEP Compound
m’Cyt m3Cyt m4Cyt msCvt
Electrophoresis, pH
Chromatography SysPm (Rc,,)
System
0.70 1.7 1.3
1.3 1.5 1.5 1.2
CRZt)
OF
WSB (%A 1.4 0.5b 1.6 1.2
( il:,,, 1.00 1.07 1.00 1.00
fl The methylated cytosines were obtained from commercial sources, except for m’Cyt. This was prepared from m4Cm by perchloric acid hydrolysis (70% acid, loo”, 45 min in a glass-stoppered tube). This procedure yielded a 260-nm-absorbing product with approximately the expected R, in WSB (Szer and Shugar, 1966). The solvents systems are: C, (Iwanami and Brown, 1968): n-butanol, H,O (85:15); D, (Iwanami and Brown, 1968): isopropanol, water, 25% ammonia (85:15:1.3); WSB, (Szer and Shugar, 1966): water-saturated butanol in NH, atmosphere. Electrophoresis was at pH 3.5, 3000 V/90 cm; samples were spotted near the anode edge. Values are referred to Cyt for convenience and reproducibility. R,‘s of Cyt in the three solvent systems were in the range 0.3-0.4; Cyt ran approximately 45 cm towards the cathode on electrophoresis. Samples of putative m5Cyt from Sindbis RNA were prepared by eluting (in 0.05 N HCl) electrophoretic segments corresponding to the putative m5Cp peaks of electropherograms such as those as Fig. 3 and 4 (peaks 4), lyophilizing the eluates, and subjecting the residues to hydrolysis in perchloric acid (0.1 ml) as above. Appropriate 260-nm markers were added to the perchloric acid prior to heating. Samples were neutralized using KOH and then desalted by spinning out the insoluble potassium perchlorate, repeatedly lyophilizing the supernatant solution, redissolving in progressively smaller volumes, and spinning out the salt, until the sample was readily soluble in 10 ~1. Samples from electrophoretic peak 4 in all cases yielded discrete methyl-labeled peaks, containing 85-90% of the recovered 3H, running precisely with m5Cyt. * Streaks.
ET
AL.
alkaline phosphatase, approximately 90% ran with m7G [the three 7-methylated guanosine congeners have similar mobilities at pH 3.5 (Hsu-Chen and Dubin, 197611. This T2-derived terminus was obtained from an RNA sample relatively rich in m2z.7G and miL*2,7G (Table 1); in fact, a bit over half of the 3H in the terminus was in these moieties. Thus, the 3H running with pm7G in Fig. 7 must represent a mixture of pm7G, pm?s7G, and pm34,8,7G; and (assuming that the end group is homogeneous with regard to the putative pppA moiety) the 3H corresponding to m7G(5’)pppA must likewise represent a mixture of 7-methylated guanosine homologues. The 3H (lo-15% of the total in various experiments) remaining at the origin after mixed venom plus phosphatase treatment (which is done at pH 8.6) probably represents a partial degradation product of 7methylated G residues. Such a neutral, nonphosphorylated peak appears after alkaline treatment of m7G-containing cellu-
Fraction
nucleotide was treated with alkaline phosphatase, approximately 95% of the 3H migrated on electrophoresis at pH 3.5 with the marker m’G(5’)pppA. When the label from such an alkaline phosphatase-treated peak was eluted and then treated with venom phosphodiesterase, approximately 90% of the 3H migrated with pm7G, and, when treated with a mixture of venom and
Number
FIG. 6. DEAE-cellulose column chromatography of the T2 digest of methyl-labeled 26 S RNA. A portion (40%) of the 26 S RNA from experiment IIa of Table 1 was subjected to TB-ribonuclease followed by DEAE chromatography as described in Materials and Methods (0.9 x 20-cm column, 260-ml gradient, 3-ml fractions). Positions of ribonuclease A digest markers are shown by arrows. Only the $H has been plotted; all (>99%) of the 14C appeared in the -2 isostich.
5’-TERMINI
OF
SINDBIS
lar mRNA (Dubin and Taylor, 1975). When methyl-labeled intracellular 42 S RNA was analyzed as for Figs. 6 and 7, similar results were obtained-
VIRUS
465
mRNA
A more definitive characterization, especially of the penultimate nucleotides and phosphate “bridges,” was provided by experiments in which cells were doubly labeled with [methyZ-3H]methionine and high levels of 32P. As shown in Fig. 8, DEAE-cellulose chromatography of T2 ribonuclease digests of 26 and 42 S RNA from such cells yielded discrete peaks of TABLE JZP CONTENT
3
OF T2 RIBONUCLEASE-RELEASED
5’-
TERMINI~ RNA
Percentage Experimental
of total Theoretical
___ p=cU
26 S 42 S
Cm from Orlgln
FIG. 7. Enzymatic analysis of a T2-derived terminus from methyl-labeled 26 S RNA. The -4 peak from Fig. 6 (bracket) was recovered as described in the text. A portion was treated with alkaline phosphatase and subjected to electrophoresis at pH 3.5 (-0-O-j. Oligonucleotide in the m?GpppA region from a sample previously processed in this manner was eluted (H,O, 3 hr, room temperature), concentrated by lyophilization, treated with venom phosphodiesterase, and run in parallel (-X-X -1. A third sample was run after treatment with mixed phosphatase-phosphodiesterase (-0-O-j.
20
0.14 0.047
32P
0.080 0.033
p = 3a 0.12 0.050
” Experimental values were estimated from the DEAE-cellulose analyses described for Fig. 8, subtracting a background from the -4 peaks as indicated by the dashed lines. Theoretical values were calculated taking chain lengths to be 5000 and 12,000 nucleotides for 26 and 42 S RNA, respectively, and assuming either that the specific activity of the middle and end phosphates of the presumed ppp bridges were equally labeled (p = (~1 or that the middle phosphate was three times as heavily labeled as the end phosphates (p = 3a). These latter alternatives are discussed in the text.
30 Froclion Number
8. DEAE-cellulose column chromatography of T2 ribonuclease digests of 13H1methyl-, 3ZP-labeled 26 and 42 S RNA. RNA was prepared as described for Figs. 1A and 2A, with the exception that infected cells (10 dishes) were labeled with 32Pi (200 &i/ml, carrier free) and [methyl-3Hlmethionine, 100 &i/4 pg/ml, 5 ml of medium per dish. Portions of each purified RNA preparation [25% of total for 26 S RNA (A); 40% for 42 S RNA (B)] were subjected to T2 digestion followed by DEAE chromatography (0.9 x 14-cm column, 200-ml gradient, 3 ml/fraction). (-O-O-), 3H; (-x-x-), 32P. FIG.
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466
Cm from Orlgin
FIG. 9. Electrophoretic and enzymatic analysis of an 13Hlmethyl-, 3ZP-labeled 5’-terminus from 26 S RNA. A portion of the 26 S -4 peak of Fig. 8A (bracket) was subjected to electrophoresis at pH 3.5 (A) in parallel with a portion previously treated with alkaline phosphatase (B). The region corresponding to the bracket in Panel B was eluted from a third (phosphatase-treated) sample run in parallel and subjected to electrophoresis after treatment with venom phosphodiesterase (Cl. Only the regions in the direction of the anode are shown; essentially no (13%) label ran towards the cathode. (-0-O-1, 3H; (-x-x-), 32P.
32P corresponding to the -4 3H peaks. The bulk of the 32P eluted, as expected, with the mononucleotides, precluding accurate determination of internal methyl label in these studies. No discrete 32P peaks corresponded to the -5 or -6 isostiches, where ppNp and pppNp residues elute. The 32P levels corresponding to the -4 peaks were somewhat higher than expected for uniformly labeled termini of the form mG(5’)pppNp, as summarized in Table 3. An explanation for this discrepancy is presented below. Figure 9A shows the electrophoretic pattern obtained for the r3Hlmethyl-, 32P-labeled 26 S -4 fraction. Approximately 95% of the 3H and 80% of the 32P migrated as a single peak slightly behind Gp; this peak
ET AL.
is presumed to represent the mG(5’)pppNp end group. As shown in Fig. 9B, phosphatase treatment of the -4 peak yielded a new [3Hlmethyl-, 32P-labeled peak running slightly slower and corresponding to marker m’GpppA. This peak contained 70% of the 32P, while 22% of the EWPappeared as Pi. The ratio of 32P in these two moieties approaches the 3:l ratio expected if the TB-released peak was indeed mG(5’)pppNp. However, this agreement is probably not meaningful. There was a low level of heterogeneously migrating nucleotides in the original sample (as seen in Fig. 9A) that might be expected to release “extra” Pi on phosphatase treatment, and we believe that the middle phosphate of the putative triphosphate bridge of the phosphatase-resistant moiety has a relatively high specific radioactivity (uide infra). These two circumstances would have opposing effects on the relative amount of 32Pi released by alkaline phosphatase. Figure 9C shows the pattern obtained when the putative 26 S mGpppA peak was eluted and treated with venom phosphodiesterase. Similar results were obtained for 42 S RNA and for a 26 S sample from a separate experiment, all as summarized in Table 4. Three main 32P-containing peaks were obtained, running with pm’G, pA, and Pi. The first two were present in approximately a 1:l ratio in all cases; however, the level of ““Pi was regularly approximately three times the theoretical TABLE VENOM
4
PHOSPHODIESTERABE
PHOSPHATASE-TREATED,
ANALYSIS
““P-LABELED
OF
5’-TERMING”
:IZP released
PmG
PA
P,
Expt 1 26 S RNA 42 S RNA
u.01 11.91
1.02 0.93
3.0 2.8
Expt 2 26 S RNA
11.01
0.91
3.1
(1 Values are normalized on the basis of the :rrP running with pm’G (referred to as pmG since this peak also contains the hypermethylated homologues of pm’G). Experiment 1 is that described in Figs. 8 and 9, and Experiment 2 was a similar, 3- to 8-hr, high-level 32P-labeling experiment.
5’-TERMINI
OF
SINDBIS
level for the presumed structure mGpppA. That this is due to phosphatase contamination of our venom preparations (cf. Keith and Fraenkel-Conrat, 1975) is unlikely, since we monitored our reactions for phosphatase activity by assaying for released m7G (cf. Fig. 7) and found none. We believe that our excess 32Pi arises from unequal specific radioactivities of CY-and P-phosphates of pppN precursors. The middle phosphate of the proposed bridge would be expected to arise from the /3phosphate of nucleoside triphosphate precursors (pppA or pppG depending on the capping mechanism involved), while the end phosphates as well as the remaining (“internal”) phosphates of the RNA molecules should arise from a-phosphates. It would not be surprising if P-phosphates of pppN pools turned over more rapidly than (Y and thus attained higher average specific activities in 5-hr-labeling studies such as those employed (cf. Sripati et al., 1976 and Brumm et al., 1956). If we assign to the middle phosphates of our bridges specific activities threefold that of the other phosphates of the RNA, the values of Tables 3 and 4 are in agreement with the inference that most 26 and 42 S RNA molecules, and perhaps all of them, terminate in mG(5’)pppAp. DISCUSSION
The mRNAs specified by most viruses of eukaryotic cells contain capped 5’-termini of the form m’G(!Y)pppPu, where the penultimate riboside is a purine riboside and is usually ribose methylated (Shatkin, 1976). Sindbis falls into a subgroup of these viruses, whose mRNAs are capped, but lack penultimate ribose methylation. This group includes in addition to Sindbis virus several plant viruses (Zimmern, 1975; Keith and Fraenkel-Conrat, 1975; Symons, 1975; Pinck, 1975; Dasgupta et al., 1976) which, like Sindbis, are positivestrand RNA viruses that multiply in the cytoplasm. The larger group, whose mRNA contains penultimate methylated ribose, consists of viruses with known or presumed nuclear phases [for example, adenovirus (Sommer et al., 1976; Moss and Koczot, 1976); SV-40 (Lavi and Shatkin,
VIRUS
mRNA
467
19751; and avian- and B77-sarcoma viruses (Furuichi et al., 1975c; Stoltzfus and Dimock, 197611 plus certain cytoplasmic viruses with virion-associated RNA-synthetic systems [cytoplasmic polyhedrosis virus (Furuichi and Miura, 1975); reovirus (Furuichi et al., 1975a); vaccinia (Wei and Moss, 1975); and vesicular stomatitis virus (VSV; Abraham et al., 1975; Rose, 197511. We propose (il that nuclear mRNA precursors, both cellular and viral, are methylated at what is to become the penultimate ribose by cellular nuclear methylases (cf. Groner and Hurwitz, 19751 and (ii) that cellular cytoplasm lacks such methylating activity unless brought in by viruses. The only cytoplasmic virus thus far studied that has a virion-associated RNA-synthetic system but whose mRNA lacks 2’-Oribose methyl groups is Newcastle disease virus, which gratifyingly is specifically deficient in ribose-methylating activity (Colonno and Stone, 1975, 1976). An unusual feature of the caps of the intracellular SV-RNA species examined, especially 26 S RNA, is the presence of substantial levels of the hypermethylated m7G congeners. We have reexamined the caps of cytoplasmic mRNA from uninfected BHK cells (cf. Dubin and Taylor, 1975) with these compounds in mind and have detected only small amounts of [3Hlmethyl in rn,2r7G and ~Q**~~‘G (5 to 10% of that in m7G); none (~2%) was found in comparable samples from CEF cells (Hsu-Chen and Dubin, unpublished observations). The only other RNA species reported to contain either congener are small nuclear RNAs of unknown function (Saponara and Enger, 1969; Ro Choi et al., 1975; Cory and Adams, 1975), which are capped by rn,‘s”.‘G in a 5’-terminal sequence very similar to that of 26 S RNA. The “standard” m’G caps of certain mRNAs have been shown to facilitate mRNA-ribosome binding, an action which may be related to the positive charge carried by m7G residues at neutral pH (see Shatkin, 1976). The hypermethylated congeners are also cationic at neutrality (HsuChen and Dubin, 19761 and thus might not be expected to grossly alter the conformation of 5’-termini. Whether the extra
466
DUBIN
methyl groups on some SV-mRNA termini have any biological significance and why their levels in 26 S RNA are so much greater than those in cellular mRNA or even in intracellular 42 S RNA are interesting questions that remain to be explored. The SV intracellular RNA species are unusual also in their internal methylated residue compositions, in particular in the prominence of m5C. There is some m5C in the cellular mRNA of BHK (Dubin and Taylor, 1975) and CEF cells (Hsu-Chen and Dubin, unpublished observations) and probably in adenovirus mRNA (Sommer et al., 1976); however, in all these cases, internal m6A is present at much higher levels than m5C. Our values for internal m6A in 26 and 42 S RNA, amounting to no more than one residue per lo-20 RNA molecules, are so low that it seems reasonable to score them as zero. Other mRNAs lack internal m6A: e.g., yeast cytoplasmic mRNA (Sripati et al., 1976) and VSVmRNA (Moyer et al., 1975). At least in mammalian systems, there appears to be a good correlation between the presence of m6A in mRNA and mRNA precursors and the existence of endonucleolytic processing mechanisms for the generation of these mRNAs (cf. Sripati et al., 1976; Sommer et al., 1976). Thus the absence of m6A from SV 26 and 42 S’RNA is in accord with the view that both species arise via separate transcriptional events (Simmons and Strauss, 1972b) and do not themselves undergo processing (Simmons and Strauss, 1974b; Cancedda et al., 1975; Clegg and Kennedy, 1975). The role, if any, of internal m5C is completely obscure. The apparent exclusion of the hypermethylated m7G congeners and m5C from virion RNA is an interesting phenomenon. One explanation for this may be that these specific methylations play a role in determining the fate of newly synthesized viral RNA molecules; that is, whether a transcript is to serve (i) as a messenger or (ii) as a template for further RNA synthesis or (iii) whether it is instead encapsidated to appear ultimately in progeny virus might be influenced by the methylation status of the transcript. Alternatively, one might
ET AL.
propose that, during the phase of rapid assembly of virions, the sequence (transcription)- (ms7G - capping) - (encapsidation)-(envelopment) is tightly linked in the infected cell (cf. Grimley et al., 1972; Friedman et al., 1972; Soderlund 1973) and that 42 S molecules not entering this linked sequence form separate pools of molecules (presumably including those on polysomes) that (i) cannot be incorporated into virions and (ii) are accessible to methylases such as the putative m7G “supermethylases” and m5C-forming enzymes. Implicit in these models is the idea that, at some stage in the generation of progeny viruses, those intracellular 42 S molecules destined to become genome are rendered inaccessible to methylases. The lower level of m5C in long-term-labeled intracellular 42 S RNA compared to 3- to 8-hr-labeled samples would then indicate that there is a relative accumulation within the cells of such “protected” RNA during the course of infection. The similarity in the 5’-terminal sequences of 26 and 42 S RNA deserves comment. We can now extend this similarity to the second nucleotide beyond the cap (Dubin, unpublished data), both species terminating in mG(5’)pppApUp. The similarity could admittedly occur fortuitously with a frequency of one in eight; if there is a biological explanation for it, however perhaps the most straightforward one would be that 26 S RNA corresponds to the extreme 5’-portion of 42 S RNA. However, studies on SFV (see Introduction) appear to rule this out. We propose that the similarity reflects common structural requirements for 26 and 42 S RNA; for example, ApUp and/or its negative-strand complement may be required for binding the same or similar RNA polymerases (cf. Clewley and Kennedy, 1976) in the generation of the two RNA species. ACKNOWLEDGMENTS This work was supported by Research Grants No. GM-14957 from the National Institute of General Medical Sciences and AI-05920 from the National Institute of Allergy and Infectious Diseases. C.-C. H. C. is a postdoctoral trainee under Institutional National Research Award No. CA-09069 from the National Cancer Institute. G. M. G. was supported
5’-TERMINI
OF
SIl gDBIS
in part by Training Grant CA-05234 from the National Cancer Institute and by a Johnson and Johnson Fellowship in Biology, 1975-1976.
REFERENCES ABRAHAM, G., RHODES, D. P., and BANERJEE, A. K. (1975). The 5’-terminal structure of the methylated mRNA synthesized in vitro by vesicular stomatitis virus. Cell 5, 51-58. BROWNLEE, G. G. (1972). “Determination of Sequences in RNA,” p. 206. American Elsevier, New York. BRUMM, A. F., POTTER, V. R., and SIEKEVITZ, P. (1956). Nucleotide metabolism. V. Incorporation of P3* into acid-soluble nucleotides of rat liver in ho. J. Bial. Chem. 220, 713-722. CANCEDDA, R., VILLA-K• MAROFF, L., LODISH, H. F., and SCHLESINGER, M. (1975). Initiation sites for translation of Sindbis virus 425 and 26s messenger RNAs. Cell 6, 215-222. CLEGG, J. C. S., and KENNEDY, S. I. T. (1975). Initiation of synthesis of the structural proteins of Semliki forest virus. J. Mol. Biol. 97, 401-411. CLEWLEY, J. P., and KENNEDY, S. I. T. (1976). Purification and polypeptide composition of Semliki Forest virus RNA polymerase. J. Gen. Virol., in press. COLONNO, R. J., and STONE, H. 0. (1975). Methylation of messenger RNA of Newcastle disease virus in vitro by a virion-associated enzyme. Proc. Natl. Acad. Sci. USA 72, 2611-2615. COLONNO, R. J., and STONE, H. 0. (1976). Newcastle disease virus mRNA lacks 2’-0-methylated nucleotides. Nature (London) 261, 611-614. CORY, S., and ADAMS, J. M. (1975). Modified 5’termini in small nuclear RNAs of mouse myeloma cells. Mol. Biol. Rep. 2, 287-294. DASGUPTA, R., HARADA, F., and KAESBURG, P. (1976). Blocked 5’-termini in brome mosaic virus RNA. J. Virol. 18, 260-267. DAVENPORT, L., TAYLOR, R., and DUBIN, D. T. (19761. Comparison of human and hamster mitochondrial transfer RNA: Physical properties and methylation status. Biochim. Biophys. Acta 447, 285-293. DEBORDE, D. C., and LIEBOWITZ, R. D. (1976). Polyadenylic acid size and position found in Sindbis virus genome and mRNA species. Virology 72, 80-88. DUBIN, D. T. (1974). Methylated nucleotide content of mitochondrial ribosomal RNA from hamster cells. J. Mol. Biol. 84, 257-273. DUBIN, D. T., and SHINE, J. (1976). The 3’-terminal sequence of mitochondrial 13s ribosomal RNA. Nucleic Acids Res. 3, 1225-1231. DUBIN, D. T., and STOLLAR, V. (1975). Methylation of Sindbis virus “26s” messenger RNA. Biochem.
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Biophys. Res. Commun. 66, 1373-1379. DUBIN, D. T., and TAYUIR, R. H. (1975). The methylation state of Poly A-containing-messenger RNA from cultured hamster cells. Nucleic Acids Res. 2, 1653-1668. FRIEDMAN, R. M., LEVIN, J. G., GRIMLEY, P. M., and B~REZESKY, I. K. (1972). Membrane-associated replication complex in arbovirus infection. J. Virol. 10, 504-515. FURUICHI, Y., and MIURA, K. (1975). A blocked structure at the 5’ terminus of mRNA from cytoplasmic polyhedrosis virus. Nature (London) 253, 374-375. FURUICHI, Y., MORGAN, M., MUTHUKRISHNAN, S., and SHATKIN, A. J. (1975a). Reovirus messenger RNA contains a methylated, blocked 5’-terminal structure: m7G(5’)ppp(5’)GmpCp. Proc. Nat. Acad. Sci. U3A 72, 362-366. FURUICHI, Y., MORGAN, M., SHATKIN, A. J., JELINEK, W., SALDITT-GEORGIEFF, M., and DARNELL, J. E. (1975b). Methylated, blocked 5’ termini in HeLa cell mRNA. Proc. Nat. Acad. Sci. USA 72, 1904-1908. FURUICHI, Y., SHATKIN, A. J., STAVNEZER, E., and BISHOP, J. M. (1975c). Blocked, methylated 5’terminal-sequences in vian sarcoma virus RNA. Nature (London) 257, 618-620. GRIMLEY, P. M., LEVIN, J. G., BEREZESKY, I. K., and FRIEDMAN, R. M. (1972). Specific membranous structures associated with the replication of group A arboviruses. J. Virol. 10, 492-503. GRONER, Y., and HURWITZ, J. (1975). Synthesis of RNA containing a methylated blocked 5’ terminus by HeLa nuclear homogenates. Proc. Nat. Acad. Sci. USA 72, 2930-2934. HEFTI, E., BISHOP, D. H. L., DUBIN, D. T., and STOLLAR, V. (1976). The B’nucleotide sequence of Sindbis virus RNA. J. Virol. 17, 149-159. HSU-CHEN, C. C., and DUBIN, D. T. (1976). Di- and tri-methylated congeners of 7-methylguanine in Sindbis virus messenger RNA. Nature (London) 264, 190-191. IWANAMI, Y., and BROWN, G. M. (1968). Methylated bases of transfer ribonucleic acid from HeLa and L cells. Arch. Biochem. Biophys. 124, 472-482. KEITH, J., and FRAENKEL-CONRAT, H. (1975). Tobacco mosaic virus RNA carries 5’-terminal triphosphorylated guanosine blocked by 5’linked 7methylguanosine. FEBS Lett. 57, 31-33. KENNEDY, S. I. T. (1976). Sequence relationships between the genome and the intracellular RNA species of standard and defective-interfering Semliki forest virus. J. Mol. Biol., in press. LAVI, S., and SHATKIN, A. J. (1975). Methylated simian virus IO-specific RNA from nuclei and cytoplasm of infected BSC-1 cells. Proc. Nat. Acad. Sci. USA 72, 2012-2016. LEVIN, J. C., and FRIEDMAN, R. M. (1971). Analysis
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of arbovirus ribonucleic acid forms by polyacrylamide gel electrophoresis. J. ViroB?, .5Q4-514. Moss, B., and K~~zoT, F. (1976). Sequence of methylated nucleotides at the 5’-terminus of adenovirus-specific RNA. J. Viral. 17, 385-392. MOWSHOWITZ, D. (1973). Identification of polysomal RNA in BHK cells infected by Sindbis virus. J. -It,--.d-Viral. 11, 535-543. MOYER, S. A., ABRAHAM, G., ADLER, R., and BANERJEE, A. K. (1975). Methylated and blocked 5’ termini in vesicular stomatitis in viuo mRNAs. Cell 5, 59-67. MUTHUKRISHNAN, S., BOTH, G. W., FURUICHI, Y., and SHATKIN, A. J. (1975). 5’-terminal 7-methylguanosine in eukaryotic mRNA is required for translation. Nature (London) 255, 33-37. PFEFFERKORN, E. R., and SHAPIRO, D. (1974). In ‘Comprehensive Virology” (Fraenkel-Conrat and Wagner, eds.), Vol. 2, pp. 171-230. Plenum Press, New York. PINCK, L. (1975). The 5’-end groups of alfalfa mosaic virus RNAs are M7G5’pppS’Gp. FEBS Lett. 59, 2428. Ro-CHOI, T. S., CHOI, Y. C., HENNING, D., McCLOSKEY, J., and BUSCH, H. (19751, Nucleotide sequence of U-2 ribonucleic acid (the sequence of the 5’-terminal oligonucleotide). J. Biol. Chem. 250, 3921-3928. ROSE, J. K. (1975). Heterogeneous 5’-terminal structures occur on vesicular stomatitis virus mRNAs. J. Biol. Chem. 250, 80988104. ROTTMAN, F., SHATKIN, A. J., and PERRY, R. P. (1974). Sequences containing methylated nucleotides at the 5’ termini of messenger RNAs: Posslible implications for processing. Cell 3, 197-199. SAPONARA, A. G., and ENGER, M. D. (1969). Occurrence of N*,N*,7-trimethylguanosine in minor RNA species of a mammalian cell line. Nature (London) 223, 1365-1366. SHAFRITZ, D. A., WEINSTEIN, J. A., SAFER, B., MERRICK, W. C., WEBER, L. A., HICKEY, E. D., and BAGLIONI, C. (1976). Evidence for role of m’G”(phosphate group in recognition of eukaryotic mRNA by initiation factor IF-M,. Nature (London) 261, 291-294. SHATKIN, A. (1976). Capping of eukaryotic mRNAs. Cell, in press. SHENK, T., and STOLLAR, V. (19731. Defective-interfering particles of Sindbis virus. I. Isolation and some chemical and biological properties. Virology 53, 162-173. SIMMONS, D. T., and STRAUSS, J. H. (1972a). Replication of Sindbis virus. I. Relative size and genetic content of 26s and 49s RNA. J. Mol. Biol. 71, 599-
ET
AL. 613. SIMMONS, D. T., and STRAUSS, J. H. (197213). Replication of Sindbis virus. II. Multiple forms of double-stranded RNA isolated from infected cells. J. Mol. B~iol. 71, 615-631. SIMMONS, D. T., and STRAUSS, J. H. (1974a). Replication of Sindbis virus. V. Polyribosomes and mRNA in infected cells. J. Virol. 14, 552-559. SIMMONS, D. T., and STRAUSS, J. H. (1974b). Translation of Sindbis virus 26s RNA and 49s RNA in lysates of rabbit reticulocytes. J. Mol. Biol. 86, 397-409. SODERLUND, H. (1973). Kinetics of formation of the Semliki Forest virus nucleocapsid. Intervirology 1, 354-361. SOMMER, S., SALDITT-GEORGIEFF, M., BACHENHEIMER, S., DARNELL, J. E., FURUICHI, Y., MORGAN, M., and SHATKIN, A. J. (1976). The methylation of adenovirus-specific nuclear and cytoplasmic RNA. Nucleic Acids Res. 3, 749-765. SRIPATI, C. E., GRONER, Y., and WARNER, J. R. (1976). Methylated, blocked 5’ termini of yeast mRNA. J. Biol. Chem. 251, 2898-2904. STOLLAR, V., STOLLAR, B. D., Koo, R., HARRAP, K. A., and SCHLESINGER, R. W. (1976). Sialic acid contents of Sindbis virus from vertebrate and mosquito cells: Equivalence of biological and immunological viral properties. Virology 69, 104115. STOLTZFUS, C. M., and DIMOCK, K. (1976). Evidence for methylation of B77 avian sarcoma virus genome RNA subunits, J. Virol. 18, 586-595. SYMONS, R. H. (1975). Cucumber mosaic virus RNA contains 7-methyl guanosine at the 5’-terminus of all four RNA species. Mol. Biol. Reports 2, 277285. SZER, W., and SHUGAR, D. (1966). Preparation and properties of some methyl-substituted cytosine ribosides and their intermediates, and poly-5-methylcytidylic acid. Actu Biochim. Polonica 13, 177192. WEI, C. M., GERSHOWITZ, A., and Moss, B. (1975). Methylated nucleotides block 5’ terminus of HeLa cell messenger RNA. Cell 4, 379-386. WEI, C. M., and Moss, B. (1975). Methylated nucleotides block 5’-terminus of vaccinia virus messenger RNA. Proc. Nat. Acad. Sci. USA 72, 318-322. WENGLER, G., and WENGLER, G. (1976). Localization of the 26-S RNA sequence on the viral genome type 42-S RNA isolated from SFV-infected cells. Virology 73, 190-199. ZIMMERN, D. (1975). The 5’-end groups of tobacco mosaic virus RNA is m7GS’pppS’Gp. Nucleic Acids Res. 2, 1189-1201.
77,
457-470
Sindbis
Virus Messenger RNA: The 5’-Termini and Methylated Residues of 26 and 42 S RNA
DONALD
Department
(1977)
T. DUBIN,’ KATHLEEN
of Microbiology,
College
VICTOR TIMKO,
STOLLAR, CHUEN-CHIN HSUCHEN, AND GREGORY M. GUILD2
of Medicine Piscataway,
and New
Dentistry of New Jersey 08854
Accepted
November
Jersey-Rutgers
Medical
School,
8,1976
We have examined the methylation status in general and the 5’-termini in particular of the main mRNA species, “26 S” and “42 S” RNA, that accumulate in Sindbis virusinfected vertebrate cells and have compared these intracellular species with virion 42 S RNA. The 5’-termini examined were all “capped” and of the structure mG(5’)pppAp. The methylated guanosine (mG) caps consisted of rn,‘,‘G and rnz2J.‘G as well as m7G in the case of the intracellular RNAs, in contrast to virion RNA, which contained only m7G. In addition, one fourth to one half of the methyl label incorporated into the intracellular RNA species occurred as “internal” m5C! residues; such residues were absent from virion RNA. In addition to differing from virion RNA, the Sindbis mRNAs differ from host cell mRNA and from the mRNA specified by most other viruses of vertebrate cells both in the structure of their 5’-termini and in the nature of their internal methylated residues. Possible implications of these differences are discussed. INTRODUCTION
Recent work has shown that messenger RNA from eukaryotic systems, both cellular and viral, is characteristically slightly, but significantly, methylated. The methylated residues tend to be clustered at 5’termini in novel, “capped” structures of the form m7G(5’)pppN,pN,p. . ., where N, and N, can be 2’-0-ribose-methylated nucleosides. In addition, small numbers of internal methylated residues, mainly m6A, are found in most cellular and some viral mRNAs [see Shatkin (1976) for a general review]. There is evidence that the m7G residue is important in mRNA-ribosome binding (Muthukrishnan et al., 1975; Shafritz et al., 1976); the other methylated residues may play roles in the processing of mRNA precursors [see, for example, Rottman et al. (1974)]. Sindbis virus (SV) is an alphavirus ’ Address reprint 2 Present address: Stanford University California 94305. Copyright All rights
requests to Dr. Dubin. Department of Biochemistry, School of Medicine, Stanford,
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
whose genome consists of a positive polarity single-stranded RNA molecule, “42 S” RNA, approximately 12,000 nucleotides long (Levin and Friedman, 1971; Simmons and Strauss, 1972a). The 42 S RNA serves a messenger function in infected cells (Mowshowitz, 1973; Simmons and Strauss, 1974a), perhaps coding for nonstructural proteins such as a viral RNA polymerase (cf. Clewley and Kennedy, 1976). Most of the intracellular 42 S RNA is packaged ultimately into nucleocapsids to serve as the genome of progeny virus (Simmons and Strauss, 1974a). The main mRNA that accumulates in SV-infected cells, however, is “26 s” RNA, a species approximately 5000 nucleotides long (Levin and Friedman, 1971; Simmons and Strauss, 1972a) that codes for the viral structural proteins (Simmons and Strauss, 197413; Cancedda et al., 1975). It contains sequences equivalent to approximately one third of 42 S RNA (Simmons and Strauss, 1972a), and evidence from experiments with the closely related alphavirus, Semliki Forest virus (SFV; Kennedy, 1976; Wengler and Wen457 ISSN
0042-6822
458
DUBIN
gler, 1976) indicates that this third is at the 3’-end of 42 S RNA. We have shown that 42 S RNA from Sindbis virions contains a single methylated residue present in the 5’-terminus m’G(5’)pppAp (Hefti et al., 1976) and have presented evidence that 26 S RNA contains both methylated caps and internal methylated residues (Dubin and Stollar, 1975). We now report detailed analyses of the 5’-terminus and methylated residues of 26 S RNA, together with parallel studies on intracellular 42 S RNA and on virion 42 S RNA. The 26 S and intracellular 42 S RNA species were found to resemble virion 42 S RNA in containing the terminus . ; however, the intracelmGW)pppAp . . lular species differed from virion RNA in two respects: (i) The caps, especially of 26 S RNA, contained di- and trimethylated congeners of m7G (as well, as m7G itself, and (ii) a substantial portion of the methyl groups of both intracellular species occurred as internal m5C residues. Some of this work was presented at the 1976 Meeting of the American Society for Microbiology (Atlantic City, N.J.). MATERIALS
AND
METHODS
Cells, medium, virus. Procedures for preparing and growing baby hamster kidney (BHK-21) and primary chick embryo fibroblast (CEF) cultures and for preparing SV stocks (SV,,,) from CEF cells have been described (Stollar et al., 1976). Infection and labeling. For “short-term” experiments, monolayer cultures in lo-cm petri dishes were infected at input multiplicities of approximately 20 PFU/cell and were labeled from 3 to 8 hr after infection in the presence of 4 pg/ml of actinomycin D (Dubin and Stollar, 1975). For “longterm” experiments, confluent monolayers in one or two roller bottles (surface area, 690 cm? were infected at input multiplicities of approximately 1 PFU/cell and were labeled from 2 to 20 hr after infection in the presence of 0.5 pg/ml of antinomycin (Hefti et al., 1976). In all cases, we added adenosine and guanosine to minimize “leakage” of 3H from [methyZ-3H]methionine into purine rings (Dubin, 1974). Purification of virus and preparation of
ET
AL.
RNA. Virus was concentrated and purified by (NH&SO, precipitation followed by centrifugation to equilibrium in sucrose-D,O gradients (Shenk and Stollar, 1973). We prepared single-stranded RNA from infected cells using a phenol-sodium dodecyl sulfate (SDS) extraction procedure followed by LiCl precipitation (Dubin and Stollar, 1975). Viral RNA was purified from pelleted virus using phenol and SDS without a lithium step (Dubin, 1974). Digestion of RNA and oligonucleotides and fractionation of digests. Digestion with 1 N HCl and electrophoretic separation of the products at pH 3.5 were as described before (Dubin and Taylor, 1975). Routine chromatographic analysis of label recovered from electrophoretic peaks (Dubin, 1974) was performed in an alkaline system (“4”) (cf. Dubin and Taylor, 1975) for adenine and methylated adenines and an acid system (Y”) (cf. Dubin and Taylor, 1975) for guanine and methylated guanines. Digestion with ribonuclease T2 (500 U/ ml) was as described before (Dubin and Taylor, 1975). DEAE-cellulase chromatography was performed using a linear gradient of NaCl (0.05 to 0.5 N) in Tris-HCl, pH 7.8, containing 7 M urea; samples were recovered from columns by readsorption to a second column and elution with triethylammonium carbonate (Davenport et al., 1976). Oligonucleotides were digested with Escherichia coli alkaline phosphatase (100 Kg/ml) in 25 ~1 of 50 mM Tris-HCl, pH 8.6, and 5 mM MgCl, for 2 hr, at 37” (Wei and Moss, 1975). Prior to electrophoresis, digests were brought to approximately pH 4 with 0.5 N HCI, and samples were mixed with equal volumes of ammonium formate buffer (0.05 N, pH 3.5) containing appropriate markers. Methylated markers were obtained from commercial sources as specified earlier (Dubin and Taylor, 1975). In addition, m7G(5’)pppA and m7G(5’)pppG were obtained from P-L Biochemicals, Inc.; m’Cyt and m5Cyt from Cycle Biochemical Corp.; and m”Cyt from Sigma Chemical Co. A sample of m32,a,7G was kindly provided by
5’-TERMINI
OF
SINDBIS
VIRUS
459
mRNA
Dr. H. Busch, and, subsequently, samples of m;2*“*7G and m,‘s7G were synthesized from commercial m2G and mZ2G (HsuChen and Dubin, 1976). The corresponding bases were obtained by 1 N HCl hydrolysis as described above. m4Cyt was obtained from a sample of m4Cm (generously provided by Dr. B. Lane), as described in the footnote to Table 2. [MethyZ-3Hlmethionine was obtained from New England Nuclear Corp. (14.1 Ci/ mmolj or Schwartz Bioresearch Corp. (4.7 or 13 Ci/mmolj. 32Pi (carrier free) and [214Cluridine (55 mCi/mmolj were obtained from New England Nuclear Corp. RESULTS
Overall Methylation S RNA
Patterns of 26 and 42
The experiment illustrated in Figs. 1 and 2 was designed to compare directly the methylation patterns of Sindbis virion RNA and of the Sindbis-specified intracellular RNA species. The latter were obtained from cells labeled from 3 to 8 hr after infection, a period corresponding to near-maximal viral mRNA synthesis (Pfefferkorn and Shapiro, 1974; Stollar, unpublished observations). Virion RNA was obtained after more prolonged incubation so as to maximize release of virus; in this and other such experiments, the corresponding (“long-term-labeled’j intracellular RNA was also examined. The density gradient pattern shown in Fig. 1A is typical of 3- to 8-hr-labeled RNA. [14Cluridine provided a general RNA label, and the 14C pattern displays the 26 and 42 S RNA peaks. The 3H from methyl-labeled methionine yielded a major 4 S peak, representing residual labeling of tRNA (which is almost 1000 times as heavily methylated as Sindbis RNA), and also a peak corresponding to 26 S RNA. Generally, no discrete methyl-labeled peak corresponded to 42 S RNA in such gradients; as shown below, this reflects the fact that 42 S RNA, although methylated, is substantially less so than 26 S RNA or than other, minor species sedimenting between 26 and 42 S RNA. There was regularly a suggestion of a
Fraction
from Tap
FIG. 1. Sedimentation analysis of [“Hlmethyl-labeled RNA from Sindbis virus-infected cells. For short-term-labeled RNA (A!, BHK cells were labeled from 3 to 8 hr after infection with [methyl3H1methionine, 250 &i/3 Kg/ml, and [‘Cluridine, 0.1 &i/ml, 10e4 M, 40 ml of medium, five dishes; for long-term-labeled RNA (Bl, a roller bottle of BHK cells was labeled from 2 to 20 hr after infection with [methyPH]methionine, 100 &i/4.5 Kg/ml, 100 ml of medium (both as described in Materials and Methods). The LiCl-precipitable fractions of the total RNA extracted from the cells were subjected to density gradient sedimentation (Dubin and Stollar, 1975). The positions of cellular RNA peaks (arrows) were determined by 260~nm absorbance. (-O-O-), 3H; (-x-x-). “C.
shoulder ahead of the 26 S RNA peak (fractions 17, 18, and 19 of Fig. 1). When RNA was recovered from this region and resedimented, most of it ran as bona fide 26 S RNA; in addition, this material resembled 26 S RNA in methylation properties. We presume that it corresponds to 33 S RNA, a species thought to be a conformational variant of 26 S RNA (Simmons and Strauss, 1974a; Kennedy, 1976). The intracellular RNA from long-term, [“HImethyl-labeled cells yielded an 3H pattern (Fig. 1Bj essentially indistinguishable from that of 3- to 8-hr-labeled RNA.
460
DUBIN
RNA was concentrated from the 42 S regions of primary gradients of intracellular RNA, as indicated in Figs. 1A and B, and resedimented in parallel with a sample of L3H]methyl-labeled virion RNA from the long-term-labeled cells. As shown in Fig. 2, peaks of [3H]methyl label corresponded in all cases to 42 S RNA. For subsequent analysis, appropriate fractions from primary gradients (as in Figs. 1 and 2C) were pooled to obtain 26 S and virion 42 S RNA and from secondary gradients (as in Figs. 2A and B) to obtain intracellular 42 S RNA. RNA samples were screened for methylated residue patterns using acid hydrolysis followed by electrophoresis at pH 3.5 coupled with chromatographic analysis of certain electrophoretic fractions (cf. Dubin and Taylor, 1975; Dubin and Stollar, 1975). As shown in Fig. 3, 26 S RNA from the short- and the long-term-labeled cells yielded similar patterns. Each contained four discrete peaks, labeled accordingly. Peak 1 contained approximately 10% of the “H (slightly more in the long-term- than the short-term-labeled samples). Some of this label was found to be in m6Ade when corresponding peaks from parallel runs were eluted and fractionated by paper chromatography. However, most was found to be in adenine, representing leakage of label into purine rings. The small amount of “H in the guanine region also appears to represent leakage, since most or all of this 3H ran with guanine on paper chromatography. Peak 2 ran between Ade and m7Gua and Peak 3 ran in the m7Gua region. The latter peak tended to be rather broad, probably due to the fact that it contained two compounds. On paper chromatography, 3H eluted from this region yielded a peak running with m7Gua and a second peak that corresponded to no conventional methylated guanine marker, as illustrated in Fig. 5. Concurrent studies have established that peak 2 is the unusual base m,ZsZ,7Gua and the minor component of peak 3 is the previously unknown base m,“,7Gua (HsuChen and Dubin, 1976). Electrophoretic peak 4 ran slightly to the cathode side of Cp, as expected for
ET
AL.
Fmcilon from Top
FIG. 2. Sedimentation
analysis of [3Hlmethyl-labeled Sindbis 42 S RNA. (A), 3- to 8-hr intracellular RNA and (B), long-term-labeled intracerlular RNA from the 42 S regions of Figs. 1A and B, respectively, pooled as indicated by brackets “b” of these figures. (Cl, RNA isolated from virus prepared from the culture described for Fig. 1B. In the absence of uridine label, we have plotted the 260~nm absorbance patterns for (B) and (C) as an indicator of overall RNA distribution (the absorbance pattern of (A) resembled that of(B)). These patterns were transcribed from ISCO monitor tracings (cf. Dubin and Shine, 1976). The rising 260-nm absorbance approaching 28 S RNA in (C) is the result of our having added carrier cellular cytoplasmic RNA (100 pg) to this preparation. Centrifugation was in “standard salt” gradients (Dubin, 19741, SW 41 rotor, 18,000 rpm, 17 hr. (-O-O-), “H; (-x-x-l, I%; c---j, 260nm absorbance.
m5Cp (Brownlee, 1972). Radioactivity from this peak was characterized by perchloric acid digestion to release the free base, followed by chromatographic analysis in several systems. As summarized in Table 2, the results show that the parent nucleotide is indeed m5Cp. Electrophoretic patterns for acid hydrolysates of 42 S RNA are shown in Fig. 4. The simplest of these is that of virion 42 S
5’-TERMINI
OF
SINDBIS
10
%2 a” z z k” I
20
IO 0 Cm from Origin
IO
FIG. 3. Electrophoretic analysis of acid hydrolysates of [:iHlmethyl-labeled 26 S RNA from short- and long-term-labeled, Sindbis-infected BHK cells. Portions (2.5%, containing approximately 1000 cpm in “H each) of the 26 S RNA from the gradients of Fig. 1 (cuts “a”) were precipitated with ethanol in the presence of 100 Fg of carrier RNA and were subjected to HCl hydrolysis and electrophoresis at pH 3.5 (3 hr, 3OOOV), as described in Materials and Methods. To facilitate comparison, results are normalized to the counts in peak 3 (m7Gua plus m,‘,‘Gua). Here and in subsequent figures, ovals represent markers run with the samples, and rectangles indicate positions of markers as determined from other runs. Only the SH has been plotted. No radioactivity ran beyond Ade, where m’Ade would appear (cf. Dubin and Taylor, 1975). (-O-O-), Short-term-labeled RNA; (-x-x-), longterm-labeled RNA.
RNA. In agreement with earlier studies (Hefti et al., 19761, 80% of the 3H ran with m’Gua, and this was found to be essentially all in m’Gua on subsequent paper chromatography (Fig. 5). Approximately 15% of the label represented leakage into purine rings, and the remainder ran in heterodisperse fashion and was not further characterized. The 3- to 8-hr-labeled, intracellular 42 S RNA bore a closer resemblance to 26 S RNA. As shown in Figs. 4 and 5 and summarized in Table 1, it contained substantial label in m5Cp and some label (albeit significantly less than 26 S RNA) in rnZZ~‘Gua and m;‘v’,‘Gua. The 42 S RNA from long-term-labeled cells, as exemplified by Fig. 4, was intermediate in its methylation complexity between the other
VIRUS
461
mRNA
two types of 42 S RNA sample, containing a small but significant portion of its methyl label in m5C. In general, no more than about 5% of the total 3H of acid hydrolysates of 26 and 42 S RNA samples ran beyond Cp towards the anode, where products arising from Nm residues appear in this system (Dubin and Taylor, 1975). The paucity of Nm residues was confirmed by DEAE analyses of T2 ribonuclease digests (see next section). Table 1 summarizes these results, together with those of two other experiments on 3- to 8-hr-labeled RNA. In one (experiment II), we compared methylated residue patterns of RNA from infected BHK cells on the one hand and CEF cells on the other. There was regularly greater leakage into purine rings with the CEF than with the BHK cells; however, the intracellular 26 and 42 S species from the two hosts proved to have very similar methylation patterns. The results of experiment III of Table 1 are presented to illustrate the range of methylation patterns we noted for the 3- to 8-hr-labeled RNA. In particular, the percentage of methyl label in m5C ranged
mr -1 20
IO Cm from Orighn
0
IO
FIG. 4. Electrophoretic analysis of acid hydrolysates of 13H]methyl-labeled 42 S RNA. Portions of the RNA recovered from the bracketed cuts of the gradients of Fig. 2 were processed as for Fig. 3. We ran 10% of each sample (approximately 500-1500 cpm). (-O-O-), Short-term-labeled intracellular 42 S RNA; (- x-x -), long-term-labeled intracellular 42 S RNA); (-0-O-1, virion 42 S RNA.
DUBIN
462
IO Cm from
2o
F
Origin
FIG. 5. Chromatographic analysis of m’Gua Region of 26 to 42 S hydrolysates. Samples were processed in parallel with each sample described in Figs. 3 and 4, and segments corresponding to the m’Gua regions of electropherograms (generally 7 to 12 cm from the origins) were eluted in 0.05 N HCI. After lyophilization, portions (one-fourth to one-half) were subjected to paper chromatography in an acid system useful for resolving methylated guanines (see Materials and Methods). The methylated guanine markers illustrated can be readily distinguished by differing fluorescences. Not illustrated are Gua and m,Gua, which run more slowly than m’Gua (cf. Dubin and Taylor, 1975), and m,*,‘Gua and rn,‘~‘~‘Gua, both of which run between m*Gua and Cp. To facilitate comparison, counts are normalized to the counts present in m’Gua. (Al, 42 S RNA; (B), 26 S RNA. (-O-O-), Short-term-labeled intracellular RNA; (-x - x -1, long-term-labeled intracellular RNA; (-0-O-1, virion RNA.
from approximately 25 to 50% for both 26 and 42 S RNA, and the ratio of methyl label in m7G congeners to that in m7G proper ranged from approximately 0.4 to 1 for 26 S RNA and 0.1 to 0.2 for 42 S RNA. It should be noted that the molar ratios of congeners to m7G are considerably lower than the above ratios, since the congeners contain two or three times the amount of methyl label per base as does m7G.
ET AL.
We have also indicated in Table 1 (last column) the ratios of 7-methylated G (i.e., m7G plus congeners) to 14C (from [14C]uridine) for 26 S RNA relative to 42 S RNA. These normalized ratios should equal the ratio of the molecular weights of 42 S:26 S RNA (ca 2.4), if there is a one to one correspondence between 7-methylated G and 5’-termini; and, in fact, they are reasonably close to this value. Another estimate for the stoichiometry of capping was obtained for the 42 S RNA from the long-term-labeled cells of Figs. 1 and 2 by comparing 3H in 7-methylated G (assuming that the specific radioactivity of RNA methyl groups is the same as that of medium methionine) to 260-nm absorbance. The resulting values were approximately 1 mG per molecule for virion 42 S RNA and 0.8 for intracellular 42 S RNA. These results, as well as those described above, are compatible with the idea that all 26 and 42 S RNA 5’-termini are capped by 7-methylated G. Structure
of the 5’-Termini
Treatment of 26 and 42 S RNA (both virion and intracellular) with periodate followed by aniline (“p-elimination”) released 85-90% of the methylated guanosine residues and only these residues from the RNA. These results (data not shown) were obtained by electrophoretic analysis (as in Figs. 3 and 4) of acid hydrolysates of the treated RNA. Since the 3’-termini are A,,, (Deborde and Liebowitz, 1976), we can conclude that the methylated guanines are at 5’-termini in “inverted” 5’-ribotide linkage. This conclusion is not trivial, especially with regard to 42 S RNA, for which a “weak” internal initiation site has been reported (Cancedda et al., 1975). (One could postulate an internal equivalent of 7methylated G caps if there were sequential “inversions”; e.g., . . . pNpN(3’?p(3’)m7G(5’)pppNpNp
. .. ;
however, the cap-equivalent illustrated lacks vicinal hydroxyls and thus would not be susceptible to p-elimination.) More direct examination of 5’-termini was accomplished by subjecting T2 ribonuclease-treated samples to DEAE-cellulose
5’-TERMINI
OF
SINDBIS TABLE
DISTRIBUTION
Experiment, labeling time, and RNA speciesb
OF 3H FROM
IN SINDBIS
Percentage
of total
26 AND 42 S RNA”
methyl
mG: 14C (42 S = 1)
label
m 1 2. 7 &a
m2’). 7 Gua
1.4 2.2
7.2 1.4
11.7 4.8
47. 55.
28. 33.
<4. <5.
2.3
4.3 4.0
6.1 2. <2.
11.3 4. <2.
45. 69.
<6. <4. <3.
-
>so.
25. 16. <4.
3. 3.
13. 3.
20. 5.
31. 52
31. 30.
<3. <4.
2.3
7. 11.
3. 2.
9. 5.
22. 6.
30. 47.
33. 39.
<3. <2.’
2.1
4. 5.
3. 4.
4. 2.
15. 4.
31. 34.
45. 53.
<2. <2.
2.1
Gua
mGAde
1.7 4.1
1. 2.
3.1 7.1 11.
3. 3. 4.
4. 7.
2. 4.
CEF, 3- to 8hr label 26 S RNA 42 S RNA
15. 25.
BHK, 3- to 8-hr label 26 S RNA 42 S RNA
7. 10.
BHK, 3- to 8hr label 26 S RNA 42 S RNA
463
mRNA
I
[Methrl-“HIMETHIONINE
Percentage of total 3H Ade
VIRUS
m’Gua
mWyt
Nm
Ia.
Ib.
BHK, 2- to 20hr label 26 S RNA 42 S RNA Virion 42 S RNA
BHK, 3- to 8 hr label 26 S RNA 42 S RNA
IIa.
IIb.
III.
” Experiment I was that described in Figs. 1 and 2. In experiment II, parallel cultures of BHK and CEF cells (five dishes each) were labeled from 3 to 8 hr after infection with [methyl-3Hlmethionine, 100 &i/l.6 kg/ml, and [‘Quridine, 0.1 &i/ml, 10m4 M, and RNA samples were processed as for experiment I. The protocol for experiment III was essentially as for experiment Ia. Percentages of 3H were estimated by electrophoresis and chromatography of acid hydrolysates, as in Figs. 3, 4, and 5. Replicate analyses of the same sample generally agreed to +lO%. The “mG:W” ratios (last column) were determined taking into account the double and triple complements of “H in the di-and trimethylated congeners of m7G; these ratios refer to intracellular RNA and are normalized to the ratio for 42 S RNA. b Unless noted otherwise, all RNA species are intracellular. (I Determined by analysis of DEAE-cellulose peaks; for reasons that are unclear, this sample yielded a falsely high level of 3H in the Nm region of electropherogram.
chromatography. An elution pattern for 26 S RNA from experiment IIa of Table 1 is shown in Fig. 6. Approximately 65% of the 3H appeared as a peak corresponding to the trinucleotide (“-4”) marker, in good agreement with expectation if all methylated guanosines occurred at 5’-termini with the structure mG(5’)pppNp. .. . (Hefti et al., 1976). Approximately 30% of the 3H eluted with the mononucleotide (“-2”) marker, and, also as expected, this was found to be mainly m5Cp. The small
amount (ca. 3%) of the 3H corresponding to the -3 isostich can account for the apparent 2’-0-ribose methyl label in acid hydrolysates of this RNA; the nature of this fraction is unknown. No more than 1% corresponded to -5, -6 region, where blocked termini of the forms mGpppNmpNp and mGpppNmpNmpNp would occur (Wei et aZ., 1975; Furuichi et al., 1975b). Figure 7 illustrates the enzymatic analysis of the 26 S -4 peak. When the oligo-
DUBIN
464 TABLE
2
CHROMATOGRAPHY AND ELECTROPHORESIS METHYLATED CYTOSINEEP Compound
m’Cyt m3Cyt m4Cyt msCvt
Electrophoresis, pH
Chromatography SysPm (Rc,,)
System
0.70 1.7 1.3
1.3 1.5 1.5 1.2
CRZt)
OF
WSB (%A 1.4 0.5b 1.6 1.2
( il:,,, 1.00 1.07 1.00 1.00
fl The methylated cytosines were obtained from commercial sources, except for m’Cyt. This was prepared from m4Cm by perchloric acid hydrolysis (70% acid, loo”, 45 min in a glass-stoppered tube). This procedure yielded a 260-nm-absorbing product with approximately the expected R, in WSB (Szer and Shugar, 1966). The solvents systems are: C, (Iwanami and Brown, 1968): n-butanol, H,O (85:15); D, (Iwanami and Brown, 1968): isopropanol, water, 25% ammonia (85:15:1.3); WSB, (Szer and Shugar, 1966): water-saturated butanol in NH, atmosphere. Electrophoresis was at pH 3.5, 3000 V/90 cm; samples were spotted near the anode edge. Values are referred to Cyt for convenience and reproducibility. R,‘s of Cyt in the three solvent systems were in the range 0.3-0.4; Cyt ran approximately 45 cm towards the cathode on electrophoresis. Samples of putative m5Cyt from Sindbis RNA were prepared by eluting (in 0.05 N HCl) electrophoretic segments corresponding to the putative m5Cp peaks of electropherograms such as those as Fig. 3 and 4 (peaks 4), lyophilizing the eluates, and subjecting the residues to hydrolysis in perchloric acid (0.1 ml) as above. Appropriate 260-nm markers were added to the perchloric acid prior to heating. Samples were neutralized using KOH and then desalted by spinning out the insoluble potassium perchlorate, repeatedly lyophilizing the supernatant solution, redissolving in progressively smaller volumes, and spinning out the salt, until the sample was readily soluble in 10 ~1. Samples from electrophoretic peak 4 in all cases yielded discrete methyl-labeled peaks, containing 85-90% of the recovered 3H, running precisely with m5Cyt. * Streaks.
ET
AL.
alkaline phosphatase, approximately 90% ran with m7G [the three 7-methylated guanosine congeners have similar mobilities at pH 3.5 (Hsu-Chen and Dubin, 197611. This T2-derived terminus was obtained from an RNA sample relatively rich in m2z.7G and miL*2,7G (Table 1); in fact, a bit over half of the 3H in the terminus was in these moieties. Thus, the 3H running with pm7G in Fig. 7 must represent a mixture of pm7G, pm?s7G, and pm34,8,7G; and (assuming that the end group is homogeneous with regard to the putative pppA moiety) the 3H corresponding to m7G(5’)pppA must likewise represent a mixture of 7-methylated guanosine homologues. The 3H (lo-15% of the total in various experiments) remaining at the origin after mixed venom plus phosphatase treatment (which is done at pH 8.6) probably represents a partial degradation product of 7methylated G residues. Such a neutral, nonphosphorylated peak appears after alkaline treatment of m7G-containing cellu-
Fraction
nucleotide was treated with alkaline phosphatase, approximately 95% of the 3H migrated on electrophoresis at pH 3.5 with the marker m’G(5’)pppA. When the label from such an alkaline phosphatase-treated peak was eluted and then treated with venom phosphodiesterase, approximately 90% of the 3H migrated with pm7G, and, when treated with a mixture of venom and
Number
FIG. 6. DEAE-cellulose column chromatography of the T2 digest of methyl-labeled 26 S RNA. A portion (40%) of the 26 S RNA from experiment IIa of Table 1 was subjected to TB-ribonuclease followed by DEAE chromatography as described in Materials and Methods (0.9 x 20-cm column, 260-ml gradient, 3-ml fractions). Positions of ribonuclease A digest markers are shown by arrows. Only the $H has been plotted; all (>99%) of the 14C appeared in the -2 isostich.
5’-TERMINI
OF
SINDBIS
lar mRNA (Dubin and Taylor, 1975). When methyl-labeled intracellular 42 S RNA was analyzed as for Figs. 6 and 7, similar results were obtained-
VIRUS
465
mRNA
A more definitive characterization, especially of the penultimate nucleotides and phosphate “bridges,” was provided by experiments in which cells were doubly labeled with [methyZ-3H]methionine and high levels of 32P. As shown in Fig. 8, DEAE-cellulose chromatography of T2 ribonuclease digests of 26 and 42 S RNA from such cells yielded discrete peaks of TABLE JZP CONTENT
3
OF T2 RIBONUCLEASE-RELEASED
5’-
TERMINI~ RNA
Percentage Experimental
of total Theoretical
___ p=cU
26 S 42 S
Cm from Orlgln
FIG. 7. Enzymatic analysis of a T2-derived terminus from methyl-labeled 26 S RNA. The -4 peak from Fig. 6 (bracket) was recovered as described in the text. A portion was treated with alkaline phosphatase and subjected to electrophoresis at pH 3.5 (-0-O-j. Oligonucleotide in the m?GpppA region from a sample previously processed in this manner was eluted (H,O, 3 hr, room temperature), concentrated by lyophilization, treated with venom phosphodiesterase, and run in parallel (-X-X -1. A third sample was run after treatment with mixed phosphatase-phosphodiesterase (-0-O-j.
20
0.14 0.047
32P
0.080 0.033
p = 3a 0.12 0.050
” Experimental values were estimated from the DEAE-cellulose analyses described for Fig. 8, subtracting a background from the -4 peaks as indicated by the dashed lines. Theoretical values were calculated taking chain lengths to be 5000 and 12,000 nucleotides for 26 and 42 S RNA, respectively, and assuming either that the specific activity of the middle and end phosphates of the presumed ppp bridges were equally labeled (p = (~1 or that the middle phosphate was three times as heavily labeled as the end phosphates (p = 3a). These latter alternatives are discussed in the text.
30 Froclion Number
8. DEAE-cellulose column chromatography of T2 ribonuclease digests of 13H1methyl-, 3ZP-labeled 26 and 42 S RNA. RNA was prepared as described for Figs. 1A and 2A, with the exception that infected cells (10 dishes) were labeled with 32Pi (200 &i/ml, carrier free) and [methyl-3Hlmethionine, 100 &i/4 pg/ml, 5 ml of medium per dish. Portions of each purified RNA preparation [25% of total for 26 S RNA (A); 40% for 42 S RNA (B)] were subjected to T2 digestion followed by DEAE chromatography (0.9 x 14-cm column, 200-ml gradient, 3 ml/fraction). (-O-O-), 3H; (-x-x-), 32P. FIG.
DUBIN
466
Cm from Orlgin
FIG. 9. Electrophoretic and enzymatic analysis of an 13Hlmethyl-, 3ZP-labeled 5’-terminus from 26 S RNA. A portion of the 26 S -4 peak of Fig. 8A (bracket) was subjected to electrophoresis at pH 3.5 (A) in parallel with a portion previously treated with alkaline phosphatase (B). The region corresponding to the bracket in Panel B was eluted from a third (phosphatase-treated) sample run in parallel and subjected to electrophoresis after treatment with venom phosphodiesterase (Cl. Only the regions in the direction of the anode are shown; essentially no (13%) label ran towards the cathode. (-0-O-1, 3H; (-x-x-), 32P.
32P corresponding to the -4 3H peaks. The bulk of the 32P eluted, as expected, with the mononucleotides, precluding accurate determination of internal methyl label in these studies. No discrete 32P peaks corresponded to the -5 or -6 isostiches, where ppNp and pppNp residues elute. The 32P levels corresponding to the -4 peaks were somewhat higher than expected for uniformly labeled termini of the form mG(5’)pppNp, as summarized in Table 3. An explanation for this discrepancy is presented below. Figure 9A shows the electrophoretic pattern obtained for the r3Hlmethyl-, 32P-labeled 26 S -4 fraction. Approximately 95% of the 3H and 80% of the 32P migrated as a single peak slightly behind Gp; this peak
ET AL.
is presumed to represent the mG(5’)pppNp end group. As shown in Fig. 9B, phosphatase treatment of the -4 peak yielded a new [3Hlmethyl-, 32P-labeled peak running slightly slower and corresponding to marker m’GpppA. This peak contained 70% of the 32P, while 22% of the EWPappeared as Pi. The ratio of 32P in these two moieties approaches the 3:l ratio expected if the TB-released peak was indeed mG(5’)pppNp. However, this agreement is probably not meaningful. There was a low level of heterogeneously migrating nucleotides in the original sample (as seen in Fig. 9A) that might be expected to release “extra” Pi on phosphatase treatment, and we believe that the middle phosphate of the putative triphosphate bridge of the phosphatase-resistant moiety has a relatively high specific radioactivity (uide infra). These two circumstances would have opposing effects on the relative amount of 32Pi released by alkaline phosphatase. Figure 9C shows the pattern obtained when the putative 26 S mGpppA peak was eluted and treated with venom phosphodiesterase. Similar results were obtained for 42 S RNA and for a 26 S sample from a separate experiment, all as summarized in Table 4. Three main 32P-containing peaks were obtained, running with pm’G, pA, and Pi. The first two were present in approximately a 1:l ratio in all cases; however, the level of ““Pi was regularly approximately three times the theoretical TABLE VENOM
4
PHOSPHODIESTERABE
PHOSPHATASE-TREATED,
ANALYSIS
““P-LABELED
OF
5’-TERMING”
:IZP released
PmG
PA
P,
Expt 1 26 S RNA 42 S RNA
u.01 11.91
1.02 0.93
3.0 2.8
Expt 2 26 S RNA
11.01
0.91
3.1
(1 Values are normalized on the basis of the :rrP running with pm’G (referred to as pmG since this peak also contains the hypermethylated homologues of pm’G). Experiment 1 is that described in Figs. 8 and 9, and Experiment 2 was a similar, 3- to 8-hr, high-level 32P-labeling experiment.
5’-TERMINI
OF
SINDBIS
level for the presumed structure mGpppA. That this is due to phosphatase contamination of our venom preparations (cf. Keith and Fraenkel-Conrat, 1975) is unlikely, since we monitored our reactions for phosphatase activity by assaying for released m7G (cf. Fig. 7) and found none. We believe that our excess 32Pi arises from unequal specific radioactivities of CY-and P-phosphates of pppN precursors. The middle phosphate of the proposed bridge would be expected to arise from the /3phosphate of nucleoside triphosphate precursors (pppA or pppG depending on the capping mechanism involved), while the end phosphates as well as the remaining (“internal”) phosphates of the RNA molecules should arise from a-phosphates. It would not be surprising if P-phosphates of pppN pools turned over more rapidly than (Y and thus attained higher average specific activities in 5-hr-labeling studies such as those employed (cf. Sripati et al., 1976 and Brumm et al., 1956). If we assign to the middle phosphates of our bridges specific activities threefold that of the other phosphates of the RNA, the values of Tables 3 and 4 are in agreement with the inference that most 26 and 42 S RNA molecules, and perhaps all of them, terminate in mG(5’)pppAp. DISCUSSION
The mRNAs specified by most viruses of eukaryotic cells contain capped 5’-termini of the form m’G(!Y)pppPu, where the penultimate riboside is a purine riboside and is usually ribose methylated (Shatkin, 1976). Sindbis falls into a subgroup of these viruses, whose mRNAs are capped, but lack penultimate ribose methylation. This group includes in addition to Sindbis virus several plant viruses (Zimmern, 1975; Keith and Fraenkel-Conrat, 1975; Symons, 1975; Pinck, 1975; Dasgupta et al., 1976) which, like Sindbis, are positivestrand RNA viruses that multiply in the cytoplasm. The larger group, whose mRNA contains penultimate methylated ribose, consists of viruses with known or presumed nuclear phases [for example, adenovirus (Sommer et al., 1976; Moss and Koczot, 1976); SV-40 (Lavi and Shatkin,
VIRUS
mRNA
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19751; and avian- and B77-sarcoma viruses (Furuichi et al., 1975c; Stoltzfus and Dimock, 197611 plus certain cytoplasmic viruses with virion-associated RNA-synthetic systems [cytoplasmic polyhedrosis virus (Furuichi and Miura, 1975); reovirus (Furuichi et al., 1975a); vaccinia (Wei and Moss, 1975); and vesicular stomatitis virus (VSV; Abraham et al., 1975; Rose, 197511. We propose (il that nuclear mRNA precursors, both cellular and viral, are methylated at what is to become the penultimate ribose by cellular nuclear methylases (cf. Groner and Hurwitz, 19751 and (ii) that cellular cytoplasm lacks such methylating activity unless brought in by viruses. The only cytoplasmic virus thus far studied that has a virion-associated RNA-synthetic system but whose mRNA lacks 2’-Oribose methyl groups is Newcastle disease virus, which gratifyingly is specifically deficient in ribose-methylating activity (Colonno and Stone, 1975, 1976). An unusual feature of the caps of the intracellular SV-RNA species examined, especially 26 S RNA, is the presence of substantial levels of the hypermethylated m7G congeners. We have reexamined the caps of cytoplasmic mRNA from uninfected BHK cells (cf. Dubin and Taylor, 1975) with these compounds in mind and have detected only small amounts of [3Hlmethyl in rn,2r7G and ~Q**~~‘G (5 to 10% of that in m7G); none (~2%) was found in comparable samples from CEF cells (Hsu-Chen and Dubin, unpublished observations). The only other RNA species reported to contain either congener are small nuclear RNAs of unknown function (Saponara and Enger, 1969; Ro Choi et al., 1975; Cory and Adams, 1975), which are capped by rn,‘s”.‘G in a 5’-terminal sequence very similar to that of 26 S RNA. The “standard” m’G caps of certain mRNAs have been shown to facilitate mRNA-ribosome binding, an action which may be related to the positive charge carried by m7G residues at neutral pH (see Shatkin, 1976). The hypermethylated congeners are also cationic at neutrality (HsuChen and Dubin, 19761 and thus might not be expected to grossly alter the conformation of 5’-termini. Whether the extra
466
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methyl groups on some SV-mRNA termini have any biological significance and why their levels in 26 S RNA are so much greater than those in cellular mRNA or even in intracellular 42 S RNA are interesting questions that remain to be explored. The SV intracellular RNA species are unusual also in their internal methylated residue compositions, in particular in the prominence of m5C. There is some m5C in the cellular mRNA of BHK (Dubin and Taylor, 1975) and CEF cells (Hsu-Chen and Dubin, unpublished observations) and probably in adenovirus mRNA (Sommer et al., 1976); however, in all these cases, internal m6A is present at much higher levels than m5C. Our values for internal m6A in 26 and 42 S RNA, amounting to no more than one residue per lo-20 RNA molecules, are so low that it seems reasonable to score them as zero. Other mRNAs lack internal m6A: e.g., yeast cytoplasmic mRNA (Sripati et al., 1976) and VSVmRNA (Moyer et al., 1975). At least in mammalian systems, there appears to be a good correlation between the presence of m6A in mRNA and mRNA precursors and the existence of endonucleolytic processing mechanisms for the generation of these mRNAs (cf. Sripati et al., 1976; Sommer et al., 1976). Thus the absence of m6A from SV 26 and 42 S’RNA is in accord with the view that both species arise via separate transcriptional events (Simmons and Strauss, 1972b) and do not themselves undergo processing (Simmons and Strauss, 1974b; Cancedda et al., 1975; Clegg and Kennedy, 1975). The role, if any, of internal m5C is completely obscure. The apparent exclusion of the hypermethylated m7G congeners and m5C from virion RNA is an interesting phenomenon. One explanation for this may be that these specific methylations play a role in determining the fate of newly synthesized viral RNA molecules; that is, whether a transcript is to serve (i) as a messenger or (ii) as a template for further RNA synthesis or (iii) whether it is instead encapsidated to appear ultimately in progeny virus might be influenced by the methylation status of the transcript. Alternatively, one might
ET AL.
propose that, during the phase of rapid assembly of virions, the sequence (transcription)- (ms7G - capping) - (encapsidation)-(envelopment) is tightly linked in the infected cell (cf. Grimley et al., 1972; Friedman et al., 1972; Soderlund 1973) and that 42 S molecules not entering this linked sequence form separate pools of molecules (presumably including those on polysomes) that (i) cannot be incorporated into virions and (ii) are accessible to methylases such as the putative m7G “supermethylases” and m5C-forming enzymes. Implicit in these models is the idea that, at some stage in the generation of progeny viruses, those intracellular 42 S molecules destined to become genome are rendered inaccessible to methylases. The lower level of m5C in long-term-labeled intracellular 42 S RNA compared to 3- to 8-hr-labeled samples would then indicate that there is a relative accumulation within the cells of such “protected” RNA during the course of infection. The similarity in the 5’-terminal sequences of 26 and 42 S RNA deserves comment. We can now extend this similarity to the second nucleotide beyond the cap (Dubin, unpublished data), both species terminating in mG(5’)pppApUp. The similarity could admittedly occur fortuitously with a frequency of one in eight; if there is a biological explanation for it, however perhaps the most straightforward one would be that 26 S RNA corresponds to the extreme 5’-portion of 42 S RNA. However, studies on SFV (see Introduction) appear to rule this out. We propose that the similarity reflects common structural requirements for 26 and 42 S RNA; for example, ApUp and/or its negative-strand complement may be required for binding the same or similar RNA polymerases (cf. Clewley and Kennedy, 1976) in the generation of the two RNA species. ACKNOWLEDGMENTS This work was supported by Research Grants No. GM-14957 from the National Institute of General Medical Sciences and AI-05920 from the National Institute of Allergy and Infectious Diseases. C.-C. H. C. is a postdoctoral trainee under Institutional National Research Award No. CA-09069 from the National Cancer Institute. G. M. G. was supported
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OF
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in part by Training Grant CA-05234 from the National Cancer Institute and by a Johnson and Johnson Fellowship in Biology, 1975-1976.
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