ABSTRACT
With anti-hnRNP monoclonal antibody 6D12 we previously showed in HeLa cells that as
early as 10 min after the onset of a heat shock at 45
o
C, a 72.5-74 kDa antigen doublet leaves the hnRNPs and strongly associates with the
nuclear matrix, the effect being reversed after a 6 h recovery at 37
o
C. cDNA cloning and sequencing enabled us to identify these antigens as hnRNP-M proteins and further to show that the correct sequence differs by an 11
amino acid stretch from the originally published sequence. We also show that
monoclonal antibodies raised against synthetic hnRNP-M peptides can directly inhibit
in vitro
splicing. Furthermore, stressing cells at 45
o
C for 10 min is sufficient to abolish the splicing capacity of subsequently
prepared nuclear extracts which, interestingly, do not contain the hnRNP-M proteins any more. Taken together, our data suggest that these proteins
are involved in splicing as well as in early stress-induced splicing arrest. Further
in situ
hybridization assays located the hnRNP-M encoding gene on human chromosome 19.
The stress response is a strategy the cell has developed to withstand an environment deviating from normal physiological conditions. This cellular
response, which may be triggered by heat shock, ultimately results in shutting
off the synthesis of most proteins and launching the synthesis of heat shock
proteins, so as to avoid injury or cell death (
1
,
2
). At the post-translational level, heat shock is known to transiently inhibit pre-mRNA splicing (
3
,
4
), thus most likely preventing errors otherwise resulting in the synthesis of abnormal proteins which would be harmful to the cell (
5
).
Pre-mRNA splicing occurs within heterogeneous nuclear RNP complexes (hnRNP) (
6
-
9
) which are associated with the nuclear matrix (
10
-
13
). Following heat shock at the upper temperature range of the stress response,
one would expect hnRNPs to be transiently altered so as to inhibit splicing.
Analysis of the general characteristics of hnRNP complexes did not show any
alteration (
14
-
16
). Screening our anti-hnRNP monoclonal antibody library by immunocytofluorescence of normal and
stressed HeLa cells essentially led to the same conclusion, except for two
antibodies which revealed differences between both types of cells (
15
,
16
). Based on this observation we further showed that heat shock rapidly induces
subtle and reversible rearrangements within hnRNP complexes. Indeed, as early
as 10 min after the onset of a heat shock at 45oC two antigen doublets of 35-37 and 72.5-74 kDa, respectively recognized by monoclonal antibodies 2H9
(
15
) and 6D12 (
16
), rapidly leave the hnRNP complexes and strongly bind to the nuclear matrix,
this effect being reversed after cells recovered for 6 h at 37oC. At this point, our data correlated with a possible role of these
antigens in turning splicing on and off.
In the present investigations centered on the 72.5-74 kDa antigens, we first addressed the question whether these antigens
really intervene in splicing and then further determined to what extent the
splicing machinery is altered by such a brief heat shock. The latter aspect is
all the more interesting since most published data describe heat-shock effects on splicing following stress periods of 1-2 h (
3
,
4
,
17
). Since the original 6D12 monoclonal antibody (IgM) was of limited use in such
functional studies, we cloned and sequenced the cDNA encoding this antigen
doublet and further raised monoclonal and polyclonal anti-peptide antibodies. We also show that these antigens are in fact hnRNP-M proteins and that their previously published sequences (
18
) contain some limited errors. Anti-peptide antibodies were then tested for their effect on
in vitro
splicing. Having in mind our previous data showing that a 10 min heat shock at
45oC causes the 72.5-74 kDa antigens to transiently leave the hnRNP complexes (
16
), we also tested the splicing capacity of nuclear extracts from normal and
stressed cells, which were further analyzed so as to detect possible
differences. Additional
in situ
hybridization allowed chromosomal localization of the hnRNP-M encoding gene.
Monoclonal antibody 6D12 (IgM) which is specific for the 72.5-74 kDa hnRNP antigens, was obtained as described (
16
), after immunizing Balb/c mice with hnRNP complexes purified from HeLa S3
cells. Both hybridoma culture supernatant and ascites fluids were used as
antibody sources. Antibody was purified from ascites fluid on an ABx column,
according to the manufacturer (J.T.Baker).
cDNA cloning was performed by using standard molecular biological techniques (
19
). A [lambda]gt11 MCF-7 cell random-primed cDNA library was immunoscreened with monoclonal
antibody 6D12. Inserts from positive plaques were used to further screen a [lambda]ZAP II HeLa cell oligo-dT primed cDNA library, so as to isolate full-size cDNAs. After self-excision in pBluescript SK
-
and synthesis of single-stranded DNA from inserts in both orientations, sequences were determined
from both strands (
20
).
The 6D12 cDNA in pBluescript SK
-
was expressed in
E.coli
as a [beta]-galactosidase fusion protein. Following IPTG induction, bacterial
proteins were analyzed by Western blotting.
Potentially immunogenic amino acid stretches of antigen 6D12 were determined
from the hydropathy (
21
) and mobility (
22
) plots drawn from the predicted primary structure. Synthetic peptides with an additional Cys residue were prepared and coupled to ovalbumin
with
m
-maleimidobenzoyl-
N
-succinimide ester.
Hybridomas were established after fusing X-63 myeloma cells with splenic cells from Balb/c mice (
16
) immunized with
peptide-ovalbumin. Monoclonal antibodies were purified from ascites liquids on
ABx columns (J.T.Baker).
HeLa S3 cells were grown in Eagle suspension medium supplemented with 10%
newborn calf serum and antibiotics (penicillin, 100 IU/ml; streptomycin, 100 [mu]g/ml). For heat treatment, cells were concentrated to 5 * 10
6
per ml and transferred to culture flasks which were immersed in a water bath
for 10 min at 45oC.
Nuclear extracts were prepared according to Dignam
et al.
(
23
) and dialyzed against buffer D for splicing assays.
For complementation assays, when investigating the role of U-snRNPs, nuclear extracts from normal cells were incubated at 30oC for 15 min with 2000 U/ml micrococcal nuclease, in the presence of
1 mM CaCl
2
. The nuclease was inactivated with 2 mM EGTA. Controls showed that this
treatment leads to complete hydrolysis of snRNAs.
Splicing substrates were prepared from three constructions: plasmid Sp4, generated by inserting almost the entire adenovirus-2 natural E1A sequence (positions 533-1342) between the
Sma
I and
Xba
I sites of vector pSP65 (
24
); plasmid Sp1, derived from the previous one and containing only part of the
E1A sequence (positions 1006-1336); a plasmid containing the rabbit [beta]-globin gene (positions 64-732) (
25
). Capped precursor RNA was synthesized using SP6 RNA polymerase in the presence
of m
7
G(5')ppp(5')G and [[alpha]-
32
P]CTP and purified as described (
24
).
Splicing reactions were carried out for 2 h at 30oC in 25 [mu]l assays including 10
5
c.p.m. of
32
P-labelled pre-mRNA (~4 ng) and 10 [mu]l nuclear extract (6-8 mg/ml protein) in the presence of 2.6 mM MgCl
2
and 60 mM KCl, as described (
24
). For studying the direct effect of antibody on splicing, nuclear extracts were
preincubated at 0oC for 30 min with 10 [mu]g purified monoclonal antibody. For controls, the nuclear extract was
incubated with an equal amount of non-relevant 5G4 antibody (IgG; from our library) or of specific anti-peptide antibody, the latter being previously either heat denatured
at 65oC for 15 min or preincubated for 2 h at room temperature in the presence of
a 50* or 100* molar excess of specific or non-specific peptide. After phenol extraction and purification,
RNA was analyzed on 5.2% polyacrylamide gels in 8 M urea.
The 6D12 cDNA coding sequence was modified so as to have the initiation codon
within an
Nde
I site and the stop codon followed by a
Bam
HI site and either cloned in a pET 3b expression vector (Novagen) or in a pET 3d
vector modified so as to encode an N-terminal His-tagged protein.
For recombinant protein purification, bacteria were freeze-thawed, resuspended
in 20 mM Tris-HCl, pH 7.9, 0.5 M NaCl and the lysate sonicated and centrifuged.
The inclusions were washed with buffer D and dissolved in 6 M urea or guanidine-HCl in buffer D; the protein was renaturated by step dialysis against the
same buffer containing 2 and 0.5 M urea or guanidine-HCl. Recombinant protein from the supernatant was prepared as follows:
His-tagged protein was purified on Ni
2+
chelation resin columns, the protein being eluted with 0.4 M imidazole in 20 mM
Tris-HCl, pH 7.9, 0.5 M NaCl and dialyzed against buffer D; non-tagged protein was immunopurified on Sepharose-antibody columns, the protein being eluted with 3.5 M NaSCN
or 4.5 M MgCl
2
and dialyzed against buffer D.
Proteins were resolved by SDS-PAGE and electrophoretically transferred to nitrocellulose sheets, the
blots being further probed with the relevant first antibody and
125
I-labelled anti-mouse Ig F(ab')
2
(
16
).
Schaffner HeLa cells (
26
) (10
5
cells/ml) in Dulbecco's modified Eagle's medium supplemented with 10% fetal
calf serum and antibiotics (100 IU/ml penicillin, 100 [mu]g/ml streptomycin) were grown on glass slides for 3 days, washed with PBS
and fixed for 4 min with 2% paraformaldehyde in PBS. For heat treatment, cell
cultures were immersed in a water bath for 10 min at 45oC prior to fixation. The cells were permeabilized for 20 min with 0.1%
Triton X-100, 0.05% NaN
3
in PBS. After rinsing with PBS/NaN
3
, the cells were incubated overnight with a mixture of 1F7 anti-D2 peptide monoclonal antibody (1 [mu]g/ml) and rabbit anti-p80 coilin antibody (1:1000) in PBS. The cells were washed with PBS/Triton/NaN
3
and incubated for 45 min with a mixture of Cy3-conjugated goat anti-mouse IgG (1:200) and FITC-conjugated donkey anti-rabbit IgG (1:50; Jackson) in PBS/Triton/NaN
3
.
After washing the cells, DNA was counterstained with Hoechst 33258. The
preparations were mounted in glycerol/PBS (4:1) containing 5% propylgallate as
an anti-oxidant. Immunofluorescence images from 0.6 [mu]m optical sections were obtained using a Leica confocal laser scanning microscope equipped with a PL APO 100/1.4 oil immersion lens and a krypton/argon laser.
Excitation wavelengths of 488 and 568 nm were selected for FITC and Cy3,
respectively. Pseudo-coloured images of the signals were generated and further superimposed
using an image processing program.
In situ
hybridization was essentially performed as described (
27
). Phytohaemagglutinin-stimulated human lymphocytes were cultured for 72 h, in the presence of 5-bromodeoxyuridine (60 [mu]g/ml) for the last 7 h. The 6D12-coding cDNA in Bluescript II SK
-
plasmid was tritium labeled by nick translation (1.5 * 10
8
d.p.m./[mu]g) and hybridized to metaphase spreads (100 ng/ml). The slides were coated
with Kodak-NTB
2
nuclear track emulsion, exposed for 18 days at 4oC and developed. R-banding was performed using the fluorochrome/photolysis/Giemsa
method.
By immunoscreening the [lambda]gt11 expression library with monoclonal antibody 6D12 we isolated a 1.1
kb cDNA fragment which was further used to screen the [lambda]ZAP II library. We finally obtained four cDNAs of 2.4 kb, each revealing
a single 2.4 kb band by Northern blotting. The 2.4 kb cDNA (GenBank accession
no. L32611) contains 2394 bp comprising a 5'-untranslated region of 9 bp, an open reading frame of 2073 bp
encoding a protein of 691 amino acids and a 3'-untranslated region including a 53 bp poly(A) tail. Database
searches (Blitz EMBO) showed that the encoded 6D12 protein is close to the
hnRNP-M1/M2 proteins described by Datar
et al.
(
18
). However, the 6D12 protein has 691 amino acids versus 690 for the M1/M2
proteins, the difference being due to three additional C (positions 80, 91 and
114) (Fig.
1
), the one at position 80 generating a unique cleavable
Bss
HII restriction site (G/CG
Our previous studies suggested that the 72.5-74 kDa antigens might be involved in turning splicing on and off (
16
). To investigate this possibility, we first had to check whether these antigens
are really involved in splicing. Therefore, we tested the ability of specific
antibodies to directly inhibit
in vitro
splicing of an 854 nt pre-mRNA synthesized from plasmid Sp4 which contains adenoviral E1A sequence.
In the salt conditions we used (see Materials and Methods), this pre-mRNA yields a 13S mRNA and a 114 nt intron.
Preincubating a nuclear extract with monoclonal antibody 6D12 had a partial but
reproducible inhibitory effect on
in vitro
splicing when compared with non-related monoclonal antibody 5G4 from our library (Fig.
2
A, lanes 3 and 4) or with IgG from non-immune mice (not shown). Since the only partial effect might be explained
by the low affinity of this antibody, we then tested two monoclonal antibodies
we raised against peptide D2 (amino acids 34-46; Fig.
1
). These antibodies, named D2-1F7 and D2-1B9 recognize the entire hnRNP-M family (
18
) and behave as does monoclonal antibody 6D12 with respect to Western blot and
immunocytofluorescence analyses (
16
). Preincubating nuclear extracts with either of the two monoclonal anti-peptide antibodies led to nearly total inhibition of 13S mRNA
accumulation. Figure
2
A (lane 5 or 7) shows the inhibition observed when using 10 [mu]g monoclonal antibody D2-1F7 (IgG
1
). Antibody D2-1B9 (IgG
1
) and also an anti-D2 rabbit polyclonal antibody led to the same result (not shown). Since
neither mature mRNA nor free exon 1 are accumulated, inhibition occurs during
spliceosome assembly. To exclude artefacts, we first preincubated nuclear
extracts with heat-denatured antibody (
6
), which had no effect on splicing (Fig.
2
A; lanes 8 and 7). Further assays in which monoclonal antibody D2-1F7 was preincubated with peptide D2 (50* or 100* molar excess) showed a significant relief of the inhibition
(Fig.
2
B; lanes 4 and 2). In contrast, non-specific peptide D9 had no effect (lane 3). Additional controls in which
nuclear extracts were preincubated with either peptide D2 or D9 alone did not
significantly alter splicing efficiency (lanes 5 and 6). Taken together, our
data show that the direct splicing inhibition we observe is due to specific
antibody-antigen recognition.
It has been shown by others that nuclear extracts from HeLa cells heat shocked
for 1-2 h at the upper temperature range of the stress response are inactive in
splicing
in vitro
; in such conditions the structure of U-snRNPs, which are essential splicing components (
28
,
29
), was found to be altered (
3
,
17
). Whether a 10 min heat shock at 45oC is already leading to inactive nuclear extracts was checked by using
three different pre-mRNAs. Figure
3
shows that splicing
in vitro
is indeed strongly reduced ([beta]-globin pre-mRNA, lane 2; Sp1 pre-mRNA, lane 4) or almost totally inhibited (Sp4 pre-mRNA, lane 8). Analysis of splicing complex
formation revealed non-specific H-complexes, but no pre-spliceosomes or spliceosomes (not shown). Further assays
showed that the splicing ability of such extracts is significantly restored by
complementation with normal cell extracts pretreated with micrococcal nuclease
to hydrolyse U-snRNPs (Fig.
3
, lanes 7-9). Thus, the lack of splicing activity of extracts from briefly stressed
cells does not seem to be due to U-snRNP alteration.
We have previously shown by immunocytofluorescence that the 72.5-74 kDa (hnRNP-M) proteins are not accessible to monoclonal antibody 6D12 in normal HeLa cells, whereas in heat shocked cells
(45oC for 10 min) the nuclei displayed a bright signal distributed as small
granules all over the interchromatin space (
16
). This behavior and this distribution were observed in HeLa cells cultured in
Dulbecco's medium supplemented with 7.5% newborn calf serum and 2.5% fetal calf
serum. However, later on during our investigations, cells were routinely grown
in culture medium supplemented with 10% fetal calf serum. Interestingly, under these conditions hnRNP-M proteins also become accessible in normal cells. In fact, monoclonal antibodies 6D12, D2-1F7 and D2-1B9 revealed the previously described small granule pattern in
both normal and heat shocked cells; in addition, up to three brightly stained
spherical nuclear bodies are also detected in part of the interphasic cells
(Fig.
6
A and D). These structures have nothing in common with the numerous irregularly
shaped nuclear speckles which are known to contain many splicing factors.
Indeed, our anti SR splicing factor 9G8 antibody (
30
), which reveals the characteristic nuclear speckled pattern, does not stain
these nuclear bodies. In fact these bodies rather resemble coiled bodies, which
are believed to contain splicing factor U2AF (
32
,
33
). This prompted us to carry out a double immunocytofluorescence assay, using a
rabbit polyclonal anti-coilin antibody to label coiled bodies (
34
,
35
) (Fig.
6
B and E) and an anti-hnRNP-M monoclonal antibody (Fig.
6
A and D). Image overlays show that the nuclear bodies revealed with the anti-hnRNP-M antigens are not coiled bodies (Fig.
6
C and F).
Southern blot analysis was performed on human cellular DNA digested with either
Eco
RI (one site in 6D12 cDNA) or
Bgl
II (no sites) and further blotted on Highbond N
+
. Hybridization under normal stringency conditions using as a probe a full 6D12
cDNA labeled with
32
P by random priming (
19
)
revealed two
Eco
RI bands of 14 and 15 kb and two
Bgl
II bands. Cellular DNAs originating from ~20 different patients all revealed this pattern, no polymorphism being
observed (not shown).
Chromosomal location of the gene encoding the hnRNP-M proteins was determined by
in situ
hybridization in human lymphocytes. Within 100 metaphasic cells examined, 153
silver grains were associated with chromosomes and among them 34 (22%) were
located on chromosome 19. This distribution was non-random as 28 grains out of 34 (82%) were located on the p13.3 band of the
short arm of chromosome 19, which allows us to map the 6D12 gene to the 19
p13.3 band of the human genome (Fig.
7
). Moreover, these results, along with the Southern blot experiments, point to a
unique hnRNP-M locus in the human genome.
Following a 10 min heat shock at 45oC, we previously showed that a 72.5-74 kDa antigen doublet transiently leaves the hnRNP population and
strongly binds the nuclear matrix, whereas the general properties of hnRNP
complexes remain unaltered (
16
). Here we study these antigens in more detail, trying to find out whether they
may intervene in stress-induced splicing arrest.
cDNA cloning led to the isolation of four 2.4 kb cDNAs, all encoding an
authentic 6D12 protein. cDNA sequencing allowed us to show that these proteins
are hnRNP-M1/M2 proteins (
18
) and also that the sequence published by Datar
et al.
(pHCM4 cDNA clone;
18
) is lacking 3 nt, thus modifying the sequence of an 11 amino acid stretch.
Curiously, like pHCM4-cDNA, our cDNAs also have short 5'-untranslated regions (9 nt), which raises the question as to
whether the N-terminus is really complete; indeed, co-migration of recombinant protein with native protein from a nuclear
extract might not be sufficient evidence, since a difference of just a few
additional amino acids is undetectable.
In contrast to our original 6D12 monoclonal antibody (IgM), three anti-D2 peptide antibodies (two monoclonals and one polyclonal) we raised
allowed direct inhibition of
in vitro
splicing of an Sp4 pre-mRNA. This demonstration relies on the utilization of three different
antibodies and further on two controls showing first that heat-denatured antibodies do no more inhibit splicing and second, and most
importantly, that the addition of specific peptide can relieve this antibody-mediated inhibition. Taken together, these data already provide further
clues in favour of either direct or indirect involvement of the hnRNP-M proteins in
in vitro
splicing, although standard depletion/complementation could not be used. Here it
is worthwhile mentioning that the difficulties we encountered reflect those
other investigators have had when trying to demonstrate a role for hnRNP
proteins in splicing. Indeed, although the structure of numerous hnRNP protein
families has been established, only two of them have been assigned a function
in splicing: hnRNP-A/B proteins, which regulate alternative splicing (
36
,
37
), and hnRNP-C proteins (
6
). Also to be mentioned is galectin-3, which has similarities to hnRNP proteins and was shown to also be
involved in splicing (
38
). The hnRNP-C proteins, which exhibit a single RNA binding domain (
39
), were further shown to interact with the polypyrimidine stretch of the 3'-end of introns (
40
) so as to display the RNA sequence in a configuration suitable for splicing
reactions (
41
), which clearly points to a chaperone activity. Since several other hnRNP
proteins also revealed strong RNA annealing activity (
36
,
42
), and therefore potential ability to modulate pre-mRNA configuration, one might think of chaperone activity as a general
concept for hnRNP protein function. Therefore, hnRNP-M proteins which preferentially bind poly(G) and poly(U) homopolymers (
18
) might also be candidates for such a function.
As it turned out that normal nuclear extracts could not be efficiently depleted
for further complementation assays, we tried to take advantage of the fact that
in vivo
, following a 10 min stress at 45oC, the hnRNP-M proteins transiently leave the hnRNP complexes and bind to the
nuclear matrix (
16
). Our assumption that this process might also be viewed as an
in vivo
depletion of hnRNP or splicing complexes was correct in that nuclear extracts
from briefly stressed cells no longer contain the hnRNP-M proteins. Since these extracts are splicing deficient, we show that the
switch of hnRNP-M proteins from hnRNP complexes to nuclear matrix (
16
) correlates with splicing inhibition, which provides further evidence
strengthening the idea that hnRNP-M proteins are required for splicing
in vitro
. Furthermore, and although our experiments essentially rely on cellular
subfractionation techniques, it remains that the heat shock was to living
cells, therefore, our results most likely reflect the
in vivo
situation, which might not be the case when nuclear extracts are submitted to
elevated temperatures (
43
). As the hnRNP-M proteins are associated with hnRNP complexes when splicing is on (normal
cells) and dissociated from these complexes when splicing is off (heat shocked
cells), our data also provide additional evidence in favour of these proteins
being involved in turning splicing on and off
in vivo
. From our data one can also appreciate the prominent role of the nuclear
matrix, which increasingly appears as a dynamic nuclear scaffold organizing and regulating gene expression (
44
), including transcription (
45
) and also splicing, in which nuclear matrix proteins were shown to be involved
(
46
,
47
).
Whether the absence of the hnRNP-M proteins can alone cause splicing inactivation is a question not yet
solved, since nuclear extracts from stressed cells could not be complemented
with recombinant protein. Several reasons linked to the recombinant protein
might explain this failure: the protein might not be in the right conformation,
it might not have the required post-translational modifications and finally it might not be representative of all hnRNP-M species. Further, we identified an additional hnRNP protein family
which is also missing in the nuclear extract from stressed cells. These are 35-37 kDa proteins, which behave as do the hnRNP-M (or 6D12) proteins (
16
), since they are associated with hnRNP complexes in normal cells and switch to
the nuclear matrix following a brief heat shock (
15
). Their structure and function will be presented elsewhere.
In contrast to what has been shown following 1-2 h heat shocks (
3
,
4
,
17
), early induced splicing arrest cannot be ascribed to U-snRNP disruption, since normal extracts, in which U-snRNPs are hydrolyzed, still restore the splicing capacity of
nuclear extracts from cells stressed for 10 min at 45oC. This is in agreement with immunocytofluorescence data showing that anti-snRNP monoclonal antibodies did not reveal any difference in the
nuclear speckled pattern between normal cells and cells stressed at 45oC for 10 min (unpublished observation), whereas after 15 min the signal is
uniformly distributed throughout the nucleoplasm (
48
). So, whether various stress conditions trigger different mechanisms to shut
off splicing is not as yet known. Assays we performed by submitting HeLa cells
to 45oC heat shocks for up to 1 h all showed the behaviour previously observed by
immunocytofluorescence, i.e. the appearance of a strong nuclear signal, lasting
for 2 h after cells were returned to 37oC and then decreasing over another 6 h until disappearance (
16
). However, we observed dramatic differences in survival capacity, since cells
stressed for longer than 15-20 min, although they apparently did well at 37oC for the next 24 h, were all dead after two days (unpublished
data). It is therefore likely that data obtained after submitting cells to long-lasting stresses at high temperature actually describe processes which are
not fully reversible and therefore might well be involved in cell death.
Finally, we incidentally observed by immunocytofluorescence that hnRNP-M protein behavior or accessibility is also sensitive to other
environmental parameters than temperature, namely serum quality or
concentration, since growing cells in 10% fetal calf serum results in a nuclear
signal in both normal and stressed cells, which also display nuclear bodies not
seen before (
16
). Although the nuclear bodies we detect are not coiled bodies, it is
nevertheless interesting to notice that, like U-snRNPs, splicing factor U2AF (
32
,
33
,
49
) and hnRNP-K and -L proteins (
50
,
51
), hnRNP-M proteins are also found in nuclear bodies. More generally, recent data
show that splicing components are also present in nuclear compartments
different from the pre-mRNA processing sites (
52
-
54
). As to the hnRNP-M proteins, variations in their nuclear distribution or accessibility may
reflect a fine tuning of their function, thus allowing the cell to adjust to
environmental changes of different kinds and of various intensity and duration.
The authors express their appreciation to G.Duval, N.Jung, V.Schultz and Y.Lutz
for producing anti-peptide polyclonal and monoclonal antibodies and to J.L.Vonesch for
carrying out confocal microscopy analysis. They thank A.Staub, P.Eberling and
F.Ruffenach for synthesizing oligonucleotides and peptides, J.M.Garnier and
T.Lerouge for technical advice and B.Boulay and J.M.Lafontaine for the
photographic work. They are grateful to Dr G.Dreyfuss (Howard Hughes Medical
Institute) for the gift of antibodies 4F4 and 3G6, to Dr M.S.Swanson
(University of Florida) for the gift of the pHCM4 cDNA clone, to Dr A.I.Lamond
(EMBL) for the gift of anti-coilin antibody and to Drs A.Hanauer and J.L.Mandel for providing human
DNA blots. This work was supported by funds from the Centre National de la
Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Centre Hospitalier Universitaire Régional and by special grants from the Association pour
la Recherche sur le Cancer. Dominique Mahé is sponsored by the Ministère de la Recherche et de l'Espace and by the Ligue contre le
Cancer.
REFERENCES
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