ABSTRACT
The monoclonal antibody CC-3 recognizes a phosphodependent epitope on a 255 kDa nuclear matrix
protein (p255) recently shown to associate with splicing complexes as part of
the [U4/U6.U5] tri-snRNP particle [Chabot
et al.
(1995)
Nucleic Acids Res
. 23, 3206-3213]. In mouse and
Drosophila
cultured cells the electrophoretic mobility of p255, faster in the latter
species, was identical to that of the hyperphosphorylated form of RNA
polymerase II largest subunit (IIo). The CC-3 immunoreactivity of p255 was abolished by 5,6-dichloro-1-
[beta]
-d-ribofuranosylbenzimidazole, which is known to cause the
dephosphorylation of the C-terminal domain of subunit IIo by inhibiting the TFIIH-associated kinase. The identity of p255 was confirmed by showing
that CC-3-immunoprecipitated p255 was recognized by POL3/3 and 8WG16, two
antibodies specific to RNA polymerase II largest subunit. Lastly, the recovery
of RNA polymerase II largest subunit from HeLa splicing mixtures was
compromised by EDTA, which prevents the interaction of p255 with splicing
complexes and inhibits splicing. Our results indicate that p255 represents a
highly phosphorylated form of RNA polymerase II largest subunit physically
associated with spliceosomes and possibly involved in coupling transcription to
RNA processing.
RNA polymerase II (RNAP II) is a large, multisubunit enzyme composed of a dozen
different polypeptides. It is found in two forms within eukaryotic cells, RNAP
IIA and RNAP IIO, according to the level of phosphorylation of its largest
subunit, designated either IIa (unphosphorylated; apparent
M
r
210 kDa) or IIo (multiphosphorylated; 240 kDa) (
1
-
2
). The phosphorylation sites of the largest subunit are located in its C-terminal domain (CTD) composed of multiple heptapeptide repeats of the
consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This domain is conserved in evolution
but its length seems to be related to the complexity of organisms (
3
). The CTD is essential
in vivo
and may play a role in the regulation of transcription or in the linkage of
transcription to other nuclear processes (
3
-
6
). Various studies suggest that the unphosphorylated CTD of RNAP IIA interacts
with the promoter to form a stable preinitiation complex and that the entry
into initiation of transcription is accompanied by the phosphorylation of the
CTD (
7
-
11
). A multiplicity of CTD kinases have been identified, including both
serine/threonine and tyrosine kinases, suggesting that the heterogeneity of CTD
phosphorylation could be extensive (
10
).
A subpopulation of subunit IIo has recently been shown to colocalize with
splicing protein SC-35 in nuclear speckled domains, and to be highly resistant to extraction
by detergents (
12
). During states of transcriptional inhibition, both subunit IIo and SC-35 coredistributed to enlarged unconnected speckled domains which are
thought to be storage and/or assembly sites for splicing factors (
12
). In this connection, previous work in our laboratories has shown that
monoclonal antibody CC-3 reacts with a phosphodependent epitope borne by a 255 kDa nuclear matrix
protein (p255) which colocalizes with splicing factor SC-35 at every stage of the cell cycle and after the cells were heat shocked
(
13
-
14
). Furthermore, this nuclear phosphoprotein was recently shown to stably
associate with spliceosomal complexes formed
in vitro
via an association with the [U4/U6.U5] tri-snRNP complex (
15
). We show here that p255 corresponds to a subset of subunit IIo carrying a
particular phosphorylated epitope specifically recognized by monoclonal
antibody CC-3. This finding signifies that a subpopulation of the largest subunit of
RNAP II is physically associated with spliceosomes. During the preparation of
this manuscript, Mortillaro
et al.
(
33
) reached the same conclusion using a different monoclonal antibody to a
hyperphosphorylated form of RNAP II large subunit.
HeLa cells were grown in 75 cm
2
culture flasks at 37oC with 5% CO
2
in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% fetal
bovine serum (FBS). Mouse NIH 3T3 were cultured in Dulbecco's modified Eagle's
medium (DMEM) supplemented with 10% FBS and
Drosophila
SL2 cells were grown in D-22 medium supplemented with 10% decomplemented FBS. In some experiments,
medium was supplemented with 5,6-dichloro-1-[beta]-d-ribofuranosylbenzimidazole (DRB), prepared
from 100 mM stock solution in dimethyl sulfoxide.
MAb CC-3 was obtained after immunization of a Balb/C mouse with pharyngeal
regions isolated from 72 h chick embryos (
16
). The CC-3 hybridoma cell line was grown in IMDM supplemented with 10% FBS and the
culture supernatant was used undiluted as a source of CC-3 antibody for immunoblotting experiments whereas ascitic fluid was used
for immunoprecipitations. The mouse monoclonal antibody POL3/3 recognizes RNAP
II largest subunit in a conserved region located outside the CTD and was kindly
provided by E.K.F. Bautz (
17
). The mouse monoclonal antibody 8WG16 (Promega, Madison, WI) binds to a peptide
epitope located in the CTD (
18
).
HeLa cells were washed with PBS and liquid nitrogen was poured over the cell
layer to reduce protease and phosphatase activities during the solubilization
step. After evaporation, the cells were solubilized with 1 ml TD buffer (0.5%
Triton X-100, 0.5% sodium deoxycholate, 50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, 50 mM sodium fluoride, 1 mM
PMSF and 2 mM sodium orthovanadate) at 4oC during 15 min with vigorous agitation at every 5 min. The suspension was
then passed through a 28G1/2 needle repetitively before centrifugation at 13
000
g
for 30 min at 4oC for clarification. A volume of supernatant equivalent to 10
6
cells was incubated with either 10 [mu]l CC-3 ascite or 2 [mu]l monoclonal antibody 8WG16 for 1 h at room temperature and then
anti-mouse IgG-agarose beads (Sigma) were added for 2 h at room temperature.
Irrelevant primary antibodies were used to control the non-specific binding to the agarose beads. Beads were washed with TD buffer
and solubilized directly in electrophoresis sample buffer. Preparations of
nuclear splicing extracts (
19
) were immunoprecipitated as described previously (
15
).
The samples were solubilized in electrophoretic sample buffer (
20
) and heated for 3 min at 95oC before loading on 5-15% polyacrylamide gradient gels (acrylamide:bisacrylamide, 30:
0.15) using a mini-protean II apparatus (BioRad). The proteins were then transferred to
nitrocellulose membranes and the position of high molecular weight markers
(myosin heavy chain, 200 kDa; human spectrin doublet, 220 and 240 kDa) was
revealed by Ponceau S staining. The membranes were saturated with PBS
containing 5% skimmed milk powder (Blotto) for 1 h at 37oC prior to incubation with monoclonal antibodies CC-3, POL3/3 or 8WG16 for 45 min at 37oC. The membranes were then extensively washed with PBS
containing 0.05% Tween-20 and incubated for 1 h with
125
I-labelled goat immunoglobulins against mouse IgG heavy chains or with
peroxidase conjugated anti-mouse IgG antibody (Promega) diluted in Blotto. Washed immunoblots were
exposed directly to Fuji RX film or immunodetection was performed using the ECL
reaction (Amersham, Oakville, Ont). In some experiments (Figs
1
and
2
), the procedure used was described previously (
21
).
Previous studies have shown that monoclonal antibody CC-3 stained nuclear speckles and nucleoplasmic material in vertebrate cells
and immunoblotted a 255 kDa protein whose reactivity was mediated by a
phosphodependent epitope (
13
,
14
). Based on its resistance to extraction with non-ionic detergents, nucleases and high ionic strength buffers, p255 was
defined as a nuclear matrix protein. Its distribution at different stages of
the cell cycle and after the cells were heat shocked was identical to that of
splicing factor SC-35 (
14
). This behaviour was reminiscent of that of cytostellin, a phosphoprotein
identified with monoclonal antibody H5, shown to distribute to nuclear regions
enriched with splicing factors (
22
,
23
). Unlike CC-3 however, H5 reactivity with intranuclear speckles appeared to fluctuate
markedly during the cell cycle (
22
) so that
a priori
a parallel between both antigens appeared fragile. Recently, however, H5
variable immunoreactivity was shown to originate from a masking effect since an
extraction with non-ionic detergents prior to fixation and H5 staining resulted in the
appearance of a speckled pattern in nearly all nuclei (
12
). The immunopurification and microsequence determination of cytostellin allowed
its identification as RNAP II largest subunit (
12
), demonstrating at the same time that a subpopulation of the RNAP II large
subunit was located in the speckled domains and was associated with a solid
phase nuclear structure.
To see if monoclonal antibody CC-3 also reacted with a subset of RNAP II large subunit, we first verified
the mobility of its antigen in immunoblotted cellular extracts from
Drosophila
cells as the heptapeptide sequence of the CTD is repeated 43 times in this
species, compared with 52 times in the mouse (
24
). As shown in Figure
1
, in murine and
Drosophila
cell lysates, antibody POL3/3 recognized two bands migrating above the 200 kDa
molecular weight marker. These two bands corresponded to the two forms of RNAP
II largest subunit (
17
,
21
). The murine polypeptides exhibited a slower electrophoretic mobility, as
expected from their longer CTD. In both cell types, monoclonal CC-3 immunoblotted a single peptide band comigrating with subunit IIo (Fig.
1
). We then compared the levels of phosphorylation of the phosphodependent CC-3 antigenic determinant with that of subunit IIo upon treatment of NIH 3T3
cells with the inhibitor of transcription DRB, which is known to promote dephosphorylation of the CTD by inhibiting the TFIIH-associated kinase (
21
). After incubation of the cells for 3 h in the presence of 100 [mu]M DRB, the IIo band almost completely disappeared when analyzed by Western
blot using antibody POL3/3 (Fig.
2
). When probed with CC-3, the intensity of p255 was also dramatically decreased in DRB-treated cells (Fig.
2
). Thus, under different circumstances p255 behaved as RNAP II largest subunit
IIo. In addition, unlike subunit IIa, p255 and most of subunit IIo remained
associated with nuclear matrix preparations following detergent extraction
(data not shown).
To confirm the identity of both proteins, p255 was immunoprecipitated with CC-3 from HeLa cellular extracts. Both the pellet and the immunodepleted
supernatant were submitted to Western blot analysis using CC-3 and antibodies to RNAP large subunit POL3/3 and 8WG16. As shown in
Figure
3
, CC-3-immunoprecipitated p255 was recognized by POL3/3 and 8WG16 (lanes
3). A CC-3-immunoreactive 180 kDa band was also found in the immunoprecipitated
fraction. It is unclear whether this band represents a p255 breakdown product
or an unrelated peptide carrying the CC-3 epitope, but it was not reacting with antibodies to RNAP large subunit.
Although p255 was completely depleted from the cell extract according to the CC-3 immunoblot (Fig.
3
, lane 2), some RNAP II large subunit unphosphorylated or intermediate forms,
visualized by antibodies POL3/3 and 8WG16, could not be precipitated by
antibody CC-3 (Fig.
3
, lanes 2). Conversely monoclonal antibody 8WG16, which recognizes a non-phosphodependent epitope on the CTD, immunoprecipitated mostly
hypophosphorylated forms of RNAP II large subunit as revealed by immunoblotting
with POL3/3 (Fig.
4
, right panel). The CC-3-reactive form could not be immunoprecipitated by 8WG16 (presumably
because the epitope is not accessible) and was entirely recovered in the
supernatant fraction (Fig.
4
, left panel). We conclude from these experiments that p255 is a highly
phosphorylated form of RNAP II largest subunit.
We thank Sylvain Bellier for valuable advice and discussions. This research was
supported by grants from the Natural Sciences and Engineering Research Council
of Canada (to M.V.), the Association pour la Recherche sur le Cancer de France
to O.B. and the National Cancer Institute of Canada (to B.C.). P.L. holds a
studentship from the Fonds FCAR (Québec). B.C. is a Chercheur-Boursier of the Fonds de la Recherche en Santé du Québec.
*To whom correspondence should be addressed. Fax: +1 418 656 7176; Email:
mvincent@rsvs.ulaval.ca
REFERENCES
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