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INTRODUCTION |
The evolutionarily conserved family of cold-shock
domain-containing proteins (CSD
proteins)1 is represented in
organisms from bacteria to man by multifunctional DNA/RNA-binding
proteins (1, 2). In bacteria, some of them known as major cold-shock
proteins are responsible for adaptation to growth at low temperatures,
and their expression is enormously activated with a temperature
decrease (3, 4). In mammalian cells, CSD proteins regulate cell
proliferation and differentiation and are involved in cell defense
systems (5-11). In the cell nucleus, CSD proteins regulate
transcription by interacting with promoters and enhancers of many genes
(9, 12-17). They are also involved in DNA replication and repair, as
well as in mRNA splicing (5, 18-20). In the cytoplasm, CSD
proteins bind mRNAs, affecting their translation fate (21-28) and
extending their lifetime (29).
In bacteria, accumulation of major cold-shock proteins at low
temperatures was shown to result mainly from strong and selective stabilization of their mRNAs (30, 31). The crucial role of eukaryotic CSD proteins in major cellular events suggests that there is
precise regulation of their expression. At present the post-transcriptional control of eukaryotic gene expression is to a
large extent attributed to the presence of specific sequences within
mRNA 5'- and 3'-untranslatable regions (UTRs), which serve as
targets for binding of certain proteins and complementary RNAs (32,
33). Here, we studied this type of regulation during in
vitro synthesis of rabbit p50, a member of the eukaryotic CSD protein family, which is the major protein of reticulocyte mRNPs and is
virtually identical to the human Y-box binding protein YB-1. To
determine a possible role of YB-1 mRNA UTRs in YB-1
post-transcriptional regulation, we examined effects of exogenous
mRNA fragments containing different UTR parts on YB-1 mRNA
translation and stability in a cell-free system from rabbit reticulocytes.
We found that addition of the 3' mRNA subfragment I, including the
last 42 nucleotides of YB-1 mRNA coding region and the first 107 nucleotides of its 3'-UTR, shut off YB-1 synthesis mainly at the
initiation stage without affecting mRNA stability. The inhibition
by this subfragment was also observed in cell-free systems translating
luciferase or endogenous globin mRNAs. In contrast, subfragment II
containing the rest of YB-1 mRNA 3'-UTR or the 5' fragment did not
affect translation. Two proteins were found to specifically bind the
inhibitory subfragment I, leading us to believe that they play an
important role in mRNA translation. We have identified these
proteins as poly(A)-binding protein (PABP) (69 kDa) and YB-1 (50 kDa)
and demonstrated that titrating out of PABP by poly(A) strongly and
specifically inhibited YB-1 both cap+poly(A)
and cap+poly(A)+ mRNA translation in a
cell-free system. Thus, PABP is capable of positively affecting YB-1
mRNA translation in a poly(A) tail-independent manner.
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EXPERIMENTAL PROCEDURES |
Plasmid Construction--
pBluescript II SK YB-1 construct
containing YB-1 cDNA, pSP36T-LucA50 construct containing 5'-UTR of
-globin mRNA fused with the luciferase coding sequence, and
pSP64/CAT were described previously (34-36)
pBluescript II SK YB-1 A50 was made as follows. The fragment containing
50-nucleotide-long poly(A) was cut from pSP36T-LucA50 construct by
digesting with HindIII and PstI and ligated
between the same sites of pBluescript II SK (Stratagene). The obtained construction was named pBluescript II SK A50. Then YB-1 gene was cut
from pBluescript II SK YB-1 by digesting with HindIII and SmaI and ligated between HindIII and
BglII of pBluescript II SK A50 (blunt ends after
BglII restriction were made using the Klenow fragment).
pBluescript II SK 3'-UTR YB-1 construct was made as follows. The 3'-UTR
fragment of YB-1 cDNA was digested with PvuII and BamHI from pBluescript II SK YB-1 and ligated between the
EcoRV and BamHI sites of pBluescript II SK (Fig.
1).
pBluescript II SK subfragment II 3'-UTR YB-1 construct was made as
follows. 5'-UTR, the coding sequence, and 3'-UTR subfragment I of YB-1
cDNA were removed from pBluescript II SK YB-1 construct by
digesting with EcoRV and BspTI, followed by
fill-in reaction with Klenow fragment and blunt end religation (Fig.
1).
In Vitro Transcription--
Luciferase
cap+poly(A)
mRNA was transcribed by SP6
RNA polymerase from pSP36T-LucA50 linearized with BglII.
Luciferase cap+poly(A)+ mRNA was
transcribed from the plasmid linearized with PstI. CAT mRNA was transcribed by SP6 RNA polymerase from pSP64/CAT
linearized with BamHI. Transcription of YB-1 mRNA and
its fragments was carried out by T7 RNA polymerase. The DNA template
for YB-1 cap+poly(A)
mRNA was pBluescript
II SK YB-1 linearized with BamHI; for YB-1 cap+poly(A)+ mRNA was pBluescript II SK
YB-1 A50 linearized with SmaI; for the 5' RNA fragment (132 nt long), pBluescript II SK YB-1 linearized with Bli736; for
the 3'-UTR fragment (273 nt), pBluescript II SK 3'-UTR YB-1 linearized
with DraI; for the subfragment I (149 nt), pBluescript II SK
3'-UTR YB-1 linearized with BspTI; and for subfragment II
(124 nt), the pBluescript II SK subfragment II 3'-UTR YB-1 linearized
with DraI.
The transcription was performed as described previously (37). Capped
transcripts were obtained by a reaction where instead of 5 mM GTP a mixture of 0.2 mM GTP and 0.8 mM m7GpppG (Amersham Biosciences) was used. To generate
32P-labeled fragments of YB-1 mRNA,
[32P]UTP (2000 Ci/mM; Radioisotop) was
added to the reaction, and the concentration of unlabeled UTP was
reduced to 0.05 mM.
In Vitro Translation Assays--
Translation of endogenous or
exogenous mRNA in a rabbit reticulocyte cell-free system was
performed as described previously (38). The incubation mixture (15 µl) contained reticulocyte lysate (7.5 µl), 10 mM
Hepes-KOH, pH 7.6, 100 mM potassium acetate, 1 mM magnesium acetate, 8 mM creatine phosphate,
0.5 mM spermidine, 0.2 mM GTP, 0.8 mM ATP, 1 mM dithiothreitol, 25 µM of each of 20 amino acids except for the labeled ones
(Leu or Met), and 6 µCi of [14C]Leu (>300 mCi/mmol;
Radioisotop) or 1 µCi of [35S]Met (>700 Ci/mmol;
Radioisotop). In the case of micrococcal nuclease (MN)-treated lysate,
the mixture was supplemented with appropriate mRNA as indicated in
the figure legends. Translation was carried out at 30 °C for 2 h. The incorporation of [14C]Leu into the protein was
measured in 5-µl aliquots as described previously (34).
[35S]Met-incorporated proteins were analyzed by 10-22%
SDS-PAGE followed by autoradiography. The relative amount of
radioactivity in the bands was determined using a Packard Cyclone
Storage Phosphor System (Packard Instrument Co., Inc.).
Sucrose Gradient Analysis--
Cell-free translation mixtures
(30 µl) with endogenous mRNA were incubated at 30 °C for 5 min, cooled, and immediately layered onto 15-40% (w/v) linear sucrose
gradients in buffer containing 10 mM Tris-HCl, pH 7.6, 100 mM NaCl, 5 mM MgCl2. Centrifugation was carried out at 45,000 rpm in an SW-60 rotor (Beckman Instruments) for 50 min. All gradients were monitored for absorbance at 254 nm
during their collection from the bottom.
Northern Blot Analysis--
Total RNA was extracted from
translation mixtures by the phenol/chloroform method. 7 µg of total
RNA from each reaction (measured by A260) was
separated by electrophoresis on a denaturing 1.5% agarose gel
containing 2.2 M formaldehyde and 1× MOPS buffer (40 mM MOPS, 10 mM sodium acetate, 1 mM
EDTA, pH 7.0). RNA was transferred onto a nylon membrane (Hybond-N,
Amersham Biosciences) and cross-linked using transilluminator
cross-linker (Vilber-Lourmat) at 0.15 J/cm2. Membrane-bound
RNA was hybridized to a 1054-nt fragment of YB-1 cDNA probe or a
600-nt fragment of
-globin cDNA probe labeled with
[32P]dATP (Multiprime DNA labeling system, Amersham
Biosciences) in 50% formamide, 5× SSC, 5x Denhard's solution, 1%
SDS, 100 µg/ml denatured salmon sperm DNA at 65 °C for 12-16 h.
The membrane was washed twice with 2× SSC, 0.1% SDS for 5 min at room
temperature, twice with 0.2× SSC, 0.1% SDS for 5 min at room
temperature, twice with 0.2× SSC, 0.1% SDS for 15 min at 42 °C,
and twice with 0.1× SSC, 0.1% SDS for 15 min at 68 °C and analyzed
by autoradiography.
Electrophoretic Mobility Shift Assay--
0.5 µl of MN-treated
rabbit reticulocyte lysate was incubated with radiolabeled RNA (10,000 cpm) in a final volume of 10 µl (10 mM Hepes-KOH, pH 7.6, 100 mM KCl, 0.5 mM dithiothreitol, 1 mg/ml bovine serum albumin) at 30 °C for 15 min. In the case of proteinase K treatment, 10 µg of proteinase K and SDS up to 0.1% were added and followed by incubation for 5 min. After incubation, the
protein-RNA complexes were analyzed by 6% PAGE in 0.5× TBE buffer (1×: 89 mM Tris, 89 mM boric
acid, 2 mM EDTA), followed by autoradiography.
UV Cross-linking Assays--
5 µl MN-treated rabbit
reticulocyte lysate was incubated with radiolabeled RNA (100,000 cpm)
in a final volume of 18 µl (10 mM Hepes-KOH, pH 7.6, 100 mM KCl) at 30 °C for 15 min. The reactions were
irradiated at 1.5 J/cm2 in a transilluminator cross-linker
(Vilber-Lourmat), incubated for 45 min at 37 °C with 0.05 unit/µl
MN and 0.5 µg/µl RNase A, and analyzed by 10-22% SDS-PAGE
followed by autoradiography.
Immunoprecipitation--
UV cross-linking mixtures (20 µl)
were preincubated with anti-YB-1 or preimmune antibodies (12 µg) in a
final volume of 200 µl (10 mM Hepes-KOH, pH 7.6, 100 mM KCl) at 25 °C for 4 h with gentle agitation.
Protein A-Sepharose beads (Amersham Biosciences) were added, and
incubation was continued for 1 h. The beads were washed three
times with 100 µl of buffer (10 mM Hepes-KOH, pH 7.6, 140 mM KCl, 0.05% Tween 20), and bound proteins were eluted with 100 mM glycin, pH 3.0, neutralized, trichloroacetic
acid-precipitated, and analyzed by 10-22% SDS-PAGE followed by autoradiography.
Depletion of YB-1 from Reticulocyte Lysate--
For
immunodepletion experiments, nuclease-treated reticulocyte lysate (30 µl) was diluted 3-fold with buffer (10 mM Hepes-KOH, pH
7.6, 100 mM KCl) and incubated with 20 µl of anti-YB-1 or
preimmune antibodies fixed on protein A-Sepharose beads for 2 h at
room temperature with gentle agitation. Then the protein A-Sepharose beads were removed by centrifugation. The efficiency of depletion of
YB-1 was controlled by immunoblotting.
Depletion of PABP from Reticulocyte Lysate--
Nuclease-treated
reticulocyte lysate (300 µl) was diluted 3-fold with buffer (10 mM Hepes-KOH, pH 7.6, 200 mM KCl) and incubated with 200 µl of poly(A)-agarose beads (Amersham Biosciences) for 2 h at 0 °C with gentle agitation. Then the poly(A)-agarose
beads were removed by centrifugation. The efficiency of depletion of PABP was controlled by immunoblotting.
Immunoblotting--
For Western blot analysis, proteins were
separated by SDS-PAGE and transferred to a nitrocellulose membrane. The
membrane was blocked overnight at 20 °C with 1% bovine serum
albumin, 1% polyvinylpyrrolidone, and 0.05% Tween 20 in buffer (10 mM Tris-HCl, pH 7.6, and 150 mM NaCl) and
probed with appropriate antibodies. Immunocomplexes were detected with
alkaline phosphatase-coupled secondary antibodies (Promega) according
to the manufacturer's recommendation.
YB-1 and PABP Purification--
YB-1 and PABP from mRNPs of
rabbit reticulocytes were obtained as described earlier (39). After
dissociation of ribosomes using 33 mM EDTA polyribosomal
mRNPs were adsorbed to the oligo(dT)-cellulose (Amersham Biosciences)
in buffer (10 mM Tris-HCl, pH 7.6, 250 mM NaCl,
2 mM EDTA, and 0.5 mg/ml heparin) at 4 °C and then the column was washed with the same buffer solution. mRNPs were eluted with
buffer (10 mM Tris-HCl, pH 7.6, 2 mM EDTA) at
37 °C, pelleted by centrifugation at 100,000 rpm for 2 h in the
type TLA-100.3 rotor (Beckman) at 4 °C and dissolved in buffer (10 mM Tris-HCl, pH 7.6, 100 mM NaCl). mRNP
proteins dissociated from mRNA using 3 M LiCl, and the
mRNA was precipitated overnight at
20 °C. The RNA precipitate
was removed by centrifugation at 12,000 rpm for 15 min at 4 °C.
Proteins from the supernatant fraction were dialyzed against buffer (10 mM Hepes-KOH, pH 7.6, 200 mM KCl) and separated from aggregates by centrifugation at 12,000 rpm for 15 min at 4 °C.
About 500 µg of mRNP proteins were applied to a Superose 6 HR/10/30
column (Amersham Biosciences) equilibrated with buffer (10 mM Hepes-KOH, pH 7.6, 200 mM KCl).
Chromatography was done at a flow rate of 0.4 ml/min using a fast
protein liquid chromatography system. The fraction volume was 1.5 ml.
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RESULTS |
3' Fragment of YB-1 mRNA Inhibits YB-1 in Vitro
Synthesis--
As we mentioned above, in eukaryotes, 5'- and 3'-UTRs
of mRNA are involved in regulation of activity, stability, and
intracellular distribution of mRNA through interaction with
specific regulatory proteins or RNAs (32, 33, 40-42). Here we studied
the effect of exogenous 5' and 3' fragments of rabbit YB-1 mRNA
(Fig. 1) on YB-1 synthesis in a rabbit
reticulocyte cell-free translation system. If these mRNA fragments
compete with YB-1 mRNA for binding to specific regulatory proteins
or RNAs, we would expect to detect a change in YB-1 mRNA activity
and/or stability.

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Fig. 1.
Diagram of fragments of YB-1 mRNA used in
experiments and nucleotide sequence of subfragment I. The stop
codon is underlined. The sequence that presumably
specifically binds YB-1 is in the shaded box.
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The addition of the 3' fragment containing the 231-nt UTR and the last
42 nt of YB-1 coding sequence to the cell-free system translating YB-1
cap+poly(A)
mRNA strongly inhibited YB-1
synthesis (Fig. 2A,
lanes 3 and 4). In contrast, the 132-nt 5'
fragment containing 5'-UTR did not affect YB-1 mRNA translation
(Fig. 2A, lanes 5 and 6). To better
localize the inhibitory signal within the 3' fragment, two its
subfragments were tested in a cell-free translation system. The
upstream 149-nt-long subfragment I caused strong inhibition of YB-1
synthesis (Fig. 2B, lanes 6 and 8),
while the downstream 124-nt-long subfragment II did not (lanes
9-11).

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Fig. 2.
Effect of 3' and 5'
fragments of YB-1 mRNA on YB-1 synthesis in the rabbit
reticulocyte cell-free translation system. Translation reactions
were carried out in 15 µl aliquots for 2 h at 30 °C in the
presence of 0.2 pmol of YB-1 mRNA
cap+poly(A) . 3' or 5' fragments were added as
indicated. [35S]Met-labeled translation products were
resolved by SDS-PAGE and visualized by autoradiography. A,
effect of 3' or 5' fragments (0.8 or 2.4 pmol) on YB-1 synthesis.
B, effect of subfragment I or II (0.8, 1.6, or 2.4 pmol) on
YB-1 synthesis.
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The addition of subfragment I also inhibited translation of luciferase
cap+poly(A)
mRNA (Fig.
3A) and natural globin
mRNA (Fig. 3B), demonstrating that this inhibition is
not specific for YB-1 mRNA translation.

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Fig. 3.
Effect of subfragments I and II on
translation of luciferase and natural globin mRNAs.
Translation reactions were carried out in 15 µl aliquots for 2 h
at 30 °C. A, effect of subfragment I or II (0.8, 1.6, and
2.4 pmol) on translation of luciferase
cap+poly(A) mRNA (0.2 pmol).
[35S]Met-labeled translation products were resolved by
SDS-PAGE and visualized by autoradiography. B, effect of
subfragment I or II (1.6, 3.2, 4.8, and 6.4 pmol) on endogenous globin
mRNA translation. Incorporation of [14C]Leu into
proteins was measured.
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Subfragment I of YB-1 mRNA Inhibits Translation Initiation
without Affecting mRNA Stability--
The stage of protein
synthesis affected by subfragment I may be identified by studying its
effect on the polysomal profile in a rabbit reticulocyte cell-free
system. Inhibition of translation at the stage of initiation is known
to be accompanied by a decay of polysomes, whereas inhibition of
elongation/termination results in larger polysomes. Subfragment I or II
was added to the cell-free translation system with endogenous mRNA,
and the mixture was incubated for 5 min and analyzed by centrifugation
in sucrose gradient. The polysomal profiles are shown in Fig.
4. The addition of subfragment I resulted
in a complete decay of polysomes (cf. Fig. 4, B
with A, presenting control with no subfragment), while with
subfragment II added, the profile was similar to that of the control
(cf. Fig. 4, C with A). The
subfragment I-induced decay of polysomes may result from inhibition of
translation at the initiation stage. However, it cannot be ruled out
that subfragment I may stimulate mRNA degradation, which would also
result in a decay of polysomes.

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Fig. 4.
Effect of subfragments I and II on the
polysomal profile and mRNA integrity in rabbit reticulocyte
lysate. Rabbit reticulocyte cell-free systems with endogenous
mRNA (15 µl of lysate) were incubated for 5 min at 30 °C in
the presence or absence (control) of subfragment I or II (12.3 pmol).
A-C, after incubation the systems were subjected to
centrifugation through 15-40% linear sucrose gradients for 50 min at
45,000 rpm in an SW-60 rotor. UV absorption profile of polysomes at 254 nm and sedimentation positions of 80 S monosomes are shown: without any
fragments (control) (A), with subfragment I (B),
and with subfragment II (C). D-F, total RNA was
isolated from the incubated systems, subjected to agarose gel
electrophoresis and Northern blot hybridization to YB-1 cDNA
(D) and -globin cDNA (E), or stained with
ethidium bromide in the gel (F). D, YB-1 mRNA
(lane 1), total RNA from lysate incubated without any
fragments (control) (lane 2), with subfragment I (lane
3), and with subfragment II (lane 4). E,
-globin mRNA (lane 1), total RNA from lysate
incubated without any fragments (control) (lane 2), with
subfragment I (lane 3), and with subfragment II (lane
4). F, YB-1 mRNA (lane 1), -globin
mRNA (lane 2), total RNA from lysate incubated without
any fragments (control) (lane 3), with subfragment I
(lane 4), and with subfragment II (lane 5).
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To verify the latter suggestion, we studied the effect of subfragments
I and II on YB-1 and
-globin mRNA stability in the translation
reaction. The total RNA was isolated from cell-free translation systems
described above after 5-min incubation, subjected to gel
electrophoresis (Fig. 4F), and analyzed (along with control YB-1 or
-globin mRNA) by Northern blot hybridization to
32P-labeled YB-1 (Fig. 4D) or
-globin
cDNA (Fig. 4E). No notable difference in mRNA
content and size was observed between experiment and control. This
allowed concluding that polysome decay caused by subfragment I did not
result from mRNA degradation but was caused by inhibition of
translation itself mostly or exclusively at the stage of initiation.
Four Proteins Specifically Interact with the 3' Fragment of YB-1
mRNA--
To ascertain the possible proteins interacting with the
3' fragment and affecting mRNA activity, we performed an
electrophoretic mobility shift experiment using reticulocyte lysate and
32P-labeled 3' fragment. Addition of the lysate to this
labeled fragment resulted in retardation of the labeled band in the gel (Fig. 5A, compare lanes
1 and 2). This retardation effect was avoided by
proteinase K treatment (Fig. 5A, lane 3) and was
sensitive to the amount of non-labeled 3' fragment added (lanes
4-6), but not to addition of other RNAs (lanes 7-12),
revealing specific binding of some lysate protein(s) to the labeled
fragment. To characterize these proteins, we UV-irradiated the mixture
containing reticulocyte lysate and the labeled 3' fragment. Four
proteins with molecular weights of about 69, 50, 46, and 44 kDa were
found to cross-link to the labeled fragment (Fig. 5B,
lane 2). The same proteins were cross-linked to the labeled
3' fragment under conditions of an active translation system with
exogenous or endogenous mRNA (2- and 15-min incubation). These
proteins, with the exception for 50-kDa protein, are efficiently
competed out by the non-labeled 3' fragment (Fig. 5B,
lanes 3-5). Other RNAs used in this experiment demonstrated
less competition (Fig. 5B, lanes 6-14) revealing that these three proteins, 69, 46, and 44 kDa, bound the 3' fragment specifically. The inability of the non-labeled 3' fragment to efficiently substitute for the labeled fragment in retarded complexes (Fig. 5A, lanes 4-6) and in UV cross-linking to
50-kDa protein (Fig. 5B, lanes 3-5) indicated
that there was an excess of 50 kDa protein in the lysate. For this
reason we could not yet conclude that the interaction of the 3'
fragment of YB-1 mRNA with 50-kDa protein was specific.

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Fig. 5.
Interaction of 3' fragment of YB-1
mRNA with proteins in rabbit reticulocyte lysate.
A, aliquots of rabbit reticulocyte lysate (0.5 µl) were
incubated for 15 min at 30 °C with 32P-labeled 3'
fragment (0.04 pmol, 5 ng) in the presence of increasing amounts of
unlabeled 3' fragment, 5' fragment of YB-1 mRNA, and -globin
mRNA (50, 100, and 200 ng). RNA-protein complexes were analyzed by
PAGE under non-denaturing conditions and visualized by autoradiography.
B, aliquots of rabbit reticulocyte lysate (5 µl) were
incubated for 15 min at 30 °C with 32P-labeled 3'
fragment (0.33 pmol, 36 ng) in the presence of unlabeled 3' fragment,
5' fragment of YB-1 mRNA, -globin mRNA, and dihydrofolate
reductase mRNA (72, 144, and 288 ng, respectively), UV
cross-linked, treated with RNase A and MN, analyzed by SDS-PAGE, and
visualized by autoradiography. DHFR, dihydrofolate
reductase.
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Next, we studied the binding specificity of 69-, 50-, 46-, and 44-kDa
proteins to subfragments I and II of the 3' fragment to determine which
of these proteins may affect mRNA translational activity. Using UV
cross-linking, we analyzed the binding of lysate proteins to the
labeled 3' fragment in the presence of increasing amounts of
non-labeled competitors: 3' fragment, subfragment I, and subfragment
II. Similarly to what was shown above, the non-labeled 3' fragment
efficiently competed out 69-, 46-, and 44-kDa proteins from the complex
containing the labeled fragment (Fig.
6A, lanes 3-5).
Moreover, we found that subfragment I competed with the labeled
fragment only for 69-kDa protein (Fig. 6A, lanes
6-8), while subfragment II competed only for 46- and
44-kDa proteins (lanes 9-11).

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Fig. 6.
Interaction of subfragments I and II of YB-1
mRNA with proteins in rabbit reticulocyte lysate.
A, aliquots of rabbit reticulocyte lysate (5 µl) were
incubated for 15 min at 30 °C with 32P-labeled 3'
fragment (0.33 pmol) in the presence of increasing amounts of unlabeled
3' fragment or each of its two subfragments (0.33, 0.65, and 1.3 pmol),
UV cross-linked, treated with RNase A and MN, analyzed by SDS-PAGE, and
visualized by autoradiography. B, aliquots of rabbit
reticulocyte lysate (5 µl) were incubated for 15 min at 30 °C with
32P-labeled 3' fragment or each of its two subfragments
(0.2 pmol), UV cross-linked, treated with RNase A and MN, analyzed by
SDS-PAGE, and visualized by autoradiography.
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Then, the 3' fragment and subfragments I and II were radioactively
labeled and incubated with the lysate separately. Analysis of proteins
UV cross-linked to each of these RNAs revealed that 69- and 50-kDa
proteins specifically bound subfragment I (Fig. 6B,
lane 5), while 46- and 44-kDa proteins bound subfragment II (lane 6). This result suggests that 50- and 69-kDa proteins
may be involved in protein synthesis and promote translation, and their
specific binding by exogenous subfragment I may cause inhibition of
translation. However, we detected much higher amounts of 50-kDa protein
compared with 69 kDa in reticulocyte lysate, which makes the latter a
more likely candidate.
50-kDa Protein Specifically Interacting with Subfragment I Is
YB-1--
Subfragment I of YB-1 mRNA was found to have the motif
AACAUC in its UTR portion (Fig. 1). According to the literature, this motif specifically interacts with Xenopus FRGY2 that
displays 64% homology to YB-1 (43). This led us to believe that 50-kDa protein specifically cross-linked to subfragment I in rabbit
reticulocyte lysate is YB-1. To verify this suggestion, YB-1 isolated
from mRNPs of rabbit reticulocytes was cross-linked to
32P-labeled subfragment I in the presence of increasing
amounts of unlabeled subfragment I or II as a competitor. Subfragment I
substituted for its labeled form in the complex with YB-1 (Fig. 7A, lanes 4-6)
much more efficiently than subfragment II (lanes 7-9),
thereby showing YB-1 specificity for subfragment I.

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Fig. 7.
Identification of 50-kDa protein specifically
interacting with YB-1 mRNA subfragment I as YB-1.
A, 2.8 pmol YB-1 from rabbit reticulocyte mRNPs (lanes
1 and 3-9) or 5 µl of rabbit reticulocyte lysate
(lane 2) was incubated for 15 min at 30 °C with
32P-labeled subfragment I (100,000 cpm) in the presence of
unlabeled subfragment I or II (0.18, 0.36, and 0.72 pmol), UV
cross-linked (except for lane 1), treated with RNase A and
MN, analyzed by SDS-PAGE, and visualized by autoradiography.
B, aliquots of rabbit reticulocyte lysate (5 µl) were
incubated for 15 min at 30 °C with 32P-labeled
subfragment I (100,000 cpm), UV cross-linked, treated with RNase A and
MN, incubated with anti-YB-1 or preimmune antibodies, and precipitated
using protein À-Sepharose beads. Proteins from eluate
(lanes 2 and 4) and flow-through (lanes
3 and 5) were analyzed by SDS-PAGE and visualized by
autoradiography. C, MN-treated rabbit reticulocyte lysate
(lane 1) was incubated with anti-YB-1 (lane 2) or
preimmune (lane 3) antibodies as a control. Protein
A-Sepharose beads were added, and the lysates were centrifuged. Equal
aliquots of the lysates were subjected to immunoblotting with anti-YB-1
antibodies and to UV cross-linking with 32P-labeled
subfragment I.
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To demonstrate that 50-kDa protein specifically cross-linked to
subfragment I in rabbit reticulocyte lysate is YB-1 as well, 32P-labeled subfragment I was added to the lysate, UV
cross-linked, treated with a nuclease mixture, incubated with anti-YB-1
antibodies, and adsorbed onto protein A-Sepharose. The adsorbed
32P-labeled proteins were analyzed by SDS-PAGE followed by
autoradiography. As seen from Fig. 7B, lane 2,
only the 50-kDa protein cross-linked to subfragment I precipitated with
anti-YB-1 antibodies, whereas 69-kDa protein did not adsorb onto
protein A-Sepharose beads (Fig. 7B, lane 3). None
of them interacted with preimmune antibodies and adsorbed onto protein
A-Sepharose (Fig. 7B, lanes 4 and 5). In another experiment, MN-treated rabbit reticulocyte lysate was incubated with anti-YB-1 antibodies, and YB-1 was precipitated with
protein A-Sepharose beads. Preimmune antibodies were used as a control.
After the precipitation, equal aliquots of all lysates were subjected
to immunoblotting and UV cross-linking to 32P-labeled
subfragment I (Fig. 7C). The 32P-band
corresponding to 50-kDa protein was absent only from the YB-1-depleted
lysate (Fig. 7C, lane 2). Hence, the 50-kDa
protein specifically interacting with subfragment I in rabbit
reticulocyte lysate is YB-1.
69-kDa Protein Specifically Interacting with Subfragment I Is
PABP--
The analysis of the subfragment I primary structure showed
that it is A-rich (34%). Some A residues are assembled in 3-5-nt clusters (Fig. 1). Besides, the molecular mass of 69-kDa protein is
close to that of PABP. To check whether 69-kDa protein specifically interacting with subfragment I has a preferential affinity for poly(A),
we studied by UV cross-linking the effect of homoribopolynucleotides on
69-kDa protein binding to subfragment I (Fig.
8A). As seen, poly(A), unlike
poly(U), poly(C), and poly(G), strongly suppressed binding of 69 kDa
protein to subfragment I, thereby demonstrating a preferential affinity
of this protein for the poly(A) sequence. Therefore, it was suggested
that 69-kDa protein is PABP. To verify this suggestion, MN-treated
rabbit reticulocyte lysate was incubated with poly(A)-agarose beads
with subsequent precipitation of PABP. The PABP depletion was
controlled by immunoblotting. As seen from Fig. 8B,
lane 2, the incubation resulted in complete depletion of
PABP. Then the PABP-depleted lysate was subjected to UV-crosslinking to
32P-labeled subfragment I (Fig. 8C, lane
3). The absence of the 32P-band indicated that the
69-kDa protein was PABP indeed. Addition of purified PABP to the
PABP-depleted lysate resulted in re-appearance of the
32P-band (Fig. 8C, lane 4) that
disappeared again upon addition of increasing amounts of unlabeled
subfragment I (Fig. 8C, lanes 5-7) but not II
(Fig. 8C, lanes 8-10). This confirms PABP
specificity to subfragment I of YB-1 mRNA.

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Fig. 8.
Identification of 69-kDa protein specifically
interacting with YB-1 mRNA subfragment I as PABP.
A, rabbit reticulocyte lysate (5 µl) was incubated for 15 min at 30 °C with 32P-labeled subfragment I (0.2 pmol,
22.5 ng) in the presence of increasing amounts of unlabeled subfragment
I or poly(A), poly(U), poly(C), and poly(G) (22.5, 225, and 450 ng), UV
cross-linked, treated with RNase A and MN, analyzed by SDS-PAGE, and
visualized by autoradiography. B, MN-treated rabbit
reticulocyte lysate (lane 1) was incubated with
poly(A)-agarose beads (lane 2) and centrifuged. Purified
PABP (lane 3) and equal aliquots of the lysates were
subjected to SDS-PAGE with Coomassie staining and to immunoblotting
with anti-PABP antibodies. C, 0.86 pmol of isolated PABP was
added to PABP-depleted rabbit reticulocyte lysate (lanes
4-10) and subjected to UV cross-linking with
32P-labeled subfragment I in the presence of unlabeled
subfragment I (lanes 5-7) or II (lanes 8-10)
(0.18, 0.36, and 0.72 pmol). MN-treated rabbit reticulocyte lysate
(lanes 1 and 2) and PABP-depleted
rabbit reticulocyte lysate (lane 3) were used as controls in
the UV cross-linking.
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Exogenous Poly(A) Specifically Inhibits Translation of
Non-polyadenilated YB-1 mRNA--
Specific binding of YB-1 and
PABP to YB-1 mRNA suggested that these proteins could selectively
affect translation of YB-1 mRNA. Here we studied the effect of PABP
titrating out on YB-1 cap+poly(A)
and
cap+poly(A)+ mRNA translation. As controls,
we used capped luciferase, CAT, and globin poly(A)+ or
poly(A)
mRNAs. mRNAs were translated in a
cell-free system with [35S]Met (Fig.
9, A-E) or
[14C]Leu (Fig. 9F) in the presence of
increasing amounts of poly(A). The proteins were separated by SDS-PAGE,
and [35S]Met translation products were detected by
autoradiography (Fig. 9, A-E). The incorporation of
[14C]Leu into the protein was measured in 5-µl aliquots
as described under "Experimental Procedures" (Fig. 9F).
Addition of poly(A) was shown to inhibit YB-1 mRNA translation much
stronger (Fig. 9, A and D) than translation of
any other mRNA (Fig. 9, B-C and E-G). This
effect did not depend on whether or not the YB-1 mRNA had a poly(A)
tail. Hence, the PABP titrating out by its binding to poly(A) inhibited
predominantly YB-1 mRNA translation. This allowed us to conclude
that PABP positively affects YB-1 mRNA translation in a poly(A)
tail-independent manner.

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Fig. 9.
Effect of exogenous poly(A) on
translation of various mRNAs in rabbit reticulocyte lysate.
Samples of cap+poly(A) mRNA
(A-C) or cap+poly(A)+ mRNA
(D-F) (80 ng) were translated in 15-µl
cell-free systems for 2 h at 30 °C in the presence of poly(A)
(25, 50, 75, and 100 ng (A-D) or 100, 150, 200, and 250 ng
(E and F). [35S]Met-labeled
translation products were resolved by SDS-PAGE and visualized by
autoradiography (A-E). The incorporation of
[14C]Leu into the protein was measured in 5-µl aliquots
as described under "Experimental Procedures" (D).
A and D, YB-1 mRNA; B and
E, Luc mRNA; C, CAT mRNA; F,
natural globin mRNA; G, translation efficiencies of
all mRNAs as dependent on the amount of added poly(A) are plotted
as a percentage of controls.
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DISCUSSION |
Our experiments demonstrated that synthesis of the major mRNP
protein YB-1 in the rabbit reticulocyte cell-free translation system
may be strongly suppressed by exogenous 3' fragment of YB-1 mRNA.
Moreover, this RNA fragment inhibited synthesis of other proteins
tested. Northern blot analysis of mRNA in a translation reaction
inhibited by addition of the 3' fragment showed mRNA intactness.
The protein synthesis inhibition was accompanied by a rapid decay of
polysomes, which shows that inhibition occurs mostly or exclusively at
the initiation stage of translation. Our idea is that 3'-UTR of YB-1
mRNA specifically interacts with protein(s) necessary to initiate
translation of all cellular mRNAs. Through this interaction, these
proteins produce an additional positive effect on YB-1 translation,
then in trans titrating out of these proteins by 3'
fragments would be expected to cause inhibition of synthesis of YB-1,
as well as other cellular proteins.
According to the literature, the majority of proteins specifically
associated with 3'-UTRs of various mRNAs cause inhibition of
mRNA translation (44, 45). However, there are recent data demonstrating the presence of enhancing sequences within 3'-UTRs, as
well as a possibility of mRNA translation activation through interaction of specific proteins with these sequences. These
observations were made for mouse protamine 1 mRNA (46, 47),
fibronectin mRNA (48), manganese superoxide dismutase mRNA
(49-51), mRNA for the
subunit of mitochondrial
H+-ATP synthase (52), human fibroblast growth factor 2 mRNA (53), and ornithine decarboxylase mRNA (54).
UV cross-linking revealed four proteins that specifically interacted
with the 3' fragment of YB-1 mRNA in rabbit reticulocyte lysate.
Their molecular masses are ~69, 50, 46, and 44 kDa. Two of them, 69- and 50-kDa proteins, specifically bind to the inhibitory subfragment I,
while 46- and 44-kDa proteins specifically interact with the rest of
YB-1 mRNA 3'-UTR. As suggested above, the association with 69- and/or 50-kDa protein is required for mRNA translation. Quantitative analysis of subfragment I required to bind all 50- and
69-kDa proteins in the lysate revealed higher amounts of 50-kDa as
compared with 69-kDa protein.
Subfragment I was shown to contain a hexamer (AACAUC) that, according
to the literature, specifically binds FRGY2, a YB-1 homolog from
Xenopus laevis (43). A reporter mRNA with
this motif is more sensitive to the inhibitory effect of FRGY2 (24). In
immunoprecipitation experiments, we have identified 50-kDa protein as
YB-1 and showed that isolated YB-1 specifically binds to subfragment I
of YB-1 mRNA. Evidently, a specific heteropolymeric sequence is
implicated in this binding, since none of the homoribopolynucleotides (including poly(G) displaying high affinity for YB-1 (55)) can compete
with subfragment I for binding to YB-1 (Fig. 8A). The nucleotide sequence of subfragment I specifically binding YB-1 is
currently under study, and soon it will be clear whether the predicted
hexameric motif AACAUC belongs to the binding site.
The analysis of nucleotide sequence of the 3' fragment also showed that
subfragment I is A-rich (34%), with As arranged in 3-5-nt clusters.
This allowed us to suggest that 69-kDa protein specifically binding to
this fragment is PABP. Experiments on competition between subfragment I
and homoribopolynucleotides for 69-kDa protein proved that this
protein really had a higher affinity for poly(A). Selective removal of
PABP from rabbit reticulocyte lysates by its adsorption onto
poly(A)-agarose beads led to a complete disappearance of the
protein specifically cross-linked to subfragment I of YB-1 mRNA,
which proved that 69-kDa protein was PABP indeed. Addition of purified
PABP to the 69-kDa protein-depleted lysate led to re-appearance of the
69 kDa band. Experiments with competitive unlabeled subfragments I and
II confirmed a higher affinity of the added PABP for subfragment I.
Since both identified proteins, YB-1 and PABP, are universal major mRNP
proteins and both are required for mRNA translation initiation
(PABP stimulates initiation of cap+poly(A)+
mRNA (56), while YB-1 stimulates both
cap+poly(A)+ and
cap
poly(A)
mRNAs (39, 57)), it becomes
clear why subfragment I inhibited translation of all mRNAs tested.
Most likely, subfragment I has a dual effect on protein synthesis: it
inhibits translation initiation due to PABP titrating out on the one
hand and due to YB-1 titrating out on the other.
Finally, we demonstrated that PABP titrating out by poly(A) strongly
and specifically inhibited translation in a cell-free system of both
poly(A)+ and poly(A)
YB-1 mRNA. Hence,
PABP specifically and positively affects YB-1 mRNA translation in a
poly(A) tail-independent manner. Inhibition of translation of other
tested mRNAs by exogenous poly(A) was far less strong and with only
a minor difference between poly(A)+ and
poly(A)
, the latter being in a good agreement with the
literature data (35). A stronger poly(A) inhibitory effect on
translation of YB-1 mRNA probably resulted from a lower PABP
affinity for a specific PABP-binding sequence of YB-1 mRNA as
compared with its affinity for the poly(A) tail.
The translational status of mRNA in the cell is determined by the
ratio between two major mRNA-associated proteins: YB-1 and PABP
(58). PABP is known to be capable of inhibiting its own synthesis
through interaction with a 61-nt A-rich element of 5'-UTR of its
mRNA (59, 60). Also, as reported here, PABP can be involved in
positive regulation of YB-1 synthesis, thereby maintaining the PABP to
YB-1 ratio optimal for translation. PABP and YB-1 specifically interact
with the same fragment of YB-1 mRNA, and therefore, overlapping of
their binding sites cannot be ruled out; then excess YB-1 might
displace PABP from its complex with YB-1 mRNA, thus negatively
regulating its own synthesis. This suggestion is currently under verification.