BIOCHEMICAL
Vol. 79, No. 3, 1977
IN VITRO
SYNTHESIS
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AND STAEILITY BOVINE
Hartmut
Land
OF RNA IN ISOLATED
NUCLEI
FROM
LYMPHOCYTES
and Klaus
P. Schgfer
Universitat Kcnstanz Fachbereich Biologie D-7750 Konstanz W.Germang Received
October
31,1977
SUMMARY: Nuclei from Concanavalin A-stimulated lymphocytes (30 h.r after Con A addition) incorporate up to 5 times more (3-H)UTP into RNA than nuclei from resting lymphocytes. The incorporation kinetics is linear for almost 60 min. 14-20s of the in vitro labeled RNA is polgadenylated. Pclg(A)(-)RNA from bothTn=f nuclei sediments from 4-5s up to more than 30s on sucrose gradients. Nuclei from stimulated cells synthesize about double the amount of RNA larger than 18s than nuclei from resting cells. The same holds for poly(A)(+)RNA. Poly(A)(-) RNA labeled during 10 min in both types of nuclei is stable during a 30 min chase. Under the same conditions pcly(A)(+)R!TA in nuclei from resting cells is degraded to about 50% during the chase whereas it is stable in nuclei from stimulated cells. INTRODUCTION Isolated nuclei offer the possibility to study and manipulate the various reactions involved in RNA synthesis and maturation, e.g. initiation of RNA chains and processing steps like modification of the 5' end ("capping"), methylation, polyadenylation and endonucleolytic cleavages (1). It can be expected that many of these reactions are affected by a change in the proliferative state of the cell. We have previously shown that the RNA synthesizing capacity of isolated nuclei reflects the physiological state of the cell since nuclei from cells which are actively synthesizing RNA --in vivo are more active in '-,in -vitro transcription than nuclei from cells which are less active in --in vivo RNA synthesis (2). Lymphocytes provide a natural example for a resting cell population.Upon stimulation by some plant lectins they can be induced Copyright 0 1977 by Academic Press, Inc. AU rights of reproduction in any form reserved.
947 ISSN
0006-291
X
Vol. 79, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
to procede from a dormant into a metabolically more active state. This change apparently leads to the expression of a cell-specific genetic program (3, 4). In this communication we compare the RNA synthesizing capacity of isolated nuclei from resting lymphocytes with that of nuclei prepared from Concanavalin A (Con A)-stimulated cells. In lymphacytes from bovine lymph nodes which are used in this work the rate of ( 3 H)uridine incorporation increases only 4-6 hours after addition of Con A (5). Consequently, no increase in the RNA synthesizing capacity of isolated nuclei was observed during this initial period (6). In the experiments to be reported below nuclei were prepared from lymphocytes 30 hours after Con A activation when the morphological changes (lymphoblast formation) and the increased ( 3 H)uridine incorporation rates (6) indicate efficient transcription activity in vivo. -MATERIALS
AND METHODS
Lymphocytes from bovine retropharyn eal lymph nodes were --Cells: prepared and cultivatsd as described (5, 6 7 . After 20 hours in a humid CO2 (5%) atmosphere, the cells were stimulated with 5,ug/ ml Con A (Boehringer, Mannheim. The cells were harvested 30 hours after Con A addition. At this time, up to 60% of all lymphocytes were converted to blast cells. An unstimulated control culture was kept under identical conditions for the same time period before the cells were collected. Isolation of nuclei (7): The cells were collected by centrifugation (5 min, 480 g) at room temperature and washed once with a 0.15 M sucrose solution containing 25mM HEPES buffer (pH 8) and 5mM CaCl (buffer A). The pellet was then cooled on ice and resuspended 3 n cold 0.25 M sucrose containing 25mM HEP S and 5mM CaCl (buffer B) to give a concentration of 4 x IO 8 cells/mlTo this guspension was added an equal volume of buffer A containing 0.5% Brij 58 (Serva, Heidelberg). The suspension was gently shaken on ice for 5 min before 25 volumes of buffer B containing 2% Dextran (Sigma) were added. The nuclei were then collected by centrifugation (6 min, 480 g). These nuclei still show some cytoplasmic material adhering to the nuclear envelope when inspected microscopically.Experiments with L-cell nuclei have shown that this together with the high concentration of serum albumin in the incubation buffer minimizes loss of enzymes, e.g. RNA polymerase (K.P.Schafer, unpublished results). RNA synthesis in isolated nuclei: Nuclei were resuspended in incubation buffer which contains: 5OmM Tris-HCl ( H 7.4); 5mN 10% (v/v B glycerol and M&L ; 6OmPI KCl; ImM dithioerythritol; 1% (w/v) bovine serum albumin (Sigma, lot A 4378). C P GTP and ATP were added in final concentrations of 0.3mM; ( 5 H)UTP (1.2 Ci/mmole) was used-.as radioactive tracer in a final concentration of 0.03mM. The standard reaction mixture also contains 3.2mM phosphoenolpyruvate and 20 u /O.25 ml If not stated otherwise, 5 x l;O et nuclei were pyruvate kinase. 948
Vol. 79, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
incubated in a 0.25 ml volume at 25'C. The reaction was stopped by addition of 1 ml ice-cold DB buffer (30mM Tris-HCl (pH 7.5), 5mM magnesium acetate, 120mM KCl, 7mM 2-mercaptoethanol) and 2 ml of 10% TCAjthe precipitates were collected on glass fiber filters, washed with TCA and counted in toluene containing 0.5% (w/v) PPO. If the in vitro synthesized RNA was to be extracted for further analysis2x nuclei were incubated in a 1 ml volume of the reaction mixture containing final concentrations of 0.5mM CTP, GTP and ATP, respectively, as well as O.O?mM of (3H)lJTP. After incubation for the appropriate time at 25 C, the sample was quickly frozen in liquid nitrogen. Isolation of RNA: The procedure used followsthat of Kirby (8). Briefly, nuclei were lysed by anionic detergents. The lysate was shaken in a phenol-cresol-hydroxychinoline-mixture at room temperature. The aqueous phase was extracted several times with chloroform; the nucleic acids were precipitated with ethanol. The dried precipitate was resuspended and treated with FU?Aasefree DNAase (lot.no. 15469, Boehringer, Mannheim) and proteinase K (Merck, Darmstadt). The enzymes were removed by chloroform extraction. Glassware was freed of RNase impurities by heating at 18O'C. Plastic material was treated with 15% H 0 Whereever necessary, buffers were treated with diethylpyroca iso 67ate and then autoclaved. Preparation of poly(A) containing RNA (9): RNA in binding buffer (700mM NaCl; 50mM Tris-HCl, pH 7.5; IOmM EDTA; 20% (v/v) formamide and 5 ug/ml potassium polyvinyl sulfate) was added to a (0.7 ml), equilibrated with binding poly(U) Sebh arose column buffer. The sample was recycled several times to allow for a complete adsor tion to the column. Flow through material was then collected in 5 ml, and the column (P~~Y(A)(-)HNA? washed with 5 ml of binding buffer. The adsorbed poly(A)(+)HNA was then eluted with IOmM potassium phosphate buffer (pH 7.5) containing IOmM EDTA, 90% (v/v) formamide and 5 ug/ml potassium polyxinyl sulfate. The eluted RNA was dialysed 4 ersus DB buffer at 4 and concentrated by ethanol precipitation. Other techniques: RNA was analysed by sedimentation through ml linear sucrose gradients (IO%-60%) for 19 hours at 21°C and 25,000 rpm in the SW 27 rotor of the Spinco centrifuge. The sucrose solution was buffered by IOmM Tris-HCl (pH 7.5) and contained IOOmM NaCl and IOmM EDTA (10). The gradients were collected from the bottom using the LKB-Uvicord and a LKB fraction collector.
I'/
FzEmJLTS
Characterization this 30
of the nuclei
communication, hours
unstimulated
after
addition cells
which
system: from
In
all
activated
of Con A, are were cultivated
949
experiments
described
lymphocytes, compared for
with the
in
prepared nuclei
same length
from
Vol. 79, No. 3, 1977
of time
in
Under
the
our
depends
BIOCHEMICAL
absence
conditions
nuclei
KC1 concentration
able
optimum
in
nuclei
at
in
cells
respectively,
a standard
(10,
stated
50-IOOmM
the
is
compar-
RNA synthesis
for
in
assay.
RNA synthe-
Maximal
was used
I-IO
other-
which
for
11).
of
incubation
of 0.2-0.3mM
when (3H)UTP
range
not
found
a concentration
the
If
was between
other
of RNA synthesis
in
buffer.
concentrations
from
was observed ATP,
salt
rate
of nuclei
were used
Optimum
sis
number
the
ml incubation
5 x IO6
to
incubation
on the
x IO6 nuclei/O.25 wise,
of Con A. of
linearly
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
GTP,
CTP and
a concentration
of
0.03mM.
Fig.
1 shows
the
precipitable be seen rate
material that
5-6
incorporation
declines
afterwards
fore
first
30 min
most
but
nuclei
from It
from for
time.
is
cells
apparent
Con A-stimulated
about
incorporation
one hour.
continues
can
incorpo-
resting also
acid
It
lymphocytes
nuclei
is not
nuclei
from for
that cells
The rate
for
at least
poly(A) in
loose,
however,
nuclei
from from
We have
weeks at
-70'
synthesize
prepared
nuclei.
capacity
RNA extracted
columns
sequences.
cells.
their
RNA labeled
Sepharose
different
unstimulated
as freshly
to poly(U)
contains
significantly
rates
of the
to polg(U)
synthesized
several
radioactive
experiments)
binds
probably
cells
the
of
constant
of six
of poly(A)(+)R.NA
kept
than
into
min.
17 _+ 3% (mean
the
of incubation
same conditions.
rate
approximately
incorporated
Con A-stimulated
(3H)UTl?
the
remains
120
from
more
under
of radioactivity as a function
nuclei
times
incubated the
amount
Con A-stimulated that
found
Sepharose. 950
synthesized that
RNA at
to polyadenylate these
and there-
The percentage.
The nuclei
from
during
nuclei
in
nuclei similar
stored
at -7O'C
RNA since did
not
bind
BIOCHEMICAL
Vol. 79, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
/
1: TIME COURSE OF RNA SYNTHESIS * UC ei (5,x 106&O ul) were incubated
IN ISOLATED NUCLEI under standard conditions (includin 6mM kC1) as described. Incorporated radioactivity g3-H 7 TJTP) was converted to pmol (3-H)UTP. 0 nuclei from Con A-stimulated cells; o nuclei from resting cells.
Fig.
2: SEDIMENTATION ANALYSIS OF IN VITRO LABELED POLY(A)(-)RNA AND POLY(A)(qc IO8 nuclei from either Con A-stimulated cells or resting cells, respectively, were incubated for 36 min under standard conditions. Total RNA was extracted, separated on polg(U) Sepharose columns in polg(A)-free and polg(A)(+)RNA. Each class of RNA was anal zed separately on IO-60% sucrose gradients. A pol~(A)(-bA B poly(A)(+)RNA 0 nuclei from Con A-stimulated cells; o nuclei from resting cells. - It should be mentioned that our experimental conditions during gradient analysis do not discriminate against aggregation of RNA.
Centrifugation
analysis
nuclei:
Nuclei
from
resting
cells,
respectively,
buffer
for
30 min
through
a poly(U)
Unbound
as well
of the
RNA synthesized
Con A-stimulated
at 25'C. Sepharose
as the
were
cells incubated
in as well in
polg(U)-bound
951
as from
incubation
The RNA was extracted column
isolated
and passed
as described. RNA were
then
sedimented
BIOCHEMICAL
Vol. 79, No. 3, 1977
through file
sucrose
gradients.
of RNA which
does
be seen
that
pared
to the
appears is
considerably
was produced
18s
labeled
preferentially Small
In
Fig.
2B the
It
is
apparent
in
weight
with
is produced
synthesized
profile
than
28s
Con A-stimulated
however,
appears
(see
of
in
larger
than
Fig.
coefficient
equal
amounts,
shown.
cells,
legend.to
sedimentation
in‘approximately
is
Con A-stimulated
coefficients
quantities
of low
also
of polg(A)(+)RNA
from
sedimentation
poly(A)(+)PNA
It
conditions.
nuclei
in high
com-
faster
from
can
28s and
cells
sediments
nuclei
It
with
cells.
RNA (4-5S),
both
in
that,
control
which
pro-
Sepharose.
Con A-stimulated
untreated
under
sedimentation
RNA sedimenting
from
sedimentation
poly(A)(+)RNA
is
to polg(U)
material
molecular
quantities
Again,
bind
synthesized
similar
18s
2 shows the
more
from
RESEARCH COMMUNICATIONS
Fig.
in nuclei
nuclei
that
cells.
not
AND BlOf’HYSlCAL
2). (~6s)
in both
types
of nuclei. Stability our
of
nuclear
would
lar
weight
It
therefore
RNA: Under did
be revealed
seemed
--in vitro
under
show unspecific
were
Nuclei
labeled of
with the
assay
tracted.
The remaining
part
RNA was then
study
used
breakdown of
from
resting for
was then of the
of UTP and incubation extracted,
separation
Sepharose
columns
gradients
(Fig.
into both
the
fate
small
of
molecu-
of RNA pulse
of an excess
(3H)UTP
Half
excess
to
conditions
ditions.
After
conditions
by an accumulation
possible
("chase").
50-fold
not
the
fragments.
substrate cells
vitro.labeled
preparations
RNA which
labeled
in
of unlabeled
and Con A-stimulated.
10 min
under
withdrawn
standard
and the
RNA ex-
assay ,was supplied continued
'for
con-
30
with
a
more
min.
too. poly(A)(-)RNA classes
and poly(A)(+)RNA of RNA were
3 and 4). 952
analyzed
on poly(u") on sucrose
Vol. 79, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I IO FRACTION
M NUMBER
FATE OF POLY(A)(-)RNA SYNTHESIZED IN VITRO nuclei were incubated under standard conditions (2 ml total volume). After 10 min 1 ml was withdrawn and ENA extracted. The remaining ml of the assay was made 2.5mM in unlabeled UTP and incubated for 30 min. RNA was then extracted as well. The poly(A)(-)RNA was isolated from both preparations as flow-through of the polg(U) Sepharose columns. (The poly(A)(+) RNA was used to obtain the gradients shown in Fig. 4.) (-)R.NA from nuclei from resting cells. (-)RNA from nuclei from Con A-stimulated cells. Gradient conditions (IO-60%) were as described under Methods. M 10 min pulse label; o---o 10 min pulse label followed by a 30 min chase. FATE OF POLY(A)(+)RNA SYNTHESIZED IN VITRO e poly(A)(+)RNA from the experiment of FE.xwas on IO-60% sucrose gradients. A)(+)RNA from nuclei from resting cells. A)(+)RNA from nuclei from Con A-stimulated ti IO min pulse label; o---o 10 min pulse label by a 30 min chase.
The pulse
labeled
ribosomal
RNA species
(Fig.
3A and B).
Con A-stimulated (Fig.
poly(A)(-)RNA
Again, cells
clearly
and a prominent we see that synthesize
peak nuclei
more
3A).
953
shows the in
the
(Fig.
RNA than
analyzed cells. followed
peaks
of
4-5s
region
3B) control
the
from nuclei
BIOCHEMICAL
Vol. 79, No. 3, 1977
Poly(A)(+)RNA
is
both
types
than
50% of the
whereas ted
pulse
labeled
of nuclei.
it
After
(Fig.
to the
poly(A)(+)RNA
has remained
cells
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
about
30 min
the
same extent
in
chase,
however,
more
has disappeared
unchanged
in
in nuclei
resting
from
nuclei
Con A-stimula-
4A and B).
DISCUSSION Under
optimum
lymphocytes from
to
myeloma
under
however,
2/3
of
of
stimulated
may reflect pared
of
30 hours maximum
about
state
after
Part
transformed
of all
nuclei
to poly(U) sequences.
resting
as well
number
is higher
that
as from in
a fraction
cients
is
labeled
ry
transcription
It
would
we observe
in
well
is
cannot to
compare
isolated
nuclei
vivo. 954
nuclei
difference cell
as com-
isolated
contains
probab-
same in nuclei the
to note
sedimentation
coeffi-
in nuclei
degradation
from
absolute
interesting
The nature
A partial
products
be interesting
F?NA with
cells.
the
we arri-
for
in
although It
case.
equally
is unknown.
is cells
latter
Con A-stimulated
DNA molecules
which
activated
of poly(A)(+)
of 5-10s
and from
the
myeloma
only (6)
remaining
and therefore
The percentage
respond
have
RNA synthesized
Sepharose
account,
(T-cells)
min
Nuclei
nuclei/
into
activity
of the
above,
ly poly(A)
cells
nuclei/30
As mentioned
at 25'.
80 pmol/106
stimulation
lymphocyte.
14-20%
min
transcription
the
Con A-stimulated
we take
our
untransformed
binds
If
15 pmole/106
of
from
about
(12).
40-60%
lymphocytes. the
to the
incorporate
about
their
nuclei
nucleotide&O
conditions
and at
ve at a figure from
cells
only
mitogen
reached
25 pmol
similar
that
the
5 x IO6
polymerize
mouse
30 min
conditions,
from
resting
of this
class
of
longer
of prima-
be excluded. the with
poly(A)-containing that
synthesized
WA -in
BIOCHEMICAL
Vol. 79, No. 3, 1977
The RNA species be compared
to nascent
investigated found
more
in
isolated
--in vivo
by Derman
that
pulse,
synthesized
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
than
RNA.
et al. 50% of
RNA,
sedimented
with
S-values
although
obtained
with
a different
with
data
the
presented
in
We see that
clearly
nuclei
Con A-stimulated
from
lymphocytes. judged
Most
from
a set
more
respect
to polg(A)(+)RNA
instability during
a uniform with
chase
degradation
has determined lymphocytes
of
the (personal
of poly(A)(+)hnRNA to completion But
she also
found
hnRNA did
not
change
half-life
is,
however,
cesses
namely
(i)
(ii)
processing
and its
export
to the
labeled
precursors
lectin
the
5
nuclei
is from
seems
that
that
half-life
of
and
intranuclear
955
human
processing and proceeds
of intermediates. of poly(A)(+) This
several
(iii)
apparent
interrelated
of poly(A)(+)hnRNA
cytoplasm,
is
S.Berger in
rapid
stimulation.
result
resting
of RNA chains
She found is
the
there
or fragments.
of poly(A)(+)hnRNA
after
in
of poly(A)(+)hnRNA
apparent
degradation
nucleus,
of
upon
around
classes
poly(A)(+)RNA
the
Again,
difference
It
species
accumulation that
pattern.
S-values
size
communication).
without
to be rRNA as
quantities
4).
all
pattern
to yield
frc,m dormant
of RNA in nuclei
(Fig.
smaller
decay
in
lymphocytes.
throughout
no accumulation
equal
class
period
results,
comparable
nuclei
appear
one striking
of this
the
in
with
almost
With
These
synthesized
sedimentation
and Con A-stimulated
cells
than
RNA species
resting
workers
a 45 set
are
R.NA is
cells
from
obvious
32.
type,
was
These
during
than
poly(A)-free
in
should
work.
RNA molecules
seems to be synthesized
cells.
labeled
cell
characteristic
of poly(A)-free
however,
RNA --in vivo
HeLa
smaller
this
of these
their
Nascent in
(13)
all
nuclei,
pro-
within
the
to poly(A)(+)mR.RA the
reutilization
breakdown
of
primary
Vol. 79, No. 3, 1977
BIOCHEMICAL
transcription
products.
poly(A)(+)RNA
from
--in
lectin
vivo
part
after
of the
cytoplasm.
the
functioning
the
isolation
What their
nucleus
to
into
of the
cell
this
isolated
Hence
channeling
of
the
pathway
or (ii)
may be
enhanced
mRNA abundance to
is
the
what
in
degree
disturbed
enzymes
which It
is
by
in
drastically procedure
should
therefore
be the
result
chains
being
nuclei
to probe
transcribed.
In
qualitative as well
nuclei
from
RNA polyme-
for
lectin
nuclei
reduces
The increase
Con A-stimulated
it
differences as their
distinguish
after
of an increased addition,
and/or
known that
as much as possible.
capacity
transcribed
(i)
however,
are
increase
Our preparation
the
the
machinery
cells.
proteins
and Con A-stimulated
(5).
respectively,
RNA synthesizing
molecules
is
nuclei
lymphocytes
of nuclear
of
nuclei.
in
(14).
transfer
cytoplasm
channeling
activities,
in
the
in regulating
and Con A-stimulated levels
the
has to be determined, of
relative
shown that
stimulation
of the
stimulation loss
It
is preserved
resting rase
the
means
the
We have
poly(A)(+)hnRNA
an important
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cells
number
of RNA
may be possible in
the
processing
set in
in
to use of RNA
resting
cells.
ACKNOWLEDGEMENT This work has been supported by tbe Deutsche Forschungsgemeinschaft. Our thanks are due to Dr. Rolf Knippers for his steady interest and stimulating criticism. This work is part of H.L.'s Diplomarbeit in biology at the University of Konstanz. REFERENCES ( 1) Perry, R. (1976) Annu.Rev.Biochem. 45, 605-609 ( .2) Schafer, K.P. (1976) Biochem.Biophys.Res.Commun. 219-226
68,
( 3) Robbins, J.H. (1964) Science 146, 1648-1654 ( 4) Hirschhorn, K. and Hirschhorn, R. (1974) Mechanisms of Cell-mediated immunity (McCluskey, R.T. and Cohen, S., eds.) pp. 115-134, John Wiley & Sons. New York
956
BIOCHEMICAL
Vol. 79, No. 3, 1977
( 5) Hauser,
H:,
Knippers,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
R. and Schafer,
K.P.
(1977)
Exp.Gell
( 6) HauszzS.H1n g?;iers R Schafer, K.P., Sons, W. and Un.&l~: H.-J. (1$76j'Exp.Cell Res. 102, 79-84 J.L. (1975) Proc.Natl.Acad.Sci. ( 7) Benz, W.C. and Strominger, USA 72, 24q3-2417 ( 8) Kirby, K.S. (1968) Methods in Enzymology (Grossmann, II. and Moldave, K., eds.) Vol. 12, part B, p. 87-99, Academic Press, New York ( 9) SaldE;zzEgi;ff, M., Jelinek, W., Darnell, J.E., : . Morgan, M. and Shatkln, A. (1976) Cell
7, i!?27-i!?37
W.F.,jr., Murphy, E.C.,jr. and Huang, R.C.G. Biochemistry 12, 3440-3446 (11) Sarma, M.H., Feman and Baglioni, C.(,l9'76) Biochim. Biophys.Acta 418, 29-38 (12) Marzluff, W.F. and Huang, R.C.C. (1975) Proc.Natl.Acad. Sci.USA 72, 1082-1086 (13) Derman, E., Goldberg, S. and Darnell, J. E. (1976) Cell (IO)
Marzluff,
(1973)
46'j-472 (14)
Jaehning, Cell
J.A., Stewart, 4, 51-57
C.C.
957
and Roeder,
R.G.
(1975)
9,
Vol. 79, No. 3, 1977
IN VITRO
SYNTHESIS
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AND STAEILITY BOVINE
Hartmut
Land
OF RNA IN ISOLATED
NUCLEI
FROM
LYMPHOCYTES
and Klaus
P. Schgfer
Universitat Kcnstanz Fachbereich Biologie D-7750 Konstanz W.Germang Received
October
31,1977
SUMMARY: Nuclei from Concanavalin A-stimulated lymphocytes (30 h.r after Con A addition) incorporate up to 5 times more (3-H)UTP into RNA than nuclei from resting lymphocytes. The incorporation kinetics is linear for almost 60 min. 14-20s of the in vitro labeled RNA is polgadenylated. Pclg(A)(-)RNA from bothTn=f nuclei sediments from 4-5s up to more than 30s on sucrose gradients. Nuclei from stimulated cells synthesize about double the amount of RNA larger than 18s than nuclei from resting cells. The same holds for poly(A)(+)RNA. Poly(A)(-) RNA labeled during 10 min in both types of nuclei is stable during a 30 min chase. Under the same conditions pcly(A)(+)R!TA in nuclei from resting cells is degraded to about 50% during the chase whereas it is stable in nuclei from stimulated cells. INTRODUCTION Isolated nuclei offer the possibility to study and manipulate the various reactions involved in RNA synthesis and maturation, e.g. initiation of RNA chains and processing steps like modification of the 5' end ("capping"), methylation, polyadenylation and endonucleolytic cleavages (1). It can be expected that many of these reactions are affected by a change in the proliferative state of the cell. We have previously shown that the RNA synthesizing capacity of isolated nuclei reflects the physiological state of the cell since nuclei from cells which are actively synthesizing RNA --in vivo are more active in '-,in -vitro transcription than nuclei from cells which are less active in --in vivo RNA synthesis (2). Lymphocytes provide a natural example for a resting cell population.Upon stimulation by some plant lectins they can be induced Copyright 0 1977 by Academic Press, Inc. AU rights of reproduction in any form reserved.
947 ISSN
0006-291
X
Vol. 79, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
to procede from a dormant into a metabolically more active state. This change apparently leads to the expression of a cell-specific genetic program (3, 4). In this communication we compare the RNA synthesizing capacity of isolated nuclei from resting lymphocytes with that of nuclei prepared from Concanavalin A (Con A)-stimulated cells. In lymphacytes from bovine lymph nodes which are used in this work the rate of ( 3 H)uridine incorporation increases only 4-6 hours after addition of Con A (5). Consequently, no increase in the RNA synthesizing capacity of isolated nuclei was observed during this initial period (6). In the experiments to be reported below nuclei were prepared from lymphocytes 30 hours after Con A activation when the morphological changes (lymphoblast formation) and the increased ( 3 H)uridine incorporation rates (6) indicate efficient transcription activity in vivo. -MATERIALS
AND METHODS
Lymphocytes from bovine retropharyn eal lymph nodes were --Cells: prepared and cultivatsd as described (5, 6 7 . After 20 hours in a humid CO2 (5%) atmosphere, the cells were stimulated with 5,ug/ ml Con A (Boehringer, Mannheim. The cells were harvested 30 hours after Con A addition. At this time, up to 60% of all lymphocytes were converted to blast cells. An unstimulated control culture was kept under identical conditions for the same time period before the cells were collected. Isolation of nuclei (7): The cells were collected by centrifugation (5 min, 480 g) at room temperature and washed once with a 0.15 M sucrose solution containing 25mM HEPES buffer (pH 8) and 5mM CaCl (buffer A). The pellet was then cooled on ice and resuspended 3 n cold 0.25 M sucrose containing 25mM HEP S and 5mM CaCl (buffer B) to give a concentration of 4 x IO 8 cells/mlTo this guspension was added an equal volume of buffer A containing 0.5% Brij 58 (Serva, Heidelberg). The suspension was gently shaken on ice for 5 min before 25 volumes of buffer B containing 2% Dextran (Sigma) were added. The nuclei were then collected by centrifugation (6 min, 480 g). These nuclei still show some cytoplasmic material adhering to the nuclear envelope when inspected microscopically.Experiments with L-cell nuclei have shown that this together with the high concentration of serum albumin in the incubation buffer minimizes loss of enzymes, e.g. RNA polymerase (K.P.Schafer, unpublished results). RNA synthesis in isolated nuclei: Nuclei were resuspended in incubation buffer which contains: 5OmM Tris-HCl ( H 7.4); 5mN 10% (v/v B glycerol and M&L ; 6OmPI KCl; ImM dithioerythritol; 1% (w/v) bovine serum albumin (Sigma, lot A 4378). C P GTP and ATP were added in final concentrations of 0.3mM; ( 5 H)UTP (1.2 Ci/mmole) was used-.as radioactive tracer in a final concentration of 0.03mM. The standard reaction mixture also contains 3.2mM phosphoenolpyruvate and 20 u /O.25 ml If not stated otherwise, 5 x l;O et nuclei were pyruvate kinase. 948
Vol. 79, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
incubated in a 0.25 ml volume at 25'C. The reaction was stopped by addition of 1 ml ice-cold DB buffer (30mM Tris-HCl (pH 7.5), 5mM magnesium acetate, 120mM KCl, 7mM 2-mercaptoethanol) and 2 ml of 10% TCAjthe precipitates were collected on glass fiber filters, washed with TCA and counted in toluene containing 0.5% (w/v) PPO. If the in vitro synthesized RNA was to be extracted for further analysis2x nuclei were incubated in a 1 ml volume of the reaction mixture containing final concentrations of 0.5mM CTP, GTP and ATP, respectively, as well as O.O?mM of (3H)lJTP. After incubation for the appropriate time at 25 C, the sample was quickly frozen in liquid nitrogen. Isolation of RNA: The procedure used followsthat of Kirby (8). Briefly, nuclei were lysed by anionic detergents. The lysate was shaken in a phenol-cresol-hydroxychinoline-mixture at room temperature. The aqueous phase was extracted several times with chloroform; the nucleic acids were precipitated with ethanol. The dried precipitate was resuspended and treated with FU?Aasefree DNAase (lot.no. 15469, Boehringer, Mannheim) and proteinase K (Merck, Darmstadt). The enzymes were removed by chloroform extraction. Glassware was freed of RNase impurities by heating at 18O'C. Plastic material was treated with 15% H 0 Whereever necessary, buffers were treated with diethylpyroca iso 67ate and then autoclaved. Preparation of poly(A) containing RNA (9): RNA in binding buffer (700mM NaCl; 50mM Tris-HCl, pH 7.5; IOmM EDTA; 20% (v/v) formamide and 5 ug/ml potassium polyvinyl sulfate) was added to a (0.7 ml), equilibrated with binding poly(U) Sebh arose column buffer. The sample was recycled several times to allow for a complete adsor tion to the column. Flow through material was then collected in 5 ml, and the column (P~~Y(A)(-)HNA? washed with 5 ml of binding buffer. The adsorbed poly(A)(+)HNA was then eluted with IOmM potassium phosphate buffer (pH 7.5) containing IOmM EDTA, 90% (v/v) formamide and 5 ug/ml potassium polyxinyl sulfate. The eluted RNA was dialysed 4 ersus DB buffer at 4 and concentrated by ethanol precipitation. Other techniques: RNA was analysed by sedimentation through ml linear sucrose gradients (IO%-60%) for 19 hours at 21°C and 25,000 rpm in the SW 27 rotor of the Spinco centrifuge. The sucrose solution was buffered by IOmM Tris-HCl (pH 7.5) and contained IOOmM NaCl and IOmM EDTA (10). The gradients were collected from the bottom using the LKB-Uvicord and a LKB fraction collector.
I'/
FzEmJLTS
Characterization this 30
of the nuclei
communication, hours
unstimulated
after
addition cells
which
system: from
In
all
activated
of Con A, are were cultivated
949
experiments
described
lymphocytes, compared for
with the
in
prepared nuclei
same length
from
Vol. 79, No. 3, 1977
of time
in
Under
the
our
depends
BIOCHEMICAL
absence
conditions
nuclei
KC1 concentration
able
optimum
in
nuclei
at
in
cells
respectively,
a standard
(10,
stated
50-IOOmM
the
is
compar-
RNA synthesis
for
in
assay.
RNA synthe-
Maximal
was used
I-IO
other-
which
for
11).
of
incubation
of 0.2-0.3mM
when (3H)UTP
range
not
found
a concentration
the
If
was between
other
of RNA synthesis
in
buffer.
concentrations
from
was observed ATP,
salt
rate
of nuclei
were used
Optimum
sis
number
the
ml incubation
5 x IO6
to
incubation
on the
x IO6 nuclei/O.25 wise,
of Con A. of
linearly
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
GTP,
CTP and
a concentration
of
0.03mM.
Fig.
1 shows
the
precipitable be seen rate
material that
5-6
incorporation
declines
afterwards
fore
first
30 min
most
but
nuclei
from It
from for
time.
is
cells
apparent
Con A-stimulated
about
incorporation
one hour.
continues
can
incorpo-
resting also
acid
It
lymphocytes
nuclei
is not
nuclei
from for
that cells
The rate
for
at least
poly(A) in
loose,
however,
nuclei
from from
We have
weeks at
-70'
synthesize
prepared
nuclei.
capacity
RNA extracted
columns
sequences.
cells.
their
RNA labeled
Sepharose
different
unstimulated
as freshly
to poly(U)
contains
significantly
rates
of the
to polg(U)
synthesized
several
radioactive
experiments)
binds
probably
cells
the
of
constant
of six
of poly(A)(+)R.NA
kept
than
into
min.
17 _+ 3% (mean
the
of incubation
same conditions.
rate
approximately
incorporated
Con A-stimulated
(3H)UTl?
the
remains
120
from
more
under
of radioactivity as a function
nuclei
times
incubated the
amount
Con A-stimulated that
found
Sepharose. 950
synthesized that
RNA at
to polyadenylate these
and there-
The percentage.
The nuclei
from
during
nuclei
in
nuclei similar
stored
at -7O'C
RNA since did
not
bind
BIOCHEMICAL
Vol. 79, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
/
1: TIME COURSE OF RNA SYNTHESIS * UC ei (5,x 106&O ul) were incubated
IN ISOLATED NUCLEI under standard conditions (includin 6mM kC1) as described. Incorporated radioactivity g3-H 7 TJTP) was converted to pmol (3-H)UTP. 0 nuclei from Con A-stimulated cells; o nuclei from resting cells.
Fig.
2: SEDIMENTATION ANALYSIS OF IN VITRO LABELED POLY(A)(-)RNA AND POLY(A)(qc IO8 nuclei from either Con A-stimulated cells or resting cells, respectively, were incubated for 36 min under standard conditions. Total RNA was extracted, separated on polg(U) Sepharose columns in polg(A)-free and polg(A)(+)RNA. Each class of RNA was anal zed separately on IO-60% sucrose gradients. A pol~(A)(-bA B poly(A)(+)RNA 0 nuclei from Con A-stimulated cells; o nuclei from resting cells. - It should be mentioned that our experimental conditions during gradient analysis do not discriminate against aggregation of RNA.
Centrifugation
analysis
nuclei:
Nuclei
from
resting
cells,
respectively,
buffer
for
30 min
through
a poly(U)
Unbound
as well
of the
RNA synthesized
Con A-stimulated
at 25'C. Sepharose
as the
were
cells incubated
in as well in
polg(U)-bound
951
as from
incubation
The RNA was extracted column
isolated
and passed
as described. RNA were
then
sedimented
BIOCHEMICAL
Vol. 79, No. 3, 1977
through file
sucrose
gradients.
of RNA which
does
be seen
that
pared
to the
appears is
considerably
was produced
18s
labeled
preferentially Small
In
Fig.
2B the
It
is
apparent
in
weight
with
is produced
synthesized
profile
than
28s
Con A-stimulated
however,
appears
(see
of
in
larger
than
Fig.
coefficient
equal
amounts,
shown.
cells,
legend.to
sedimentation
in‘approximately
is
Con A-stimulated
coefficients
quantities
of low
also
of polg(A)(+)RNA
from
sedimentation
poly(A)(+)PNA
It
conditions.
nuclei
in high
com-
faster
from
can
28s and
cells
sediments
nuclei
It
with
cells.
RNA (4-5S),
both
in
that,
control
which
pro-
Sepharose.
Con A-stimulated
untreated
under
sedimentation
RNA sedimenting
from
sedimentation
poly(A)(+)RNA
is
to polg(U)
material
molecular
quantities
Again,
bind
synthesized
similar
18s
2 shows the
more
from
RESEARCH COMMUNICATIONS
Fig.
in nuclei
nuclei
that
cells.
not
AND BlOf’HYSlCAL
2). (~6s)
in both
types
of nuclei. Stability our
of
nuclear
would
lar
weight
It
therefore
RNA: Under did
be revealed
seemed
--in vitro
under
show unspecific
were
Nuclei
labeled of
with the
assay
tracted.
The remaining
part
RNA was then
study
used
breakdown of
from
resting for
was then of the
of UTP and incubation extracted,
separation
Sepharose
columns
gradients
(Fig.
into both
the
fate
small
of
molecu-
of RNA pulse
of an excess
(3H)UTP
Half
excess
to
conditions
ditions.
After
conditions
by an accumulation
possible
("chase").
50-fold
not
the
fragments.
substrate cells
vitro.labeled
preparations
RNA which
labeled
in
of unlabeled
and Con A-stimulated.
10 min
under
withdrawn
standard
and the
RNA ex-
assay ,was supplied continued
'for
con-
30
with
a
more
min.
too. poly(A)(-)RNA classes
and poly(A)(+)RNA of RNA were
3 and 4). 952
analyzed
on poly(u") on sucrose
Vol. 79, No. 3, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I IO FRACTION
M NUMBER
FATE OF POLY(A)(-)RNA SYNTHESIZED IN VITRO nuclei were incubated under standard conditions (2 ml total volume). After 10 min 1 ml was withdrawn and ENA extracted. The remaining ml of the assay was made 2.5mM in unlabeled UTP and incubated for 30 min. RNA was then extracted as well. The poly(A)(-)RNA was isolated from both preparations as flow-through of the polg(U) Sepharose columns. (The poly(A)(+) RNA was used to obtain the gradients shown in Fig. 4.) (-)R.NA from nuclei from resting cells. (-)RNA from nuclei from Con A-stimulated cells. Gradient conditions (IO-60%) were as described under Methods. M 10 min pulse label; o---o 10 min pulse label followed by a 30 min chase. FATE OF POLY(A)(+)RNA SYNTHESIZED IN VITRO e poly(A)(+)RNA from the experiment of FE.xwas on IO-60% sucrose gradients. A)(+)RNA from nuclei from resting cells. A)(+)RNA from nuclei from Con A-stimulated ti IO min pulse label; o---o 10 min pulse label by a 30 min chase.
The pulse
labeled
ribosomal
RNA species
(Fig.
3A and B).
Con A-stimulated (Fig.
poly(A)(-)RNA
Again, cells
clearly
and a prominent we see that synthesize
peak nuclei
more
3A).
953
shows the in
the
(Fig.
RNA than
analyzed cells. followed
peaks
of
4-5s
region
3B) control
the
from nuclei
BIOCHEMICAL
Vol. 79, No. 3, 1977
Poly(A)(+)RNA
is
both
types
than
50% of the
whereas ted
pulse
labeled
of nuclei.
it
After
(Fig.
to the
poly(A)(+)RNA
has remained
cells
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
about
30 min
the
same extent
in
chase,
however,
more
has disappeared
unchanged
in
in nuclei
resting
from
nuclei
Con A-stimula-
4A and B).
DISCUSSION Under
optimum
lymphocytes from
to
myeloma
under
however,
2/3
of
of
stimulated
may reflect pared
of
30 hours maximum
about
state
after
Part
transformed
of all
nuclei
to poly(U) sequences.
resting
as well
number
is higher
that
as from in
a fraction
cients
is
labeled
ry
transcription
It
would
we observe
in
well
is
cannot to
compare
isolated
nuclei
vivo. 954
nuclei
difference cell
as com-
isolated
contains
probab-
same in nuclei the
to note
sedimentation
coeffi-
in nuclei
degradation
from
absolute
interesting
The nature
A partial
products
be interesting
F?NA with
cells.
the
we arri-
for
in
although It
case.
equally
is unknown.
is cells
latter
Con A-stimulated
DNA molecules
which
activated
of poly(A)(+)
of 5-10s
and from
the
myeloma
only (6)
remaining
and therefore
The percentage
respond
have
RNA synthesized
Sepharose
account,
(T-cells)
min
Nuclei
nuclei/
into
activity
of the
above,
ly poly(A)
cells
nuclei/30
As mentioned
at 25'.
80 pmol/106
stimulation
lymphocyte.
14-20%
min
transcription
the
Con A-stimulated
we take
our
untransformed
binds
If
15 pmole/106
of
from
about
(12).
40-60%
lymphocytes. the
to the
incorporate
about
their
nuclei
nucleotide&O
conditions
and at
ve at a figure from
cells
only
mitogen
reached
25 pmol
similar
that
the
5 x IO6
polymerize
mouse
30 min
conditions,
from
resting
of this
class
of
longer
of prima-
be excluded. the with
poly(A)-containing that
synthesized
WA -in
BIOCHEMICAL
Vol. 79, No. 3, 1977
The RNA species be compared
to nascent
investigated found
more
in
isolated
--in vivo
by Derman
that
pulse,
synthesized
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
than
RNA.
et al. 50% of
RNA,
sedimented
with
S-values
although
obtained
with
a different
with
data
the
presented
in
We see that
clearly
nuclei
Con A-stimulated
from
lymphocytes. judged
Most
from
a set
more
respect
to polg(A)(+)RNA
instability during
a uniform with
chase
degradation
has determined lymphocytes
of
the (personal
of poly(A)(+)hnRNA to completion But
she also
found
hnRNA did
not
change
half-life
is,
however,
cesses
namely
(i)
(ii)
processing
and its
export
to the
labeled
precursors
lectin
the
5
nuclei
is from
seems
that
that
half-life
of
and
intranuclear
955
human
processing and proceeds
of intermediates. of poly(A)(+) This
several
(iii)
apparent
interrelated
of poly(A)(+)hnRNA
cytoplasm,
is
S.Berger in
rapid
stimulation.
result
resting
of RNA chains
She found is
the
there
or fragments.
of poly(A)(+)hnRNA
after
in
of poly(A)(+)hnRNA
apparent
degradation
nucleus,
of
upon
around
classes
poly(A)(+)RNA
the
Again,
difference
It
species
accumulation that
pattern.
S-values
size
communication).
without
to be rRNA as
quantities
4).
all
pattern
to yield
frc,m dormant
of RNA in nuclei
(Fig.
smaller
decay
in
lymphocytes.
throughout
no accumulation
equal
class
period
results,
comparable
nuclei
appear
one striking
of this
the
in
with
almost
With
These
synthesized
sedimentation
and Con A-stimulated
cells
than
RNA species
resting
workers
a 45 set
are
R.NA is
cells
from
obvious
32.
type,
was
These
during
than
poly(A)-free
in
should
work.
RNA molecules
seems to be synthesized
cells.
labeled
cell
characteristic
of poly(A)-free
however,
RNA --in vivo
HeLa
smaller
this
of these
their
Nascent in
(13)
all
nuclei,
pro-
within
the
to poly(A)(+)mR.RA the
reutilization
breakdown
of
primary
Vol. 79, No. 3, 1977
BIOCHEMICAL
transcription
products.
poly(A)(+)RNA
from
--in
lectin
vivo
part
after
of the
cytoplasm.
the
functioning
the
isolation
What their
nucleus
to
into
of the
cell
this
isolated
Hence
channeling
of
the
pathway
or (ii)
may be
enhanced
mRNA abundance to
is
the
what
in
degree
disturbed
enzymes
which It
is
by
in
drastically procedure
should
therefore
be the
result
chains
being
nuclei
to probe
transcribed.
In
qualitative as well
nuclei
from
RNA polyme-
for
lectin
nuclei
reduces
The increase
Con A-stimulated
it
differences as their
distinguish
after
of an increased addition,
and/or
known that
as much as possible.
capacity
transcribed
(i)
however,
are
increase
Our preparation
the
the
machinery
cells.
proteins
and Con A-stimulated
(5).
respectively,
RNA synthesizing
molecules
is
nuclei
lymphocytes
of nuclear
of
nuclei.
in
(14).
transfer
cytoplasm
channeling
activities,
in
the
in regulating
and Con A-stimulated levels
the
has to be determined, of
relative
shown that
stimulation
of the
stimulation loss
It
is preserved
resting rase
the
means
the
We have
poly(A)(+)hnRNA
an important
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cells
number
of RNA
may be possible in
the
processing
set in
in
to use of RNA
resting
cells.
ACKNOWLEDGEMENT This work has been supported by tbe Deutsche Forschungsgemeinschaft. Our thanks are due to Dr. Rolf Knippers for his steady interest and stimulating criticism. This work is part of H.L.'s Diplomarbeit in biology at the University of Konstanz. REFERENCES ( 1) Perry, R. (1976) Annu.Rev.Biochem. 45, 605-609 ( .2) Schafer, K.P. (1976) Biochem.Biophys.Res.Commun. 219-226
68,
( 3) Robbins, J.H. (1964) Science 146, 1648-1654 ( 4) Hirschhorn, K. and Hirschhorn, R. (1974) Mechanisms of Cell-mediated immunity (McCluskey, R.T. and Cohen, S., eds.) pp. 115-134, John Wiley & Sons. New York
956
BIOCHEMICAL
Vol. 79, No. 3, 1977
( 5) Hauser,
H:,
Knippers,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
R. and Schafer,
K.P.
(1977)
Exp.Gell
( 6) HauszzS.H1n g?;iers R Schafer, K.P., Sons, W. and Un.&l~: H.-J. (1$76j'Exp.Cell Res. 102, 79-84 J.L. (1975) Proc.Natl.Acad.Sci. ( 7) Benz, W.C. and Strominger, USA 72, 24q3-2417 ( 8) Kirby, K.S. (1968) Methods in Enzymology (Grossmann, II. and Moldave, K., eds.) Vol. 12, part B, p. 87-99, Academic Press, New York ( 9) SaldE;zzEgi;ff, M., Jelinek, W., Darnell, J.E., : . Morgan, M. and Shatkln, A. (1976) Cell
7, i!?27-i!?37
W.F.,jr., Murphy, E.C.,jr. and Huang, R.C.G. Biochemistry 12, 3440-3446 (11) Sarma, M.H., Feman and Baglioni, C.(,l9'76) Biochim. Biophys.Acta 418, 29-38 (12) Marzluff, W.F. and Huang, R.C.C. (1975) Proc.Natl.Acad. Sci.USA 72, 1082-1086 (13) Derman, E., Goldberg, S. and Darnell, J. E. (1976) Cell (IO)
Marzluff,
(1973)
46'j-472 (14)
Jaehning, Cell
J.A., Stewart, 4, 51-57
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957
and Roeder,
R.G.
(1975)
9,
