Volume3 no.6 June1976
Nucleic Acids Research
Globin mRNAk 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 Received 8 March 1976
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, tat 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 ' . About 20 per cent of the ds RNA prepared from a nuclear pre-mRNA (hnRNA)1,2 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 3 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 MEIHODS 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 erythroid precursor-cells was incubated for 15 min at 37°C with H uridine (0. 85 mCI
C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
1487
Nucleic Acids Research /2* 109cells, specific activity, 20-30 CI/mmole, UVVVR, Praha, CSSR), and nuclear 5 RNA was subsequently extracted by the hot phenol fractionation method . According to this method, pre-rRNA is obtained in the 400C fraction and pre-mRNA in the
fractions extracted above 550C 65°C and 85°C fractions were combined and ultracentrifuged through a 12 ml linear 5-20% (w/v) sucrose gradient in a buffer containing 10mM Tris-HCl (pH8), 0.1 M NaCa, 1 mM EDTA, 0. 5% SDS in the Spinco SW 40 .
rotor at 230.
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 TI RNAase (Calbiochem) as 7 described previously . After incubation, the mixture was treated with pronase (Reanal, 100 pg/ml), applied to a Sephadex G-75 column (lx80 cm) and eluted with 7 0.2M sodium acetate buffer . Only the material eluted in the void volume was collected, and treated again with pronase (100 pg/ml) at 250 for 30 min. Thereafter SDS was added to 1% and the mixture was shaken with phenol for 30 min at 600 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 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 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,(1000-2000 cpm radioactive material) were incubated at 650 C for the time necessary to obtain the appropriate Cot value . To calculate the Cot 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 1488
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 pg) of radioactive material was taken, mixed with mRNA and annealed in 2xSSC at 650 for different time intervals to make ds RNA-driven -4 Cot equal to lxlO to 3xlO -4 . The proportion of acid-insoluble radioactive material stable to RNAase was determined as described previously . For preparative isolation of hybridized sequences, about 1O,000 to 15,000 cpm of melted radioactive ds RNA (with specific activity 50,0)) cpm/pg) was mixed with 600 pg of globin mRNA and annealed in 3 ml of 2xSSC at 650 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. 200 pg of pronase (Reanal) were added to stop the reaction.
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 NaaI -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 electrophoresis of RNA. RNA samples were analysed on polyacrylamide gel using a modification of the Loening method as described in the legend to fig.4. Electrophoresis in formamide was carried out according to Pinder Val a 12 (20 nucleotides long) and oligo(U)10 .5S ribosomal 4S transfer, 4 tRNA et al. 1489
Nucleic Acids Research were
used
as
markers.
The gels containing non-labeled markers
with "Stains All" (Eastman-Kodak).
sliced into 2 or
mm
were
stained
The gels containing radioactive material
were
fractions, dissolved in "Tlssue solubilizer" (Amersham/Searle)
in "Aquasol" (New England Nuclear, Boston, Mass.) and counted in toluene.
RESULTS Characterization of ds RNA prepared from pre-mRNA of rabbit bone
Nuclear pre-mRNA
was
fractionation procedure
isolated from bone and the ds
marrow
marrow
cells.
cells using the hot phenol
sequences were
prepared from the high-molecular 7
weight
fraction of
pre-mRNA
as
described
previously
.It should be
pointed
out that
4s
.1,
co
~400 QC
30
Stice No Fig.l_. 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. 1490
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. lshows. 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 short incubation under hybridization conditions (1-2 hr at 650C), the molecular weight of melted ds RNA did not decrease (data not shown). Kinetic studies on the renaturation of the melted ds RNA (fig 2) showed that about 20% of the sequences (in different experiments from 15 to 25%) renature at low Cot valuesL from 10 to 2x-O2, while the rest of the material renatures at higher Cot values. These results are quite similar to those obtained with mouse liver and carcinoma ds RNA.2 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.
0
Go
2
._
-48
ca
2O
40Q
40fo
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. 1491
Nucleic Acids Research The ds RNA Cot was chosen sufficiently low (1-3xl1O Hybridization of melted ds RNA with globin mRNA.
prepared from rabbit reticulocytes in the usual
way
to
minimize its renaturation.
Unlabeled globin mRNA
was
by polysome pelleting, isolation of
polysomal poly(A) RNA followed by purification of 9S poly(A) RNA. This RNA was electrophoretically pure. It can be completely bound to poly(U)-Sepharose and programmes globin synthesis in a heterogeneous cell-free
system6.
A mixture of an excess of globin mRNA with melted ds RNA
formation
was
was
annealed and hybrid
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
RNAase-stable under the conditions used. The addition of mRNA immediately followed by RNAase treatment did not increase the amount of RNAasesequences were
stable material while after annealing with mRNA, the
material increased significantly.
For
an
amount of
RNAase-stable
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
It was observed previously that the In agreement with ' . species but not tissue specific 2,3
amount of RNAase stable material.
hybridization reaction
was
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
poly(U)- Sepharose through the poly(A)-end of mRNA.
the binding of hybrids to
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 did not exceed 2%.
annealed with tRNA
was not
added, the binding to poly(U)-Sepharose
The same background values were obtained when ds RNA was or
with
a
vast excess of poly(A) or when mRNA was added to
ds RNA just before passing through the poly(U)-Sepharose.
of ds RNA observed in
our
experiments after annealing
The highest binding
with mRNA was 30 to 35%o
(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. One may suggest that the hybrid contains the duplex region as well as
1492
Nucleic Acids Research
PC
60
a -1-
-
40
42
3
*r
5
6
7
a
9
Fractton No 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
Exp. N0
Ratio of mRNA to ds RNA (w/w)
b ds RNA sequences hybridized(per cent total )
Technique used for analysis
Rabbit bone marrow ds RNA + rabbit globin mRNA
1 2 3 4
1. 3x 103
5
6
6.3x 103 6.3xl1
7 8
6.3 x 103 6.3x 10
17 30
9
4 1. 8 x 10
1
RNAase
10
1.5xle
1.5
RNAase
6 7
1.3x103
6.3x103
9 28
6.3 x 10
3
Rabbit bone marrow ds RNA + rabbit liver mRNA
Mouse carcinoma ds RNA + rabbit globin mRNA Mouse liver ds RNA + rabbitglobin mRNA
37 11
RNAase RNAase
RNAase poly(U)-Sepharose
~~~~~~~~~~binding b
3
RNAase poly(U)-Sepharose binding
In the majority of experiments ds RNA-driven Cot was about 3 x 10 (it was calculated on the basis of specific radioactivity of pre-mRNA, from which ds RNA was isolated). In exp. N05 the ds RNA-driven Cot was 5 times 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 subtracted from the total counts obtained (see also the reassociation curve in Fig.2, and the control curve in Fig.3).
1493
Nucleic Acids Research the unpaired tail of ds RNA.
Such
an
RNAase treatment of the hybrid bound
interpretation is supported by the fact that to
the poly(U)- Sepharose column, digests
from 30 to 75% of the labeled material. The size of ds RNA-mRNA
from ds RNA
was
can see
one quarter
tRNAVal and oligo(U)10
from Fig.4 that the RNAase-stable material
were
migrates
peak in the region of chains of about 30 nucleotides long.
narrow
material (original melted ds RNA as was
sequence
originated
determined using polyacrylamide gel electrophoresis.
RNA, 5S ribosomal RNiA,
One
The size of the hybridised
hybrid.
mentioned above,
location (in the region of
was
or
Transfer
used
as a
as
rather
Undigested
RNA eluted from the poly(U)-Sepharose column)
located with chains 80-150 nucleotides long.
sequences
5S
markers.
The
same
of 80-150 nucleotides long) was observed for
4S
ftRNA WC
E
0 ioo
0~~ ~~~o3
Sice
No
Fig.4. Polyacrylamide gel electrophoresis of the sequence of 3H-labeled ds RNA hybridized with non-labeled glcbin mRNA. Preparative isolation and purification of The gel hybridized sequences was performed as described in Materials and Methods. 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 950 C for 2 min, chilled and applied to the gels. Electrophoresis was perforned at 100 V for 1.5 hr at 240C. 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. :
1494
Nucleic Acids Research RNAase-stable material of ds RNA annealed without mRNA (data Therefore all values used in Fig.4
were
not
shown).
obtained after substracting this background.
DISCUSSION The results presented in this
paper
demonstrate the existence of
tides long) region in the double-stranded hairpin-like
sequence
is complementary to
In
a
portion of the globin mRNA.
that polysomal poly(A) mRNA of
length of the complementary nucleotides well
as
10
mouse can
sequences was
our
also hybridize
a
short (- 30 nucleo-
of pre-mRNA which
previous study it
to mouse ds
was
shown
RNA, and the
heterogeneous, varying from 10
to
60
The following hypothetical scheme could be drawn from this result 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
\
Pze Rn/fiA '
pos
~ ~ ~ 5'e~ ~ -Rt (A) An 4 MMVu
mRA' saQgence
2i
Ceeavage 8B ds RNAase JO dose
s
ejIRease
of mRNA and
430 nucteot.es Alts t*ansJez to cytopt4sm
Etiminaton o ho6 8zonch
fo
we.
J\end gy/\
modif cation
JO nmcteotives
moture
nRVA
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.
1495
Nucleic Acids Research pairs is localized tive part
on
the border line between the mRNA
of the pre-mRNA.
region, and destroys
a
sequence
and the non-informa-
The processing enzyme recognizes the double-helical
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 their experimental data. Several points of this model should be considered. in the hairpin is unknown.
hybridize to
some
the palindrome
First, the size of the unpaired loop
It was shown previously that the
of the palindromes present in DNA
seems to
be
very
15 .
mouse
ds RNA could
The size of the unpaired loop in
small since this structure is not cleaved with DNAase
16
-RAi is transcribed S1 . However, it has not been proved that ds RNA in the pre-mRNA from the palindrome itself. It could be also transcribed from the complementary sequences identical to palindromic sequences but separated by a long spacer. This rncie
question is under investigation in
our
laboratory. only a few mRNA molecules contain
Second, it is not clear whether all,
or
complementary to ds RNA.
experiments, the mRNA/ds RNA ratio
In
our
sequences was
3
(I.3-6.3)x10 . 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 complementar 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 explain the failure to find internal reiteration heterogeneity in mRNAs elucidate this question, detailed kinetic experiments are necessary.
17-18 .
To
Third, poly(U)-Sepharose experiments showed that a high proportion of ds RNA 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
nucleotides) sequence which is
can
common to most
a short
(- 30
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. 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
1496
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 We thank N. N. Dobbert for expert technical assistance and Drs. I. UVndretsov, V. Schick and V. Scheinker for the gift of tRNA, 4 ttRNA Vaand 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,O.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 Plnder, J.C., Stainov, D.Z., and Gratzer, W.B. (1974) Biochemistry 13, 5373 13 Naora, H., and Whitelam,J.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 1497
Nucleic Acids Research 18 19 20 21 22
1498
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.Acad.Sci. USA 70, 3296 Hercules, K., Schweiger, M., and Sauerbier, W. (1974) Proc.Nat.Acad.Sci. USA 71, 840.
Nucleic Acids Research
Globin mRNAk 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 Received 8 March 1976
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, tat 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 ' . About 20 per cent of the ds RNA prepared from a nuclear pre-mRNA (hnRNA)1,2 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 3 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 MEIHODS 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 erythroid precursor-cells was incubated for 15 min at 37°C with H uridine (0. 85 mCI
C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
1487
Nucleic Acids Research /2* 109cells, specific activity, 20-30 CI/mmole, UVVVR, Praha, CSSR), and nuclear 5 RNA was subsequently extracted by the hot phenol fractionation method . According to this method, pre-rRNA is obtained in the 400C fraction and pre-mRNA in the
fractions extracted above 550C 65°C and 85°C fractions were combined and ultracentrifuged through a 12 ml linear 5-20% (w/v) sucrose gradient in a buffer containing 10mM Tris-HCl (pH8), 0.1 M NaCa, 1 mM EDTA, 0. 5% SDS in the Spinco SW 40 .
rotor at 230.
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 TI RNAase (Calbiochem) as 7 described previously . After incubation, the mixture was treated with pronase (Reanal, 100 pg/ml), applied to a Sephadex G-75 column (lx80 cm) and eluted with 7 0.2M sodium acetate buffer . Only the material eluted in the void volume was collected, and treated again with pronase (100 pg/ml) at 250 for 30 min. Thereafter SDS was added to 1% and the mixture was shaken with phenol for 30 min at 600 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 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 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,(1000-2000 cpm radioactive material) were incubated at 650 C for the time necessary to obtain the appropriate Cot value . To calculate the Cot 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 1488
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 pg) of radioactive material was taken, mixed with mRNA and annealed in 2xSSC at 650 for different time intervals to make ds RNA-driven -4 Cot equal to lxlO to 3xlO -4 . The proportion of acid-insoluble radioactive material stable to RNAase was determined as described previously . For preparative isolation of hybridized sequences, about 1O,000 to 15,000 cpm of melted radioactive ds RNA (with specific activity 50,0)) cpm/pg) was mixed with 600 pg of globin mRNA and annealed in 3 ml of 2xSSC at 650 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. 200 pg of pronase (Reanal) were added to stop the reaction.
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 NaaI -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 electrophoresis of RNA. RNA samples were analysed on polyacrylamide gel using a modification of the Loening method as described in the legend to fig.4. Electrophoresis in formamide was carried out according to Pinder Val a 12 (20 nucleotides long) and oligo(U)10 .5S ribosomal 4S transfer, 4 tRNA et al. 1489
Nucleic Acids Research were
used
as
markers.
The gels containing non-labeled markers
with "Stains All" (Eastman-Kodak).
sliced into 2 or
mm
were
stained
The gels containing radioactive material
were
fractions, dissolved in "Tlssue solubilizer" (Amersham/Searle)
in "Aquasol" (New England Nuclear, Boston, Mass.) and counted in toluene.
RESULTS Characterization of ds RNA prepared from pre-mRNA of rabbit bone
Nuclear pre-mRNA
was
fractionation procedure
isolated from bone and the ds
marrow
marrow
cells.
cells using the hot phenol
sequences were
prepared from the high-molecular 7
weight
fraction of
pre-mRNA
as
described
previously
.It should be
pointed
out that
4s
.1,
co
~400 QC
30
Stice No Fig.l_. 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. 1490
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. lshows. 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 short incubation under hybridization conditions (1-2 hr at 650C), the molecular weight of melted ds RNA did not decrease (data not shown). Kinetic studies on the renaturation of the melted ds RNA (fig 2) showed that about 20% of the sequences (in different experiments from 15 to 25%) renature at low Cot valuesL from 10 to 2x-O2, while the rest of the material renatures at higher Cot values. These results are quite similar to those obtained with mouse liver and carcinoma ds RNA.2 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.
0
Go
2
._
-48
ca
2O
40Q
40fo
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. 1491
Nucleic Acids Research The ds RNA Cot was chosen sufficiently low (1-3xl1O Hybridization of melted ds RNA with globin mRNA.
prepared from rabbit reticulocytes in the usual
way
to
minimize its renaturation.
Unlabeled globin mRNA
was
by polysome pelleting, isolation of
polysomal poly(A) RNA followed by purification of 9S poly(A) RNA. This RNA was electrophoretically pure. It can be completely bound to poly(U)-Sepharose and programmes globin synthesis in a heterogeneous cell-free
system6.
A mixture of an excess of globin mRNA with melted ds RNA
formation
was
was
annealed and hybrid
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
RNAase-stable under the conditions used. The addition of mRNA immediately followed by RNAase treatment did not increase the amount of RNAasesequences were
stable material while after annealing with mRNA, the
material increased significantly.
For
an
amount of
RNAase-stable
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
It was observed previously that the In agreement with ' . species but not tissue specific 2,3
amount of RNAase stable material.
hybridization reaction
was
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
poly(U)- Sepharose through the poly(A)-end of mRNA.
the binding of hybrids to
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 did not exceed 2%.
annealed with tRNA
was not
added, the binding to poly(U)-Sepharose
The same background values were obtained when ds RNA was or
with
a
vast excess of poly(A) or when mRNA was added to
ds RNA just before passing through the poly(U)-Sepharose.
of ds RNA observed in
our
experiments after annealing
The highest binding
with mRNA was 30 to 35%o
(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. One may suggest that the hybrid contains the duplex region as well as
1492
Nucleic Acids Research
PC
60
a -1-
-
40
42
3
*r
5
6
7
a
9
Fractton No 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
Exp. N0
Ratio of mRNA to ds RNA (w/w)
b ds RNA sequences hybridized(per cent total )
Technique used for analysis
Rabbit bone marrow ds RNA + rabbit globin mRNA
1 2 3 4
1. 3x 103
5
6
6.3x 103 6.3xl1
7 8
6.3 x 103 6.3x 10
17 30
9
4 1. 8 x 10
1
RNAase
10
1.5xle
1.5
RNAase
6 7
1.3x103
6.3x103
9 28
6.3 x 10
3
Rabbit bone marrow ds RNA + rabbit liver mRNA
Mouse carcinoma ds RNA + rabbit globin mRNA Mouse liver ds RNA + rabbitglobin mRNA
37 11
RNAase RNAase
RNAase poly(U)-Sepharose
~~~~~~~~~~binding b
3
RNAase poly(U)-Sepharose binding
In the majority of experiments ds RNA-driven Cot was about 3 x 10 (it was calculated on the basis of specific radioactivity of pre-mRNA, from which ds RNA was isolated). In exp. N05 the ds RNA-driven Cot was 5 times 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 subtracted from the total counts obtained (see also the reassociation curve in Fig.2, and the control curve in Fig.3).
1493
Nucleic Acids Research the unpaired tail of ds RNA.
Such
an
RNAase treatment of the hybrid bound
interpretation is supported by the fact that to
the poly(U)- Sepharose column, digests
from 30 to 75% of the labeled material. The size of ds RNA-mRNA
from ds RNA
was
can see
one quarter
tRNAVal and oligo(U)10
from Fig.4 that the RNAase-stable material
were
migrates
peak in the region of chains of about 30 nucleotides long.
narrow
material (original melted ds RNA as was
sequence
originated
determined using polyacrylamide gel electrophoresis.
RNA, 5S ribosomal RNiA,
One
The size of the hybridised
hybrid.
mentioned above,
location (in the region of
was
or
Transfer
used
as a
as
rather
Undigested
RNA eluted from the poly(U)-Sepharose column)
located with chains 80-150 nucleotides long.
sequences
5S
markers.
The
same
of 80-150 nucleotides long) was observed for
4S
ftRNA WC
E
0 ioo
0~~ ~~~o3
Sice
No
Fig.4. Polyacrylamide gel electrophoresis of the sequence of 3H-labeled ds RNA hybridized with non-labeled glcbin mRNA. Preparative isolation and purification of The gel hybridized sequences was performed as described in Materials and Methods. 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 950 C for 2 min, chilled and applied to the gels. Electrophoresis was perforned at 100 V for 1.5 hr at 240C. 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. :
1494
Nucleic Acids Research RNAase-stable material of ds RNA annealed without mRNA (data Therefore all values used in Fig.4
were
not
shown).
obtained after substracting this background.
DISCUSSION The results presented in this
paper
demonstrate the existence of
tides long) region in the double-stranded hairpin-like
sequence
is complementary to
In
a
portion of the globin mRNA.
that polysomal poly(A) mRNA of
length of the complementary nucleotides well
as
10
mouse can
sequences was
our
also hybridize
a
short (- 30 nucleo-
of pre-mRNA which
previous study it
to mouse ds
was
shown
RNA, and the
heterogeneous, varying from 10
to
60
The following hypothetical scheme could be drawn from this result 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
\
Pze Rn/fiA '
pos
~ ~ ~ 5'e~ ~ -Rt (A) An 4 MMVu
mRA' saQgence
2i
Ceeavage 8B ds RNAase JO dose
s
ejIRease
of mRNA and
430 nucteot.es Alts t*ansJez to cytopt4sm
Etiminaton o ho6 8zonch
fo
we.
J\end gy/\
modif cation
JO nmcteotives
moture
nRVA
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.
1495
Nucleic Acids Research pairs is localized tive part
on
the border line between the mRNA
of the pre-mRNA.
region, and destroys
a
sequence
and the non-informa-
The processing enzyme recognizes the double-helical
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 their experimental data. Several points of this model should be considered. in the hairpin is unknown.
hybridize to
some
the palindrome
First, the size of the unpaired loop
It was shown previously that the
of the palindromes present in DNA
seems to
be
very
15 .
mouse
ds RNA could
The size of the unpaired loop in
small since this structure is not cleaved with DNAase
16
-RAi is transcribed S1 . However, it has not been proved that ds RNA in the pre-mRNA from the palindrome itself. It could be also transcribed from the complementary sequences identical to palindromic sequences but separated by a long spacer. This rncie
question is under investigation in
our
laboratory. only a few mRNA molecules contain
Second, it is not clear whether all,
or
complementary to ds RNA.
experiments, the mRNA/ds RNA ratio
In
our
sequences was
3
(I.3-6.3)x10 . 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 complementar 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 explain the failure to find internal reiteration heterogeneity in mRNAs elucidate this question, detailed kinetic experiments are necessary.
17-18 .
To
Third, poly(U)-Sepharose experiments showed that a high proportion of ds RNA 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
nucleotides) sequence which is
can
common to most
a short
(- 30
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. 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
1496
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 We thank N. N. Dobbert for expert technical assistance and Drs. I. UVndretsov, V. Schick and V. Scheinker for the gift of tRNA, 4 ttRNA Vaand 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,O.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 Plnder, J.C., Stainov, D.Z., and Gratzer, W.B. (1974) Biochemistry 13, 5373 13 Naora, H., and Whitelam,J.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 1497
Nucleic Acids Research 18 19 20 21 22
1498
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.Acad.Sci. USA 70, 3296 Hercules, K., Schweiger, M., and Sauerbier, W. (1974) Proc.Nat.Acad.Sci. USA 71, 840.
