1 Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska
Institutet, SE-17177 Stockholm, Sweden
2 Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow
Region 142292, Russia
* Author for correspondence (e-mail: bertil.daneholt{at}cmb.ki.se)
Accepted 7 January 2003
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Summary |
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Key words: Y-box protein, YB-1, Pre-mRNP, hnRNP, RNP assembly
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Introduction |
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The nascent pre-mRNA molecule is immediately associated with proteins to
form a ribonucleoprotein complex, which is usually called pre-mRNP or hnRNP
(heterogeneous nuclear RNP) (for reviews, see
Dreyfuss et al., 1993;
Krecic and Swanson, 1999
). The
proteins of the complex, designated hnRNP proteins, comprise about 30 major
proteins and a large number of minor proteins. The hnRNP complex forms a 5-10
nm RNP fibril, often referred to as a perichromatin fibril, which can be
further packed into spherical particles, the perichromatin granules
(Puvion-Dutilleul, 1983
;
Fakan, 1994
). The hnRNP
proteins unfold the RNA and keep it extended, thereby facilitating
interactions with proteins or protein assemblies. The hnRNP proteins show a
general RNA-binding ability and a certain degree of sequence preference. The
proteins are not randomly distributed along the RNA, and a given transcript is
likely to harbour a specific subset of hnRNP proteins
(Matunis et al., 1993
;
Wurtz et al., 1996
). It is
known that hnRNP proteins affect splicing and 3'-end processing, retain
hnRNA in the nucleus or mediate RNA transport from nucleus to cytoplasm.
Furthermore, some of the hnRNP proteins are involved in processes such as
translation, mRNA degradation, and transport of mRNA within cytoplasm (for
reviews, see Krecic and Swanson,
1999
; Nakielny and Dreyfuss,
1999
). It is evident, therefore, that the pre-mRNP/mRNP complexes
should be looked upon as more-or-less well-organised substrates for both
cotranscriptional and post-transcriptional processes. Although the population
of hnRNP proteins as a whole has been extensively studied, there is still
little information on the composition and organisation of RNA-binding proteins
along specific transcripts and also on how this RNP structure is established
and affects cotranscriptional and subsequent events.
The Balbiani ring (BR) genes in the larval salivary glands of the dipteran
Chironomus tentans form a system that is suitable for analysing the
cotranscriptional assembly of a pre-mRNP particle (for a review, see
Daneholt, 2001). The salivary
gland cells contain four polytene chromosomes, and the transcriptionally
active regions appear as chromosome puffs. Two giant puffs, BR1 and BR2,
harbour active genes that are 35-40 kb in size and encode large secretory
proteins. The assembly of the exceptionally large BR pre-mRNP particles can be
visualised using the electron microscope, the completed BR mRNP product can be
followed to and through the nuclear pore, and the loading of the BR transcript
into polysomes just outside the nuclear pore can be seen. By immunoelectron
microscopy the behaviour of several RNA-binding proteins has been studied
during the assembly and transport of this specific pre-mRNP particle
(Daneholt, 2001
). One protein,
the hnRNP A1-like protein, hrp36, shows a remarkable flow pattern: it is added
to the nascent BR transcript and accompanies the mRNA not only into the
cytoplasm but also into polysomes (Visa et
al., 1996a
). It is not known whether hrp36 affects protein
synthesis but it seems reasonable that the loading of hrp36 concomitant with
transcription influences not only intranuclear events but also subsequent
cytoplasmic events.
To further explore the possibility that cotranscriptional loading of
pre-mRNA does influence the nuclear and cytoplasmic fate of the transcript, we
studied the behaviour of the C. tentans equivalent of an abundant
cytoplasmic protein known to regulate translation in somatic cells of
vertebrates, the mRNP protein p50 (YB-1) (for a review, see
Evdokimova and Ovchinnikov,
1999). Along with the poly(A) binding protein 1 (PABP1), it
constitutes the most abundant protein in mRNP particles
(Jain et al., 1979
), and it is
also abundant in polysomes (Minich et al.,
1993
). The p50 protein binds strongly to mRNA with little or no
sequence preference (Minich et al.,
1993
; Evdokimova et al.,
1995
). Furthermore, like the hnRNP proteins, it melts secondary
structures in the RNA, keeping the mRNA available for interactions
(Evdokimova et al., 1995
). At
high concentrations it blocks translation
(Minich et al., 1993
;
Davydova et al., 1997
), whereas
it stimulates translation at lower concentrations
(Minich and Ovchinnikov,
1992
), presumably by modulating the structure of the mRNP complex.
The p50 protein exerts its effect at the level of initiation
(Evdokimova et al., 1998
) by
promoting the binding of 40S ribosomal subunits at the initiation codon of
mRNA (Pisarev et al., 2002
).
The p50 protein can also act as a potent cap-dependent mRNA stabiliser
(Evdokimova et al., 2001
). A
p50-like protein has also been found in hnRNPs in the cell nucleus
(Sommerville and Ladomery,
1996a
), and YB-1 can affect pre-mRNA processing
(Chansky et al., 2001
).
Finally, p50-like proteins are also present in germ cells
(Tafuri et al., 1993
;
Kwon et al., 1993
). We
conclude that the available information on p50 (YB-1) suggests that this
protein should be a good candidate for studying whether a general RNA-binding
protein, functioning in the cytoplasm, is already loaded onto pre-mRNA
concomitant with transcription.
In the present study, we identified a C. tentans Y-box protein by cDNA cloning. The protein, designated Ct-p40/50 (or p40/50 for short), shows high sequence homology to p50 (YB-1) and has a similar domain organisation. A specific polyclonal antibody was raised against p40/50, and the intracellular distribution of the protein was revealed by immunocytology, immunoelectron microscopy and UV crosslinking. It could be concluded that p40/50 is added to the BR transcript cotranscriptionally, is loaded along the entire RNA molecule and probably remains bound to the mRNA from gene to polysomes. The importance of the cotranscriptional loading process in early programming of the cytoplasmic fate of the mRNA is discussed in this paper.
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Materials and Methods |
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cDNA cloning
A C. tentans salivary gland expression gt11cDNA library
was screened according to Alzhanova-Ericsson et al.
(Alzhanova-Ericsson et al.,
1996
) using a p50 polyclonal antibody. A positive clone with a
short p50-like sequence was identified and used as template for PCR
amplification to generate a DNA probe. This probe was used to screen a C.
tentans salivary gland oligo dT-primed
ZAP (Stratagene, La Jolla,
CA) cDNA library to obtain full-length cDNA clones. Two positive clones were
selected to determine the sequence of the two isoforms of p40/50.
DNA sequencing and sequence analysis
The cDNA inserts of positive clones were sequenced and analysed as
previously described (Sun et al.,
1998).
Bacterial expression and purification of p40
The p40 cDNA sequence from the ZAP cDNA library was amplified by
PCR (Boehringer Mannheim Roche Diagnostics Scandinavia, Bromma, Sweden). The
oligonucleotides were designed to introduce a NcoI restriction site
at the 5' end and a HindIII site at the 3' end of the PCR
product. The p40 fragment was inserted into the expression vector pET21d,
which was transformed into Nova Blue cells (Novagen, Darmstadt, Germany).
These cells were grown at 37°C in SOC (LB-Broth, 10 mM MgSO4,
20 mM glucose and 10 mM MgCl2) medium for 60 minutes after heat
shock at 42°C for 40 seconds, and grown over night at 37°C on LB-Broth
agar plates with ampicillin. The positive clones were sequenced, and the
correct unmutated clones were transformed as above into BL21(DE3)pLysS cells
(Novagen), using LB-Broth agar plates with ampicillin and chloramphenicol.
Expression of the fusion protein was induced by the addition of 1 mM IPTG
(Boehringer Mannheim), and the bacteria were grown for another 2.5 hours
before chilling and pelleting. The bacteria were then washed, pelleted and
stored at 70°C.
The p40 protein was purified under denaturing conditions. The frozen bacteria were thawed and incubated in 8 M urea, 0.1 M NaH2PO4, 0.01 M Tris-HCl (pH 8.0) for 2 hours at room temperature. After pelleting the cell debris, the p40 protein was purified from the supernatant using the Ni-NTA system (Qiagen, GmbH, Germany). The supernatant was incubated with the Ni-NTA resin for 90 minutes and washed in 8 M urea, 0.1 M NaH2PO4, 0.01 M Tris-HCl (pH 5.9); the protein was eluted in the same buffer adjusted to pH 4.5. The eluted fraction was dialysed against PBS over night and concentrated using Centriprep MWCO 10 (Amicon, Millipore, Bedford, MA). The p40 protein was used to raise a polyclonal antibody in rabbits.
Affinity purification of antibody
The p40 protein solution was coupled to NHS-activated Sepharose 4B
(Amersham Biosciences, Uppsala, Sweden) according to the manufacturer's
protocol. After incubation with antibody serum, the Sepharose beads were
washed with 0.5 M NaCl, 10 mM NaH2PO4 (pH 6.8) in PBS to
remove unbound proteins. The antibody was eluted with 100 mM glycine-HCl (pH
3.0), which was neutralised immediately with one-tenth volume of 1 M
NaH2PO4 (pH 8.0). The eluted fractions were dialysed
against PBS and stored in PBS with 0.02% sodium azide.
SDS-PAGE and western blot analysis
Isolated salivary glands were transferred to sample buffer (25% glycerol,
2% SDS, 14.4 mM ß-mercaptoethanol, 0.1% bromophenol blue, 600 mM
Tris-HCl, pH 6.8) and frozen in liquid nitrogen. The frozen sample was
homogenised with a mini-homogeniser and boiled.
Extracts were prepared from whole or fractionated tissue culture cells. To obtain a total cell extract, tissue culture cells were pelleted, washed in PBS and resuspended in sample buffer, then vortexed and boiled. To obtain nuclear and cytoplasmic extracts, the cells were washed twice in cold PBS, resuspended in cold TNM buffer (10 mM triethanolamine-HCl, pH 7.0, 100 mM NaCl and 1 mM MgCl2) containing 0.2% NP40 and Complete protease inhibitor cocktail (Roche Diagnostics Scandinavia, Bromma, Sweden), homogenised in a glass tissue grinder provided with a tight-fitting pestle and centrifuged at 2000 g for 10 minutes at 4°C. The resulting supernatant constituted the cytoplasmic extract and the proteins were precipitated in acetone. The pellet, which contained the nuclei, was washed once in TNM buffer with the protease inhibitor cocktail and further resuspended in PBS with the protease inhibitor cocktail, sonicated, RNase A treated (final concentration 30 ng/ml) and centrifuged at 14,000 g at 4°C. The supernatant, designated the nuclear extract, was precipitated in acetone. The extracts were dissolved in sample buffer before electrophoresis.
The samples were analysed by electrophoresis in a 10% or 12% polyacrylamide
gel containing 0.1% SDS. Western blot analysis was performed as described
previously by Sun et al. (Sun et al.,
1998). Both the anti-p40/50 serum and pre-immune serum were used
in a 1:10,000 dilution.
Immunocytology of salivary glands
Immunostaining on semi-thin cryosections of salivary glands was performed
as previously described (Visa et al.,
1996a). The salivary glands were fixed in 4% formaldehyde,
cryoprotected with 2.3 M sucrose, frozen and sectioned with an Ultracut S/FC4S
(Reichert, Leica, Austria). The sections were blocked in 2% BSA in
PBS-glycine. The first antibody was either the anti-p40/50 serum or the
pre-immune serum (negative control) diluted 1:150, and an antibody against
rabbit immunoglobulin G conjugated to 6 nm gold particles (Amersham) diluted
1:50 was used as secondary antibody. The immunogold labelling was
silver-enhanced with IntenSETMM (Amersham).
Immunocytology of isolated polytene chromosomes
The isolation of polytene chromosomes from salivary glands, the RNase
treatment of the chromosomes and the immunocytological analysis have been
previously described (Sun et al.,
1998). The affinity-purified anti-p40/50 antibody was used diluted
1:50.
Immunoelectron microscopy of salivary glands
Immunoelectron microscopy was essentially carried out according to the
cryomethod described by Visa et al. (Visa
et al., 1996a). The specimens were prepared as described above for
light microscopy, with the addition of 0.1% glutaraldehyde in the fixation
buffer. Ultrathin cryosections were treated with 10% new-born calf serum
before incubation with the antibody solutions. The affinity-purified
anti-p40/50 antibody and the pre-immune serum were used at a 1:500 dilution.
After immunolabelling, the sections were stained with 2% aqueous uranyl
acetate and embedded in 4% polyvinyl alcohol (9-10 kDa, Aldrich, WN) provided
with 0.4% aqueous uranyl acetate. The specimens were examined and photographed
in a Philips CM 120 microscope at 80 kV.
Immunoelectron microscopy of isolated polytene chromosomes
The polytene chromosomes were prepared as described above, and the
immunoreaction was carried out the same way, except that the anti-p40/50 serum
and the pre-immune serum were used at a 1:500 dilution. The specimens were
post-fixed, dehydrated, sectioned and stained as previously described
(Björkroth et al.,
1988).
UV crosslinking of RNAprotein complexes
UV irradiation was performed as previously described
(Pinol-Roma et al., 1989) and
specified by Visa et al. (Visa et al.,
1996a
).
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Results |
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The p40/50 protein shows high sequence homology to the rabbit reticulocyte
p50 protein (and human YB-1) and other proteins of the Y-box protein family
(Wolffe et al., 1992;
Wolffe, 1994
;
Sommerville and Ladomery,
1996a
; Matsumoto and Wolffe,
1998
; Graumann and Marahiel,
1998
; Sommerville,
1999
; Evdokimova and
Ovchinnikov, 1999
). The p50 isoform of p40/50, which is about the
same length as the rabbit p50 protein (317 and 324 amino acids, respectively),
has an amino acid sequence that is 47% identical to that of the rabbit p50
protein. Furthermore, p40/50 displays the same three-domain organisation as
rabbit p50 and other Y-box proteins: an N-terminal domain, a central
cold-shock domain (CSD) and a C-terminal domain
(Fig. 1B).
The N-terminal domain is rich in alanine and proline, as is the same domain
in rabbit p50 (Fig. 1A). It has
been proposed that this domain could be responsible for the ability of rabbit
p50 to bind to actin filaments in the cytoplasm
(Ruzanov et al., 1999).
The CSD of p40/50 comprises 74 amino acids (55-128; boxed region in
Fig. 1A) and is 81% identical
to the CSD in rabbit p50, suggesting that this domain in particular is highly
conserved during evolution. Two RNA binding motifs, RNP1 and RNP2, are present
in the p40/50 protein like in other CSDs
(Matsumoto and Wolffe, 1998).
The CSD is known to be responsible for the nucleic acid binding activity of
p50 and other Y-box proteins (Wolffe et
al., 1992
; Graumann and
Marahiel, 1998
).
The C-terminal domain of p40/50 is rich in glycine and arginine and
contains many RG repeats (Fig.
1A), which is a characteristic feature of invertebrate Y-box
binding proteins, e.g. the Drosophila yps protein
(Thieringer et al., 1997), the
Schistosoma SMYB1 protein (Franco
et al., 1997
) and the planarian DjY1 protein
(Salvetti et al., 1998
).
However, its similarity to the C-terminal domain of the mammalian p50 protein
is also striking both have alternating acidic and basic regions and
several clusters of arginine (Fig.
1A). The alternating charge distribution is usually not observed
in invertebrate Y-box binding proteins (e.g.
Thieringer et al., 1997
).
Thus, the C-terminal domain of Ct-p40/50 shares features with both vertebrate
and invertebrate Y-box binding proteins.
The two p40/50 isoforms differ in their length of the C-terminal tail but
both show the same high content of glycine and arginine throughout the entire
length of the tail (Fig. 1A).
It has been proposed that the C-terminal domain can contribute to RNA binding
through an interaction between its positive charge (which is due to the
presence of arginine clusters), and the negatively charged RNA phosphate
groups (Bouvet et al., 1995;
Ladomery and Sommerville,
1994
; Murray,
1994
). A high glycine content with several RGG repeats is also
likely to promote binding to RNA in particular, in combination with
other types of RNA-binding domains (Burd
and Dreyfuss, 1994
). Furthermore, the C-terminal domain is thought
to be responsible for proteinprotein interactions due to the
alternating modules of acidic and basic amino acids
(Wolffe et al., 1992
;
Swamynathan et al., 1998
).
Finally, the C-terminal domain also seems to mediate self-multimerisation by
proteinprotein interactions (Wolffe
et al., 1992
; Evdokimova and
Ovchinnikov, 1999
).
Generation of a specific anti-p40/50 antibody
The rabbit p50 antibody recognised not only the C. tentans p40/50
protein but also a few additional proteins in western blot analysis (data not
shown). Therefore, we had to raise a more specific antibody for studying the
intracellular distribution of p40/50 in C. tentans. The ZAP
p40 cDNA sequence was amplified by PCR and inserted into a pET21 expression
vector. The p40 isoform was expressed, purified and used to raise a polyclonal
antibody. The specificity of the antibody was tested by western blot analysis
of salivary gland and tissue culture cell extracts using the anti-p40/50 serum
(Fig. 2A). In the salivary
gland extract (lane 2), two bands were recorded and the corresponding proteins
were designated p40 and p50 according to their apparent molecular masses in
kDa. The more rapidly migrating protein probably corresponds to the p40
isoform of p40/50, given that it migrated in the gel as the recombinant p40
isoform (lane 1). The more slowly migrating protein was identified as the p50
isoform by a p50-specific antibody (D.N., T.S., Elizaveta Kovrigina, L.O. and
B.D., unpublished). The apparent molecular masses (40 and 50 kDa) are
evidently considerably higher than the values predicted for the two isoforms
from the amino acid sequences (28 and 34 kDa, respectively). Such an aberrant
migration is probably due to a high content of charged amino acids and has
been observed for p50 and other Y-box proteins (e.g.
Murray et al., 1992
). Apart
from the two p40/50 bands, the anti-p40/50 antibody recognised no other
proteins. In the tissue culture cell extract, only the p40 isoform was
observed (lane 3). We conclude that the newly raised polyclonal p40/50
antibody is specific to the p40/50 protein.
|
To reduce the background in subsequent immunocytological and immunoelectron microscopy experiments, the serum antibody was affinity-purified on p40-Sepharose. The purified antibody showed the same specific immunolabelling pattern as the anti-p40/50 serum in western blots (Fig. 2B and data not shown).
p40/50 is present in the nucleus and abundant in the cytoplasm
To elucidate the cellular distribution of p40/50, we first studied nuclear
and cytoplasmic extracts from C. tentans tissue culture cells by
western blotting (Fig. 2B). The
protein is predominantly located in the cytoplasm (note that only 1/10 of the
cytoplasmic extract was loaded onto the gel in
Fig. 2B), but it is also
present in the cell nucleus.
To further study the intracellular distribution of p40/p50, salivary gland cryosections were immunolabelled with the p40/50 antibody and a gold-conjugated secondary antibody, silver-enhanced, and studied under the light microscope (Fig. 3A). The cytoplasm (c) was heavily stained, confirming that the p40/50 protein is abundant in the cytoplasm. A weaker and nonuniform immunolabelling was detected in the nucleus (n). This staining seemed to correspond to the positions of the chromosomes and the nucleoplasm, whereas the nucleoli (nu) were unstained. The pre-immune serum was used as negative control and did not show any labelling (Fig. 3B). Thus, the immunocytology study of the salivary gland cells confirmed the cell fractionation result that p40/50 is abundant in the cytoplasm and more sparsely in the nucleus. However, the distribution in the nucleus is uneven; p40/50 is located on the polytene chromosomes and in the nucleoplasm but seems to be absent from the nucleoli.
|
p40/50 is present in Balbiani rings and other chromosomal puffs and
is associated with RNA
As our study was crucially dependent on the appearance of p40/50 in the
Balbiani rings (BRs), we continued to investigate the localisation of p40/50
on the isolated giant salivary gland chromosomes using immunocytology.
Fig. 4 shows the
immunolabelling of two of the four chromosomes: chromosome IV with the giant
puffs BR1, BR2 and BR3 (Fig.
4A), and chromosome I (B). The BRs and a large number of smaller
puffs along the chromosomes were stained
(Fig. 4A,B), but not the
nucleoli on chromosomes II and III (data not shown). The pre-immune serum was
used as negative control (Fig.
4C,D). To determine whether p40/50 was associated with RNA in the
BRs and the other puffs, isolated chromosomes were also treated with RNase
(Fig. 4E,F). The immunosignal
was essentially abolished by this treatment, showing that the presence of
p40/50 in the puffs was dependent on RNA. Thus, p40/50 is likely to be
associated with RNA at a large number of transcription sites, including the
Balbiani rings.
|
p40/50 is associated with BR mRNA during assembly and transport
To further study the relation of p40/50 to the specific transcription
product generated in the BRs, the BR RNP particles, we performed
immunoelectron microscopy on cryosections from salivary glands. Gold particles
(arrows), indicating the position of the p40/50 proteins, were detected in the
BRs (Fig. 5A), the nucleoplasm
(Fig. 5B) and abundantly in the
cytoplasm (Fig. 5C). The
immunosignal in the BRs, nucleoplasm and cytoplasm was determined relative to
that in a pre-immune serum control, and it amounted to 10x, 5x and
75x, respectively, that of the control. The signal in the nucleoli was
close to background. Thus, the overall distribution of the immunolabelling
examined at the ultrastructural level confirmed that p40/50 was present in
chromosome puffs, in the nucleoplasm and abundantly in the cytoplasm, but
absent from the nucleoli.
|
The ultrastructural analysis was then focused on the BR RNP particles. As
shown schematically in Fig. 5,
lower part, the BR RNP products can be seen in statu nascendi in the BRs and
after release from the genes as granules, 50 nm in diameter, in the
nucleoplasm. The particles unfold when passing through the nuclear pore, and
the exiting mRNP fibre associates immediately with ribosomes to form
polysomes, which anchor to the endoplasmic reticulum (ER) (for a review, see
Daneholt, 2001). Both growing
RNP particles (Fig. 5A) and
released particles (Fig. 5B)
are immunolabelled (barred arrows in Fig.
5A,B, respectively). The label in the cytoplasm is to a large
extent, but not exclusively, localised at the tubular ER, but as the unfolded
mRNP fibril cannot be identified in the cytoplasm, it is not possible to show
directly that the immunolabel is due to p40/50 bound to mRNP in polysomes. We
conclude that p40/p50 becomes bound to BR pre-mRNA co-transcriptionally and
remains bound to the released BR RNP particles in the nucleoplasm.
p40/50 is added along the BR transcript during transcription
To further investigate the behaviour of p40/p50 during the assembly of the
BR particle on the gene, polytene chromosomes were isolated and studied by
immunoelectron microscopy. A pre-embedding immunolabelling procedure was used,
giving essentially no background labelling (50x lower than the specific
immunosignal).
An overview of a BR region is shown in Fig. 6. Proximal (p), middle (m) and distal (d) portions of the BR genes have been indicated in the figure and should be compared with the schematic drawing presented in Fig. 6B. In the proximal region the RNP filament is folded into an RNP fibre, whereas in the middle and distal regions the 5' end of the filament is being further packed into a globular structure, which is growing in size along the gene.
|
The immunolabel is present in all three segments of the BR gene
(Fig. 6A). We also show
examples of proximal (Fig. 6C),
middle (Fig. 6D,E) and distal
(Fig. 6F,G) regions, along with
interpretations. We noted that immunolabel is present in all segments and to
approximately the same extent. As the label appears already early in the
proximal region (Fig. 6C),
p40/50 is present at or close to the 5' end of the transcript. It is,
however, not restricted to this region (cf. e.g. the exclusive 5' end
labelling of the cap-binding protein CBP20 in Visa et al.
(Visa et al., 1996b). The gold
particles seem rather to be randomly bound to the growing BR particles,
indicating that p40/50 appears along the entire RNA molecule. As the degree of
labelling is not increased along the gene it seems likely that the epitope
becomes to a large extent concealed during the packing of the RNP filament
into a compact particle. The p40/p50 labelling pattern would then be similar
to that shown earlier for the hnRNP A1-like hrp36
(Visa et al., 1996a
), which is
known to be distributed along the RNA molecule
(Kiseleva et al., 1997
). It is
not excluded that all the p40/50 protein is added early during transcription
and subsequently continuously redistributed along the growing transcript, but
we regard this as less probable. Thus, we conclude that p40/p50 is added to
the RNA transcript early during transcription and is probably further added to
the growing transcript in parallel with the growth of the transcript.
p40 is bound to poly(A) RNA both in nucleus and cytoplasm
To determine whether p40/50 is connected to poly(A) RNA both in the nucleus
and the cytoplasm, we carried out UV crosslinking experiments with C.
tentans tissue culture cells following the procedure devised by
Pinol-Roma et al. (Pinol-Roma et al.,
1989). The cultured cells were irradiated with UV light and
separated into cytoplasmic and nuclear fractions. The samples were treated
with 0.5% SDS at 65°C, and the poly(A) RNA with its covalently bound
proteins was isolated with oligo(dT) chromatography. The RNA was degraded with
RNase, and the proteins further studied by SDS-PAGE and western blot analysis.
The previously studied hnRNP protein hrp36
(Visa et al., 1996a
) binds to
mRNA and serves as a positive control in this study. Non-UV-irradiated cells
were studied in parallel. The results showed that poly(A) RNA bound to the
p40/50 protein in both the nuclear and the cytoplasmic fractions after UV
treatment (Fig. 7A). Neither
hrp36 nor p40/50 copurified with RNA when the UV irradiation was omitted
(Fig. 7B). Thus, the p40/50
protein is directly bound to poly(A) RNA both in the nucleus and in the
cytoplasm of living cells.
|
General conclusion
The immunoelectron microscopy results showed that p40/50 is added along the
BR transcript concomitant with transcription, and it accompanies the completed
and released BR mRNA into the nucleoplasm. The p40/50 protein is abundant in
the cytoplasm and, to a considerable extent, located at the ER, where BR mRNA
resides in the cytoplasm. As the crosslinking experiment showed that p40/50 is
bound to poly(A) RNA both in the nucleus and the cytoplasm, it seems plausible
that p40/50 remains associated with the mRNA also during the exit into the
cytoplasm and during the protein synthesis at the ER.
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Discussion |
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The Y-box binding proteins are capable of binding to one or more types of
nucleic acids: single- or double-stranded DNA or RNA
(Murray, 1994;
Ladomery and Sommerville,
1994
). We have strong evidence that Ct-p40/50 is bound to RNA. The
p40/50 protein appears to be associated with transcriptionally active regions,
the chromosome puffs, and it is released from isolated chromosomes on RNase
treatment, suggesting that p40/50 is associated with RNA. In addition, the
protein appears in growing RNP particles on the BR genes and remains
associated with the released BR RNP particles in the nucleoplasm. Furthermore,
the recombinant Ct-p40 and Ct-p50 proteins bind to BR RNA in gel-shift
experiments (D.N., T.S., Elizaveta Kovrigina, L.O. and B.D., unpublished).
Finally, our crosslinking experiments showed that p40/50 is bound to both
nuclear and cytoplasmic poly(A) RNA in vivo. There is no p40/50 in the
nucleolus, suggesting that it is not present on RNA polymerase I transcripts.
Thus, p40/50 is bound to RNA polymerase II transcripts in C.
tentans.
The Ct-p40/50 and p50 (YB-1) are not only structurally similar but also show the same cellular distribution, i.e. they are both predominantly cytoplasmic. Furthermore, they are both bound to poly(A) RNA in the cytoplasm and are present in polysomes. It seems likely, therefore, that Ct-p40/50 has a role in translation initiation similar to that of rabbit p50 (see Introduction).
We conclude that the C. tentans p40/50 protein resembles the rabbit p50 protein and is a typical member of the Y-box binding protein family. Like rabbit p50, the p40/50 protein binds to RNA polymerase II transcripts and is predominantly located in the cytoplasm; presumably, it is involved in the regulation of translation.
p40/50 accompanies mRNA from gene to polysomes
Western blot analysis and immunocytology showed that the p40/50 protein is
present both in the nucleus and in the cytoplasm. The immunocytology analysis
of isolated polytene chromosomes further argued that the protein is already
added to pre-mRNA during transcription, suggesting that p40/50 is coupled to
the mRNA sequence during the whole lifespan of the mRNA. Such a hypothesis
could be directly tested in the C. tentans system by analysis of a
specific transcription product, the BR mRNP particles.
By applying immunoelectron microscopy, we found that the p40/50 protein
becomes associated with the growing transcripts on the BR genes already in the
proximal portion of the gene, and is added all along the growing transcript.
In this context, it should be pointed out that the Y-box proteins FRGY2a/b
have also been detected on nascent transcripts along lampbrush loops in
Xenopus oocytes (Sommerville and
Ladomery, 1996b). In C. tentans, the p40/50 protein could
be observed bound to the BR particles when these appear in the nucleoplasm.
The BR mRNA is further translocated as an RNP complex through the nuclear
pores, and appears in the cytoplasm, where the BR mRNA is immediately engaged
in protein synthesis (Mehlin et al.,
1992
). The giant BR mRNA-containing polysomes are associated with
the rough ER and are responsible for the synthesis of the major protein
product of the salivary gland cells, the giant-sized secretory proteins
(Case and Wieslander, 1992
).
The unfolded BR mRNP fibril could not be visualised in the cytoplasm, and
therefore, it is not possible to relate p40/50 to the BR RNA-containing
polysomes directly by using the electron microscope. However, we could observe
that p40/50 is located mostly at the rough ER in the salivary gland cells.
Furthermore, as the UV crosslinking experiment showed that p40/50 is coupled
to poly(A) RNA in both the nucleus and the cytoplasm of tissue culture cells,
it seems likely that the ER-associated p40/50 is bound to the BR mRNA involved
in the synthesis of secretory proteins. We conclude that the p40/50 protein is
probably coupled to the BR RNA all the way from the gene via the nuclear pores
to the polysomes located on the ER.
The fact that many maybe all RNA polymerase II transcripts are loaded cotranscriptionally with p40/50 suggests that not only BR mRNA is associated with p40/50 in the cytoplasm. We noted that cytoplasmic poly(A) RNA readily forms crosslinks with p40/50 in tissue culture cells, further supporting such a notion. Moreover, p40/50 is present in all the larval tissues studied (salivary glands, stomach, intestine, colon, Malpighian tubules and imaginal disks) (D.N., T.S., Elizaveta Kovrigina, L.O. and B.D., unpublished). Thus, not only BR mRNA, but probably many more mRNAs leave the nucleus associated with p40/50.
p40/50 and cotranscriptional assembly of pre-mRNP
As the immunolabelling of the polytene chromosomes showed that p40/50 is
added onto a large number of growing primary transcripts, it is believed to
participate in the formation of pre-mRNP (hnRNP) complexes in general. The
detailed analysis of the long nascent transcripts on the BR genes further
showed that p40/50 is being added along the growing transcripts and is not
restricted to the 5' end region, for example. This is a distribution
similar to that previously recorded for several important hnRNP proteins
(Daneholt, 2001). In fact,
there are several striking similarities between the p40/50 homologue, the
reticulocyte p50 protein and the A/B type hnRNP proteins the
predominant RNA-binding proteins in the pre-mRNP complexes
(Dreyfuss et al., 1993
;
Krecic and Swanson, 1999
).
Although p50 and A/B type hnRNP proteins have different amino acid sequences,
they both have at least one RNA-binding domain of the RNP consensus type
(CS-RBD, also called RNA recognition motif, RRM) and a C-terminal auxiliary
domain with both RNA- and protein-binding ability (for a review, see
Dreyfuss et al., 1993
). Like
hnRNP proteins, p50 melts secondary structures, facilitates annealing of
complementary RNA and shows little or no sequence specificity for RNA (see
Introduction). It seems likely, therefore, that p40/50, like the A/B type
hnRNP proteins, is bound to many different RNA sequences and packages the RNA
into an RNP fibril, which will constitute the substrate on which the various
molecular machines, such as those of splicing, transport and translation, are
likely to operate.
Initially, it was believed that the hnRNP proteins were essentially
functioning in a more global manner as general packaging proteins, but more
and more information is accumulating to suggest that the hnRNP proteins exert
more specific roles apart from packaging the RNA (e.g.
Krecic and Swanson, 1999). For
example, the hnRNP A1 can influence the outcome of alternative splicing
(Mayeda and Krainer, 1992
;
Mayeda et al., 1994
) and
probably also mediates mRNA transport
(Michael et al., 1995
).
Another hnRNP protein, hnRNP C, contains a nuclear retention signal, and thus
could act as a regulator of export of RNA
(Nakielny and Dreyfuss, 1996
),
whereas others, including hnRNP A1, K and E, enter cytoplasm and affect
protein synthesis, mRNA localisation and mRNA stability
(Krecic and Swanson, 1999
). In
the BR system, the hnRNP A1-like hrp36 enters polysomes and remains there
during translation, which suggests that it plays a defined role during
translation (Visa et al.,
1996a
). It has been suggested that general RNA-binding proteins
like hnRNPA1 can improve the efficiency of translation by suppressing false
initiations of translation along the message
(Svitkin et al., 1996
). Thus,
it seems that the hnRNP proteins are not only packaging proteins they
can also play many, and even multiple, roles in gene expression. Regarding p50
(YB-1), there is evidence that the protein can affect splicing in vertebrate
cells (Chansky et al., 2001
).
However, the most important role of p50 seems to be to regulate translation
(see Introduction). As many mRNAs, maybe all, are concerned
(Evdokimova et al., 1995
;
Davydova et al., 1997
), p50
exerts a global effect on translation and presumably it acts by governing the
organisation of the mRNA in polysomes and free mRNP particles (see
Evdokimova and Ovchinnikov,
1999
). It is plausible, but remains to be shown, that p40/50
behaves like p50 (YB-1).
The general picture emerging from analysis of hnRNP proteins and which is
further strongly supported by the present study of C. tentans p40/50
is that the post-transcriptional fate of an mRNA could, to a large extent, be
determined when the primary transcription product, the pre-mRNP complex, is
assembled (Daneholt, 2001). A
similar conclusion has been reached by Wolffe and co-workers in studies of the
oocyte-specific Y-box protein FRGY2 in Xenopus (for a review, see
Matsumoto and Wolffe, 1998
).
Clearly, the RNP structure regulates many co- and post-transcriptional events
in the cell nucleus (Dreyfuss et al.,
1993
; Krecic and Swanson,
1999
). However, as some RNA-binding proteins like p40/50 and the
hnRNP A1-like hrp 36, and perhaps shuttling hnRNP proteins in general,
accompany mRNA into cytoplasm, it is possible that at least some structural
features in pre-mRNP are maintained in cytoplasmic mRNP, governing the
functional options for mRNA (see Matsumoto
and Wolffe, 1998
). Thus, the present study of p40/50 highlights
the possibility that the cotranscriptional loading of RNA-binding proteins
onto pre-mRNA could represent an early programming of the transcript and
influence not only post-transcriptional processes in the nucleus but also the
fate of mRNA in the cytoplasm.
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