J. Cell Sci. 6, 53-72 (1970)
Printed in Great Britain
53
INTRANUCLEAR ACCUMULATION OF RNA
RESEMBLING THE SMALLER RIBOSOMAL
RNA COMPONENT
M. E. BRAMWELL*
Sir William Dunn School of Pathology, University of Oxford, England
SUMMARY
A study was made of the nuclear RNA in HeLa cells with particular reference to the rapidly
labelled fractions. It was found that if cells were incubated at a high density, that is, under
'step-down' conditions, there was a rapid accumulation of RNA in the nucleus. The fraction
of the nuclear RNA which includes rapidly labelled RNA and which binds tightly to columns
of methylated albumin on kieselguhr increased in amount and reached levels which permitted
enough of the material to be isolated for direct measurement of its base composition. This
was found to be very similar to that of 16s ribosomal RNA.
When cells growing logarithmically were treated with low concentrations of actinomycin D
and then incubated in the presence of ["HJuridine it was found that an RNA fraction which
bound tightly to methylated albumin on kieselguhr again accumulated in the nucleus. This
fraction resembled that which accumulated under 'step-down' conditions. It contained over
85 % of the total radioactivity in the nuclear RNA and again had a base composition very
similar to 16s ribosomal RNA. Since nucleolar RNA synthesis was inhibited by the concentrations of actinomycin D used, it appeared that an RNA closely resembling 16s ribosomal
RNA was synthesized outside the nucleolus.
Sedimentation patterns on sucrose density gradients and thermal denaturation profiles lent
support to the view that the RNA which binds tightly to columns of methylated albumin on
kieselguhr probably represents 'nascent' 16s ribosomal RNA.
INTRODUCTION
Certain rapidly labelled fractions of RNA (rl-RNA) isolated from animal cells have
been found to bind very strongly to columns of methylated albumin on kieselguhr
(MAK) (Ellem & Sheridan, 1964; Ellem, 1966; Bramwell & Harris, 1967; Lingrel,
1967; Lichtenstein, Piker & Shapot, 1967; Roberts & D'Ari, 1968). These fractions
can be removed only by drastic means. Other workers have found labelled fractions
which are eluted after the main ribosomal RNA peaks (r-RNA), but still within the
normal salt gradient (Yoshikawa, Fukada & Kawade, 1964; Kimura, Tornado &
Sibatini, 1965; Kubinski & Koch, 1966). In plants, also, short periods of labelling
result in the bulk of the radioactivity eluting after the main ribosomal RNA peaks
(Chroboczek & Cherry, 1965; Ingle, Key & Holm, 1965; Jachymczyk & Cherry,
1968).
These rapidly labelled fractions are thought to contain precursors of r-RNA and
•Member of staff, British Empire Cancer Campaign Cell Biology Unit.
54
M. E. Bramwell
messenger RNA (m-RNA). Base compositions determined by [32P]-labelling have
shown that the fractions become progressively more 'DNA-like' the more difficult
they are to remove from the column (Yoshikawa et al. 1964; Ellem & Sheridan, 1964;
Ingle et al. 1965; Ellem, 1966; Kubinski & Koch, 1966; Lichtenstein et al. 1967;
Roberts & D'Ari, 1968). In addition, the RNA isolated from plants and fractionated
in this manner has been shown to have a high template activity in cell-free amino acid
incorporating systems, compared with the bulk of the RNA (Jachymczyk & Cherry,
1968).
RNA possessing the properties of rapid labelling, 'DNA-like' base composition
and high template activity (suggested criteria for identifying m-RNA) has been isolated using other methods, which usually involve hot phenol extractions over a
temperature range of 45-95 °C (Georgiev & Mantieva, 1962; Brawerman, 1963;
Brawerman, Gold & Eisenstadt, 1963; Scherrer, Latham & Darnell, 1963; Krsmanovic, Kanazir & Errera, 1965; Kimura et al. 1965; Dukes, Sekeris & Schmid, 1966;
Hadjiolov, 1966; Soeiro, Birnboim & Darnell, 1966; Arion, Mantieva & Georgiev,
1967; Mizuno, Collin & Hof, 1967; Morrison &McCluer, 1967). The general experience
seems to have been that those RNA fractions in which the three properties mentioned
above are most pronounced are also the most difficult fractions to extract from
animal cells. It is, therefore, very likely that such RNA fractions can be equated with
those which bind strongly to MAK columns.
Under 'step-down' conditions, for example, amino-acid starvation, in Escherichia
coli, there is a preferential increase in the amount of m-RNA, as judged by increased
template activity and increased ability to hybridize with DNA (Hayashi & Spiegelman, 1961; Mandel & Borek, 1962; Willson & Gros, 1964; Bellamy, 1966; Friesen,
1968; Sarkar & Moldave, 1968; Venetianer, Berberich & Goldberger, 1968). Similarly, animal cells in a stationary phase of growth have been shown to accumulate a
'DNA-like' RNA fraction (Kubinski & Koch, 1966). Treatment of both bacteria and
animal cells with low levels of actinomycin D leads to an accumulation of an RNA
fraction with the properties ascribed to m-RNA and a cessation of r-RNA synthesis
(Georgiev, Samarina, Lerman & Smirnov, 1963; Samarina, 1964; Harel et al. 1964;
Perry, Srinivasan & Kelley, 1964; Trakatellis, Axelrod & Montjar, 1964; Kubinski &
Koch, 1966; Samarina, Lukanidin, Molnar & Georgiev, 1968). The fact that an RNA
fraction with the properties ascribed to m-RNA can be preferentially synthesized
under certain conditions, coupled with the fact that the bulk of the rapidly-labelled
RNA in HeLa cell nuclei binds to MAK columns very strongly, suggested a method
by which the rl-RNA could be isolated and its base composition measured directly
without recourse to the 'p 2 P]-pulse' procedure, which has been shown by several
workers to be of doubtful value (Spencer, 1962; Harris et al. 1963; Roberts, 1965;
Marbaix, Burny, Huez & Chantrenne, 1966; Hadjiolov, Venkov, Dolapchiev &
Genchev, 1967).
Intranuclear accumulation of RNA
55
MATERIALS AND METHODS
Cultivation of cells
HeLa cells were grown in suspension culture as described by Harris & Watts (1962).
Step-dovm conditions
The cells were spun out of the suspension medium at a low speed and resuspended in a
smaller volume of a modified Eagle's medium (Eagle et al. 1956). The effect of increasing the
cell density by a factor of 5 to 10 produced the necessary 'step-down' conditions. Cells
were then labelled and harvested as required.
Isolation of cell nuclei
The nuclei were isolated as described previously (Bramwell & Harris, 1967), using the
'Tween' method of Fisher & Harris (1962).
Extraction of nuclear RNA
HeLa cell nuclei obtained from 1 1. of suspension culture medium were washed twice with
buffered saline (0-15 M NaCl + o-oi M tris-HCl, pH 7-4) and suspended in distilled water
(10 ml). One ml of 20 % (w/v) sodium dodecyl sulphate was added and the suspension stirred.
The nuclei lysed rapidly to form a viscous solution. An equal volume of aqueous phenol was
added and the mixture rotated at 5 °C for 30 min. After centrifugation, the supernatant was
precipitated with 2 volumes of cold ethanol containing a few drops of a 2 M NaCl solution.
The pellet was washed twice with ethanol and drained. Five ml of io" 1 M MgCL solution were
added, together with 125/tg deoxyribonuclease. The pellet dissolved rapidly leaving an
opalescent solution. This was made 2 % with respect to sodium dodecyl sulphate and extracted
twice with aqueous phenol. The final supernatant was mixed with 2 volumes of ethanol, spun
down and the pellet extracted with 2 M NaCl at o CC to remove glycogen and DNA fragments.
Extraction of ribosomal RNA
This was done as described previously, by extraction of the 'Tween' supernatant with
aqueous phenol and 2 % (w/v) sodium dodecyl sulphate (Bramwell & Harris, 1967). For
comparative purposes, 16s RNA was also extracted from the small subunit of the ribosome.
The Tween supernatant was made 0-4 M with respect to NaCl and samples put on to linear
sucrose gradients (30-5% w/v) containing 0-25 M NaCl + o-oi M tris-HCl, pH 7-4. After
centrifugation for 15 h at 22500 rev/min in the SW 25 rotor of a Spinco L-50 centrifuge, 15drop fractions were collected on a fraction collector. The 30 S peak fractions were pooled, made
2 % (w/v) with respect to sodium dodecyl sulphate and shaken with an equal quantity of phenol
saturated with water. After centrifugation, the supernatant was mixed with cold ethanol to
precipitate the RNA. The pellet was washed twice with ethanol, drained and either used
immediately or stored at — 18 °C.
Preparation of methylated albumin on kieselguhr
The MAK was prepared as described by Mandell & Hershey (i960), except that Hyflo
Super-Cel (Johns-Manville, New York, N.Y., U.S.A.) was used in place of crude kieselguhr.
Extraction of MAK-bound RNA
A single-layer MAK column was prepared and washed with 0-15 M NaCl + o-oi M tris-HCl,
pH 74. A solution of nuclear RNA (n-RNA) in the same buffer was passed through the
column, which was further washed with the 0-15 M NaCl buffer. This procedure resulted in
complete adsorption of the RNA to the MAK. A large proportion of the n-RNA could be
56
M.E. Bramwell
eluted by washing the column exhaustively with 2 M NaCl + o-oi M tris-HCl, pH 7-4. This
fraction is designated 2 M NaCl-eluted n-RNA. The fraction of the RNA which remains
tightly bound to the MAK after successive washings with 2 M NaCl is designated MAK-bound
RNA. Conditions of the elution are rather critical: complete removal of an RNA sample,
known to contain a minimum of MAK-bound RNA, could only be achieved at a temperature
of 35-40 CC with a step-wise increase of the salt content from 015 M to 2 M NaCl.
For recovery of the MAK-bound RNA two methods were employed, depending on the purpose
of the experiment. For base-composition determinations, the MAK containing the bound RNA
was washed once with 015 M NaCl to reduce the salt concentration. The MAK pellet was
then resuspended in 0 3 M KOH of sufficient volume to yield a smooth suspension. At this
point it should be noted that the Hyflo Super-Cel has an actual volume which is only about
1/10 of its apparent volume, so that 2 M KOH was added to allow for the dilution effect of the
liquid contained in the MAK pellet. After digestion for 18 h at 37 °C the suspension was centrifuged and the supernatant removed. The pellet was washed once with a small volume of
water, which was pooled with the supernatant. Perchloric acid was added to a concentration of
10% and the solution left for 30 min in an ice-bath. Insoluble protein was removed by centrifugation and the solution brought to pH 6-7 with 2 M KOH. Before chromatography, the
volume was reduced by evaporation.
For recovery of intact MAK-bound RNA, the MAK pellet was shaken with a mixture of
2 % (w/v) sodium dodecyl sulphate solution and phenol saturated with water for 30 min.
After centrifugation, the supernatant fraction was diluted with 2 volumes of cold ethanol and
left overnight at —18 °C. The precipitate which formed was collected by centrifugation and
dissolved in a small volume of 2 % sodium dodecyl sulphate solution. Re-extraction with
aqueous phenol was necessary since there was still some protein present. The clear supernatant
was again treated with 2 volumes of ethanol and left overnight at — 18 °C. The precipitate
was washed twice with ethanol, drained and stored at — 18 C C.
Measurement of MAK-bound RNA and 2 M NaCl-eluted RNA
The precipitate of n-RNA was dissolved in 2 ml of 0-15 M NaCl + o-oi M tris-HCl, pH 7-4.
Samples were taken for measurement of total radioactivity and optical density. To the remaining solution 1 ml of a suspension of MAK was added. This was repeated until the n-RNA had
been completely adsorbed to the MAK as judged by the loss of radioactivity and optical density
from the supernatant liquid. Any trace of unadsorbed material was later subtracted from the
measurement of the total amount in the sample. The supernatant liquid was decanted off after
centrifugation and 5 ml of 2 M NaCl solution added. The mixture was stirred for 5 min in a
water-bath at 37 °C and again centrifuged. The supernatant now contained the n-RNA
extractable with 2 M NaCl (2 M NaCl-eluted RNA) and the measurement of optical density
and volume allow the amount of 2 M NaCl-eluted RNA to be calculated. (As noted earlier, the
volume of the MAK must be taken into account, since the actual volume it occupies is only
10% of its apparent volume.) The amount of RNA still bound to the MAK (MAK-bound
RNA) is, therefore, the difference between the total and the 2 M NaCl-eluted RNA.
Base analysis
This was performed according to the method of Katz & Comb (1963). Success in this method
is dependent on complete removal of all protein contamination, which otherwise elutes with
uridylic acid. Particular attention was, therefore, paid to the purity of each sample analysed,
and only those with 220—260 nm ratios less than 1 were used.
Sucrose gradients
Linear sucrose gradients from 20 to 2 % (w/v) were prepared in appropriate buffers. Samples
of 0-2-0-3 ml of solution were applied to the top of the gradients. Centrifugation was carried
out in the SW 39 or 50 rotors of a Spinco Model L-50 ultracentrifuge at 5 °C for an appropriate
time. Fractions were collected on an LKB UltroRac fraction collector and were diluted for
measurement of extinction at 260 nm.
Intranuclear accumulation of RNA
57
Measurement of radioactivity
The Nuclear Chicago liquid-scintillation system 725 (Nuclear Chicago Corp., Des Plaines,
111., U.S.A.) was used. Samples 0-1-0-5 m l i n volume were added to counting vials containing
15 ml of scintillation liquid (600 ml of ethanol + 1 1. of toluene containing o-i g of 1,4-bis(5-phenyloxazol-2-yl) benzene and o-6 g of 2,5-diphenyloxazole).
Spectrophotometry
Measurements of the extinction of solutions were made in a Zeiss PMQII spectrophotometer. Heating profiles were made with a water-heated cell holder, the temperature being
measured by means of a thermistor probe fitted into the blank cell (Grant Instrument Co.,
Cambridge, England).
Reagents
EDTA (reagent grade), phenol (analytical reagent grade) and sucrose (analytical reagent
grade) were obtained from British Drug Houses Ltd., Poole, Dorset. The phenol was redistilled
before use. Sodium dodecyl sulphate was obtained from Koch-Light Laboratories Ltd.,
Colnbrook, Bucks, and TRIZMA buffer from Sigma Chemical Co. Ltd., London, S.W.6.
Bovine plasma albumin was obtained from the Armour Pharmaceutical Co. Ltd., Eastbourne,
Sussex. Deoxyribonuclease I was an electrophoretically purified enzyme from the Worthington
Biochemical Co., Freehold, N.J., U.S.A. This was further purified by extraction with 200 fig
of bentonite (B.D.H.) which was added to a solution of 5 mg of the enzyme in 5 ml of distilled
water. The purified enzyme contained no ribonuclease activity.
[5- 3 H]Cytidine, specific activity 250 Ci/mM, [5- 3 H]uridine, specific activity 265 Ci/mM
and [S-'^CJadenine, specific activity 277 mCi/mM were obtained from the Radiochemical
Centre, Amersham, Bucks.
Autoradiography
Autoradiographs were made of cells labelled in suspension culture. A small sample of cells
was taken from the culture vessel, washed twice with saline and flattened on to glass slides in a
cytocentrifuge (Shandon-Elliot, S.C.A.-0020, Shandon Scientific Co. Ltd., London, N.W. 10).
After fixation in methanol for 10 min the slides were washed with distilled water and extracted
with 5 % trichloroacetic acid at 5 °C for 45 min. Stripping film (Kodak AR 10) was applied
in the usual way and the slides were exposed for several days.
OBSERVATIONS
Effect of' step-down' on the amount of nuclear RNA
HeLa cells growing in suspension culture (1-5 1. at a cell density of 1 x io6/ml) were
centrifuged at low speed and resuspended in 200 or 500 ml of the modified Eagle's
medium containing 2/tCi/ml of [5-3H]uridine. Samples (40 or 100 ml) were taken at
the times indicated. n-RNA was extracted and the amount measured from the extinction at 260 nm. Figure 1 shows the effect of transferring the cells to the poorer
environment ('step-down'). The amount of n-RNA has increased 3-4 fold in 80 min
at the higher cell density (7-5 x io6 cells/ml) and has more than doubled at the lower
density (3 xio 6 cells/ml). Incubation at the higher density thus results in a greater
increase in n-RNA content.
This situation was analysed further by making use of the property of MAK to
bind certain fractions of RNA very tightly. Figure 2 shows the result of such an
analysis. The n-RNA has been separated into MAK-bound RNA and 2 M NaCl-
58
M. E.
Bramwell
eluted RNA and the amount of RNA present in each fraction determined. Both
fractions increase in amount with time and then decrease. The amount of MAK-bound
RNA increases most: at zero time, the MAK-bound RNA accounts for less than i %
of the total n-RNA, but as incubation proceeds this proportion increases almost
linearly to reach over 50% (Fig. 3). This must surely reflect a profound change in
1-1
20
r
09
0-7
\
OS
0-3
0-1 -
45
90
135
20
180
40
60
80
100
120
Time after transfer to
'step-down'conditions (min)
Time after transfer to
'step-down' conditions (min)
Fig. 2
Fig. 1
Fig. 1. Accumulation of nuclear RNA under 'step-down' conditions. The cells were
grown in suspension medium to a density of 1 x io 6 cells/ml, centrifuged at low speed
and resuspended in modified Eagle's medium at a density of 3 x io 6 cells/ml ( • ) , or
7-5 x io 9 cells/ml (O)Fig. 2. Accumulation of nuclear RNA fractions under 'step-down' conditions. The
cells were grown in suspension medium to a density of 1 x 10' cells/ml, centrifuged
at low speed and resuspended at a density of 7-5 x io 8 cells/ml in modified Eagle's
medium. O, 2 M NaCl-eluted RNA; • , MAK-bound RNA.
50 1—
40
30
20
10
0
20
40
60
80
100
120
Time after transfer to 'step-down' conditions (min)
Fig. 3. Accumulation of MAK-bound RNA under 'step-down' conditions. The cells
were grown in suspension medium to a density of 1 x 1 0 ' cells/ml, centrifuged at
low speed and resuspended at a density of 7-5 x 10' cells/ml in modified Eagle's
medium.
Intranuclear accumulation of RNA
59
the metabolism of the cell and suggests that a block has occurred which results in the
accumulation of MAK-bound RNA.
It was previously found that, after a short period of radioactive labelling under
conditions which were essentially 'step-down', the bulk of the labelled RNA in the
HeLa cell nucleus was bound to MAK very tightly (Ellem & Sheridan, 1964; Ellem,
1966; Bramwell & Harris, 1967). Figure 4 shows the amount of radioactivity incorporated into the MAK-bound and 2 M NaCl-eluted RNA fractions in the present experiment. There is again an increase in both fractions followed by a decrease, which is
more marked in the case of the MAK-bound RNA. Figure 5 shows the increase in
specific activity of the RNA fractions over the course of the experiment. The MAKbound RNA has the higher specific activity, but, in both fractions, the specific activity
continues to rise even after the total amount of n-RNA has begun to decrease. This
suggests that there is an increased rate of degradation of n-RNA, rather than a reduction in the rate of its synthesis after 80 min.
:? 30
18
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o
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i_ oc
x! ,6
^ 10
7 S
o
£
20
40
60
80
100
Time after transfer to
'step-down' conditions (min)
120
0
20
60
90
120
Time after transfer to 'step-down'
conditions (min)
Fig- 4
Fig. 5
Fig. 4. Incorporation of radioactivity into nuclear RNA fractions under 'step-down'
conditions. The cells were grown in suspension medium to a density of 1 x 10' cells/
ml, centrifuged at low speed and resuspended at a density of 75 x 10" cells/ml in
modified Eagle's medium containing 2 /iCi/ml of [5-3H]uridine. # , MAK-bound
RNA; O, 2 M NaCl-eluted RNA.
Fig. 5. Specific activity of nuclear RNA fractions accumulated under 'step-down'
conditions. The cells were grown in suspension medium to a density of 1 x 10'
cells/ml, centrifuged at low speed and resuspended at a density of 75 x 10s cells/ml
in modified Eagle's medium containing 2/iCi/ml of [5-3H]uridine. 9 , MAK-bound
RNA; O, 2 M NaCl-eluted RNA.
MAK-bound RNA in cells growing logarithmically
The zero time measurements of the amount of MAK-bound RNA in cells in 'stepdown ' indicated that this fraction contained less than 1 % of the total optical density,
but the amount of radioactivity incorporated was not enough to permit meaningful
measurement. Clarification of this point was achieved by the following experiment.
M. E. Bramwell
6o
To i 1. of HeLa cells growing logarithmically in Earle's medium 20 /iCi of [8- u C]adenine were added. Samples (200 ml) were taken at the times indicated in Fig. 6.
The n-RNA was extracted as before and separated into the 2 M NaCl-eluted and
MAK-bound fractions. The results show that over the period of the experiment
about 80% of the radioactivity was eluted with 2 M NaCl whereas only about 20%
was bound to the MAK. This occurred even at 30 min which is, in terms of the
generation time of the cell, almost equivalent to a pulse label. By comparison, more
12 r
10
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u
—
1I 6
o
:s 4
X
I
O
0
30
60
90
120
Time after addition of [S-'^CJademne (mm)
Fig. 6. Incorporation of radioactivity into the nuclear RNA fractions obtained from
logarithmically growing cells in Earle's medium containing o-2 /iCi/ml of [8-14C]adenine. • , MAK-bound RNA; O, 2 M NaCl-eluted RNA.
than 60% of the radioactivity was bound to MAK in 30 min in the 'step-down'
situation. Under conditions previously employed (Bramwell & Harris, 1967), in
which cells were labelled at a much higher density than in the present experiments
(2 x io7 as compared with 7-5 x io6 cells/ml), over 90% of the radioactivity was
MAK-bound.
These results strongly suggest that the MAK-bound RNA represents an initial
transitory stage in RNA maturation, and that any interference with the cell results in
a slowing down of the maturation process and an accumulation of the primary products
of the RNA synthetic system. Since RNA cannot be extracted from cells without their
being centrifuged and washed, it is possible that the 20% of the radioactive n-RNA
which is found to be MAK-bound in growing cells may accumulate during the ' stepdown ' produced by the procedures necessary for extraction of the RNA. The amount
of MAK-bound RNA in the growing cell may be much less. This view is supported
by the fact that the proportion of the radioactive RNA which is MAK-bound remains
constant over the whole period of the experiment. On kinetic grounds, the proportion
of radioactivity in the precursor material should decrease relative to the product with
Intranuclear accumulation of RNA
61
time. On the other hand, it might be expected that the physical conformation of
m-RNA molecules would not be influenced to any great extent by environment, as it
is supposed that these molecules do not undergo the same secondary modifications
and shape changes as do the r-RNA precursors. If the MAK-bound RNA in cells in
logarithmic growth is m-RNA, it represents less then i % of the total n-RNA and
less than o-2% of the total cell RNA.
Effect of actinomycin D on MAK-bound RNA
To i 1. of HeLa cells growing logarithmically in Earle's medium, actinomycin D
was added to a concentration of 0-05 /ig/ml and, after 30 min, 20 /iCi of [8-14C]adenine.
This interval allows the actinomycin D to penetrate the cell (Perry, 1963). Samples
were taken at 30-min intervals thereafter, n-RNA was extracted and fractionated into
7
r
25
20
15
10
5
30
60
90
120
Time after addition of
actinomycin D (min)
Fig. 7
0
30
60
90
120
150
Time after addition of actinomycin D (min)
Fig. 8
Fig. 7. Incorporation of radioactivity into the nuclear RNA fractions obtained from
logarithmically growing cells in Earle's medium after the addition of actinomycin D
(0-05 /ig/ml). [8-14C]Adenine was added to a concentration of O'2 /iCi/ml 30 min after
the addition of the actinomycin D. 0 , MAK-bound RNA; O, 2 M-eluted RNA.
Fig. 8. Accumulation of MAK-bound RNA in the presence of 0-05 /ig/ml of actinomycin D.
2 M NaCl-eluted RNA and MAK-bound RNA as before. Figure 7 shows the result
of such an experiment in terms of the amount of radioactivity incorporated into each
fraction. It is apparent that there is a much greater proportion of radioactivity incorporated into the MAK-bound RNA than into the 2 M NaCl-eluted RNA, and the
situation is very similar to that in the 'step-down' experiment shown in Fig. 4, except
that there is no decrease in incorporation after the initial increase. The result is, thus,
the reverse of that obtained in the absence of actinomycin D (Fig. 6).
In terms of optical density there is a very slight overall increase in RNA content
in the nucleus, but nothing comparable to that obtained in 'step-down' conditions.
The greatest increase is in the MAK-bound RNA, as before, which rises from less
M. E. Bramwell
62
than i % to about 20% of the total n-RNA (Fig. 8). At this low level of actinomycin
D, there is some incorporation of radioactivity into cytoplasmic ribosomal RNA
(Fig. 9). The sedimentation pattern of the cytoplasmic RNA is rather polydisperse,
but the most prominent peak is in the 16s component with a shoulder at 28s. Compared with the control (Fig. 10) the pattern of labelling is clearly aberrant and the
level much reduced (9% of the total radioactivity is incorporated into high-molecularweight RNA, compared with 60% in the control). This incorporation into ribosomal
RNA is abolished at a concentration of actinomycin D of o-i /6g/ml (Fig. 10).
0-5
200
04
160 S-f-
20 3
28 s
5 O
120 n S
1-4
O 1-
10
-LI
02
80 1 I
0-1
40
16s
30 >
20 o
0-6
10 x
0-2
2
6
10 14 18
Fraction no.
Fig. 9
22
n
O
6
10
14
18
22
Fraction no.
Fig. io
Fig. 9. Sucrose density-gradient sedimentation pattern of the radioactivity incorporated into cytoplasmic ribosomal RNA in 90 min in the presence of actinomycin D at a
concentration of 0-05 /ig/ml. The gradient contained 50 mM NaCl and 10 mM EDTA
at pH 7 4 . Centrifugation was for 5 h at 37500 rev/min at 5 °C. O, E.e0; # , radioactivity.
Fig. 10. Sucrose density-gradient sedimentation pattern of the radioactivity incorporated into cytoplasmic ribosomal RNA in 90 min in the presence of actinomycin D
at a concentration of 01/tg/ml. The gradient contained 50 mM NaCl and 10 mM
EDTA at pH 7-4. Centrifugation was for 5 h at 37500 rev/min at 15 °C. O, E«60;
# , radioactivity in control sample;
, radioactivity in the presence of actinomycin
D.
The residual incorporation of radioactivity into the RNA in the nucleus in the
presence of low levels of actinomycin D has been shown to be largely ' extranucleolar'
and to have a 'polydisperse' pattern of sedimentation in a sucrose gradient (Perry,
1963). On these grounds it has been suggested that continued synthesis of m-RNA
takes place in the presence of actinomycin D (Perry et al. 1964). In the present
experiments the.RNA which accumulated in the presence of actinomycin D was also
found to have a 'polydisperse' sedimentation in conventional sucrose gradients
(Fig. 11).
The accumulation of this RNA in the nucleus again permitted a direct measurement of its base composition. The measurements shown below indicate that a ' DNAlike' RNA does not accumulate in the presence of actinomycin D.
Intranuclear accumulation of RNA
63
The base composition of MAK-bound RNA
In 'step-down' conditions and in the presence of actinomycin D the amount of
MAK-bound RNA increases sufficiently to permit direct measurement of its base
composition. The results of the determinations are set out in Table 1. It can be seen
-, 3 5
8
12
16
Fraction no.
24
Fig. 11. Sucrose density-gradient sedimentation pattern of the radioactivity incorporated into total cell RNA in 4 h in the presence of actinomycin D at a concentration of
o-i /tg/ml. The gradient contained 50 min NaCl and 10 mM EDTA at pH 7-4. Centrifugation was for 4 h at 37500 rev/min at 10 °C. O, £250; •> radioactivity.
Table 1. The base composition of MAK-bound RNA
MAK-bound RNA accumulated
under 'step-down' conditions (min)
1
AMP
UMP
GMP
CMP
No. of
80
no
US
18-6
2I-I
180
247
309
24'0
25-8
311
298
25-8
238
26-4
1
2
2
MAK-bound
RNA accumu- 16s RNA
lated in the isolated from
presence of
the small
o-i /tg/ml of
subunit of
actinomycin D the ribosome
199
236
209
29-8
26-7
3
290
24-1
26-0
3
determinations
that the MAK-bound RNA which accumulates under the two conditions has the same
base composition, within the limits of experimental error. Comparison with the
cytoplasmic components isolated from the ribosomal subunits reveal a striking similarity between the MAK-bound RNA and the 16s r-RNA. Two possibilities exist to
64
M. E. Bramwell
account for this: (i) MAK-bound RNA is a mixture of many species of high molecular
weight RNA and its overall base composition is coincidentally similar to one of these
species; (2) MAK-bound RNA consists of immature 16s RNA.
In considering the first alternative, if it is assumed that both 28s and 16s RNA are
synthesized at similar rates, the amount of m-RNA or 'DNA-like' RNA needed to
produce the required shift to the observed 16s r-RNA-like base composition would be
about equal to the combined amount of 28 + 16s RNA synthesized. This is conceivable,
but one would also have to assume that, in the presence of actinomycin D, all three
species were synthesized in the ' extranucleolar' region of the nucleus, since nucleolar
incorporation of radioactivity is greatly reduced in this situation (Perry, 1963; Table 2).
28 s
30
25
>- <=
-r*
10
g .9
02
28s
0 1<J-
16s
0 5 x 2
o £
0 25 *" 2.
0 05
30-1
10
14 18
Fraction no.
2
6
10
14
18
Fraction no.
22
25
22
20
£• =
1 0
x 2
0-6
0-2
6
10 14
18
Fraction no.
Fig. 12
22
10
14 18
Fraction no
Fig. 13
22
Fig. 12. Sucrose density-gradient sedimentation pattern of (A) MAK-bound RNA
extracted from HeLa cells after incubation in 'step-down' conditions for 6omin;
(B) cytoplasmic ribosomal RNA as a control. The gradients contained 5 mM NaCl
and 10 mM EDTA at pH 7 4 . Centrifugation was for 3 h at 48000 rev/min at 5 °C.
Fig. 13. Sucrose density-gradient sedimentation pattern of the radioactivity incorporated into MAK-bound RNA extracted from HeLa cells after incubation in 'stepdown' conditions for 35 min. The gradients contained: (A) 5 mM NaCl and 10 mM
EDTA at pH 7-4; (B) 51T1M EDTA-tris buffer pH 7-0. Centrifugation was for 5 h
at 37500 rev/min at 5 °C. O, £290; •> radioactivity.
The second possibility, that MAK-bound RNA is, in fact, immature 16s RNA,
seems the more likely for the following reasons. (1) The base composition of MAKbound RNA is very similar to that of 16s ribosomal RNA over a range of times in
the 'step-down' environment, and in the presence of actinomycin D. (2) MAK-bound
Intranuclear accumulation of RNA
65
RNA and rl-RNA from HeLa cells can be sedimented in sucrose gradients as a
single peak with a sedimentation coefficient of about 16s (Fig. 13B, Bramwell &
Harris, 1967). (3) End-group analysis of rl-RNA from L cells indicates that its mean
chain length is very similar to that of 16s ribosomal RNA (Tamaoki & Lane, 1967).
(4) Comparative chain length analysis of rl-RNA and 16s ribosomal RNA from HeLa
cells, by means of a specific exonuclease, has also shown them to be equal (Riley,
1969). That MAK-bound RNA can be indentified with rl-RNA is indicated by the
similarity in sedimentation behaviour (Figs. 12A, 13A) of the two materials, and by
the fact that rl-RNA under severe 'step-down' conditions binds very tightly to MAK.
The physical state of MAK-bound RNA
The physical state of MAK-bound RNA may be studied in several ways. From a
consideration of the properties of polyuridylic acid and denatured DNA, which have
little secondary structure and which bind tightly to MAK columns (Hershey, Goldberg, Burgi & Ingraham, 1963; Asano, 1965), it would be expected that MAK-bound
RNA would have a high molecular weight and a low degree of secondary structure.
The first point is established by its insolubility in 2 M NaCl and its high sedimentation
rate in sucrose gradients. Fig. 12 A shows the sedimentation pattern of MAK-bound
RNA extracted from cells incubated under 'step-down' conditions for 60 min. The
predominant feature is the peak of RNA with a sedimentation coefficient greater than
the 28s RNA in the control gradient (Fig. 12B). This is followed by more polydisperse
material. Mature 28s RNA, which normally is found in the nuclear RNA as a whole,
does not bind to the MAK. The pattern of optical density in Fig. 12A resembles
that of the radioactivity incorporated into MAK-bound RNA after pulse labelling of
a sample of HeLa cells under 'step-down' conditions (Fig. 13A). In this case, the
period in 'step-down' was short and there is therefore very little RNA present in
terms of optical density. When the MAK-bound RNA was dissolved in 5 mM EDTA
and centrifuged on a sucrose gradient containing 5 mM EDTA only, the pattern seen
in Fig. 13 B was obtained. A symmetrical peak of radioactivity is now seen which
sediments at slightly less than 16s. The initial sedimentation pattern is not, therefore,
due to the presence of larger amounts of some other kind of RNA which sediments
rapidly and with which the radioactivity might co-sediment. The conversion of
rapidly sedimenting 'polydisperse' RNA to a single component sedimenting in the
16s position has been previously described for rl-RNA (Bramwell & Harris, 1967).
As pointed out by these authors, this effect is reversible and cannot therefore be due
to the action of contaminant ribonucleases as suggested by Fujisawa & Muramatsu
(1968).
Another way of showing differences in secondary structure between groups of
polynucleotides is to compare the heating profiles obtained by measuring the variation of optical density produced by changes in temperature. Figure 14 shows such a
profile obtained from the sample of MAK-bound RNA shown in Fig. 12A. The upper
heating curve indicates a lower degree of hypochromic change than is normal for
RNA (19-0% compared with 27-0%). However, on cooling, a lower absorbance
than the initial value was obtained, suggesting an increase in hypochromicity. As
r
C E I. 6
M. E. Bramwell
66
shown in Fig. 14, a second curve was obtained on reheating, which coincided with
the first only at the highest temperatures. One explanation of this might be that the
effect was due to contamination by a protein which became denatured on heating and
precipitated out, thus lowering the overall absorbance. However, there was no visible
evidence of protein precipitation, and the maximum difference between the two curves
occurs below 50 °C, which is not normally a temperature at which proteins denature.
Alternatively, the effect may be due to an annealing process which occurs over the range
20-50 °C. Thus, prior to extraction from the cells, the MAK-bound RNA may exist
in an extended form and, after extraction, intermolecular and intramolecular hydrogen
10 r-
0-9
07
20
40
60
80
100
Temperature (°C)
Fig. 14. Heating profiles of MAK-bound RNA in 0-13 M NaCl and 10 mM tris-HCl,
pH 70. O, 1st heating; • , value obtained after cooling; # , 2nd heating.
bonding may occur in a random fashion. This would prevent the formation of more
specific and stable regions which are known to exist after the extensive modification
of shape which takes place when an extended polynucleotide chain is converted into
a ribosomal subunit.
Comparison of the heating profiles of MAK-bound RNA and 16s r-RNA (Fig. 15)
shows that MAK-bound RNA has a lower Tm and a more gradual transition than
16s r-RNA. The central portion of the curve which shows a relatively sharp rise
in 16s r-RNA (45-65 °C) is absent in the MAK-bound RNA, suggesting that the
latter possesses fewer regions containing long stretches of hydrogen-bonded base
pairs. Methylation is thought to play a part in stabilizing regions of RNA, and it is
interesting to note that Tamaoki & Lane (1967) have shown that the degree of methy-
Intranuclear accumulation of RNA
67
lation of rl-RNA from L cells is only 25% of that in 16 or 28s r-RNA. But all three
species of RNA contain a common methylated sequence (Tamaoki & Lane, 1968). It
has also been found that 'extranucleolar' RNA from rat liver cells (Muramatsu &
Fujisawa, 1968) and from HeLa cells (Zimmerman & Holler, 1967) has a much lower
degree of methylation than nucleolar RNA.
10 r
u
0-9
H 08
U
07
15 20
30
40
50
60
Temperature (°C)
70
80
90
100
Fig. 15. Heating profiles of MAK-bound RNA and 16s ribosomal RNA compared.
O, 16s r-RNA in 0-15 M NaCl and 10 mM tris-HCl, pH 7-0; • , MAK-bound RNA
in 0-13 M NaCl and 10 mM tris-HCl, pH yo, 2nd heating.
Autoradiography
In order to determine the location of the RNA which accumulates in the nucleus
in 'step-down' and in the presence of actinomycin D, the pattern of labelling in the
HeLa cell nucleus was examined autoradiographically. Samples of cells in various
growth conditions were taken after labelling with [5-3H]cytidine for 15 min and
autoradiographs prepared. Table 2 shows the proportion of grains over the nucleolus
compared with those over the rest of the nucleus. In cells growing logarithmically it is
apparent that the bulk of the rl-RNA is found in the nucleolus. However, after incubation in 'step-down' conditions for 75 min, followed by a 15-min pulse label, the
bulk of the counts are in the 'extranucleolar' region. In cells which have not yet
achieved logarithmic growth, the results are intermediate between the two. Prolonged
incubation of HeLa cells in the presence of actinomycin D and [5-3H]cytidine results
exclusively in 'extranucleolar' labelling.
The proportions of grains found outside the nucleolus under these three different
situations closely parallels the proportions of radioactively labelled RNA which binds to
MAK tightly after similar treatments. There appears, therefore, to be a direct corre5-2
68
M. E. Bramwell
lation between ' extranucleolar' RNA and MAK-bound RNA, indicating that both in
'step-down' and in the presence of actinomycin D, MAK-bound RNA accumulates
outside the nucleolus.
Table 2. Distribution of labelled RNA within the nucleus, under different conditions
Mean no.
of grains
over the
' extranucleolar'
region
' extranucleolar'
region (%)
RNA (%)
7-5 ±0-64
3-6±o-6
35-6
19-5
3'5±°-4
257 ±1-76
88-o
90-0
0-25 ±o-io
21-3 ± 1-06
98-8
82-7
Mean no.
of grains
over nucleolus
' Log-growth'
15 min label
' Step-down'
15 min label
Actinomycin D
o-i /ig/ml for 3 h
MAKbound
DISCUSSION
The main conclusions to be drawn from this paper are: (1) there is a rapid accumulation of MAK-bound RNA in the nucleus of HeLa cells when they are transferred
to a 'step-down' situation and when they are treated with low concentrations of
actinomycin D; (2) the MAK-bound RNA has a base composition very similar to
16s ribosomal RNA; (3) this MAK-bound RNA is synthesized and accumulates
outside the nucleolus. There are two possible interpretations of these conclusions:
(a) that MAK-bound RNA is the precursor of 16s ribosomal RNA, and that it is
made outside the nucleolus; (b) that MAK-bound RNA represents a mixture of precursor RNA molecules and that the overall base composition is coincidentally similar
to 16s ribosomal RNA.
The evidence against the first view and in favour of the second is mainly derived
from measurements of the apparent base composition of rl-RNA bound to MAK
columns, made by means of the [32P]-pulse method (Yoshikawa et al. 1964; Ellem &
Sheridan, 1964; Ingle et al. 1965; Ellem, 1966; Kubinski & Koch, 1966; Lichtenstein et al. 1967; Roberts & D'Ari, 1968), and from similar measurements of the
rl-RNA isolated from whole cells (Brawerman, 1963; Hadjivassiliou & Brawerman,
1965; Houssais & Attardi, 1966; Watson & Ralph, 1967). The results obtained by the
use of this method indicate that rl-RNA has a base composition quite unlike the bulk
of the RNA in the cell, but somewhat similar to that of the DNA. However, the
[32P]-labelling procedure has been shown to be of doubtful value for short pulses
(Spencer, 1962; Harris et al. 1963; Roberts, 1965; Marbaix et al. 1966; Hadjiolov
et al. 1967). Even so, in certain cases, the base composition of the rl-RNA determined
in this way appears to be very similar to 16s r-RNA (Hadjiolov, Venkov & Dolapchiev, 1965; Venkov & Hadjiolov, 1967; Mizuno et al. 1967; Beiderbeck & Richter,
1968). It has also been observed that after longer periods of labelling with [32P] the
MAK-bound RNA in HeLa cells has an apparent base composition very similar to
16s r-RNA (Ellem, 1966).
Intranuclear accumulation of RNA
69
That the rl-RNA can be equated with a fraction of the MAK-bound RNA is clear
from the fact that, under 'step-down' conditions, the bulk of the rl-RNA binds tightly
to columns of MAK. But the whole of the MAK-bound RNA behaves very similarly
to rl-RNA on sucrose density-gradient centrifugation, showing a characteristically
polydisperse sedimentation pattern, which is resolved to a single peak at about 16s
on gradients containing 51T1M EDTA (Bramwell & Harris, 1967). This fact, together
with the evidence that rl-RNA from animal cells has a mean chain length equal to
16s r-RNA (Tamaoki & Lane, 1967; Riley, 1969) argue in favour of the view, suggested by its base composition, that MAK-bound RNA is a precursor to 16s r-RNA.
The fact that RNA with a base composition similar to 16s r-RNA may be synthesized outside the nucleolus, is clearly contrary to a current view that both 16 and 28 s
r-RNA are synthesized co-ordinately in the nucleolus (Ritossa & Spiegelman, 1965;
Birnstiel, Wallace, Sirlin & Fischberg, 1966; Brown, 1966). These experiments are,
however, based on the technique of RNA-DNA hybridization the validity of which
has been recently questioned in the case of animal cells (Melli & Bishop, 1969). These
authors have shown that only a very small proportion (5 %) of the total DNA in the
cell nucleus will readily form hybrids with RNA, even when the RNA used for
hybridization is known to be complementary in sequence to at least 50% of the DNA.
The small fraction of the DNA which does hybridize with RNA appears to contain
a high proportion of repeating sequences, as judged by the speed of its renaturation
after melting. It is this property which apparently confers on this DNA the ability
to form hybrids with all types of high molecular weight RNA including 'DNA-like'
RNA. Although ribosomal RNA cistrons may well be present in this small fraction
of DNA, the possibility that they occur elsewhere on the genome cannot now be
excluded. Indeed, it has been shown that in Chinese hamster cells RNA can be synthesized on a large number of different chromosomes at sites which are clearly
identifiable as micronucleoli (Phillips & Phillips, 1969). Another objection to coordinate synthesis of 16 and 28s RNA at the one site is the fact that 16s r-RNA always
labels before 28s r-RNA. Moreover, the differential effects of certain compounds on
the synthesis of the two ribosomal RNA components point to these being synthesized
separately (Ennis, 1966; Wagner & Roizman, 1968; Mayo, Andrean & De Kloet, 1968).
The fact that the initial precursor of 16s RNA and not 28s RNA binds to MAK
so strongly may reflect a fundamental difference in the rate at which the secondary
and tertiary structures are imposed on the two species of molecule. On the other hand,
the degree to which MAK binds RNA molecules depends on molecular weight,
secondary structure and base composition. Base composition is, of course, closely
related to the secondary structure. A higher G + C/A + U ratio would confer a
higher degree of secondary structure on the molecule and consequently ensure earlier
elution from MAK. Since the proportion of A + U is much higher in the 16s RNA
than in 28s RNA it will have a less stable secondary structure and will therefore be
eluted later, but one would expect this to be cancelled out to some extent by the
difference in molecular weight. In cells growing logarithmically there is very little
MAK-bound RNA, but in an unfavourable situation, it accumulates. The present
experiments suggest that this accumulation represents 16s RNA which has not
70
M. E. Bramwell
matured. If 16s RNA is synthesized outside the nucleolus it may, therefore, have to
pass into the nucleolus to be matured. The 16s RNA which accumulates outside the
nucleolus in 'step-down' and in the presence of actinomycin D may thus fail to be
matured. The 28 s RNA, on the other hand, appears to be synthesized inside the
nucleolus and might, therefore, mature more rapidly.
There is at present no decisive evidence against the view that the rapidly labelled,
'polydisperse', MAK-bound RNA is nascent 16s r-RNA, and it appears that this
RNA is synthesized in the 'extranucleolar' regions of the nucleus.
I thank Professor Henry Harris for his advice and criticism and Mrs Jane Tully for her
excellent technical assistance.
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{Received 30 April 1969)