BIOCHEMICAL
Vol. 66, No. 4,1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
METHYLATION OF SINDBIS Donald College
Department and Dentistry Piscataway,
of Medicine
Received
August
T. Dubin
26,
VIRUS "26s" and Victor
MESSENGER RNA Stollar
of Microbiology of New Jersey - Rutgers New Jersey 08854
Medical
School
1975
The main virus-specific messenger RNA species of Sindbis virusinfected hamster cells, the "26s" RNA, has been examined with regard to Internal methylated residues and terminal methylated methylation status. The internal methyl residues were present, in approximately equal amounts. groups were almost all in 5-methylcytosine residues and the terminal methyl groups were mainly in 7-methylguanine residues. Evidence is presented that these latter occur in "capped" 5'-termini with the novel structure m7G(5')pppNp. SUMMARY.
INTRODUCTION. slightly
but
--in vitro
under
virus
(7),
Recent significantly
of the cellular
and all
We have,
in addition,
contains
a single
Sindbis “26s”
virus-coded RNA (12,
(l-5),
of cytoplasmic (8),
and vesicular
of the viral 5'-termini found
methyl
(11).
shown that
methylated
virus
at "capped"
m7G(5')pppAp
have
the direction
vaccinia
clustered
studies
group
We now report mRNA species
occurs methylation
in
infected
cellular
mRNA is
the mRNA synthesized
polyhedrosis
virus
stomatitis groups
the general
the virion which
as is
mRNA methyl
with that
mammalian
reo-
virus
(9).
Some
were
found
to be
structure
("42s")
(6),
m7G(5')pppNmp?
RNA of Sindbis
virus
in the 5'-terminus properties hamster
cells,
of the main the so-called
13).
METHODC. Growth of BHR-21 cells as monolayers, infection with Sindbis virus (14), harvesting of infected cells, and phenol extraction of RNA, were essentially as previously described (15). The RNA was also subjected to LiCl precipitation (4", 18h, in 2M LiCl; ref. 16). B-elimination and DEAE cellulose column chromatography were performed according to published procedures (17, 8). Nucleotides were recovered from column fractions by BaC12 precipitation (18). Acid hydrolysis was accomplished by heating in 1N HCl at 100' for 45 min. and digestion with ribonuclease T2 (Sigma Chemical Co.) was according to *Unless otherwise noted, nucleic acid components: nucleoside-3'-phosphate; side.
standard abbreviations (10) have been used for Np, e.g., PN, nucleoside-5'-phosphate; m7G, 7-methylguanosine; Nm, 2'-O-methylnucleo-
1373
Vol. 66, No. 4, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Furuichi et al (19), omitting prior denaturation of the RNA. Paper electrophoresis was performed in 0.05 N ammonium formate buffer, pH 3.5, on Whatman 3MM sheets at 3OOOV/90 cm, for 3h. Samples were spotted mid-way between the anode and cathode. Methylated base standards were obtained from Cycle Chemical Corporation, and [methyl-3Hlmethionine (specific (specific activity 55.5mci/mmole) activity 10.5 ci/mmole) and [2- 14Cluridine from New England Nuclear Corp. RESULTS. general
Hamster
cells
RNA label)
and
beginning were hibit purified 13),
added
3h after
RNA.
In view
with
with
RNA.
Fig.
(to virus. purine
of the expectation
was a well-defined that
label
Adenosine rings
viral
(to
RNA methyl
as expected
gradient for
provide
a
groups),
and guanosine
and actinomycin
1 shows a density
peak of 14 C appeared it
14 C-uridine
5h with
Sindbis
of 3H into
entry
A major
for
[methyl-3H]methionine
of cellular
and associated
labeled
infection
to minimize
labeling
were
to inpattern
of
26s mENA (12,
3H peak. mBNA would
contain
methylated
14 C-Uridine-labeled Fig. 1. Density Gradient Sedimentation of 3H-Methyl, RNA from Sindbis Virus-Infected Cells. Five BHK monolayers in lOOmm-petri plates were infected at zero time with Sindbis virus (14), 20 pfu/cell. After lh at 20' for adsorption, the virus suspension was replaced with Eagle's medium (24) containing the non-essential amino acids, adenosine and guanosine (lo-4M), actinomycin D (4 pg/ml) and a lowered level of methionine (0.3pg/ml). At 3h [methyl-3K]methionine (200 uci/3.5pg/ml, final) and [2-14Ciuridine At 8h the cells were harvested and processed as (O.lpc/ml, 10w4M) were added. described in the text. The purified RNA was subjected to density gradient sedimentation (15-30% sucrose in Tris-HCl buffer, pH 7.5, containing 0.1 N NaCl, 0.001 M EDTA and 0.2% sodium dodecyl sulfate) in an SW 27.1 rotor; Samples of 20 1.11were transferred from each 0.6 ml 23,000 RPM, 17h at 20'. fraction to scintillation vials containing T ton X-100 and New England counter. Nuclear Formula 949, and assayed for 3H and rf C in a scintillation The positions of cytoplasmic RNA markers were determined on the basis of 33, (-0-O-J ; 260 mn absorbancy tracings using an ISCO absorbance monitor. 14c, (-x-x-).
1374
Vol. 66, No. 4,1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Froctian Number
Fig. 2. DEAE Cellulose Chromatography of T2 Eibonuclease Digests of Sindbis Virus 26s RNA. An aliquot (10% of the total) of the 26s cut of Fig. 1 was subjected to B-elimination, followed by T2 ribonuclease digestion in parallel with a second, intact, aliquot. The digests were subjected to DEAE cellulose chromatography as described in Methods and 0.4 ml portions of each 2.0 ml fsc~:~;~~,'~~;-r;;;cJ $2 an tlAquasolfl scintillant. Only the k is pl;;ezd; C appeared in the mononucleotide fractions. arrows indicate the mid-points of the indicated isostiches from pancreatic ribonuclease digests of unlabeled cytoplasmic RNA run with the labeled digests. RNA; B, B-eliminated RNA. A, Intact
5'-termini
such as those
following
experiment.
oxidation
followed
should
release
linkage,
as is
"B-eliminated" with
compounds
with
and Nl is
absent
standard
lose
by aniline
from the the
case for
internal
treatment
m'G in
was then
structure
in
samples (3'-5')
of the
Introduction,
RNA.
("B-elimination"). present
the proposed
digested This
with
linkage
to prior
appear
As shown in Fig.
ZA, intact
chromatography.
One corresponded
This
structure
ENA yielded
( 8, 19).
5'-termini
n can be zero,
one,
g-elimination.
to the mononucleotide
The
in parallel
"capped"
two main peaks
procedure 5'-terminal
T2 ribonuclease,
as mononucleotides
the
to periodate
in "inverted"
enzyme releases
Nl(5')ppp(Nmp)nNp; subjected
we performed
26s ENA was subjected
RNA any ribosides
of intact the
in the
An aliquot
aliquot
an aliquot
described
or two
Residues (19,
as
in
20).
on DEAE-cellumarker
and con-
Vol. 66, No. 4, 1975
tained
BIOCHEMICAL
50% of the
(the
" -4"
second
peak)
3H.
subjecting
hydrolysis
under
toward
(5).
the cathode,
tives
hydrolysis
N2, N$di-,
guanine,
pyrimidine
adenines
also
phoretic
pattern
is
run
obtained
(Fig.
for
-4 peak from DEAE-cellulose. nificant ure,
3H is approx.
adenine
("peak
7-methylguanine;
Detailed
chromatographic
N6-methyladenine
the presumed
indicate
(30%) and adenine amount
of "leakage"
constituent end group
(70%); of peak (Fig.
peak
of 3H into
carbon
1376
of the methylated 3 shows the electro-
("2")
RNA and of containing
ahead
l-4
the Figin the
of
to 7-methylCp (peak
will
skeletons;
"4").
be published
a mixture
presumably
of arising
peak 3 is Peak 2 and
5-methylcytosine.
The 38 recovered contained
the sig-
RNA ran
just
behind
peak 1 is
the latter
3, open circles)
fastest,
corresponding
of peaks
7-methyl-
As shown in
slightly
3 are unidentified.
for
of intact
(70X),
and peak 4 is
tested
strip
length).
that
guanines
of intact of the
("3")
the origin
studies
Fig.
move deriva-
except
and most
hydrolysate
peak
of ribosc
ribose
the next
5).
total
characterizations our
7-methylguanine
the lesser
of the
off
and pyrimidine
and methyl
Adenine
by
at pH 3.5.
as a mixture
the rest;
5% ran as a small
ran just
Briefly,
elsewhere.
appear
the region
35% ran as a major
and 40-45%
mainly
(l/3
1");
guanine;
from a small
Only
illustrated
purines
hydrolysate
10% of the 3H of the acid
region
free
3 and ref.
an acid
was
examined
by electrophoresis
and the methylated
3.
aliquot
at pH 3.5 the purines
than
shown in Fig.
from 42s virion
was further
move as a group
faster
as a group
derived
residues
nucleotides
Guanine
moves slightly
position
2B).
residues
the anode.
N$,-dimethylguanine,
(Fig.
releases
and 7- methylguanine)
which
of this
On electrophoresis
while
move towards
The elution
followed
our conditions
derivatives
marker
of the B-eliminated
methylated
2'-0-ribose-methylated
phosphate
(l-,
to acid
to the trinucleotide
of the end-group
of the -4 peak
of the various
samples
nucleotides;
that
3H.
The DEAE-pattern
(90%) devoid
The nature
Acid
(11).
corresponded
43% of the
resembles
RNA, m7G(5')pppAp essentially
The other
and contained
peak closely
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
only
from
electrophoretic
Vol. 66, No. 4, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fig. 3. Electrophoretic Analysis of Acid Hydrolysates of Sindbis Virus 26s RNA and of the T2 Eibonuclease-derived End-Group therefrom. Samples of intact 26s RNA and of the labeled oligonucleotide from the -4 isostich of Fig. 2A (bracketed) were subjected to acid hydrolysis followed by electrophoresis at pH 3.5 as described in the text. The ovals indicate markers run with the hydrolysates and the bars indicate parallel markers. Symbols are as follows: A, adenine; 6MA, N6-methyladenine; 7MG, 7-amthylguanine; DMG, Ni-dimethylguanine; G, guanine. The markers for the two hydrolysates ran indistinguishably. One-cm segments were counted in an "NCS" scintillant. The 14C represents the label in Cp in the hydrolysate of intact RNA; no 14C was detectable in the end-group hydrolysate. 3H from hydrolysate of intact RNA, (-O-O-); 14C from hydrolysate of intact RNA, (-x-x-); 38 from -4 peak, (-O-O-).
peaks
2 and 3, in the
5% of the
total
derivatives
again the
3H ran beyond
would
bracket),
same relative
do (5).
when analyzed
no 2'-O-methyl g-eliminated
reaction
the same way, were
completion
(cf
the
mainly
observed.
ref.
20),
failure
small
(Fig.LB,
2 and 3;
residual
of the than
phosphate
sample
peaks
Thus the
rather
No more than
as 2'-O-methyl
%-eliminated
yielded
reflects
RNA.
label
in
B-elimination amounts
of
termini.
The present occur
as internal
other
half
occur
data
indicate
residues, in structures
5'-termini
DISCUSSION.
in
as the intact
the anode,
The -4 peak from
-4 peak presumably
N(5')pppNmpNp
nucleoside
Cp towards
derivatives
to reach
capped
amounts
mN(5')pppNp,
that almost with where
half all
of the methyl of which
properties
are
groups
5-methylcytosine.
resembling
mN is mainly
of 26s RNA
m7G
those
The of the
and the penultimate
is unmodified, Certain
facets
of the 26s RNA methylation
1377
pattern
are
similar
to
Vol. 66, No. 4, 1975
patterns
found
ilar.
BIOCHEMICAL
for
mRNA from other
The presence
of this this
feature
residue
required
One striking viral
systems
in the
Introduction
However,
find
of internal internal
failure
of internal
other
mature
methylation 1) the
2) Sindbis
with these
mkWA differs
from
play
to examine
The pattern distinctive HeLa (4)
other
of internal
and mouse (3)
the role(s)
methylated
cytoplasmic
no internal,
BHK cytoplasmic
internal
methylated
(i.e.,
(23)
than
far
other
ab-
examined
viruses
for
in that
as SV40 mPNA) in that
it
may be correlated (e.g., It would
ref.
4),
be of
regard.
in Sindbis
26s RNA is also
of m5Cp residues. as of SV-40
residues
from
and
in this
Sindbis-specific
1378
indicates
26s RNA differs
as suggested
mRNA, but
obscure.
to
of mRNA polarity);
properties
residues
failure
the absence
mRNA thus
mRNA, as well
of the
mRNA is
respect
residues
base-methylated
The significance of the
1" viruses
proportion
rather
from the
if,
monkey
systems
mRNA precursors.
in the prominence
m5Cp in uninfected
of m6Ap (5).
These
report
mRNA.
mRNA (as well
Nm residues
"class
Perhaps
virus
differs
in processing
, particularly
(211, revealed found
a role
m6Ap.
methylases,
and "plus"
(22).
of penultimate
a high
and viral,
also
methyla-
another
contained
viral
that
the --in vitro
of internal
RNA-synthetic
Sindbis
(20)
on SV40-infected
In this
cellular
in the cytoplasm
residues
interest
virus
from
manuscript
all
certain
cytoplasmic,
single-stranded
the absence
viral
of the
RNA is
synthesized
albeit
evidence
ribosomes.
finding
(21),
Nm residues.
Sindbis
our
of this
from "native"
eukaryotic
status.
virion
residues,
feature
with
the generality
and those
mRNA also
to encapsidate
5'-terminal
results is
in --in vitro
distinctive
of penultimate
our
preparation
methylation
m/G extends
in accord
mRNA appeared
methylated
and some are dissim-
by eukaryotic
SV40-specific
methylation
Another
is
viral
of the virions
sence
all
during
The cytoplasmic
(75%)
translation between
on --in vivo-synthesized cells.
mRNA, and is
for
systems, with
"capped"
difference cited
ted residues.
eukaryotic
of 5'-termini
of eukaryotic is
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
besides at levels pattern,
Analyses
of
cytoplasmic m6Ap. of only
mRNA
We have l/4
and in
in ml?NA in general,
that fact
remain
BIOCHEMICAL
Vol. 66, No. $1975
The present cumber citly
infected cipate
of provocative in the
Sindbis
results,
questions, Others
foregoing.
mRNA play cell? that
together
a role Are the
ongoing
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
with
studies
will
cited
some of which include:
in subverting pertinent
those
have been
work,
touched
Does the distinctive the protein-synthetic
methylases help
from other
cell-
to answer
a
upon implimethylation
machinery
or virus-coded? these
raise
of of the
We anti-
questions.
by Grant No. GM-14957 of the NatACKNOWLEDGMENTS. This work was supported ional Institute of General Medical Sciences and Grant No. AI-05920 of the National Institute of Allergy and Infectious Diseases, U.S.P.H.S. We thank Mrs. K. Timko for her expert technical assistance. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
Perry, R.P. and Kelley, D.E. (1974) Cell, 37-42. Desrosiers, R., Friderici, K., and Rottman, F. (1974) Proc. Natl. Acad. Sci., U.S.A. 71, 3971-3975. Adams, J.M. and Cory, S. (1975) Nature, 255, 28-33. Furuichi, Y., Morgan, M., Shatkin, A.J., Jelinek, W., Salditt-Georgieff, M., and Damell, J.E. (1975) Proc. Natl. Acad. Sci., U.S.A. 72, 1904-1908. Dubin, D.T. and Taylor, R.H. (1975) Nucleic Acids Res., in press. Furuichi, Y. and Miura, K.I. (1975) Nature, 253, 374-375. Furuichi, Y., Morgan, M., Muthukrishnan, S., and Shatkin, A.J. (1975) Proc. Natl. Acad. Sci., U.S.A., 72, 362-366. Wei, C.M. and Moss, B. (1975) Proc. Natl. Acad. Sci., U.S.A., 72, 318-322. Abraham, G., Rhodes, D.P., and Banerjee, A.K. (1975) Cell, 5, 51-58. IUPAC-IUB Commission on Biochemical Nomenclature (1971) J. Mol. Biol., 55, 299-310. Hefti, E., Bishop, D.H.L., Dubin, D.T. and Stollar, V. (1975) J. Virol., submitted for publication. Mowshowitz, D. (1973) J. Virol., 11, 535-545. Simmons, D.T. and Strauss, J.H. (1974) J. Virol., 14, 552-559. Stollar, V., and Shenk, T.E. (1973) J. Virol., 11, 592-595. Stollar, V., Shenk, T.E., and Stollar, B.D. (1972) Virology, 47, 122-132. Baltimore, D. (1966) J. Mol. Biol., 18, 421-428. Fraenkel-Conrat, H. & Steinschneider, A. (1968), in Methods in Enzymology, Grossman and Moldave, Ed., Vol. XIIB, pp. 243-246, Academic Press, New York. D.H.L. (1973) J. Virol., 11, 487-501. ROY, P., and Bishop, Furuichi, Y., Muthukrishnan, S., and Shatkin, A.J. (1975) Proc. Natl. Acad. Sci., U.S.A., 72, 742-745. Muthukrishnan, S., Both, G.W., Furuichi, A.J., and Shatkin, A.J. (1975) Nature, 255, 33-37. Lavi, S. and Shatkin, A. (1975) Proc. Natl. Acad. Sci., U.S.A., 72, 2012-2016. Pfefferkom, E.R. and Shapiro, D. (1974) Comprehensive Virology, Fraenkel-Conrat and Wagner, Ed., Plenum Press, New York. 2, 171-230. Shatkin, A.J. (1974) Ann. Rev. Biochem., 41, 643-665. Eagle, H. (1959) Science, 130, 432-437.
1379
Vol. 66, No. 4,1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
METHYLATION OF SINDBIS Donald College
Department and Dentistry Piscataway,
of Medicine
Received
August
T. Dubin
26,
VIRUS "26s" and Victor
MESSENGER RNA Stollar
of Microbiology of New Jersey - Rutgers New Jersey 08854
Medical
School
1975
The main virus-specific messenger RNA species of Sindbis virusinfected hamster cells, the "26s" RNA, has been examined with regard to Internal methylated residues and terminal methylated methylation status. The internal methyl residues were present, in approximately equal amounts. groups were almost all in 5-methylcytosine residues and the terminal methyl groups were mainly in 7-methylguanine residues. Evidence is presented that these latter occur in "capped" 5'-termini with the novel structure m7G(5')pppNp. SUMMARY.
INTRODUCTION. slightly
but
--in vitro
under
virus
(7),
Recent significantly
of the cellular
and all
We have,
in addition,
contains
a single
Sindbis “26s”
virus-coded RNA (12,
(l-5),
of cytoplasmic (8),
and vesicular
of the viral 5'-termini found
methyl
(11).
shown that
methylated
virus
at "capped"
m7G(5')pppAp
have
the direction
vaccinia
clustered
studies
group
We now report mRNA species
occurs methylation
in
infected
cellular
mRNA is
the mRNA synthesized
polyhedrosis
virus
stomatitis groups
the general
the virion which
as is
mRNA methyl
with that
mammalian
reo-
virus
(9).
Some
were
found
to be
structure
("42s")
(6),
m7G(5')pppNmp?
RNA of Sindbis
virus
in the 5'-terminus properties hamster
cells,
of the main the so-called
13).
METHODC. Growth of BHR-21 cells as monolayers, infection with Sindbis virus (14), harvesting of infected cells, and phenol extraction of RNA, were essentially as previously described (15). The RNA was also subjected to LiCl precipitation (4", 18h, in 2M LiCl; ref. 16). B-elimination and DEAE cellulose column chromatography were performed according to published procedures (17, 8). Nucleotides were recovered from column fractions by BaC12 precipitation (18). Acid hydrolysis was accomplished by heating in 1N HCl at 100' for 45 min. and digestion with ribonuclease T2 (Sigma Chemical Co.) was according to *Unless otherwise noted, nucleic acid components: nucleoside-3'-phosphate; side.
standard abbreviations (10) have been used for Np, e.g., PN, nucleoside-5'-phosphate; m7G, 7-methylguanosine; Nm, 2'-O-methylnucleo-
1373
Vol. 66, No. 4, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Furuichi et al (19), omitting prior denaturation of the RNA. Paper electrophoresis was performed in 0.05 N ammonium formate buffer, pH 3.5, on Whatman 3MM sheets at 3OOOV/90 cm, for 3h. Samples were spotted mid-way between the anode and cathode. Methylated base standards were obtained from Cycle Chemical Corporation, and [methyl-3Hlmethionine (specific (specific activity 55.5mci/mmole) activity 10.5 ci/mmole) and [2- 14Cluridine from New England Nuclear Corp. RESULTS. general
Hamster
cells
RNA label)
and
beginning were hibit purified 13),
added
3h after
RNA.
In view
with
with
RNA.
Fig.
(to virus. purine
of the expectation
was a well-defined that
label
Adenosine rings
viral
(to
RNA methyl
as expected
gradient for
provide
a
groups),
and guanosine
and actinomycin
1 shows a density
peak of 14 C appeared it
14 C-uridine
5h with
Sindbis
of 3H into
entry
A major
for
[methyl-3H]methionine
of cellular
and associated
labeled
infection
to minimize
labeling
were
to inpattern
of
26s mENA (12,
3H peak. mBNA would
contain
methylated
14 C-Uridine-labeled Fig. 1. Density Gradient Sedimentation of 3H-Methyl, RNA from Sindbis Virus-Infected Cells. Five BHK monolayers in lOOmm-petri plates were infected at zero time with Sindbis virus (14), 20 pfu/cell. After lh at 20' for adsorption, the virus suspension was replaced with Eagle's medium (24) containing the non-essential amino acids, adenosine and guanosine (lo-4M), actinomycin D (4 pg/ml) and a lowered level of methionine (0.3pg/ml). At 3h [methyl-3K]methionine (200 uci/3.5pg/ml, final) and [2-14Ciuridine At 8h the cells were harvested and processed as (O.lpc/ml, 10w4M) were added. described in the text. The purified RNA was subjected to density gradient sedimentation (15-30% sucrose in Tris-HCl buffer, pH 7.5, containing 0.1 N NaCl, 0.001 M EDTA and 0.2% sodium dodecyl sulfate) in an SW 27.1 rotor; Samples of 20 1.11were transferred from each 0.6 ml 23,000 RPM, 17h at 20'. fraction to scintillation vials containing T ton X-100 and New England counter. Nuclear Formula 949, and assayed for 3H and rf C in a scintillation The positions of cytoplasmic RNA markers were determined on the basis of 33, (-0-O-J ; 260 mn absorbancy tracings using an ISCO absorbance monitor. 14c, (-x-x-).
1374
Vol. 66, No. 4,1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Froctian Number
Fig. 2. DEAE Cellulose Chromatography of T2 Eibonuclease Digests of Sindbis Virus 26s RNA. An aliquot (10% of the total) of the 26s cut of Fig. 1 was subjected to B-elimination, followed by T2 ribonuclease digestion in parallel with a second, intact, aliquot. The digests were subjected to DEAE cellulose chromatography as described in Methods and 0.4 ml portions of each 2.0 ml fsc~:~;~~,'~~;-r;;;cJ $2 an tlAquasolfl scintillant. Only the k is pl;;ezd; C appeared in the mononucleotide fractions. arrows indicate the mid-points of the indicated isostiches from pancreatic ribonuclease digests of unlabeled cytoplasmic RNA run with the labeled digests. RNA; B, B-eliminated RNA. A, Intact
5'-termini
such as those
following
experiment.
oxidation
followed
should
release
linkage,
as is
"B-eliminated" with
compounds
with
and Nl is
absent
standard
lose
by aniline
from the the
case for
internal
treatment
m'G in
was then
structure
in
samples (3'-5')
of the
Introduction,
RNA.
("B-elimination"). present
the proposed
digested This
with
linkage
to prior
appear
As shown in Fig.
ZA, intact
chromatography.
One corresponded
This
structure
ENA yielded
( 8, 19).
5'-termini
n can be zero,
one,
g-elimination.
to the mononucleotide
The
in parallel
"capped"
two main peaks
procedure 5'-terminal
T2 ribonuclease,
as mononucleotides
the
to periodate
in "inverted"
enzyme releases
Nl(5')ppp(Nmp)nNp; subjected
we performed
26s ENA was subjected
RNA any ribosides
of intact the
in the
An aliquot
aliquot
an aliquot
described
or two
Residues (19,
as
in
20).
on DEAE-cellumarker
and con-
Vol. 66, No. 4, 1975
tained
BIOCHEMICAL
50% of the
(the
" -4"
second
peak)
3H.
subjecting
hydrolysis
under
toward
(5).
the cathode,
tives
hydrolysis
N2, N$di-,
guanine,
pyrimidine
adenines
also
phoretic
pattern
is
run
obtained
(Fig.
for
-4 peak from DEAE-cellulose. nificant ure,
3H is approx.
adenine
("peak
7-methylguanine;
Detailed
chromatographic
N6-methyladenine
the presumed
indicate
(30%) and adenine amount
of "leakage"
constituent end group
(70%); of peak (Fig.
peak
of 3H into
carbon
1376
of the methylated 3 shows the electro-
("2")
RNA and of containing
ahead
l-4
the Figin the
of
to 7-methylCp (peak
will
skeletons;
"4").
be published
a mixture
presumably
of arising
peak 3 is Peak 2 and
5-methylcytosine.
The 38 recovered contained
the sig-
RNA ran
just
behind
peak 1 is
the latter
3, open circles)
fastest,
corresponding
of peaks
7-methyl-
As shown in
slightly
3 are unidentified.
for
of intact
(70X),
and peak 4 is
tested
strip
length).
that
guanines
of intact of the
("3")
the origin
studies
Fig.
move deriva-
except
and most
hydrolysate
peak
of ribosc
ribose
the next
5).
total
characterizations our
7-methylguanine
the lesser
of the
off
and pyrimidine
and methyl
Adenine
by
at pH 3.5.
as a mixture
the rest;
5% ran as a small
ran just
Briefly,
elsewhere.
appear
the region
35% ran as a major
and 40-45%
mainly
(l/3
1");
guanine;
from a small
Only
illustrated
purines
hydrolysate
10% of the 3H of the acid
region
free
3 and ref.
an acid
was
examined
by electrophoresis
and the methylated
3.
aliquot
at pH 3.5 the purines
than
shown in Fig.
from 42s virion
was further
move as a group
faster
as a group
derived
residues
nucleotides
Guanine
moves slightly
position
2B).
residues
the anode.
N$,-dimethylguanine,
(Fig.
releases
and 7- methylguanine)
which
of this
On electrophoresis
while
move towards
The elution
followed
our conditions
derivatives
marker
of the B-eliminated
methylated
2'-0-ribose-methylated
phosphate
(l-,
to acid
to the trinucleotide
of the end-group
of the -4 peak
of the various
samples
nucleotides;
that
3H.
The DEAE-pattern
(90%) devoid
The nature
Acid
(11).
corresponded
43% of the
resembles
RNA, m7G(5')pppAp essentially
The other
and contained
peak closely
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
only
from
electrophoretic
Vol. 66, No. 4, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fig. 3. Electrophoretic Analysis of Acid Hydrolysates of Sindbis Virus 26s RNA and of the T2 Eibonuclease-derived End-Group therefrom. Samples of intact 26s RNA and of the labeled oligonucleotide from the -4 isostich of Fig. 2A (bracketed) were subjected to acid hydrolysis followed by electrophoresis at pH 3.5 as described in the text. The ovals indicate markers run with the hydrolysates and the bars indicate parallel markers. Symbols are as follows: A, adenine; 6MA, N6-methyladenine; 7MG, 7-amthylguanine; DMG, Ni-dimethylguanine; G, guanine. The markers for the two hydrolysates ran indistinguishably. One-cm segments were counted in an "NCS" scintillant. The 14C represents the label in Cp in the hydrolysate of intact RNA; no 14C was detectable in the end-group hydrolysate. 3H from hydrolysate of intact RNA, (-O-O-); 14C from hydrolysate of intact RNA, (-x-x-); 38 from -4 peak, (-O-O-).
peaks
2 and 3, in the
5% of the
total
derivatives
again the
3H ran beyond
would
bracket),
same relative
do (5).
when analyzed
no 2'-O-methyl g-eliminated
reaction
the same way, were
completion
(cf
the
mainly
observed.
ref.
20),
failure
small
(Fig.LB,
2 and 3;
residual
of the than
phosphate
sample
peaks
Thus the
rather
No more than
as 2'-O-methyl
%-eliminated
yielded
reflects
RNA.
label
in
B-elimination amounts
of
termini.
The present occur
as internal
other
half
occur
data
indicate
residues, in structures
5'-termini
DISCUSSION.
in
as the intact
the anode,
The -4 peak from
-4 peak presumably
N(5')pppNmpNp
nucleoside
Cp towards
derivatives
to reach
capped
amounts
mN(5')pppNp,
that almost with where
half all
of the methyl of which
properties
are
groups
5-methylcytosine.
resembling
mN is mainly
of 26s RNA
m7G
those
The of the
and the penultimate
is unmodified, Certain
facets
of the 26s RNA methylation
1377
pattern
are
similar
to
Vol. 66, No. 4, 1975
patterns
found
ilar.
BIOCHEMICAL
for
mRNA from other
The presence
of this this
feature
residue
required
One striking viral
systems
in the
Introduction
However,
find
of internal internal
failure
of internal
other
mature
methylation 1) the
2) Sindbis
with these
mkWA differs
from
play
to examine
The pattern distinctive HeLa (4)
other
of internal
and mouse (3)
the role(s)
methylated
cytoplasmic
no internal,
BHK cytoplasmic
internal
methylated
(i.e.,
(23)
than
far
other
ab-
examined
viruses
for
in that
as SV40 mPNA) in that
it
may be correlated (e.g., It would
ref.
4),
be of
regard.
in Sindbis
26s RNA is also
of m5Cp residues. as of SV-40
residues
from
and
in this
Sindbis-specific
1378
indicates
26s RNA differs
as suggested
mRNA, but
obscure.
to
of mRNA polarity);
properties
residues
failure
the absence
mRNA thus
mRNA, as well
of the
mRNA is
respect
residues
base-methylated
The significance of the
1" viruses
proportion
rather
from the
if,
monkey
systems
mRNA precursors.
in the prominence
m5Cp in uninfected
of m6Ap (5).
These
report
mRNA.
mRNA (as well
Nm residues
"class
Perhaps
virus
differs
in processing
, particularly
(211, revealed found
a role
m6Ap.
methylases,
and "plus"
(22).
of penultimate
a high
and viral,
also
methyla-
another
contained
viral
that
the --in vitro
of internal
RNA-synthetic
Sindbis
(20)
on SV40-infected
In this
cellular
in the cytoplasm
residues
interest
virus
from
manuscript
all
certain
cytoplasmic,
single-stranded
the absence
viral
of the
RNA is
synthesized
albeit
evidence
ribosomes.
finding
(21),
Nm residues.
Sindbis
our
of this
from "native"
eukaryotic
status.
virion
residues,
feature
with
the generality
and those
mRNA also
to encapsidate
5'-terminal
results is
in --in vitro
distinctive
of penultimate
our
preparation
methylation
m/G extends
in accord
mRNA appeared
methylated
and some are dissim-
by eukaryotic
SV40-specific
methylation
Another
is
viral
of the virions
sence
all
during
The cytoplasmic
(75%)
translation between
on --in vivo-synthesized cells.
mRNA, and is
for
systems, with
"capped"
difference cited
ted residues.
eukaryotic
of 5'-termini
of eukaryotic is
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
besides at levels pattern,
Analyses
of
cytoplasmic m6Ap. of only
mRNA
We have l/4
and in
in ml?NA in general,
that fact
remain
BIOCHEMICAL
Vol. 66, No. $1975
The present cumber citly
infected cipate
of provocative in the
Sindbis
results,
questions, Others
foregoing.
mRNA play cell? that
together
a role Are the
ongoing
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
with
studies
will
cited
some of which include:
in subverting pertinent
those
have been
work,
touched
Does the distinctive the protein-synthetic
methylases help
from other
cell-
to answer
a
upon implimethylation
machinery
or virus-coded? these
raise
of of the
We anti-
questions.
by Grant No. GM-14957 of the NatACKNOWLEDGMENTS. This work was supported ional Institute of General Medical Sciences and Grant No. AI-05920 of the National Institute of Allergy and Infectious Diseases, U.S.P.H.S. We thank Mrs. K. Timko for her expert technical assistance. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
Perry, R.P. and Kelley, D.E. (1974) Cell, 37-42. Desrosiers, R., Friderici, K., and Rottman, F. (1974) Proc. Natl. Acad. Sci., U.S.A. 71, 3971-3975. Adams, J.M. and Cory, S. (1975) Nature, 255, 28-33. Furuichi, Y., Morgan, M., Shatkin, A.J., Jelinek, W., Salditt-Georgieff, M., and Damell, J.E. (1975) Proc. Natl. Acad. Sci., U.S.A. 72, 1904-1908. Dubin, D.T. and Taylor, R.H. (1975) Nucleic Acids Res., in press. Furuichi, Y. and Miura, K.I. (1975) Nature, 253, 374-375. Furuichi, Y., Morgan, M., Muthukrishnan, S., and Shatkin, A.J. (1975) Proc. Natl. Acad. Sci., U.S.A., 72, 362-366. Wei, C.M. and Moss, B. (1975) Proc. Natl. Acad. Sci., U.S.A., 72, 318-322. Abraham, G., Rhodes, D.P., and Banerjee, A.K. (1975) Cell, 5, 51-58. IUPAC-IUB Commission on Biochemical Nomenclature (1971) J. Mol. Biol., 55, 299-310. Hefti, E., Bishop, D.H.L., Dubin, D.T. and Stollar, V. (1975) J. Virol., submitted for publication. Mowshowitz, D. (1973) J. Virol., 11, 535-545. Simmons, D.T. and Strauss, J.H. (1974) J. Virol., 14, 552-559. Stollar, V., and Shenk, T.E. (1973) J. Virol., 11, 592-595. Stollar, V., Shenk, T.E., and Stollar, B.D. (1972) Virology, 47, 122-132. Baltimore, D. (1966) J. Mol. Biol., 18, 421-428. Fraenkel-Conrat, H. & Steinschneider, A. (1968), in Methods in Enzymology, Grossman and Moldave, Ed., Vol. XIIB, pp. 243-246, Academic Press, New York. D.H.L. (1973) J. Virol., 11, 487-501. ROY, P., and Bishop, Furuichi, Y., Muthukrishnan, S., and Shatkin, A.J. (1975) Proc. Natl. Acad. Sci., U.S.A., 72, 742-745. Muthukrishnan, S., Both, G.W., Furuichi, A.J., and Shatkin, A.J. (1975) Nature, 255, 33-37. Lavi, S. and Shatkin, A. (1975) Proc. Natl. Acad. Sci., U.S.A., 72, 2012-2016. Pfefferkom, E.R. and Shapiro, D. (1974) Comprehensive Virology, Fraenkel-Conrat and Wagner, Ed., Plenum Press, New York. 2, 171-230. Shatkin, A.J. (1974) Ann. Rev. Biochem., 41, 643-665. Eagle, H. (1959) Science, 130, 432-437.
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