Vol. 66, No. 1,1975
METHYLATION
BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS
OF C Y T O P L A S M I C A D E N O V I R U S LATE IN P R O D U C T I V E
S t e p h e n M. Fernandez
RNA S Y N T H E S I Z E D
EARLY AND
INFECTION
and Heschel J. Raskas
D e p a r t m e n t s of Pathology and M i c r o b i o l o g y W a s h i n g t o n U n i v e r s i t y School of M e d i c i n e St. Louis, M i s s o u r i 63110 R e c e i v e d June 23,1975 SUMMARY
[3H]-methyl m e t h i o n i n e was i n c o r p o r a t e d into poly A containing c y t o p l a s m i c RNA s y n t h e s i z e d d u r i n g p r o d u c t i v e infection w i t h a d e n o v i r u s 2. P o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s of c y t o p l a s m i c RNA lacking poly A d e m o n s t r a t e d that the m e t h i o n i n e m e t h y l was not i n c o r p o r a t e d into purine rings: P r e p a r a t i o n s labeled w i t h [3H]-uridine c o n t a i n e d peaks of c e l l u l a r 5S RNA and viral 5.5S RNA in a d d i t i o n to tRNA. In contrast RNA labeled w i t h [3H]m e t h y l m e t h i o n i n e y i e l d e d only tRNA, with no d e t e c t a b l e incorp o r a t i o n into the u n m e t h y l a t e d 5S and 5.5S RNAs. The poly A c o n t a i n i n g RNA labeled w i t h [3H]-methyl m e t h i o n i n e was a n n e a l e d to a d e n o v i r u s DNA; 39% of RNA s y n t h e s i z e d early in infection formed hybrid and m o r e than 35% of late ~NA was virus specific.
INTRODUCTION
Recent studies have d e m o n s t r a t e d lated r i b o n u c l e o t i d e s (6-10) mRNAs. from reovirus
Structural RNA
proposed
analyses of RNAs t r a n s c r i b e d
in v i t r o
(5) have shown
of these viral RNAs c o n t a i n a blocked,
structure.
Similar
structures
for the 5' termini of mRNAs
m o u s e L cells
(6,7).
with reovirus
and V S V viral RNAs
(12) indicate
that m e t h y l a t i o n
translation
(1-5) and e u k a r y o t i c
(i) and v a c c i n i a virus DNA
that the 5' termini methylated
in animal virus
the existence of m e t h y -
have also been
isolated
from HeLa and
In vitro studies of p r o t e i n
Copyright © 19 75 by Academic Press, Inc. All rights of reproducnon in any form reserved.
(ii) and r e t i c u l o c y t e m R N A
of viral m R N A s
in vivo. 67
synthesis
is r e q u i r e d
for
Vol. 66, No. 1, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Because of the possible roles of methylated nucleotides in mRNA metabolism and translation, experiments were performed to determine if mRNA transcribed from a nuclear DNA virus is methylated.
Cultured human cells
2 were labeled with
(KB) infected with adenovirus
[3H]-methyl methionine,
and the label in
RNA was characterized by hybridization to viral DNA.
The
results provide evidence that viral RNA synthesized at both early and late times in infection is methylated.
METHODS
Cell culture and virus infection Exponentially growing KB suspension cultures were concentrated to 1.2 x 107 cells/ml and infected with adenovirus 2 as previously described (13,14). To label RNA synthesized early in infection, the culture was diluted to 9 x i0 cells/ml after the 1 hr adsorption period. One hour later the culture was centrifuged again at room temperature and resuspended at 9 x 105 cells/ml in Joklik minimum essential medium containing no serum, 20 ~M L-methionine (1/5 the normal methionine concentration), 20 mM sodium formate, 20 ~M adenosine and guanosine each, and 30 ~Ci/ml of [3H]-methyl methionine (ii Ci/mmole, New England Nuclear Corp.) ( 8 ) . For all early RNA preparations, cycloheximide was present in the medium at concentrations of 25 ~g/ml (15) For the preparation of late RNA, cultures were labeled with 20 ~Ci/ml of [-H]-methyl methionine in the same medium, beginning 18 hours after infection. Control RNAs were prepared by labeling for 3 hrs in the presence of 12.5 ~Ci/ml [3H]-uridine (40 Ci/mmol, New England Nuclear). Cell fractionation,
RNA purification and hybridization
Purification of cytoplasmic RNA, selection of poly A containing molecules by oligo-dT-cellulose chromatography, and hybridization to adenovirus 2 DNA were performed as previously described (15). Electrophoresis of RNA Electrophoresis of RNA was performed with polyacrylamide gels cross-linked with ethylene diacrylate (14); after electrophoresis, gels were frozen, sliced, solubilized with 0.i ml concentrated NH4OH and counted in a scintillation counter.
RESULTS AND DISCUSSION Different portions of the adenovirus 2 genome code for
68
Vol. 66, No. 1, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cytoplasmic viral RNA synthesized early and late in productive infection.
At early times, before viral DNA synthesis begins,
approximately 25% of the viral genome is transcribed into cytoplasmic RNA; late in infection transcripts of a much larger portion of the genome are present as functional mRNA
(16-18).
Most if not all the viral mRNAs synthesized at early and late times contain poly A
(17,19,20).
To isolate early RNA, cultures were labeled in the presence of cycloheximide,
an inhibitor of protein synthesis, which
prevents the onset of viral DNA synthesis
(21).
Early RNA was
prepared from cultures labeled 2-5 hrs after infection, and late RNA was isolated from cultures labeled 18-21 hrs. if the
To determine
[3H]-methyl methionine present in purified RNA preparations
was indeed incorporated into RNA molecules,
cytoplasmic RNA pre-
parations were treated with either DNAse or pronase
(Table i).
Neither treatment reduced the acid-precipitable radioactivity whereas RNAse or KOH degraded the incorporated to acid-soluble material
[3H]-methyl label
(Table i).
A lower percent of RNA preparations labeled with
[3H]-methyl
methionine bound to oligo-dT-cellulose as compared to control RNAs labeled with
[3H]-uridine
(Table 2).
This observation is
consistent with evidence that the level of methylation in mRNA is lower than that in ribosomal and transfer RNA that do not bind to oligo-dT-cellulose
(22).
(8),
Some of the
label in RNA not binding to oligo-dT-cellulose might be methionyl-tRNA.
To test this possibility,
RNA molecules [3H]-
[3H]-
the RNA excluded
from oligo-dT-cellulose was deacylated by incubating in 1.8M Tris HCI, pH 8.1, for 100' at 37 ° (9); this procedure reduced the acid-precipitable radioactivity 25-35%. Electrophoretic analysis of the
69
[3H]-methyl labeled RNA
Vol. 66, No. 1, 1975
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table PROPERTIES PREPARED
OF
[3H]-METHYL
FROM CULTURES
1
LABEL
HARVESTED
INCORPORATED E A R L Y A N D LATE
Percent Treatment
Early
None
100
KOH
INTO RNA IN I N F E C T I O N
Resistant
RNA
Late RNA
i00
0.91
None
2.1
100
DNAse
I
100
99.5
RNAse A
80.5
0.54
Pronase
1.3
94.5
89.5
C u l t u r e s w e r e labeled w i t h [3H]-methyl m e t h i o n i n e as d e s c r i b e d in the M e t h o d s section. C y t o p l a s m i c R N A was isolated by phenolc h l o r o f o r m - i s o a m y l alcohol e x t r a c t i o n and d i s s o l v e d in i0 m M Tris b u f f e r pH 8.0. E a r l y c y t o p l a s m i c RNA (input 16,219 cpm) and late c y t o p l a s m i c ~NA (input 2588 cpm) were i n c u b a t e d w i t h RNAse A (i0 ~g/ml); p r o n a s e (i0 ~g/ml), or DNAse I (5 ~ g / m l in 5 m M Mg) at 37 ° for 1 hr. A l i q u o t s w e r e also d i g e s t e d w i t h 1 N KOH for 1 hr at 80 ° . F o l l o w i n g each t r e a t m e n t the a c i d - p r e c i p i t a b l e r a d i o a c t i v i t y was determined.
which
did not b i n d to o l i g o - d T - c e l l u l o s e
there was not s i g n i f i c a n t methyl
into purine
is e x t e n s i v e (23,24,25) Neither
synthesis
as well
t R N A peak
with
(Fig. result
Late
[3H]-methyl
synthesis
labeling
infection
is virus
that
there
specified
of c e l l u l a r
(26,27).
methionine
IA) ; r a d i o a c t i v e when
in a d e n o v i r u s
is m e t h y l a t e d
evidence
of the m e t h i o n i n e
of a 5.5S R N A w h i c h
as c o n t i n u e d
of these RNAs
in i n f e c t i o n
expected
groups.
incorporation
provided
5S RNA
RNA labeled
contained
(25).
late
a radioactive
5S and 5.5S RNA were not present,
with a precursor
70
utilized
only
for
the
Vol. 66, No. 1,1975
BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS
Table 2 Y I E L D OF
[3H]-METHYL L A B E L E D AND
P L A S M I C RNAs S Y N T H E S I Z E D
Labeling Time
Precursor
late
uridine
late
[3H]-URIDINE
LABELED CYTO-
E A R L Y AND LATE IN P R O D U C T I V E
Yield 8 (cpm/3xl0 cells)
INFECTION
Percent binding to o l i g o - d T cellulose
32.5 x l06
8.3
methionine
4.4 x 106
2.1
late
methionine
2.3 x 106
3.1
early
uridine
25.4 x 106
17.2
early
methionine
5.3 x 106
0.3
early
methionine
7.5 x 106
1.2
E a r l y RNAs were isolated from cultures labeled w i t h either [3H]-uridine (12.5 ~Ci/ml) or [3H]-methyl m e t h i o n i n e (30 ~Ci/ml) from 2-6 hours after infection. L a t e r RNAs w e r e p r e p a r e d from cultures labeled 18-21 hours after infection w i t h either [3H]u r i d i n e (12.5 ~Ci/ml) or [3HI-methyl m e t h i o n i n e (20 ~Ci/ml).
the m e t h y l groups of nucleotides. labeled w i t h
[3H]-uridine
RNA in a d d i t i o n to tRNA
A control
contained
(Fig. IB).
a single peak of 5.5S and 5S Since ribosomal
is reduced more than 85% by the infection amounts
of 28S and 18S RNA were d e t e c t e d
[3H]-uridine
and
virus
demonstrated
[3H]-methyl m e t h i o n i n e
specified
(Fig. 2).
to filters c o n t a i n i n g
preparation
(28), only small in RNA labeled w i t h both
incorporated
chromatography
increasing
[3H]-uridine
labeled w i t h
that a s i g n i f i c a n t
por-
into m R N A was
Poly A c o n t a i n i n g m o l e c u l e s were
s e l e c t e d by o l i g o - d T - c e l l u l o s e
RNA labeled w i t h
RNA synthesis
[3H]-methyl methionine.
RNA-DNA hybridization tion of the
RNA p r e p a r a t i o n
and then a n n e a l e d
amounts of viral DNA.
annealed
45%
(Fig. 2A) ; a p a r a l l e l
[3H]-methyl m e t h i o n i n e
71
Early
annealed
39%
Vol. 66, No. 1, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A 285 18S
B 28S 18S
4S
10 I ~
8
x
~
S
2 i
~4
2
20
lo
Figure i:
Of the poly A c o n t a i n i n g
times in infection,
30
I
18 hrs after infection, to 16 ~g of viral DNA not s u f f i c i e n t
(Fig. 2C).
from cultures
late in infection
is virus
RNA annealed
it is likely that into m R N A
specified.
the i n c o r p o r a t i o n
adenovirus
and location of the m e t h y l a t e d
72
(19,20).
labeled at
label incorporated
demonstrate
label into c y t o p l a s m i c
at late
Since this amount of DNA was
hybridization,
[3H]-methyl
These o b s e r v a t i o n s
structure
specified
35% of the poly A c o n t a i n i n g
for e x h a u s t i v e
a high p e r c e n t of the synthesized
RNA s y n t h e s i z e d
at least 80% is virus
[3HI-methyl RNA was p r e p a r e d
methyl
20
P o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s of n o n p o l y a d e n y l a t e d RNA from infected cells labeled w i t h [3H]-methyl m e t h i o n i n e or [3H]-uridine. C y t o p l a s m i c RNA p r e p a r e d from cultures labeled late in infection was fractionated by c h r o m a t o g r a p h y on o l i g o - d T - c e l l u l o s e . The e x c l u d e d RNA was c o n c e n t r a t e d by ethanol p r e c i p i t a t i o n and a n a l y z e d on 3.2% p o l y a c r y l a m i d e gels c r o s s - l i n k e d w i t h ethylene diacrylate. E l e c t r o p h o r e s i s was at room t e m p e r a t u r e for 4 hrs, 5 mA/gel. The positions of [14C] ribosomal RNA and tRNA markers are indicated. (A) [3H]-methyl RNA; (B) [3H]-uridine RNA.
(Fig. 2B).
When
30 40 10 SLICE NUMBER
RNA.
of
However,
nucleotides,
[3H]-
the
as w e l l
Vol. 66, No. 1,1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
C
,4OO r~ 300
J
~ 20o x
~00 ½
Figure
2:
4
study
to r i b o s o m a l was
the amount
remain
in the p r e s e n c e
ribosomal
least
(28).
adenovirus
In this
derived
contain
methylated
labeled
processing of specific of viral
regions
nucleotides. [3H]-uridine
same extent
viral mRNAs
to d e t e r m i n e
from d i f f e r e n t
with
and
to adenovirus
to exit
nucleotides
mRNA
as w e l l
RNA r e l a t i v e
and late
is i n h i b i t e d
at
specific
viral
mRNA
(31) all
the fact that early
DNA suggests
methionine that m o s t
adenovirus (32-35),
an i m p o r t a n t
as in its translation.
73
in a d e n o v i r u s
of the genome
from the nucleus m a y play
a drug w h i c h
if individual
[3H]-methyl
Since
RNA
we have not used
However,
m u s t be methylated. prior
(29,30),
study,
In the
for the early
RNA synthesis
initial
2 DNA fragments
species
to the
in viral
of c y c l o h e x i m i d e ,
RNA synthesis
28S and 18S r i b o s o m a l
85%
to be determined.
of m e t h y l a t i o n
infection
RNAs
12 16 ~g DNA
R N A could not be determined,
synthesized
inhibits
8
H y b r i d i z a t i o n of c y t o p l a s m i c poly A c o n t a i n i n g RNAs l a b e l e d w i t h [3H]-uridine or [3H]-methyl methionine. C y t o p l a s m i c RNA was p r e p a r e d from c u l t u r e s labeled either early or late in p r o d u c t i v e infection. The poly A c o n t a i n i n g RNA was p u r i f i e d by o l i g o - d T - c e l l u l o s e c h r o m a t o g r a p h y and a n n e a l e d to increasing amounts of a d e n o v i r u s 2 DNA. Hybridizations were performed with 6.5 m m c e l l u l o s e n i t r a t e filters at 66°C for 20 hrs. Panel A shows the results of h y b r i d i z a t i o n of 860 cpm early RNA labeled w i t h [3H]-uridine. For the h y b r i d i zation of [3H]-methyl labeled early RNA 758 cpm were used (B) and for [3H]-methyl labeled late RNA the input was 915 cpm (C).
as the level of m e t h y l a t i o n , present
J.
role
annealed
early
RNAs u n d e r g o the m e t h y l a t i o n in the m a t u r a t i o n
Vol. 66, No. 1, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.
Furuichi, Y., Morgan, M., Muthukrishnan, S. and Shatkin, A.J. (1975) Proc. Natl. Acad. Sci. 72, 362-366. Abraham, G., Moyer, S.A., Adler, R. and Banerjee, A.K. (1975) Fed. Proc. 34, 708. Furuichi, Y. and Miura, K. (1975) Nature 253, 374-375. Lavi, S. and Shatkin, A.J. (1975) Fed. Proc. 34, 526. Wei, C.M. and Moss, B. (1975) Proc. Natl. Acad. Sci. 72, 318-322. Wei, C.M., Gershowitz, A. and Moss, B. (1975) Cell 4, 379-386. Perry, R.P., Kelley, D.E., Friderici, K. and Rottman, F. (1975) Cell 4, 387-394. Perry, R.P. and Kelley, D.C. (1974) Cell i, 37-42. Desrosiers, R., Friderici, K. and Rottman, F. (1974) Proc. Natl. Acad. Sci. 71, 3204-3207. Adams, J.M. and Cory, S. (1975) Nature 255, 28-33. Both, G.W., Banerjee, A.K. and Shatkin, A.J. (1975) Proc. Natl. Acad. Sci. 72, 1189-1193. Muthukrishnan, S., Both, G.W., Furuichi, Y. and Shatkin, A.J. (1975) Nature 255, 33-37. Raskas, H.J. and Okubo, C.K. (1971) J. Cell Biol. 49, 438-449. Tal, J., Craig, E.A. and Raskas, H.J. (1975) J. Virol. 15, 137-144. Craig, E.A. and Raskas, H.J. (1974) J. Virol. 26-32. Tibbetts, C., Pettersson, U., Johansson, K. and Philipson, L. (1974) J. Virol. 13, 370-377. Craig, E.A. and Raskas, H.J. (1974) J. Virol. 14, 751-757. Sharp, P.A., Gallimore, P.H. and Flint, S.J. (1974) Cold Spring Harbor Symposium Volume 39, 457-474. Lindberg, U., Persson, T. and Philipson, L. (1972) J. Virol. i0, 909-919. Bhaduri, S., Raskas, H.J. and Green, M. (1972) J. Virol. 10, 1126-1129. Horowitz, M.S., Brayton, C. and Baum, S.G. (1973) J. Virol. ii, 544-551. Aviv, H. and Leder, P. (1972) Proc. Natl. Acad. Sci. 69, 1408-1412. Ohe, K., Weissman, S.M. and Cooke, N.R. (1969) J. Biol. Chem. 244, 5320-5332. Price, R. and Penman, S. (1972) J. Mol. Biol. 70, 435-447. Weinmann, R., Raskas, H.J. and Roeder, R.G. (1974) Proc. Natl. Acad. Sci. 71, 3426-3430. Ohe, K. and Weissman, S.M. (1971) J. Biol. Chem. 246, 6991-7009. Forget, B.G. and Weissman, S.M. (1967) Science 158, 1695-1699. Raskas, H.J., Thomas, D.C. and Green, M. (1970) Virology 40, 893-902. Craig, N.C. and Perry, R.P. (1970) 45, 554-564. Willems, M., Penman, M. and Penman, S. (1969) J. Cell Biol. 41, 177-187. Tal, J., Craig, E.A., Zimmer, S. and Raskas, H.J. (1974) Proc. Natl. Acad. Sci. 71, 4057-4061. Parsons, J.T., Gardner, J. and Green, M. (1971) Proc. Natl. Acad. Sci. 68, 557-560. Wall, R., Philipson, L. and Darnell, J.E. (1972) Virology 50, 27-34. Brunner, M. and Raskas, H.J. (1972) Proc. Natl. Acad. Sci. Ii, 3101-3104. Craig, E.A., Zimmer, S. and Raskas, H.J. (1975) J. Virol. 15, 1202-1213.
74
METHYLATION
BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS
OF C Y T O P L A S M I C A D E N O V I R U S LATE IN P R O D U C T I V E
S t e p h e n M. Fernandez
RNA S Y N T H E S I Z E D
EARLY AND
INFECTION
and Heschel J. Raskas
D e p a r t m e n t s of Pathology and M i c r o b i o l o g y W a s h i n g t o n U n i v e r s i t y School of M e d i c i n e St. Louis, M i s s o u r i 63110 R e c e i v e d June 23,1975 SUMMARY
[3H]-methyl m e t h i o n i n e was i n c o r p o r a t e d into poly A containing c y t o p l a s m i c RNA s y n t h e s i z e d d u r i n g p r o d u c t i v e infection w i t h a d e n o v i r u s 2. P o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s of c y t o p l a s m i c RNA lacking poly A d e m o n s t r a t e d that the m e t h i o n i n e m e t h y l was not i n c o r p o r a t e d into purine rings: P r e p a r a t i o n s labeled w i t h [3H]-uridine c o n t a i n e d peaks of c e l l u l a r 5S RNA and viral 5.5S RNA in a d d i t i o n to tRNA. In contrast RNA labeled w i t h [3H]m e t h y l m e t h i o n i n e y i e l d e d only tRNA, with no d e t e c t a b l e incorp o r a t i o n into the u n m e t h y l a t e d 5S and 5.5S RNAs. The poly A c o n t a i n i n g RNA labeled w i t h [3H]-methyl m e t h i o n i n e was a n n e a l e d to a d e n o v i r u s DNA; 39% of RNA s y n t h e s i z e d early in infection formed hybrid and m o r e than 35% of late ~NA was virus specific.
INTRODUCTION
Recent studies have d e m o n s t r a t e d lated r i b o n u c l e o t i d e s (6-10) mRNAs. from reovirus
Structural RNA
proposed
analyses of RNAs t r a n s c r i b e d
in v i t r o
(5) have shown
of these viral RNAs c o n t a i n a blocked,
structure.
Similar
structures
for the 5' termini of mRNAs
m o u s e L cells
(6,7).
with reovirus
and V S V viral RNAs
(12) indicate
that m e t h y l a t i o n
translation
(1-5) and e u k a r y o t i c
(i) and v a c c i n i a virus DNA
that the 5' termini methylated
in animal virus
the existence of m e t h y -
have also been
isolated
from HeLa and
In vitro studies of p r o t e i n
Copyright © 19 75 by Academic Press, Inc. All rights of reproducnon in any form reserved.
(ii) and r e t i c u l o c y t e m R N A
of viral m R N A s
in vivo. 67
synthesis
is r e q u i r e d
for
Vol. 66, No. 1, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Because of the possible roles of methylated nucleotides in mRNA metabolism and translation, experiments were performed to determine if mRNA transcribed from a nuclear DNA virus is methylated.
Cultured human cells
2 were labeled with
(KB) infected with adenovirus
[3H]-methyl methionine,
and the label in
RNA was characterized by hybridization to viral DNA.
The
results provide evidence that viral RNA synthesized at both early and late times in infection is methylated.
METHODS
Cell culture and virus infection Exponentially growing KB suspension cultures were concentrated to 1.2 x 107 cells/ml and infected with adenovirus 2 as previously described (13,14). To label RNA synthesized early in infection, the culture was diluted to 9 x i0 cells/ml after the 1 hr adsorption period. One hour later the culture was centrifuged again at room temperature and resuspended at 9 x 105 cells/ml in Joklik minimum essential medium containing no serum, 20 ~M L-methionine (1/5 the normal methionine concentration), 20 mM sodium formate, 20 ~M adenosine and guanosine each, and 30 ~Ci/ml of [3H]-methyl methionine (ii Ci/mmole, New England Nuclear Corp.) ( 8 ) . For all early RNA preparations, cycloheximide was present in the medium at concentrations of 25 ~g/ml (15) For the preparation of late RNA, cultures were labeled with 20 ~Ci/ml of [-H]-methyl methionine in the same medium, beginning 18 hours after infection. Control RNAs were prepared by labeling for 3 hrs in the presence of 12.5 ~Ci/ml [3H]-uridine (40 Ci/mmol, New England Nuclear). Cell fractionation,
RNA purification and hybridization
Purification of cytoplasmic RNA, selection of poly A containing molecules by oligo-dT-cellulose chromatography, and hybridization to adenovirus 2 DNA were performed as previously described (15). Electrophoresis of RNA Electrophoresis of RNA was performed with polyacrylamide gels cross-linked with ethylene diacrylate (14); after electrophoresis, gels were frozen, sliced, solubilized with 0.i ml concentrated NH4OH and counted in a scintillation counter.
RESULTS AND DISCUSSION Different portions of the adenovirus 2 genome code for
68
Vol. 66, No. 1, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cytoplasmic viral RNA synthesized early and late in productive infection.
At early times, before viral DNA synthesis begins,
approximately 25% of the viral genome is transcribed into cytoplasmic RNA; late in infection transcripts of a much larger portion of the genome are present as functional mRNA
(16-18).
Most if not all the viral mRNAs synthesized at early and late times contain poly A
(17,19,20).
To isolate early RNA, cultures were labeled in the presence of cycloheximide,
an inhibitor of protein synthesis, which
prevents the onset of viral DNA synthesis
(21).
Early RNA was
prepared from cultures labeled 2-5 hrs after infection, and late RNA was isolated from cultures labeled 18-21 hrs. if the
To determine
[3H]-methyl methionine present in purified RNA preparations
was indeed incorporated into RNA molecules,
cytoplasmic RNA pre-
parations were treated with either DNAse or pronase
(Table i).
Neither treatment reduced the acid-precipitable radioactivity whereas RNAse or KOH degraded the incorporated to acid-soluble material
[3H]-methyl label
(Table i).
A lower percent of RNA preparations labeled with
[3H]-methyl
methionine bound to oligo-dT-cellulose as compared to control RNAs labeled with
[3H]-uridine
(Table 2).
This observation is
consistent with evidence that the level of methylation in mRNA is lower than that in ribosomal and transfer RNA that do not bind to oligo-dT-cellulose
(22).
(8),
Some of the
label in RNA not binding to oligo-dT-cellulose might be methionyl-tRNA.
To test this possibility,
RNA molecules [3H]-
[3H]-
the RNA excluded
from oligo-dT-cellulose was deacylated by incubating in 1.8M Tris HCI, pH 8.1, for 100' at 37 ° (9); this procedure reduced the acid-precipitable radioactivity 25-35%. Electrophoretic analysis of the
69
[3H]-methyl labeled RNA
Vol. 66, No. 1, 1975
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table PROPERTIES PREPARED
OF
[3H]-METHYL
FROM CULTURES
1
LABEL
HARVESTED
INCORPORATED E A R L Y A N D LATE
Percent Treatment
Early
None
100
KOH
INTO RNA IN I N F E C T I O N
Resistant
RNA
Late RNA
i00
0.91
None
2.1
100
DNAse
I
100
99.5
RNAse A
80.5
0.54
Pronase
1.3
94.5
89.5
C u l t u r e s w e r e labeled w i t h [3H]-methyl m e t h i o n i n e as d e s c r i b e d in the M e t h o d s section. C y t o p l a s m i c R N A was isolated by phenolc h l o r o f o r m - i s o a m y l alcohol e x t r a c t i o n and d i s s o l v e d in i0 m M Tris b u f f e r pH 8.0. E a r l y c y t o p l a s m i c RNA (input 16,219 cpm) and late c y t o p l a s m i c ~NA (input 2588 cpm) were i n c u b a t e d w i t h RNAse A (i0 ~g/ml); p r o n a s e (i0 ~g/ml), or DNAse I (5 ~ g / m l in 5 m M Mg) at 37 ° for 1 hr. A l i q u o t s w e r e also d i g e s t e d w i t h 1 N KOH for 1 hr at 80 ° . F o l l o w i n g each t r e a t m e n t the a c i d - p r e c i p i t a b l e r a d i o a c t i v i t y was determined.
which
did not b i n d to o l i g o - d T - c e l l u l o s e
there was not s i g n i f i c a n t methyl
into purine
is e x t e n s i v e (23,24,25) Neither
synthesis
as well
t R N A peak
with
(Fig. result
Late
[3H]-methyl
synthesis
labeling
infection
is virus
that
there
specified
of c e l l u l a r
(26,27).
methionine
IA) ; r a d i o a c t i v e when
in a d e n o v i r u s
is m e t h y l a t e d
evidence
of the m e t h i o n i n e
of a 5.5S R N A w h i c h
as c o n t i n u e d
of these RNAs
in i n f e c t i o n
expected
groups.
incorporation
provided
5S RNA
RNA labeled
contained
(25).
late
a radioactive
5S and 5.5S RNA were not present,
with a precursor
70
utilized
only
for
the
Vol. 66, No. 1,1975
BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS
Table 2 Y I E L D OF
[3H]-METHYL L A B E L E D AND
P L A S M I C RNAs S Y N T H E S I Z E D
Labeling Time
Precursor
late
uridine
late
[3H]-URIDINE
LABELED CYTO-
E A R L Y AND LATE IN P R O D U C T I V E
Yield 8 (cpm/3xl0 cells)
INFECTION
Percent binding to o l i g o - d T cellulose
32.5 x l06
8.3
methionine
4.4 x 106
2.1
late
methionine
2.3 x 106
3.1
early
uridine
25.4 x 106
17.2
early
methionine
5.3 x 106
0.3
early
methionine
7.5 x 106
1.2
E a r l y RNAs were isolated from cultures labeled w i t h either [3H]-uridine (12.5 ~Ci/ml) or [3H]-methyl m e t h i o n i n e (30 ~Ci/ml) from 2-6 hours after infection. L a t e r RNAs w e r e p r e p a r e d from cultures labeled 18-21 hours after infection w i t h either [3H]u r i d i n e (12.5 ~Ci/ml) or [3HI-methyl m e t h i o n i n e (20 ~Ci/ml).
the m e t h y l groups of nucleotides. labeled w i t h
[3H]-uridine
RNA in a d d i t i o n to tRNA
A control
contained
(Fig. IB).
a single peak of 5.5S and 5S Since ribosomal
is reduced more than 85% by the infection amounts
of 28S and 18S RNA were d e t e c t e d
[3H]-uridine
and
virus
demonstrated
[3H]-methyl m e t h i o n i n e
specified
(Fig. 2).
to filters c o n t a i n i n g
preparation
(28), only small in RNA labeled w i t h both
incorporated
chromatography
increasing
[3H]-uridine
labeled w i t h
that a s i g n i f i c a n t
por-
into m R N A was
Poly A c o n t a i n i n g m o l e c u l e s were
s e l e c t e d by o l i g o - d T - c e l l u l o s e
RNA labeled w i t h
RNA synthesis
[3H]-methyl methionine.
RNA-DNA hybridization tion of the
RNA p r e p a r a t i o n
and then a n n e a l e d
amounts of viral DNA.
annealed
45%
(Fig. 2A) ; a p a r a l l e l
[3H]-methyl m e t h i o n i n e
71
Early
annealed
39%
Vol. 66, No. 1, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A 285 18S
B 28S 18S
4S
10 I ~
8
x
~
S
2 i
~4
2
20
lo
Figure i:
Of the poly A c o n t a i n i n g
times in infection,
30
I
18 hrs after infection, to 16 ~g of viral DNA not s u f f i c i e n t
(Fig. 2C).
from cultures
late in infection
is virus
RNA annealed
it is likely that into m R N A
specified.
the i n c o r p o r a t i o n
adenovirus
and location of the m e t h y l a t e d
72
(19,20).
labeled at
label incorporated
demonstrate
label into c y t o p l a s m i c
at late
Since this amount of DNA was
hybridization,
[3H]-methyl
These o b s e r v a t i o n s
structure
specified
35% of the poly A c o n t a i n i n g
for e x h a u s t i v e
a high p e r c e n t of the synthesized
RNA s y n t h e s i z e d
at least 80% is virus
[3HI-methyl RNA was p r e p a r e d
methyl
20
P o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s of n o n p o l y a d e n y l a t e d RNA from infected cells labeled w i t h [3H]-methyl m e t h i o n i n e or [3H]-uridine. C y t o p l a s m i c RNA p r e p a r e d from cultures labeled late in infection was fractionated by c h r o m a t o g r a p h y on o l i g o - d T - c e l l u l o s e . The e x c l u d e d RNA was c o n c e n t r a t e d by ethanol p r e c i p i t a t i o n and a n a l y z e d on 3.2% p o l y a c r y l a m i d e gels c r o s s - l i n k e d w i t h ethylene diacrylate. E l e c t r o p h o r e s i s was at room t e m p e r a t u r e for 4 hrs, 5 mA/gel. The positions of [14C] ribosomal RNA and tRNA markers are indicated. (A) [3H]-methyl RNA; (B) [3H]-uridine RNA.
(Fig. 2B).
When
30 40 10 SLICE NUMBER
RNA.
of
However,
nucleotides,
[3H]-
the
as w e l l
Vol. 66, No. 1,1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
C
,4OO r~ 300
J
~ 20o x
~00 ½
Figure
2:
4
study
to r i b o s o m a l was
the amount
remain
in the p r e s e n c e
ribosomal
least
(28).
adenovirus
In this
derived
contain
methylated
labeled
processing of specific of viral
regions
nucleotides. [3H]-uridine
same extent
viral mRNAs
to d e t e r m i n e
from d i f f e r e n t
with
and
to adenovirus
to exit
nucleotides
mRNA
as w e l l
RNA r e l a t i v e
and late
is i n h i b i t e d
at
specific
viral
mRNA
(31) all
the fact that early
DNA suggests
methionine that m o s t
adenovirus (32-35),
an i m p o r t a n t
as in its translation.
73
in a d e n o v i r u s
of the genome
from the nucleus m a y play
a drug w h i c h
if individual
[3H]-methyl
Since
RNA
we have not used
However,
m u s t be methylated. prior
(29,30),
study,
In the
for the early
RNA synthesis
initial
2 DNA fragments
species
to the
in viral
of c y c l o h e x i m i d e ,
RNA synthesis
28S and 18S r i b o s o m a l
85%
to be determined.
of m e t h y l a t i o n
infection
RNAs
12 16 ~g DNA
R N A could not be determined,
synthesized
inhibits
8
H y b r i d i z a t i o n of c y t o p l a s m i c poly A c o n t a i n i n g RNAs l a b e l e d w i t h [3H]-uridine or [3H]-methyl methionine. C y t o p l a s m i c RNA was p r e p a r e d from c u l t u r e s labeled either early or late in p r o d u c t i v e infection. The poly A c o n t a i n i n g RNA was p u r i f i e d by o l i g o - d T - c e l l u l o s e c h r o m a t o g r a p h y and a n n e a l e d to increasing amounts of a d e n o v i r u s 2 DNA. Hybridizations were performed with 6.5 m m c e l l u l o s e n i t r a t e filters at 66°C for 20 hrs. Panel A shows the results of h y b r i d i z a t i o n of 860 cpm early RNA labeled w i t h [3H]-uridine. For the h y b r i d i zation of [3H]-methyl labeled early RNA 758 cpm were used (B) and for [3H]-methyl labeled late RNA the input was 915 cpm (C).
as the level of m e t h y l a t i o n , present
J.
role
annealed
early
RNAs u n d e r g o the m e t h y l a t i o n in the m a t u r a t i o n
Vol. 66, No. 1, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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