Studies on Components of RNA in Cardiac
Muscle with Emphasis on the
Rapidly Labeled 4 to 18 S Fraction
By B. I. Posner and B. L. Fanburg
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ABSTRACT
Studies on RNA from cardiac muscle of rats were performed by labeling the
RNA with 32 P, extracting it with phenol, and purifying it prior to fractionating
and analyzing it on a sucrose density gradient. The major components of RNA
found had sedimentation values of 28, 18, and 4 S. Early 32P-labeIed RNA
ranged in size from 4 to > 45 S. Characterization of RNA from 4 to 18 S
showed it to be more rapidly labeled and to have a higher turnover rate than
28 S RNA, to be located, at least in part, in the microsomes, and to have a
base composition lower in guanine and cytosine content than 28 S RNA. Labeling of 4 to 18 S and 28 S RNA was equally inhibited by actinomycin. The possibility that 4 to 18 S RNA may represent a portion of messenger RNA in
cardiac muscle is considered.
ADDITIONAL KEY WORDS 32P-labeIed RNA
phenol extraction
actinomycin
subcellular particles
sucrose-density-gradient analysis
fractionation of RNA
base analysis of RNA
messenger RNA
turnover rate of RNA
• The messenger hypothesis postulates that
RNA molecules transfer information from
DNA to ribosomes (1). Studies of bacteria
have demonstrated species of RNA thought
to function in this capacity: the important
characteristics are rapidity of labeb'ng by
RNA precursor molecules, decreased stability relative to that of ribosomal RNA, a base
composition that resembles that of DNA,
heterogeneity of molecular size, a capacity for
hybrid formation with homologus DNA, localization in polysomes, and a stimulatory effect
on amino-acid incorporation in vitro (2-11).
A heterogeneous, rapidly labeled RNA
has been demonstrated in a number of mammalian tissues (12-27) as well as in germinating plants (28) and yeast cells (29). No
comparable material has been characterized in
striated muscle although its existence in this
From the New England Medical Center Hospitals
and the Department of Medicine, Tufts University
School of Medicine, Boston, Massachusetts.
This investigation was supported by grants (5T1
HE 5391 and 2R01 HE 06924) from the U. S. Public
Health Service.
Accepted for publication July 16, 1966.
CircuUiio* Kisurcb, Vol. XIX, Octoitr 1966
tissue has been suggested (30). The heart is
more suitable than skeletal muscle for the
study of RNA synthesis because considerably
more cardiac muscle RNA can be labeled in
vivo (Fig. 1). In this communication, we wish
to record the existence of a heterogeneous,
rapidly labeled RNA fraction in cardiac muscle of the rat and to delineate some of its
pertinent properties.
Methods
Animals. Male albino rats, weighing 90 to 120
g except when otherwise noted, were used; they
were maintained on Purina chow ad lib. until they
were killed.
Materials. Phenol, 8-hydroxyquinoline, bentonite, and diphenylamine (special indicator grade)
were purchased from the Fisher Scientific Company. Sodium dodecyl sulfate and yeast RNA
(type XI) were obtained from the Sigma Chemical Company. 3 -P was obtained from the New
England Nuclear Corporation.
Labeling of RNA. The tail veins of rats lightly
anesthetized with ether were injected with carrierfree HR3-PO4 diluted in 0.5 to 1.0 ml of 0.9S
NaCl. Animals were killed by decapitation at
varying times after injection of the label. The
heart, and sometimes the skeletal muscle and the
liver, were removed rapidly, washed in cold wa805
806
POSNER, FAN BURG
10,000
3POO
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10
20
TIHE(HRS)
FIGURE 1
Specific activity of RNA and radioactivity in acidsoluble pools of cardiac and skeletal muscle. Rats
were injected with 1 fie of 3lP/g body weight, and
RNA was extracted as described in the section
on Methods except that 0.05% sodium dodecyl sulfate
was used and DNAase treatment was omitted. Acidsoluble pools were determined as indicated in Methods.
Each point in the figure represents a value obtained
from extraction of two rat hearts and an equal weight
of hind-leg skeletal muscle from the same animah.
RNA specific activity: heart, o o; skeletal muscle,
•
•. Radioactivity in pools of acid-soluble material:
heart, o o; skeletal muscle, •
•.
ter, blotted, and when appropriate, weighed. The
tissue was then fractionated or subjected directly to RNA extraction.
Extraction of RNA from whole tissue. The tissue was minced with scissors in ice-cold 20 mM
Tris buffer (pH 7.2), rinsed three times, and
homogenized in 20 volumes of this buffer (usually two hearts were combined for each sample)
by a Virtis 45 homogenizer run at a speed setting
of 50 for 30 sec.
The homogenate was made 0.5% with respect
to sodium dodecyl sulfate (unless otherwise
noted), shaken, and allowed to stand on ice for
5 min. An equal volume of once redistilled 88%
phenol containing 0.1% 8-hydroxyquinoline was
added, and the mixture was shaken mechanically
on ice for 30 min. The sample was then centrifuged at 10,000 X g for 10 min at 0°C in a
refrigerated centrifuge and the aqueous upper
phase was carefully removed with a pipette and
extracted a second time for 15 min with an
equal volume of phenol. One-tenth volume of
20% sodium acetate (pH 5.2) and 2.5 volumes of
95% ethanol were added to the final aqueous
phase. The mixture was allowed to stand at least
2 hours in the freezer at — 20 °C and then centrifuged at 2,000 rpm for 10 min, giving a white
precipitate.
A procedure adapted from Hiatt (13) was
used to remove the DNA extracted with the
RNA. The white precipitate was dissolved in 2.0
ml of medium containing 0.01 M Tris (pH 7.2),
0.02 M MgSO4 and 10 ^g/ml of DNAase I
(Worthington) and allowed to stand at room
temperature for 20 min. After adding 8 ml of
cold distilled water, sodium dodecyl sulfate was
added to a final concentration of 0.5%, and the
mixture was extracted with 5 ml of 88% phenol.
After this mixture was mechanically shaken on
ice for 15 min, the aqueous upper phase was
separated in a refrigerated centrifuge at 10,000
X g for 10 min. The upper phase was treated
with sodium acetate and 95% ethanol as before to
precipitate the nucleic acids.
RNA was freed of oligodeoxyribonucleotides
by die following procedure. The precipitate was
dissolved in 2% sodium acetate (pH 5.2), and an
equal volume of 4 M potassium acetate was added.
Ice-cold 95% ethanol was added drop by drop
during mixing of the solution until the final concentration was 25%. The solution, usually cloudy
at this point, was kept at —20°C for 30 min,
and the precipitate, collected by centrifugation,
was subjected to the above procedure a second
time. The final precipitate was washed three
times with 95% ethanol and then dissolved in an
appropriate volume of 2% sodium acetate (pH
5.2).
This precipitation procedure removes not only
oligodeoxyribonucleotides but also a considerable
amount of RNA with a sedimentation velocity of
4 S. Thus when RNA is precipitated from 2%
sodium acetate with final concentrations of ethanol
of > 70%, considerably more 4 S material is seen
after zone centrifugation of the final RNA specimen.
Fractionation of heart muscle. All procedures
were carried out at 0 to 4°C unless noted otherwise. The hearts were cut into small pieces with
scissors and homogenized for 10 sec in 2 volumes
of medium containing 50 mM Tris (pH 7.6),
5 mM MgClo, 0.2 M sucrose, and 0.1 g/100 ml
bentonite in a Virtis 45 homogenizer at speed
setting 50 followed by 2 or 3 strokes in a tightfitting, all-glass, hand homogenizer. The homogenate was centrifuged for 30 min at 13,000 X g in a
high-speed attachment of an International centrifuge, and the supernatant was carefully removed by pipette and centrifuged at 105,000 X g
for 3 hours in a no. 50 Spinco rotor to give a microsomal fraction. This procedure is similar to that
employed by Breuer et al. (31) for skeletal
muscle in which the ribosomal preparations were
active in incorporating amino acids.
CircmUtion Rts—rcb, Vol. XIX, Octobtr 1966
RAPIDLY LABELED RNA IN CARDIAC MUSCLE
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The 13,000 X g sediment was resuspended by
brief hand homogenization in about 25 volumes
of 20 ITIM Tris buffer (pH 7.2), and this suspension was centrifuged at 600 X g for 10 min. The
600 X g sediment was resuspended and recentrifuged as above. The final 600 X g sediment will
be called the "myofibrillar-nuclear" fraction.
Extraction of RNA from fractionated heart
muscle. RNA was extracted from the myofibrillarnuclear fraction, resuspended in 20 ITIM Tris buffer (pH 7.2), by the same procedure used for the
extraction of RNA from whole tissue. In order to
extract the microsomal fraction, the pellet was
resuspended in 15 ml of 20 ITIM Tris (pH 7.2),
and the suspension made 1.056 with sodium dodecyl sulfate, shaken vigorously, and kept on ice
for 20 min. The suspension was then extracted
with phenol, as for whole tissue, except that the
first precipitate of nucleic acid was not treated
with DNAase but was purified by dissolving it in
2% sodium acetate (pH 5.2) and reprecipitating
it with 3 volumes of 95% ethanol. This procedure
was repeated, and the final precipitate was
washed once with 9556 ethanol and then dissolved
in an appropriate volume of 2% sodium acetate
(pH5.2).
Analysis by sucrose density gradient. Munro
et al.'s adaptation (32) of Britten and Roberts'
method (33) was used. RNA, 150 to 250 /xg in
0.2 to 0.3 ml of 2% sodium acetate, was applied
to a 5 to 20% sucrose gradient, the sucrose for this
gradient having been dissolved in 2% sodium acetate (pH 5.2). The material was centrifuged for
3Ji hours in a SVV-39 Spinco rotor. At the end of
this time the bottom of the tube was punctured
with a no. 22 needle, and the drops were collected in numbered tubes of distilled water.
Determination of RNA and radioactivity. RNA
in the gradient samples was determined by measuring optical density at 260 mfj, in a Beckman
DB spectrophotometer. The RNA solution was
put into counting vials containing 556 naphthalene,
0.7% 2-5-diphenyloxazole (PPO), and 0.00556 1,4frw-2-(5-phenyloxazolyl) benzene (POPOP) in
dioxane and counted in a Packard tri-carb liquid
scintillation counter. The volumes of fluid used
produced no significant quenching of the counts,
and the efficiency of counting was better than 95%
for ™V.
Acid-soluhle pool of radioactive material. Two
milliliters of ice-cold 10% trichloracetic acid were
added to 1 ml of whole-muscle homogenate. The
supernatant was collected by centrifugation, and
the precipitate was washed once with 2 ml of
1056 trichloracetic acid. An aliquot of the combined supernatants was counted, and the counts
were expressed per gram wet weight of tissue.
Analysis of base composition of RNA. The distribution of aiP in the component bases of 8 -PCircmlslion Rtltrcb, Vol. XIX, Octobtr 1966
807
labeled RNA was determined. Following separation by the density gradient, various areas of the
gradient were pooled; unlabeled yeast RNA was
added as carrier and the RNA was precipitated
by making the ice-cold solution 5% with respect
to trichloracetic acid. The precipitate was then
washed twice with ether to remove the trichloracetic acid and hydrolyzed at a RNA concentration
of 2 to 3 mg/ml for 16 hours at room temperature
in 0.5 M KOH. After hydrolysis, the pH was adjusted to approximately 3.4 with perchloric acid,
and the K perchlorate precipitate was removed
by centrifugation. An aliquot of the supernatant
(usually 0.1 to 0.2 ml) was applied to Whatman
no. 3 chromatography paper, and the nucleoside
-2'-3'-monophosphates were separated by electrophoresis at 1,000 for 2)i to 3 hours according
to the method of Markham and Smith (34).
This procedure was carried out at room temperature with precooled buffer (0.03 M sodium citrate
pH 3.5) and precooled CC14. The four nucleotide
spots, identified by ultraviolet light, were cut
out and eluted with 0.1 N HC1 for 2 hours at
room temperature, and the radioactivity in the
supernatant was determined. Counts recovered
were 96% to 100% of those applied.
Results
Conditions for RNA extraction from heart
muscle. Table 1 illustrates representative results of the extraction of nucleic acid from
heart muscle. In the absence of sodium dodecyl
sulfate, no DNA was extracted with the RNA.
As the concentration of sodium dodecyl sulfate increased to O.55J, extraction of both RNA
and DNA increased. At concentrations greater
than 0.55?, no further increase in RNA extracted
was found. When the concentration of sodium dodecyl sulfate was below 0.0535, the
amount of DNA extracted was negligible. The
presence of MgCl 2 and both an increase or
decrease in pH from 7.2 produced a significant decrease in the RNA extracted while
the DNA extracted increased. It would appear
that conditions for the extraction of maximal
amounts of RNA would be those in which the
buffer is at pH 7.2 and the final concentration
of sodium dodecyl sulfate is 0.5%. These conditions are consistent with those developed
in other tissues (13, 36, 37) to assure the extraction of rapidly labeled RNA with high
specific activity into the aqueous phase of the
phenol extractions. With the concentration of
sodium dodecyl sulfate at 0.55?, the propor-
808
POSNER, FANBURG
a sucrose density gradient, and DNA was removed as detailed above.
In vivo labeling of RNA in heart and skele-
tion of DNA in the nucleic acid extract is
large enough to interfere with adequate fractionation of RNA by centrifugation against
TABLE 1
Extraction of Nucleic acid* from Cardiac Muscle of 200 to 300 g Male Rats
Extraction conditions
Total nucleic acid
p.g/g muscle
(wet wt.)
RNA
11% li muscle
(wet wt.)
DNA
in total
nucleic acid
*/
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2% sodium acetate (pH = 5.2)
+ 0.05% SDS
0.02 M Tris (pH = 8.0)
+ 0.05% SDS
580
487
16.0
1060
765
27.5
0.02 M Tris (pH = 7.2)
+ 0.05% SDS
+ 0.5% SDS
+ 0.05% SDS + 0.005 M MgCL,
+ 0.05% SDS + 0.05 M MgCl2
595
860
1520
960
540
595
845
1140
630
320
0
1.7
25.0
34.2
41.0
'Extraction of nucleic acid was carried through the steps prior to DNA removal with the
modifications noted in the table. The nucleic acid yield was estimated from optical density
at 260 m/i using E J*m = 20. DNA was determined by the diphenylamine reaction (35),
and RNA estimated by the difference between these values. Yields from the hearts of smaller
rats (90 to 120 g) were found to be larger, consistent with the higher concentration of nucleic
acid in the hearts of small as compared to larger rats. SDS = sodium dodecyl sulfate.
2000
B.
OP
30
—
29
-
20
-
.19
-
180
-
190
120
\ A
90 ^
JO -
60
•
0
30
S
i
n
20
I
i
10
i
1
20
FRACTION NUMBER
FIGURE 2
Sedimentation values for components of RNA in cardiac muscle. Livers from rats injected with
1 fic/g body weight of "P and hearts from unlabeled animals were extracted to obtain RNA,
and the RNA was applied to a 5 to 20% sucrose gradient as described in Methods. A. Labeled rat
liver. B. Mixture of labeled RNA from rat liver and unlabeled RNA from rat hearts in a 1:10
ratio. Optical density (OD), •
• ; counts/min, o—o.
CircmUtwn R.ittrcb, Vol. XIX, Oaobtr 1966
RAPIDLY LABELED RNA IN CARDIAC MUSCLE
809
32
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tal mttscle. Injection of P intravenously resulted in greater labeling of RNA in heart
than in skeletal muscle (Fig. 1). Thus, the
ratio of specific activity of RNA in heart
to that in skeletal muscle 6 hours after H2P
was injected was 4.7:1.0. The corresponding
radioactivity of acid-soluble pools from both
heart and skeletal muscle are fairly constant
for the first 6 hours after injection of :12P, while
the specific activity of the RNA of these tissues is increasing at a constant rate. The ratio
of acid-soluble pools of radioactive material
in the heart to that in skeletal muscle approximates the ratio of the specific activity of
heart RNA to skeletal muscle RNA in this first
6-hour period. This suggests that the difference in degree of RNA labeling in these
two tissues may be a function of the difference in 3-P uptake by the tissues.
Major components of RNA in cardiac muscle.
The major components of RNA extractable
from cardiac muscle have essentially the same
sedimentation velocities as those extracted
from liver. To demonstrate this, rat-liver RNA
highly labeled with 32P extracted 24 hours
after the injection of 32P [when the radioactivity and optical density profiles are identical (Fig. 2A) ], was added to unlabeled RNA
extracted from rat heart to give a mixture containing liver RNA/heart RNA in a ratio of
1/10. A sample was then analyzed on a sucrose
density gradient and, as shown in Fig. 2R,
the profiles for radioactivity and optical density superimpose. We conclude that the three
major peaks of the optical density profile of
heart RNA have the values of approximately
28, 18 and 4 S; i.e., the values reported for
the major components of liver RNA (38, 39).
The pattern of labeling of tissue in the whole
heart preparations. Figure 3 illustrates the pattern of labeling of RNA at various times after
injection of 3-P. After 1 hour (Fig. 3A) the
labeled material is polydisperse, and areas of
particularly high specific activity occur in the
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FIGURE 3
Pattern of labeling of RNA components at various times following 32 P injection. RNA was
extracted from whole heart tissue at various times following 32P injection, and fractionated
on a linear 5 to 20% sucrose gradient. A, 10 ixc/g body weight S*P, extracted after 1 hour.
B, 5 iic/g body weight "P, extracted after 4 hours. C, 1 fic/g body weight, extracted after 12
hours. D, 1 iic/g body weight, extracted after 24 hours. Specific activity was calculated by
taking the ratio of counts/min (CPM) to optical density (OD) at each point. Optical density,
• — • ; counts/min, o—o; specific activity, A—A. Shaded areas discttssed in Figure 5.
CircuUnom Rnurcb,
Vol. XIX, Octobtr
1966
810
POSNER, FANBURG
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region between the 4 S and 18 S peaks
and in the >28 S region. After 4 hours (Fig.
3B), RNA in the 4 to 18 S region is highly
labeled relative to that of heavier RNA components. By 12 hours (Fig. 3C), there is a
small but definite decrease in the specific
activity of the 4 to 18 S material relative to
that of the 28 S RNA although the labeling
pattern resembles that at 4 hours. By 24
hours (Fig. 3D) all the RNA has the same
specific activity.
The labeling of microsomal and myofibriUarnuclear fractions. Figure 4A shows that 6 hours
after the administration of S2V that portion of
the RNA which is most highly labeled in the
microsomes is the RNA with sedimentation
velocities between 4 and 18 S. At t-his time
significant labeling of 28 S RNA is also evident. The pattern and extent of labeling of
the corresponding myofibrillar-nuclear frac-
tion (Fig. 4B) are comparable to those of the
microsomal fraction.
Time course of labeling of 4 to 18 S and
28 S RNA. The yield of microsomes from cardiac muscle is extremely low (40); since the
pattern of labeling of whole tissue is comparable to that of the microsomal and myofibrillar-nuclear fractions (Figs. 3 and 4), the
time course of labeling was studied in whole
tissue. Because of the higher specific activity
in the region between 4 and 18 S, both in
whole-tissue RNA as well as microsomal
RNA, and because much attention has been
focused on RNA of this size range in other
tissues (12-27), we compared the specific
activity time course of labeling of RNA in
this region to that in the 28 S region (Fig.
5). These curves demonstrate that the
initial rate of increase of specific activity of
4 to 18 S RNA is greater than that of 28 S
720
290
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1 -600
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21
, 1
25
FRACTION NUMBER
FIGURE 4
Patterns of labeling of microsomal and "myofibrillar-nuclear" fractions. Twenty rats were
injected with 2 iic/g body weight of "P; 6 hours later they were killed, and the hearts from
10 unlabeled animals were added to those which had received 32P. SubceUular fractionation
into microsomal (A) and "myofibrillar-nuclear" (B) fractions and RNA extraction were carried
out as described in Methods. Optical density, • — • ; counts/min, o — o .
CircuUtio* Rti—rch, Vol. XIX, Oaoitr 1966
RAPIDLY LABELED RNA IN CARDIAC MUSCLE
FIGURE 5
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
Time course of labeling of RNA with sedimentation
velocities of 28 S and 4 to 18 S. RNA was extracted
from whole heart tissue at various times following the
injection of 1 ixc/g body weight 3tP and was fractionated on a linear 5 to 20% sucrose gradient. The specific
activity was determined for the two shaded areas in
Figure 3B by taking the ratio of total counts/min
(CPU) to total optical density (OD) in the appropriate
regions of the gradient. It was found that the ratio of
4 to 18 S RNA specific activity (counts/min per OD)
to 28 S RNA specific activity at 6 hours after "P administraton was as follows: whole tissue, 2.0; "myofibrillar-nuclear" fraction, 2.1; microsomal fraction, 2.8.
It should be noted that the area in Figure 3B does
not include the peak points at 4 and 18 S. Each point
in the figure was obtained from the extraction of two
hearts and is a representative value as borne out by
other determinations at these times. RNA 28 S, o—o;
RNA 4 to 18 • — • .
RNA. It is also clear that the specific activity
of the 4 to 18 S RNA reaches a plateau by 6
hours, whereas that of 28 S RNA increases
in a biphasic manner over the whole 24hour period of observation and approximates
specific activity of 4 to 18 S RNA by about
24 hours. The presence of overlap between
rapidly labeled RNA components of high
specific activity in the 4 to 18 S region and
18 S ribosomal RNA means that the specific
activity of 4 to 18 S RNA is an underestimation of the true specific activity of the rapidly
labeled RNA species which sediment in this
portion of the sucrose gradient. The removal
of S RNA by precipitation from 10* NaCl (41,
42) only slightly affects the specific activity
of RRA in 4 to 18 S region.1 The change
in specific activity with time in the 4 to 18 S
region, which is in contrast to that of the 28 S
^Specific activity of 28 S RNA: before NaCl 400,
after NaCl 408. Specific activity of RNA between 4
and 18 S: before NaCl 574, after NaCl 658.
CircmUtum Rtiurcb,
Vol. XIX, Octobtr
1966
811
region, is similar to observations made on
the labeling of RNA fractions of this size
in mouse-liver polysomes (26).
Effect of actinomycin on the labeling of
heart muscle RNA. Actinomycin, when given
2 hours prior to the administration of 32P,
inhibited equally the labeling of 28 S RNA and
RNA in the 4 to 18 S region (Table 2).
Base composition of S2P-labeled RNA in
cardiac muscle. Base composition of s2P-labeled RNA in cardiac muscle was obtained as
noted in Methods and is shown in Table 3.
It can be seen that at 24 hours, 28 S RNA has
a distinctly lower ratio of adenine plus uracil
to guanine plus cytosine than any other type
of RNA on the gradient. This base composition resembles that noted for 28 S ribosomal
RNA obtained from other tissues (15, 17, 19,
21, 24, 43). After only 4 hours of labeling, the
content of guanine is relatively lower and that
of uracil relatively higher, suggesting the
presence of non-ribosomal rapidly labeled
RNA in the 28 S region. The presence of this
rapidly labeled RNA may account for the
greater rate of labeling of 28 S RNA in the
first 6-hour period, and hence for the biphasic
nature of the time curve for specific activity
(Fig. 5). RNA in the 4 to 18 S region, which
is highly labeled at 4 hours (Fig. 3B), shows
a considerably different base composition from
that at 28 S, and small differences from RNA
in the 4 S and 18 S regions.
Discussion
The results of these studies demonstrate
that there is in cardiac muscle some species
of RNA that do not correspond to the three
major RNA constituents obtained after sucrose
gradient centrifugation. RNA labeled for the
relatively short period of time of 1 hour (Fig.
3A) is heterogeneous, ranging from 4 to 45 S,
This is similar to what has been observed when
RNA from pulse-labeled bacteria is carefully
extracted (7, 9), and when RNA from several
different mammalian tissues is examined very
soon after being labeled (12-15, 17-20).
As seen in Figure 3, RNA in the 4 to 18 S
area remained highly labeled relative to
other RNA species over a considerable
812
POSNER, FANBURG
TABLE 2
Effect of Actinomycin on Specific Activity of 28 S and 4 to 18 S RNA*
RNA
sedimentation velocity
...
. .
, count»/min
Specific
activity (
:—r—= :— )
r
opticil density
Control
Actinomycin
28
4-18
270
480
Inhibition
%
55
110
79.4
77.1
•Rats were given actinomycin (1.5 Mg/g body weight) intraperitoneally, and 2 hours later
were injected with 1 ixc 3 2 P/g body weight. Four hours after 32 P injection, the hearts were
removed and extracted as described in Methods. Control animals were killed 4 hours after
32
P. The above data are representative of several experiments. No further suppression of RNA
labeling was obtained with 3 i±g of actinomycin/g body weight.
TABLE 3
Base Composition of "P-Labeled RNA Fractions
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
RNA
sedimentation velocity* Hours after
labeling
S
28
28
18
4-18
4-18
4
24
4
24
4
24
24
c
24.9
26.7
21.5
23.6
23.0
23.6
Base composition ( % ) t
A
G
15.8
17.1
16.2
19.2
20.3
20.4
35.7
28.2
28.0
27.1
28.0
24.6
U
A+U
G+C
23.6
27.7
33.9
30.1
28.7
31.5
.65
.82
1.02
.98
.96
1.08
*28 S and 4 to 18 S RNA represent the shaded areas (A and B respectively) indicated in
Figure 3B. 18 S RNA was collected from the area under the second optical density peak and
4 S RNA from that under the third peak.
fC = cytosine; A = adenine; G = guanine; U = uracil.
period of the time of observation. Other workers, who have ascribed a "messenger" function to this species of RNA, have studied its
properties in polysomes. It was found impractical to analyze the kinetics of labeling of
RNA in heart polysomes since both the extent of labeling of RNA in and the yield of
polysomes from this tissue were very low
relative to that found in tissues such as liver.
It has been observed that during the fractionation of skeletal (44) or cardiac (45)
muscle, the bulk of tissue RNA remains adherent to the myofibrils. We have made a
similar observation in our preparation (unpublished data). It is thus likely that the microsomes obtained from cardiac muscle represent only a small portion of that originally
present in the tissue.
We have attempted a kinetic analysis of
labeling of whole-tissue RNA. This does not
permit the use of the methods such as those
described by Staehlin et al. (25) for determining specific activity of messenger RNA.
The specific activity was determined simply
by the ratio of counts per minute to optical
density. This calculation probably underestimates the true specific activity of highly
labeled RNA in the 4 to 18 S region. Despite
the approximate nature of the calculation, the
time course of labeling between 4 and 18 S
is distinctly different from that at 28 S. The
difference would not seem to be attributable
to the presence of 4 S RNA since its removal,
by precipitation of RNA from 10% NaCl, does
not significantly alter the specific activity of
the RNA sedimenting between 4 and 18 S.
If it is assumed that the kinetics of labeling
of 28 S and 18 S ribosomal RNA are identical
in heart tissue, as they are in mouse liver (26),
then it would appear that the difference in
the time course of labeling of the 4 to 18 S
region as compared to that of the 28 S region
in these experiments is largely a function of
the heterogeneous material in this region.
As shown in Figure 5, RNA with a sedimentation velocity of 4 to 18 S is more rapidly
CircuUuon Research, Vol. XIX, October 1966
813
RAPIDLY LABELED RNA IN CARDIAC MUSCLE
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labeled than 28 S ribosomal RNA and attains
a relatively constant specific activity sooner
(6 hours for 4 to IS S RNA; 24 hours for 28 S
ribosomal RNA). This behavior is similar to
that found by Trakatellis et al. (26) in their
studies on rapidly labeled RNA in liver polysomes. If we assume that the relative size of
the different populations of molecules remains
constant during the period of observation (and
that they both utilize the same pool of precursors), then the turnover of 4 to 18 S RNA is
more rapid than that of 28 SribosomalRNA.
RNA in either region is equally susceptible to
inhibition by actinomycin (Table 2).
The base composition of highly labeled 4
to 18 S RNA differed from that of 28 S
ribosomal RNA at both 4 and 24 hour labeling
times. After 4 hours the predominant material in the 4 to 18 S region was rapidly
labeled, rather heterogeneous, RNA whose
base composition resembled that of 18 S and
4 S RNA but differed distinctly from that of
28 S ribosomal RNA. Although there may be
some overlapping of radioactivity from the 4 S
and 18 S RNA within the 4 to 18 S region,
the relatively higher specific activity between
the 4 S and 18 S peaks (Fig. 3B) indicates
that most of the counts are in a species distinct
from the 4 S and 18 S components. The 4 S
and 18 S peak points were omitted in the
analyses of the 4 to 18 S area to minimize the
contaminants from these two RNA species.
The 4 to 18 S RNA has a ratio of adenine
plus uracil to guanine plus cytosine that is
closer to rat DNA than is that of 28 S RNA
(24, 46). That 18 S and 28 S ribosomal RNA
differ from one another in base composition
has been previously noted in studies on other
systems (15,43,47).
RNA with a sedimentation velocity between
4 and 18 S in cardiac muscle of rats is very
similar to a material of similar size which has
been described in several different mammalian tissues (19-27) and which has been characterized in considerable detail in rat-liver
polysomes (24-27). This material has been
referred to as "messenger" RNA because its
properties—rapidity of labeling, relatively fast
turnover time, DNA-like base composition, loCtrcubium Resircb, Vol. XIX, Ociobtr 1966
cation in polysomes—are consistent with those
of a messenger as hypothesized by Jacob and
Monod (1). Template activity (48) has also
been described for this fraction in rat liver
(49) although recent studies cast doubt on
accepting this as an established characteristic (50, 51). Some of the rapidly labeled
material in the 4 to 18 S region may represent
breakdown products of larger molecules or
species of RNA whose function is as yet
undefined (52).
If RNA in the 4 to 18 S region does, at
least in part, subserve a messenger function,
it is probably not that species coding for myosin. If we assume that the smallest subunit
of the myosin molecule has a molecular weight
of 2 X 105 (53), this would require a RNA
template with a molecular weight of about 1.8
X10°. The empirical formulation of Spirin
(54) or Gierer (55), which relates molecular
weight and S coefficient obtained on sucrosegradient centrifugation of single-stranded
RNA, would imply that the subunit's messenger would have to be 35 to 43 S in size.
It is evident (Fig. 3A) that heavier RNA molecules in this size range are present. Some of
these may subserve a template function and
could conceivably carry the code for a large
protein such as the myosin subunit.
Acknowledgment
We are grateful to Dr. Maurice S. Raben and Dr.
Asher Korner for their very helpful advice, support,
and encouragement and to Mr. Mark Roffman, Mr.
William Lieberman, and Miss Jean Frieden for
their technical assistance.
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B. I. POSNER and B. L. FANBURG
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Circ Res. 1966;19:805-815
doi: 10.1161/01.RES.19.4.805
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