Nucleic Acids Research
Volume 3 no.6 June 1976
Globin mRNA contains a sequence complementary to double-stranded region of
nuclear pre-mRNA.
Aleksei P. Ryskov, Olga V.Tokarskaya, Georgii P.Georgiev, Charles Coutelle* and
Berndt Thiele*
Institute of Molecular Biology, Academy of Sciences of the USSR, Moscow, USSR
ABSTRACT
Melted ds RNA isolated from rabbit bone marrow pre-mRNA was hybridized with
excess of globin mRNA which was prepared from rabbit reticulocytes. 7-9% of ds
sequences became RNAase-stable and about 30% of the sequences could be bound to
poly(U)-Sepharose through poly(A) of mRNA. The size of RNAase-stable hybrid is
about 30 nucleotides, that is one fourth of the length of one strand of the ds RNA.
INTRODUCTION
It was previously shown that the cytoplasmic mRNA from mouse liver or Ehrlich
carcinoma cells could form RNAase-stable hybrids with melted ds RNA sequences
1 2
prepared from a nuclear pre-mRNA (hnRNA) ' . About 20 per cent of the ds RNA
sequences were involved in a hybridization reaction.
It was suggested that the
double-stranded hairpin-like structures formed the border-lines between mRNA
sequences and the non-informative part of the pre-mRNA.. In the course of processing,
part of the ds region would be destroyed resulting in the separation of the mRNA.
A part of one branch of ds RNA would remain bound to mRNA and could hybridize with
the ds RNA from pre-mRNA .
In this paper, similar experiments were made with the individual mRNA (globin mRNA)
using different techniques of hybrid detection.
The results obtained are similar to
those described earlier with total mRNA of mouse cells.
The size of the hybridized
ds RNA sequences in the complexes of mRNA with ds RNA was determined.
MATERIALS AND METHODS
Isolation of pre-mRNA. On the 5th day of recovery from phenylhydrazine anaemia an
enrichment with nucleated erythroid cells up to 70% can be obtained in the bone marrow
4
of rabbits, using partial in vivo synchronization . The cell suspension enriched in
3
erythroid precursor-cells was incubated for 15 min at 37°C with H uridine (0.85 mCi
© Information Retrieval Limited 1 Falconberg Court London W1V5FG England
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Received 8 March 1976
Nucleic Acids Research
/2-10 9 cells, specific activity, 20-30 Q/mmole, UWVR, Praha, CSSR), and nuclear
RNA was subsequently extracted by the hot phenol fractionation method .
According
to this method, pre-rRNA is obtained in the 40°C fraction and pre-mRNA in the
fractions extracted above 55°C .
65°Cand 85°C fractions were combined and ultra-
centrifuged through a 12 ml linear 5-20% (w/v) sucrose gradient in a buffer containing
lOmM Tris-HCl (pH8), 0.1 M Nad, 1 mM EDTA, 0.5% SDS in the Spinco SW 40
The fraction were collected, the aliquots were analyzed and all
fractions heavier than 35S were pooled and precipitated with ethanol.
Isolation and purification of ds RNA.
Pre-mRNA dissolved in 2xSSC was treated
with a mixture of pancreatic RNAase (Sigma) and TT RNAase (Galbiochem) as
described previously .
After incubation, the mixture was treated with pronase
(Reanal, 100 fjg/ml), applied to a Sephadex G-75 column (1x80 cm) and eluted with
0.2M sodium acetate buffer .
Only the material eluted in the void volume was
collected, and treated again with pronase (100 ug/ml) at 25° for 30 min.
Thereafter
SDS was added to 1% and the mixture was shaken with phenol for 30 min at 60°C.
The water phase was treated with a mixture of phenol-chloroform (1:1 v/v) and
chloroform at room temperature and the material was precipitated with ethanol in
the presence of carrier tRNA.
This procedure completely frees ds RNA samples
from RNAase contamination.
Isolation of globin mRNA of rabbit. Globin 9S mRNA of rabbit reticulocytes was
Q
isolated according to the technique described by Evans and Lingrel from polysomal
RNA by two cycles of ultracentrifugation in sucrose gradients followed by two cycles
of chromatography of the material in the 9S peak on poly(U)-Sepharose 4B (Pharmacia).
The procedure for producing a phenylhydrazine-induced anemia in rabbits and for
9
preparing a lysate of the reticulocyte rich blood was described previously . Total
poly(A) RNA of rabbit liver was isolated from total cytoplasm or from polyribosomes
9a
free from cytoplasmic informosomes and monoribosomes as described elsewhere .
Reassociation of denatured ds RNA.
ds RNA was dissolved in a small volume of
water and denatured by heating in small teflon tubes. One tenth of the volume of 20xSSC
was added and the samples,5 to 10 pl,(10OO-2000 cpm radioactive material) were
incubated at 65°C for the time necessary to obtain the appropriate Cot value.
To
calculate the Q)t value, it was assumed that the specific radioactivity of ds RNA was
equal to that of heavy pre-mRNA.
After annealing, the samples were diluted with
2xSSC and the proportion of RNAase-stable acid insoluble radioactive material was
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rotor at 23°.
Nucleic Acids Research
estimated as described elsewhere
10
.
In control experiments, the proportion of
RNAase-stable material without annealing of denatured ds RNA was determined.
Hybridization experiments.
For hybridization, ds RNA was melted by heating;
about 1000 cpm (~0.02 (jg) of radioactive material was taken, mixed with mRNA
and annealed in 2xSSC at 65° for different time intervals to make ds RNA-driven
-4
-4
Cot equal to 1x10 to 3x10 . The proportion of acid-insoluble radioactive
For prepar-
ative isolation of hybridized sequences, aboutlO.OOO to 15,000 cpm of melted
radioactive ds RNA (with specific activity 50,000 cpm/^ig) was mixed with 600 pg of
globin mRNA and annealed in 3 ml of 2xSSC at 65° for 12 min.
At the end of the
annealing, 30 pg of pancreatic RNAase (Sigma) were added and the mixture was
incubated for 30 min at room temperature.
to stop the reaction.
200 pg of pronase (Reanal) were added
The RNAase-resistant material was deproteinized with 0.5%
SDS and phenol at 60°C with a mixture of phenol+chloroform (1:1 v/v), with chloroform at room temperature and then precipitated with ethanol in the presence of
tRNA as carrier.
The precipitated material was dissolved in water, denatured
by heating and used in electrophoretic experiments.
In control experiments, the
same procedure was applied to the same amount of ds RNA annealed without mRNA
under the same conditions.
Detection of hybrid complexes on poly(U)-Sepharose.
Hybridization mixture
containing melted ds RNA and globin mRNA was annealed as described above and
applied to a poly(U)-Sepharose 4B (Pharmacia) at room temperature in 0.4 M Nad
-0.01 M EDTA - 0.01 M Tris-HCl (pH 7.5) - 0.2% SDS (buffer 1).
The column
was washed with the same buffer and the bound material eluted with the buffer 1
lacking NaCl (buffer 2) at 50°C.
The acid-insoluble radioactive material was
collected on Millipore or fiberglass filters and counted in a toluene scintillator.
In control experiments, the same procedure was applied to ds RNA alone, to ds
RNA mixed with globin mRNA just before chromatography without annealing, and
to ds RNA annealed with a vast excess (40 to 80 ug) of tRNA or commercial poly(A)
(Reanal).
Polyacrylamide gel electrophoregis of RNA.
RNA samples were analysed on poly-
acrylamide gel using a modification of the Loening method
as described in the
legend tc fig.4. Electrophoresis in formamide was carried out according to Hnder
e t a l . 1 . 5S ribosomal 4S transfer, i tRNA a (20 nucleotides long) and oligo(U)10
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material stable to RNAase was determined as described previously .
Nucleic Acids Research
were used as markers.
The gels containing non-labeled markers were stained
with "Stains All" (Eastman-Kodak).
The gels containing radioactive material were
sliced into 2 mm fractions, dissolved in "Tissue solubilizer" (Amersham/Searle)
or in "Aquasol" (New England Nuclear, Boston, Mass.) and counted in toluene.
RESULTS
Characterization of ds RNA prepared from pre-mRNA of rabbit bone marrow cells.
fractionation procedure , and the ds sequences were prepared from the high-molecular
weight fraction of pre-mRNA as described previously .
It should be pointed out that
300
3- 200
20
30
Slice No
Fig. 1. Polyacrylamide gel electrophoresis in formamide of the melted ds RNA
from pre-mRNA of rabbit bone marrow cells, ds RNA was isolated, purified and
melted as described in Material and Methods. The gels contained 12%acrylamide,
with a ratio of acrylamide to bisacrylamide of 30 : 1 in de-ionized formamide
containing 0.02 M diethylbarbituric acid (pH 9.0). Seven cm gels were prepared
and allowed to polymerise for 2 hr at room temperature. RNA samples in 98%
formamide were heated at 95°C for 2 min, chilled and applied to the gels. Electrophoresis was performed at 100 V for 5 hr at 24° C with an electrode solution of 0.02 M
sodium chloride. The direction of migration was from left to right, and the position
of non-labeled markers run in the parallel gel are indicated by the arrow.
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Nuclear pre-mRNA was isolated from bone marrow cells using the hot phenol
Nucleic Acids Research
a loop in the hairpin is digested by RNAase during ds RNA isolation and therefore the
melting converts ds RNA into single-stranded RNA.
These ds RNA sequences were
characterized in respect to size and renaturation kinetics.
Fig.l shows that the melted ds RNA from bone marrow cells displays a rather
heterogeneous distribution upon polyacrylamide gel electrophoresis with a maximum
in the region of chains of 80-150 nucleotides long.
It was also observed that during
of melted ds RNA did not decrease (data not shown).
Kinetic studies on the renatura-
tion of the melted ds RNA (fig 2) showed that about 20% of the sequences (in different
-4
-2
experiments from 15 to 25%) renature at low Cot values from 10 to 2x10 , while the
rest of the material renatures at higher Cot values.
2
These results are quite similar to those obtained with mouse liver and carcinoma ds RNA.
One can conclude that about 20% of total ds RNA is represented by a material which is
rather homogeneous in sequence and consists of ds RNA of one or a few kinds.
From data represented in fig.2, the conditions for the hybridization reaction were chosen.
3
o
£
«f
a
Fig.2. The renaturation curve of denatured ds RNA from pre-mRNA of rabbit
bone marrow cells, ds RNA was isolated, purified, melted and renatured as
described in Materials and Methods.
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short incubation under hybridization conditions (1-2 hr at 65°C), the molecular weight
Nucleic Acids Research
The ds RNA Cot was chosen sufficiently low
(1-3)K10
-4
Hybridization of melted ds RNA with globin mRNA.
, to minimize its renaturation.
Unlabeled globin mRNA was
prepared from rabbit reticulocytes in the usual way by polysome pelleting, isolation of
polysomal poly(A) RNA followed by purification of 9S poly(A) RNA.
electrophoretically pure.
This RNA was
It can be completely bound to poly(U)-Sepharose and programmes
globin synthesis in a heterogeneous cell-free system .
formation was detected using two techniques (table 1).
The first one was the detection of RNAase-stable material.
In control experiments
which werecarried out in the absence of mRNA, 5 to 10 per cent of the melted ds
sequences were RNAase-stable under the conditions used.
The addition of mRNA
immediately followed by RNAase treatment did not increase the amount of RNAasestable material while after annealing with mRNA, the amount of RNAase-stable
3
material increased significantly. For an mRNA/ds RNA ratio of about 10 the
difference in respect to the control figure reached 7-9%.
A further increase of the mRNA/ds RNA ratio did not lead to an increase in the
amount of RNAase stable material.
It was observed previously that the
2 3
hybridization reaction was species but not tissue specific ' . In agreement with
these data the globin mRNA of rabbit failed to hybridize with the mouse Ehrlich
carcinoma ds RNA, whereas mRNA prepared from rabbit liver efficiently binds
bone marrow ds RNA (Table 1).
Another technique used in detecting hybrid complexes was the binding of hybrids to
poly(U)- Sepharose through the poly(A)-end of mRNA.
Fig. 3 demonstrates that a
significant proportion of the ds RNA binds to poly(U)-sepharose after annealing with
mRNA.
In control experiments where mRNA was not added, the binding to poly(U)-Sepharose
did not exceed 2%.
The same background values were obtained when ds RNA was
annealed with tRNA or with a vast excess of poly(A) or when mRNA was added to
ds RNA just before passing through the poly(U)-Sepharose.
The highest binding
of ds RNA observed in our experiments after annealing with mRNA was 30 to 35%
(Table 1).
It should be pointed out that the proportion of the poly(U)-Sepharose bound material
was much higher than that of RNAase- stable material after annealing under the same
conditions.
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One may suggest that the hybrid contains the duplex region as well as
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A mixture of an excess of globin mRNA with melted ds RNA was annealed and hybrid
Nucleic Acids Research
Fig.3. Detection of hybrid complexes on poly(U)-Sepharose column. About 1,000
cpm of radioactive material of melted ds RNA was passed through a 0.5 ml poly(U)Sepharose column after annealing with 75 pg of globin mRNA (-O-), or alone
(-A-) as described in Materials and Methods. mRNA driven Cot was about 9 at
mRNA/ds RNA ratio of 6500(w/w).
Table 1
Hybridization of ds RNA sequences with mRNA
Nature of RNAs used
In hybridization reaction
Rabbit bone marrow ds RNA
+ rabbit globin mRNA
Exp.N 0
Ratio of mRNA to
ds RNAa (w/w)
ds RNA sequences
.
hybridized(per cent total )
1
2
3
4
1.3x10^
1.3x10,
6.3x10^
6.3x 10
5
6
6.3x10,
6.3x 10
37
11
7
8
6.3x10,
6.3 x 10
17
30
9
1.8 x 10
1
RNAase
10
1.5 x 104
1.5
RNAase
6
7
9
28
3
Rabbit bone marrow ds RNA
+ rabbit liver mRNA
Mouse carcinoma ds RNA +
rabbit globin mRNA
Mouse liver ds RNA +
rabbit globin mRNA
Technique used
for analysis
RNAase
RNAase
RNAase
poly(U)-Sepharose
binding
- "- "-"-"RNAase
poly( U)-Sepharose
binding
In the majority of experiments ds RNA-driven C^t was about 3 x 1 0 (it was calculated on the basis of specific radioactivity
of pre-mRNA, from which ds RNA was isolated). In exp.N°5 the ds RNA-driven Cot was 5 dmes higher than in the other
experiments.
Background equals to 5-10% of total counts in the RNAase experiments, and 2% in the poly(U)-Sepharose binding experiments.
The background was detected in each experiment and siijtracted from the total counts obtained (see also the reassociation
curve in Fig.2, and the control curve in Fig.3).
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Fraction A/o
Nucleic Acids Research
the unpaired tail of ds RNA.
Such an interpretation is supported by the fact that
RNAase treatment of the hybrid bound to the poly(U)-Sepharose column, digests
from 30 to 75% of the labeled material.
The size of ds RNA-mRNA hybrid.
The size of the hybridised sequence originated
from ds RNA was determined using polyacrylamide gel electrophoresis.
RNA, 5S ribosomal RNA, one quarter tRNA
a
and oligo(U)
Transfer
were used as markers.
narrow peak in the region of chains of about 30 nucleotides long.
Undigested
material (original melted ds RNA or RNA eluted from the poly(U)-Sepharose column)
as was mentioned above, was located with chains 80-150 nucleotides long.
The same
location (in the region of sequehces of 80-150 nucleotides long) was observed for
200
E
ex.
j
•3 100
o
o
4
10
20
Sties Ho
Fig.4. Polyacrylamide gel electrophoresis of the sequence of ^H-labeled ds RNA
hybridized with non-labeled globin mRNA. Preparative isolation and purification of
hybridized sequences was performed as described in Materials and Methods. The gel
contained 15% acrylamide, with a ratio of acrylamide to bisacrylamide of 375 : 1 in
0.09 M Tris-borate buffer (pH 8.3) containing 2 mM EDTA. Seven cm gels were
prepared and allowed to polymerise for 1 hr at room temperature. RNA samples in
distilled water were heated at 95°C for 2 min, chilled and applied to the gels. Electrophoresis was performed at 100 V for 1.5 hr at 24°C. The direction of migration was
from left to right, and the positions of non-labeled markers run in the parallel gel are
indicated by the arrows.
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One can see from Fig.4 that the RNAase-stable material migrates as a rather
Nucleic Acids Research
RNAase-stable material of ds RNA annealed without mRNA (data not shown).
Therefore all values used in Fig.4 were obtained after substracting this background.
DISCUSSION
The results presented in this paper demonstrate the existence of a short (~ 30 nucleotides long) region in the double-stranded hairpin-like sequence of pre-mRNA which
is complementary to a portion of the globin mRNA.
In our previous study it was shown
length of the complementary sequences was heterogeneous, varying from 10 to 60
nucleotides
. The following hypothetical scheme could be drawn from this result as
2 3 13
well as from the previous data obtained with total mRNAs ' ' (Fig. 5).
We suggest that in the giant pre-mRNA,a long hairpin structure of about 100-150 base
100-200 tase p
r
fy(
ve fxnt
30 6a%t
m
t
mRNA sequence
| |
FH
VNA
I Release of mRNA and
|
/Wotare
t
mRNA
Fig. 5. A hypothetical model for the localization and functioning of double-stranded
regions of pre-mRNA as a "separator" of mRNA from non-informative part of the
precursor.
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that polysomal poly(A) mRNA of mouse can also hybridize to mouse ds RNA, and the
Nucleic Acids Research
pairs is localized on the border line between the mRNA sequence and the non-informative part of the pre-mRNA.
The processing enzyme recognizes the double-helical
region, and destroys a significant part of the hairpin.
The rest of the hairpin
survives and a piece of the right branch of the hairpin about 30 nucleotides long
(for globin mRNA of rabbit) remains at the 5'-end of mRNA moving with it into the
14
cytoplasm. A similar possibility is also suggested by Crippa et al. on the basis of
Several points of this model should be considered.
in the hairpin is unknown.
First, the size of the unpaired loop
It was shown previously that the mouse ds RNA could
hybridize to some of the palindromes present in DNA
.
The size of the unpaired loop in
the palindrome seems to be very small since this structure is not cleaved with DNAase
16
SI . However, it has not been proved that ds RNA in the pre-mRNA is transcribed
from the palindrome itself.
It could be also transcribed from the complementary
sequences identical to palindromic sequences but separated by a long spacer.
This
question is under investigation in our laboratory.
Second, it is not clear whether all, or only a few mRNA molecules contain sequences
complementary to ds RNA. In our experiments, the mRNA/ds RNA ratio was
3
(1.3-6.3)xlO . Assuming that the complementary region comprises about 5% of mRNA
and 15% of total ds RNA nucleotides, this ratio should be decreased to about 400 to 2000.
This ratio is high enough and even if only some of the mRNA molecules have complementary
sequences, hybridization should still take place.
There is a possibility that further
processing of mRNA leads to elimination of the rest of the hairpin in mRNA.
The
possible absence of the complementary sequence in most of the mRNAs could
17-18
explain the failure to find internal reiteration heterogeneity in mRNAs
.
To
elucidate this question, detailed kinetic experiments are necessary.
Third, poly(U)-Sepharose experiments showed that a high proportion of ds RNA can
hybridize with mRNA..
It was higher than the content of rapidly renaturing ds RNA.
This could be explained by assumption that ds RNA regions contain a short (~30
nucleotides) sequence which is common to most of the ds regions and to most of the
different pre-mRNAs, while the other part of the ds region is characterized by a
higher sequence heterogeneity.
This possibility also has to be checked experi-
mentally .
2 19
A possible role of hairpin-like loops as signals for enzymes of processing '
has
found support in experiments with procaryotic systems, where it was shown that
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their experimental data.
Nucleic Acids Research
pre-mRNA and pre-rRNA are processed to mature molecules with the aid of RNAase
20-22
III which is specific for double-stranded RNA
. A very rapid attack of the
precursor molecules by the enzyme prevented detection of these precursors before
the mutants containing defective RNAase became available. A similar situation may
exist also in the case of eucaryotic cells.
ACKNOWLEDGEMENTS
V. Schick and V. Scheinker for the gift of tRNA, \ tRNAVal
and
oligo(U)10-.
* Central Institute of Molecular Biology, Academy of Sciences of the DDR,
Institute of Physiological and Biological Chemistry, Humboldt University,
Berlin, GDR.
REFERENCES
1
Ryskov, A.P., Limborska, S.A., and Georgiev, G.P. (1973) Mol.Biol.
Reports 1, 215.
2
Georgiev, G.P., Varshavsky, A.Ja., Ryskov, A. P., and Church, R.B.
(1973) Cold Spring Harbor Symp.Quant.Biol. 38, 869.
3
Ryskov, A.P., Kramerov, D.A., Limborska, S.A., and Georgiev, G.P.
(1975) Molek.Biol. 9, 6.
4
Coutelle, Ch., Reineke, H.H., Steindamm, E., Meurer, W., Grieger, M.,
and Rosenthal, S. (in press). Haematologia (Budapest).
5
Georgiev, G.P., Ryskov, A.P., Coutelle, Ch., Mantieva, V.L., and
Avakyan, E.R. (1972) Biochim.Biophys. Acta 259, 259.
6
Coutelle, Ch., Thiele, B., Ladhoff, A., Ryskov, A. P., and Papies, B. (1974)
FEBS Symp. 63.
7
Ryskov, A.P., Saunders, G.F., Farashyan, V.R., and Georgiev, G.P. (1973)
Biochim.Biophys. Acta 312, 152.
8
Evans, M.J., and Lingrel, J.B. (1969) Biochemistry 8, 3000
9
Ladhoff.A.M., Thiele, B., Coutelle, Ch. (1975) Eur.J.Biochem., 58, 431
9a Brykina, E.V., Podobed,0. V., Chernovskaya, T.V., and Lerman, M.I. (1974)
Mol.Biol. Report 1, 417
10 Ryskov,A.P., Tokarskaya, O.V., and Georgiev, G.P. (in press) Mol.Biol.
Reports.
11 Loening, J.E. (1967) Biochem. J. 102, 251.
12 Hnder, J.C., Stainov, D.Z., and Gratzer, W.B. (1974) Biochemistry 13, 5373
13 Naora, H., and WhitelamJ.M. (1975) Nature 256, 756.
14 Crippa, M., Meza, I., and Dina, D. (1973) Cold Spring Harbor Symp. Quant.
Biol. 38, 933.
15 Ryskov, A. P., Church, R.B., Baiszar, D., Georgiev, G.P. (1973) Mol.Biol.
Reports 1, 119
16 Church, R.B., and Georgiev, G.P. (1973) Mol.Biol. Reports 1, 21
17 Klein, W.H., Murohy, W., Attardi, G., Britten, R.J., and Davidson, B.H.
(1974) Proc. Nat.Acad.Sci. USA 71, 1885
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We thank N.N.Dobbert for expert technical assistance and Drs.I. Undretsov,
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18
19
20
21
22
Campo, M.S., and Bishop, J.O. (1974) J.Mol.Biol. 90, 649
Jelinek, W., and Darnell, J.D. (1972) Proc. Nat.Acad. Sci. USA 69, 2537
Nikolaev, N., Silengo, L . , and Schlessinger, D. (1973) Proc.Nat.Acad. Sci.
USA 70, 3361
Dunn, J.J., and Studier, F.W. (1973) Proc. Nat. A cad. Sci. USA 70, 3296
Hercules, K., Schweiger, M., and Sauerbier, W. (1974) Proc.Nat.Acad.Sci.
USA 71, 840.
Downloaded from http://nar.oxfordjournals.org/ at Penn State University (Paterno Lib) on March 3, 2016
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