Department of Pathology and * Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510
In the preceding study we found that Sm
snRNPs and SerArg (SR) family proteins co-immunoprecipitate with Pol II molecules containing a hyperphosphorylated CTD (Kim et al., 1997). The association between Pol IIo and splicing factors is maintained
in the absence of pre-mRNA, and the polymerase need
not be transcriptionally engaged (Kim et al., 1997
). The
latter findings led us to hypothesize that a phosphorylated form of the CTD interacts with pre-mRNA splicing components in vivo. To test this idea, a nested set of CTD-derived proteins was assayed for the ability to alter the nuclear distribution of splicing factors, and to interfere with splicing in vivo. Proteins containing heptapeptides 1-52 (CTD52), 1-32 (CTD32), 1-26 (CTD26), 1-13 (CTD13), 1-6 (CTD6), 1-3 (CTD3), or
1 (CTD1) were expressed in mammalian cells. The
CTD-derived proteins become phosphorylated in vivo,
and accumulate in the nucleus even though they lack a
conventional nuclear localization signal. CTD52 induces a selective reorganization of splicing factors from discrete nuclear domains to the diffuse nucleoplasm, and significantly, it blocks the accumulation of
spliced, but not unspliced, human
-globin transcripts.
The extent of splicing factor disruption, and the degree
of inhibition of splicing, are proportional to the number
of heptapeptides added to the protein. The above results indicate a functional interaction between Pol II's
CTD and pre-mRNA splicing.
The preceding paper provides succinct background
information about the COOH-terminal domain
(CTD)1 of RNA polymerase II (Pol II) (Kim et al.,
1997 Multiple groups have reported that the unphosphorylated CTD binds to transcription factors, such as TATAbinding protein (TBP), TFIIF and TFIIE (Kim et al., 1997 Little attention has been paid to the phosphorylation
state of the CTD after the polymerase disengages from
chromatin in vivo. Recently, a fraction of Pol IIo was immunolocalized in 20-50 discrete nuclear domains ("speckles"), which are enriched with serine/arginine dipeptide
repeat motif (SR) splicing proteins and Sm snRNPs (Bregman et al., 1995 Below, we show that overexpression of CTD-derived
proteins results in the dispersal of Sm snRNPs and SR
splicing factors from a speckled pattern to a diffuse nucleoplasmic distribution. This property is selective, since
other types of nuclear domains remain intact. Next, we
show that CTD-derived proteins block the accumulation of spliced, but not unspliced, human Plasmids Expressing Flag-tagged CTD-derived Proteins
Epitope-tagged CTD expression plasmids were created using standard
techniques (Sambrook et al., 1989 One control plasmid, pF-CTDless.3, expresses the Flag-tagged NH2terminal 282 amino acids of Pol II LS. This segment was generated by
PCR amplification, using human Pol II LS cDNA as the template. The oligos for this reaction were p337U (5 Plasmids Expressing The
Plasmids Expressing Human Plasmids that co-express Flag-tagged CTD-derived proteins and human
Antibodies
For a description of mAbs H5, H14, Y12, and B1C8 see preceding paper
(Kim et al., 1997 Cell Culture and Transient Plasmid Transfections
See preceding paper (Kim et al., 1997 SDS-PAGE and Immunoblotting
See preceding paper (Kim et al., 1997 Immunofluorescence Microscopy and Image Analysis
See preceding paper (Kim et al., 1997 Quantitative RT-PCR and RNase Protection Assays
Quantitative RT-PCR was carried out as follows: Total RNA was prepared from HeLa cells 1-2 d after transfection using UltraSpec RNA®
(Biotecx, Houston, TX), and digested with RNase-free RQ1 DNase (Promega) to remove contaminating DNA. The RNA was phenol extracted, ethanol precipitated, and dissolved in water. A reverse primer (449 nucleotides downstream from the HindIII site) that hybridizes to
the second exon of the The RNase protection assay was carried out using the RPA II Ribonuclease Protection Kit (Ambion, Austin, TX) according to the manufacturer's procedures. Briefly, a HindIII-BamHI fragment containing exon 1, intron 1, and most of exon 2 of human The results of the preceding study (Kim et al., 1997 Plasmids Expressing Unidirectionally Truncated
CTD Sequences
We constructed plasmid vectors that express a variety of
CTD-containing fusion proteins shown in Fig. 1 (Materials
and Methods). The expression and intracellular distribution of each fusion protein has been documented by
immunoblotting, immunoprecipitation, and immunostaining with antibodies directed at the Flag epitope or CTD-derived Proteins Accumulate in the Nucleus
The CTD-derived fusion proteins must gain access to the
nucleus to interact with splicing factors. At the beginning
of the study we immunolocalized each fusion protein in
CV1 or HeLa cells to confirm that our experimental approach meets this requirement. Plasmids expressing each
of the 13 proteins illustrated in Fig. 1 were transfected into
cells (Materials and Methods). 2 d later, the cells were
fixed and double immunostained with: (a) an antibody directed at the indicator portion of the fusion protein (antiFlag or anti- A representative experiment is shown in Fig. 2 C. In this
case, CV1 cells were transfected with pF-CTD52, fixed
and double stained with anti-Flag mAb M2 and anti-CTD
mAb H14. A cell expressing the F-CTD52 protein is
shown at the top of each panel, and an untransfected cell is
shown at the bottom. Interestingly, F-CTD52 is distributed almost exclusively in the nucleus, even though it lacks
a conventional nuclear localization signal (Fig. 2 C, left
panel). The F-CTD52 protein is present in the diffuse nucleoplasm, but it is most concentrated in ~50 discrete,
nonnucleolar sites (Fig. 2 C, left panel, arrows). In addition, the transfected cell nucleus is much more intensely
stained by mAb H14 than the untransfected cell nucleus
(Fig. 2 C, right panel). The nuclear "dots" are also intensely stained by mAb H14 (Fig. 2 C, right panel, arrows), and mAb H5 (Du, L., and S.L. Warren, unpublished results) antibodies, both of which recognize CTD phosphoepitopes (Kim et al., 1997
The CTD-derived Proteins Are Phosphorylated In Vivo
All observations indicating an association between Pol II
LS and splicing factors suggest a mechanism involving a
hyperphosphorylated CTD (Bregman et al., 1995 An analysis of the Flag-tagged proteins is presented in
Fig. 2 A. mAbs H14 and H5 blot a ~240-kD protein corresponding to endogenous Pol IIo in all of the extracts (Fig.
2 A, right panels, IIo). In cells transfected with the pFCTD
series of plasmids, mAbs H5 and H14 14 blot a nested set
of fusion proteins. In this experiment, mAb H5 immunoblots F-CTD26, F-CTD32, and F-CTD52 (Fig. 2 A, lanes
15-17), and mAb H14 immunoblots pF-CTD6, pF-CTD13, pF-CTD26, pF-CTD32, and pF-CTD52 (Fig. 2 A, lanes
21-25). As expected, the stepwise removal of heptapeptide repeats incrementally increases the electrophoretic
mobility of the proteins. However, the apparent mol wt of
each fusion protein significantly exceeds its predicted size.
For example, F-CTD52 migrates as a 120/130-kD doublet,
even though it has a predicted mol wt of ~66 kD (Fig. 2 A,
lanes 17 and 25). Repeated immunoblotting experiments
reveal that many of the CTD-derived proteins migrate as
closely spaced doublets (Du, L., and S.L. Warren, unpublished results).
The anomalous SDS-PAGE mobilities of the CTD-derived
proteins, and our observation that alkaline phosphatase
treatment of the filters abolishes mAb H14 and H5 immunoreactivity (Du, L. and S.L. Warren, unpublished results),
indicate that the CTD-derived proteins are phosphorylated. Together with our previous studies showing that
mAbs H5 and H14 recognize distinct phosphoepitopes on
the CTD of native Pol II (Kim et al., 1997 Some of the Flag-tagged CTD proteins are immunoblotted weakly, or not at all, by anti-Flag mAb M2 (Fig. 2 A,
lanes 2-8). However, all of the Flag-tagged CTD-derived
proteins are expressed in HeLa or CV1 cells, since antiFlag mAb M2 stains the nucleus in cells transfected by pFCTD1 (Fig. 4 A), pF-CTD3 (Fig. 4 D), pF-CTD6 (Fig. 4 G),
pF-CTD13 (Fig. 4 J), pF-CTD26 (Fig. 4 M), pF-CTD32 (data not shown), and pF-CTD52 (Fig. 3 A and G). We
have repeatedly immunostained the Flag-tagged CTD-derived
proteins with mAb M2, but it has not been easy to reproducibly detect many of the CTD-derived proteins by immunoblotting with the same antibody. The basis for this
discrepancy is not understood. One factor may be low
transfection efficiencies; expression of CTD-derived proteins in a small fraction of cells is difficult to detect by immunoblotting, but easy to detect by in situ methods such as
immunostaining.
Some short CTD-derived proteins are not immunoblotted by mAbs H5 and H14 (Fig. 2 A). Nevertheless, we believe that all of the FCTD proteins are phosphorylated in
the cell, as indicated by enhanced mAb H14 immunostaining of transfected cell nuclei (see Fig. 4). The inability of
mAb H5 to immunoblot F-CTD1, F-CTD3, F-CTD6, and
F-CTD13, and the inability of mAb H14 to immunoblot F-CTD1 and F-CTD3, may be explained by three factors:
(1) Transfection efficiencies vary widely from experiment
to experiment and from plasmid to plasmid. (2) Fusion
proteins with only a few heptapeptides have fewer potential phosphorylation sites, and hence fewer mAb H5- and
H14-binding sites, than proteins with long CTD segments (e.g., F-CTD52 has ~50-fold more phosphorylation sites
than F-CTD1). (3) Finally, it is possible that downstream
heptapeptides are better kinase substrates than upstream
heptapeptides. In this regard, it is interesting to note that
repeats 1-3 diverge from the YSPTSPS consensus sequence more than other repeats in the CTD.
An immunoblot of selected Expression of F-CTD52 or The F-CTD52 protein is phosphorylated on CTD epitopes
(Fig. 2 A, lanes 17 and 25), and it enters the nucleus where
it is frequently, but not always, observed in discrete nuclear dots (Fig. 2 C, arrows). One possible explanation for
this distribution is that CTD52 targets the Flag peptide to
splicing factor domains, perhaps reflecting its ability to associate with Sm snRNPs and SR family splicing proteins,
which are most concentrated in the speckles. To further explore this idea, we sought evidence that the F-CTD52
containing dots overlap or colocalize with speckle domains. Thus, CV1 cells were transfected with pF-CTD52,
and the cells were double immunostained with anti-Flag
mAb M2 (IgG) and mAb B1C8 (IgM), which recognizes an SR-related splicing protein in the speckle domains
(Blencowe et al., 1994 This experiment yielded a striking (and unexpected) result: the B1C8 splicing factor is distributed in a speckled
pattern in untransfected cell nuclei (Fig. 3 B, left side of
panel, arrows), but it is distributed in a nearly uniform, diffuse nuclear pattern in every cell expressing the F-CTD52
protein (Fig. 3 B, right side of panel). Control proteins
such as F-CTDless.1 accumulate in the nucleus, but they
have little effect on the distribution of B1C8 (Fig. 3 E,
right side of panel, arrows).
Next we sought to confirm that the CTD is responsible
for the redistribution of B1C8. For this purpose, CV1 cells
were transfected with p We asked whether F-CTD52 alters the distribution of
proteins located in other types of nuclear domains. ND55
(55 kD) is one of several proteins localized in ~10 highly
circumscribed nuclear dots, referred to as "N10 domains"
or "PML bodies" (Ascoli and Maul, 1991 We had originally predicted that CTD heptapeptides
would behave like SR domains, which target indicator proteins to the splicing factor domains (Li and Bingham,
1991 Addition of Heptapeptide Repeats to the Fusion Protein
Leads to an Incremental Disruption of B1C8 Speckles
Our next goal was to determine how many heptapeptide
repeats are required to induce the redistribution of B1C8.
Therefore, we performed the following "heptapeptide titration" experiment: CV1 cells were transfected with a
nested set of Flag-tagged CTD-derived proteins: pFCTD26, pF-CTD13, pF-CTD6, pF-CTD3, and pF-CTD1.
2 d later, the cells were fixed and double immunostained
with anti-Flag mAb M2 (IgG) and mAb B1C8 (IgM) (Fig. 4).
First, consider the results obtained with F-CTD26. Immunostaining with mAb M2 reveals four transfected cell
nuclei (Fig. 4 M). Note that mAb M2 staining is almost exclusively intranuclear, and the level of FCTD26 expression
varies widely among the four cells (Fig. 4 M, transfected
nuclei marked by white dots). Diffuse mAb M2-immunoreactivity is observed in all four nuclei, but two nuclei
also contain discrete dots harboring the F-CTD26 protein (Fig. 4 M, arrows). The nucleus expressing the highest
level of F-CTD26 has a completely dispersed pattern of
B1C8 staining (Fig. 4, M and N, lower right corner). The
nucleus expressing the second highest level of F-CTD26
has a nearly complete dispersal of B1C8 staining (Fig. 4, M
and N, upper right corner). The two nuclei expressing low
levels of F-CTD6 have a partial dispersal of the B1C8
staining pattern as indicated by the multiple diminutive B1C8 speckles (Fig. 4, M and N, middle of panel). Finally,
the two untransfected nuclei each contain ~20 prominent
B1C8-speckles (Fig. 4 N, thick arrows). These results indicate that the upstream half of the CTD retains the ability
to disrupt the distribution of B1C8, and the degree of
B1C8 disruption is proportional to the level of CTD-
derived protein in the nucleus. Similar results were obtained
with F-CTD32 (Du, L., and S.L. Warren, unpublished results).
Next, consider the results obtained with F-CTD13. Immunostaining with anti-Flag mAb M2 reveals a transfected cell nucleus (Fig. 4, J-L, right) and an untransfected
cell nucleus (Fig. 4, J-L, left). mAb M2 staining is almost
exclusively intranuclear; the F-CTD13 protein is distributed in ~75 discrete dots, as well as the diffuse nucleoplasm (Fig. 4 J, right). The nucleus expressing F-CTD13
has a dispersed pattern of B1C8 staining (Fig. 4 K, right) and the control nucleus has a typical speckled pattern (Fig.
4 K, left). Thus, removal of 75% of the heptapeptides from
the CTD does not abolish the B1C8-disrupting property of
the fusion protein.
Consider the results obtained with F-CTD6 and F-CTD3.
Three representative nuclei expressing low, medium, and
high levels of the F-CTD3 protein are presented (Fig. 4 D).
Again, mAb M2 staining is almost exclusively intranuclear, and the distribution of F-CTD3 is diffuse with a few
discrete dots (Fig. 4 D, arrow). The nucleus expressing a
low level of F-CTD3 retains a prominent speckled pattern
of B1C8 staining (Fig. 4 E, left, arrowhead). Nuclei expressing higher levels of F-CTD3 protein have a partial disruption of B1C8 staining, as indicated by diminutive
speckles (Fig. 4 E, center and right). Partial disruption of
B1C8 stained speckles is observed in a nucleus expressing
F-CTD6. Note that the transfected nucleus has diminutive
B1C8 speckles (Fig. 4, G-I, center of panel).
Finally, consider the results obtained with F-CTD1. A
representative transfected cell nucleus reveals mAb M2
staining in a diffuse and punctate distribution (Fig. 4 A).
Most nuclei expressing the F-CTD1 protein have prominent B1C8 containing speckles, as shown here (Fig. 4 B,
arrowhead). When the anti-B1C8 and anti-Flag images are
merged, one observes a close spatial relationship between the B1C8-speckles and F-CTD1 dots (Fig. 4 C, arrowhead
and thin arrow). Close examination of a nucleus expressing F-CTD6 reveals a similar phenomenon (Fig. 4 I).
Many of the overexpressed CTD proteins form discrete
dots, and in nuclei containing intact B1C8 speckles the
CTD-rich dots do not coincide with the speckles. Quantitative image analysis is needed to determine whether the CTD-rich dots are organized randomly with respect to the
B1C8 speckles, or whether they reproducibly form at
the periphery of the speckles. It is possible that the CTD-rich
dots revealed by mAb M2 staining might be aggregated,
Flag-tagged CTD proteins, which are randomly distributed
in the nucleus.
The effect of CTD length (i.e., number of heptapeptide
repeats) on B1C8-speckles was quantitated as follows:
CV1 cells were transfected with each of the Flag-tagged
CTD-derived plasmids in Fig. 1. The cells were fixed and
double stained with anti-Flag mAb M2 and a mAb directed against B1C8 as described in Fig. 4. The pattern of
B1C8 staining in each transfected cell nucleus was scored
as "intact" (20-50 prominent speckles) or "disrupted" (diffuse pattern or diminutive speckles). Multiple sets of
experiments were conducted, and 150-250 nuclei were
scored for each plasmid (see Materials and Methods).
The scoring results are presented in Fig. 5. Intact B1C8
speckles were observed in >90% of control (untransfected) nuclei (Fig. 5, light gray bar). Intact speckles were
observed in ~76% of nuclei expressing a control protein,
F-CTDless.1. The significance of this reduction is uncertain, but it is interesting to note that F-CTDless.1 contains
a heptapeptide-like sequence on its COOH terminus,
which was derived from the region upstream of the CTD
(Materials and Methods). These heptapeptide-like sequences are deleted in F-CTDless.2, and interestingly, intact speckles were observed in ~86% of nuclei expressing
this control protein. Expression of F-CTDless.3, which has
no heptapeptide-like sequences, does not reduce the frequency of intact B1C8 speckles (Du, L., and S.L. Warren,
unpublished results). Intact B1C8 speckles were observed
in ~70% of cell nuclei expressing F-CTD1, and significantly, the addition of 2-4 heptapeptides markedly increases the B1C8 disrupting activity: only ~30% of nuclei
expressing F-CTD3 or F-CTD6 have intact B1C8 speckles.
The addition of 7, 20, or 26 heptapeptides to F-CTD6
does not further reduce the frequency of nuclei with intact B1C8 speckles, but the longer CTD segments (e.g.,
F-CTD13, F-CTD26, and F-CTD32) induce a more severe
disruption of the B1C8 speckles than short CTD segments
(not reflected by the histogram in Fig. 5). Significantly,
F-CTD52 induces a complete disruption of the B1C8
speckled pattern in nearly 100% of the transfected nuclei.
A similar trend was observed with a nested set of CTD sequences linked to
Multiple SR Splicing Factors and Sm snRNPs
Redistribute from a Speckled to a Diffuse Pattern in
Nuclei Expressing CTD-derived Proteins
B1C8 is one of many SR family splicing proteins in speckle
domains (reviewed by Fu, 1995 Speckle domains are also enriched with other classes of
splicing factors, such as Sm snRNPs and U-rich snRNAs
(reviewed by Fu, 1995 The results are presented in Fig. 6, A-F. Three untransfected cell nuclei are immunostained relatively weakly with
mAb H5 (Fig. 6 A). In contrast, one nucleus expressing
F-CTD52 is intensely immunostained (Fig. 6 A, upper
right). Sm antigens are observed in speckle domains of the
untransfected nuclei, but they are diffusely distributed in
the transfected cell nucleus (Fig. 6 B, upper right). The
Expression of CTD-derived Fusion Proteins Disrupts
Speckle Domains, but Not Coiled Bodies
Coiled bodies (CBs) are dot-like nuclear domains that
contain certain snRNPs and snRNAs that are also present
in the speckle domains (Lamond and Carmo-Fonseca,
1993 To ascertain whether the CTD-derived proteins disrupt
the organization of CBs, each Flag-tagged CTD-derived
protein was expressed transiently in CV1 cells, which were
fixed and double immunostained with anti-p80 coilin and
anti-Flag mAb M2. Our results indicate that the distribution of p80-coilin is unaffected by CTD-derived proteins
F-CTD52, F-CTD32, F-CTD26, and F-CTD13. In the example presented here, CBs are observed in a control cell
nucleus as well as three nuclei expressing F-CTD13 (Fig. 6,
G-I, double arrows).
Expression of F-CTD52 Blocks the
Accumulation of Spliced, but Not Unspliced, The transfection experiments described above, in conjunction with the accompanying study (Kim et al., 1997 First, HeLa cells were transfected with pF-CTDless.1
This result was confirmed using an RNase protection assay (Fig. 8 B, lanes 2-4). Here, a 343-nt protecting RNA
probe (Fig. 8 B, lane 8) was designed to hybridize with 203 nucleotides of the second To control for possible cis effects between the The last series of experiments used a thalassemic Finally, we asked whether the removal of heptapeptide
repeats from F-CTD52 progressively decreases the inhibitory
effect on in vivo splicing. To test this idea, HeLa cells were
transfected with plasmids that co-express the The experiments in the preceding paper, including the key
observation that anti-CTD phosphoepitope-specific mAbs
H5 and H14 release Pol IIo from immunoprecipitates prepared with splicing factor antibodies, suggested that Pol
II's CTD may interact with certain splicing components.
As an initial test of this hypothesis, we transfected cells
with CTD-containing proteins that lack DNA-binding and
catalytic domains, and asked whether they would localize
specifically in the splicing factor domains. Originally, we
had predicted that the CTD might behave like SR domains, which target indicator proteins to speckle domains
(Li and Bingham, 1991 The ability of CTD-derived proteins to disrupt splicing
factor domains led us to ask whether these proteins can
specifically affect pre-mRNA splicing in vivo. Overexpression of the F-CTD52 protein blocks the accumulation of
spliced Removal of heptapeptides diminishes two in vivo properties of the Flag-tagged fusion proteins: their ability to inhibit splicing (Fig. 8) and their ability to disrupt speckle domains (Figs. 4 and 5). The correlation between the speckle
disruption and inhibition of splicing leads to the question
of whether pre-mRNA splicing takes place in the speckles.
Studies from other investigators indicate that certain Pol
II transcripts are produced and spliced in nucleoplasmic
sites outside of the SR protein-rich speckle domains
(Zhang et al., 1994 Two recent studies provide independent evidence that
Pol IIo is associated with splicing factors. Yuryev and colleagues used a yeast two-hybrid screen to identify CTD interacting proteins in rat cells (Yuryev et al., 1996 Pol II transcription and pre-mRNA splicing are known
to be closely associated processes in evolutionarily diverse
eukaryotic species (reviewed by Beyer and Osheim, 1991 Nascent pre-mRNAs may contain all of the information
required to recruit splicing factors. According to this
model, transcription and splicing machinery would be
linked exclusively by the pre-mRNA that is synthesized by
the polymerase. Alternatively, the basal Pol II transcription machinery may participate directly in the recruitment
and assembly of splicing factors on the nascent pre-mRNAs, as proposed in a speculative, but prescient model (Greenleaf, 1993 The results presented here provide compelling experimental support for the model proposed by Greenleaf
(1993) and references therein). Here, it is necessary to supplement this background with relevant genetic analyses of
the CTD. Previous studies showed that removal of more
than half of the CTD is lethal in yeast (Nonet et al., 1987
),
Drosophila (Zehring et al., 1988
), and mammalian cells,
indicating that the upstream half of the CTD is essential for cell viability. In addition, a positive and incremental effect on gene expression and cell growth is achieved as heptapeptides are added to the upstream half of the CTD
(Nonet et al., 1987
; Scafe et al., 1990
). These genetic studies indicated that partial truncation of the CTD leads to
partial functional deficits in gene expression, but the molecular basis of these effects is poorly understood. Consistent with a transcriptional role for Pol II's CTD, mouse
Pol II molecules containing five or fewer CTD heptapeptide repeats cannot respond to enhancer-driven activators
in vivo (Gerber et al., 1995
).
).
The ability of the unphosphorylated CTD to interact with
general transcription factors and the suppresser of RNA
polymerase B (SRB) mediator complex suggests a transcriptional role for the CTD, and it is consistent with the
idea that phosphorylation of the CTD releases the polymerase from the promoter-bound transcription factors
(discussed by Koleske and Young, 1995
; Dahmus, 1996
). Although there is increasing evidence indicating a transcriptional role for the CTD, it remains unclear whether
CTD phosphorylation regulates transcription, or whether
it merely coincides with transcriptional initiation (see Kim
et al., 1997
and references therein). Indeed, it is possible
that CTD is a multifunctional domain with roles in transcription as well as other processes, which may not be revealed by genetic selection (viability) or in vitro transcription assays.
; Blencowe et al., 1996
; Zeng, 1997). In addition, Pol IIo, SR proteins and Smith antigen-containing
small nuclear ribonucleo proteins (Sm snRNPs) become
sequestered in dot-like nonchromosomal domains during
mitosis, when transcription is inactive (Warren et al., 1992
;
Bregman et al., 1994
). These immunolocalization experiments revealed Pol IIo molecules in the same nonchromosomal location as certain splicing factors, but it was the
preceding study which showed for the first time that splicing factors are associated with Pol IIo in the absence of premRNA, and at times when the polymerase is not engaged
in transcription (Kim et al., 1997
). The latter findings,
together with the observation that anti-CTD phosphoepitope-specific mAbs H5 and H14 can release Pol IIo
from the splicing factors in vitro (Kim et al., 1997
),
strongly imply that Pol IIo's association with the splicing
factors is mediated by the hyperphosphorylated CTD. Indeed, the results of the latter study prompted us to ask
whether the CTD interacts with the pre-mRNA splicing
process in vivo.
-globin transcripts in
vivo. Interestingly, the stepwise addition of heptapeptide
repeats to a fusion protein potentiates its ability to disrupt
the splicing factor domains, and to inhibit splicing in vivo.
These results, in conjunction with the preceding study,
strongly suggest that the highly conserved and repetitive
CTD links splicing components to a key subunit of RNA
polymerase II, thereby helping to coordinate the processes
of transcription and splicing.
Materials and Methods
). Full-length CTD coding sequences
were obtained from a human Pol II LS cDNA isolated and sequenced by
(Du, L., unpublished results). The Pol II LS cDNA was authenticated by
comparison to EMBL sequence X63564 (Wintzerith et al., 1992
). A 2.1-kb
BamHI fragment containing the COOH-terminal domain plus 146 bp of
3
-untranslated mRNA was subcloned into the BamHI site of pcDNA3AB,
an expression vector derived from pcDNA3 (Invitrogen, San Diego, CA).
pcDNA3AB has a Flag® epitope (AspTyrLysAspAsp AspAspLys; Kodak)
immediately upstream of the multiple cloning site (Morrow, J., personal
communication). The full-length Flag-tagged CTD expression plasmid is
termed "pF-CTD52" to indicate the presence of 52 heptapeptide repeats.
pF-CTD52 is predicted to express a fusion protein comprised of an NH2terminal Flag peptide® attached to 636 amino acids derived from the
COOH terminus of human Pol II LS. The latter segment includes residues
1335-1588 (immediately upstream of the CTD) and residues 1589-1970, which contain 52 tandemly repeated heptapeptides. pF-CTD32 (analogous to pF-CTD52, but lacking heptapeptides 33-52) was derived from a
BamHI/EcoRI cDNA clone isolated from a human fetal liver library (Stratagene, La Jolla, CA); sequence analysis revealed this fragment to be
truncated within the 32nd repeat of the CTD coding sequence (Du, L.,
and S. Warren, unpublished results). pF-CTD26 (identical to pF-CTD52,
but lacking heptapeptides 27-52) was made as follows: the 2.1-kb BamHI
fragment of human Pol II LS cDNA (from nt 4001 to nt 6059 of coding region) was digested with SpeI. The resulting 1.3-kb BamHI-SpeI fragment
was subcloned into the BamHI-XbaI sites of pcDNA3AB. pF-CTD13,
pF-CTD6, pF-CTD3, and pF-CTD1 were generated by PCR mutagenesis.
The forward primer for each of these reactions was p4204U (5
AAGAGGTGGTGGACAAGATGGATG-3
), an oligonucleotide that hybridizes to a 24-nucleotide sequence 183-160 base pairs upstream of the
BamHI site located at nucleotides 4001-4006 within the coding portion of
the human Pol II LS cDNA. Reverse primers included: p5394 (5
-GCGAATTCGCTGGGAGAGGTGGGCGAATAGCT-3
) for pF-CTD13; p5264 (5
-GCGAATTCGGACTGGTTGGAGAATAGGATGGA-3
) for
pF-CTD6; p5205 (5
-GCGAATTCAAGAGGGACTCTGGGGTGTGTAGCC-3
) for pF-CTD3, and p1CTD (5
-GCGAATTCAGCTTGGACTAGTGGGTGAGTAGCTGGGAGACATGGCGCCACCTGGTGA3
) for pF-CTD1. The PCR products were digested with EcoRI (encoded in downstream primers) and BamHI (present in Pol II cDNA 183-160 nucleotides downstream of the upstream primer). The PCR products were
subcloned into the BamHI/EcoRI sites of pcDNA3AB.
-GCGAATTCGGCTTTTTGTAGTGAGGTTTG-3
) and p1209L (5
GCGAATTCGTCAGCC-AGTTTGTGAGTCAGGTC-3
). The amplified segment of DNA was digested with
EcoRI and subcloned into the EcoRI site of pcDNA2AB. Another control plasmid, pF-CTDless.1, expresses a Flag-tagged ~25-kD segment of
Pol II immediately upstream of the CTD. This control sequence corresponds to a 714-bp BamHI-SmaI fragment derived from the Pol II LS
cDNA, which was subcloned into the BamHI-EcoRV sites of pcDNA3AB.
A third control, pF-CTDless.2, expresses a Flag-tagged ~22-kD segment
of Pol II LS, which contains a 500-bp fragment immediately downstream
from the BamHI site. This fragment was generated by PCR amplification
using oligos p4204U (see above) and p4869L (5
-GCGAATTCAGCCGGTGGGTCCAGCAGC-3
). The PCR product was digested with BamHI
and EcoRI and subcloned into pcDNA3AB. This F-CTDless.2 protein is
similar to F-CTDless.1, but lacks a heptapeptide-like sequence (MFFGSAPSPMGGISPAMTPWNQGATPAYGAWSPSVGSGMTPGA A G F S P S A - ASDASGFSPGYSPAWSPTPGSPGSPGPSSPYIPSPGGA), which precedes
the CTD.
Galactosidase-linked
CTD Proteins
Gal-CTD fusion constructs were made as follows: First, the stop
codon at the end of the
Galactosidase gene was replaced with restriction
sites EcoNI, BamHI, and SalI, which were recombinantly added to the
COOH terminus. This PCR reaction used pSV
(Promega, Madison, WI)
as a template, and two primers: a downstream adapter oligo which contains a SalI site (pMCS, 5
-GCGTCGACTCTAGAATTCGCGGATCCTCCTGAAGGTTTTTGACACCAGACCAACTGG-3
) and an internal
oligo p3047 (5
-GGATTGGTGGCGACGACTCCTGGA-3
). The 130-bp
PCR product was digested with EspI and SalI and inserted back into
pSV
that had been cut with EspI and SalI to make pSV
MCS. Next, the
Gal coding sequence was excised from pSV
MCS with SmaI and BamHI,
and subcloned into pcDNA3 that has been cut with HindIII, filled in with
Klenow and cut with BamHI. The resulting vector, pcDNA
Gal, preserves all the cloning sites downstream of BamHI from pcDNA3. Finally,
CTD-26, CTD-32, and CTD-52 fragments with BamHI-EcoRI ends were
subcloned into the corresponding sites of pcDNA
Gal to generate the
Gal-CTD series (Fig. 1 shows only p
Gal-CTD52, p
Gal-CTDless, and
Gal).
Fig. 1.
Fusion proteins derived from Pol II's CTD. The largest subunit of RNA Polymerase II (Pol II LS) is illustrated schematically (top). An expanded view of the CTD shows 52 heptapeptide repeats represented by variably shaded boxes. Lightly shaded boxes represent consensus repeats (YSPTSPS) and more darkly shaded boxes represent variant repeats (see Corden et al., 1985; Wintzerith et al.,
1992). The CTD coding sequence was unidirectionally truncated from the COOH terminus and recombinantly fused to the Flag® peptide (Flag symbol) or
Galactosidase (oval symbol). The resulting fusion proteins are described by nomenclature that begins with the
NH2 terminus and ends with the COOH terminus, including the number of heptapeptide repeats. Symbols are summarized in the key
(for details see Materials and Methods).
[View Larger Version of this Image (19K GIF file)]
-Globin Genes and
Recombinant CTD-derived Proteins
-globin genes are generically termed "pF-CTDx
-globin [+/
]," where
"F" refers to the Flag peptide coding sequence, "CTD" refers to the sequence of the CTD-derived protein, "x" refers to the number of heptapeptide repeats, "
-globin" refers to the
-globin gene, and the "[+]"
and "[
]" signs designate the relative orientation of the two transcription
units. The plasmids were constructed as follows: A 2.7-kb HindIII-FspI
fragment containing the 2.3-kb human
-globin gene plus an SV40 enhancer element was excised from pUC
128SV (Caceres et al., 1994
), filled
in with Klenow fragment of DNA polymerase I, and subcloned into CTD
expression plasmids pF-CTD1, pF-CTD6, pF-CTD13, and pF-CTD52,
each of which had been digested with EcoRV. For controls,
-globin
genes were subcloned into pF-CTDless.1, pF-CTDless.3, and p
gal as illustrated in Fig. 7.
Fig. 7.
Plasmids expressing human -globin transcripts and CTD-derived fusion proteins. (A) A wildtype human
-globin gene
with a downstream SV40 enhancer (SV40E) was inserted
into an EcoRV site in multiple plasmids that express
Flag-tagged proteins or
Gal (Fig. 1). For brevity the illustration depicts the insertion
of various protein-encoding
sequences into a site upstream of the
-globin gene.
The Flag-tagged proteins and
Gal coding sequences are
under the control of the
CMV promoter (CMVp) (for
details see Materials and
Methods).
-Globin introns
are represented by open
boxes, exons by black boxes,
and noncoding flanking sequences open boxes at the
ends of the gene.
-Globin
and CMV promoters are indicated by bent arrows. The
resulting constructs are generically termed "FusionProtein
-globin [+]." The plus
sign indicates that the two
genes are oriented in the
same direction. The primers (P1 and P2) hybridize with complementary (cDNA) sequences within exons 1 and 2, respectively. PCR amplification with P1 and P2 yields 170-nt and 300-nt DNA fragments corresponding to spliced and unspliced transcripts, respectively. The 343-nt RNA probe used
for RNase protection is shown below the
-globin gene. The open box on this probe represents a nonhybridizing portion derived from
pBluescript, and the black bar hybridizes with a 276-nt segment of the unspliced
-globin transcript. The 276-nt segment spans an intronexon boundary including 203 nucleotides of exon 2 and 73 nucleotides of intron 1. Therefore, the spliced and unspliced
-globin transcripts protect 203 and 276 nucleotide segments of the probe, respectively. (B) A wild-type human
-globin gene with a downstream
SV40 enhancer (SV40E) was also inserted in the opposite orientation of the EcoRV site in the plasmids expressing Flag-tagged proteins
or
Gal. The resulting constructs are generically termed "FusionProtein
-globin [
]." The minus sign indicates that the two genes are
oriented in the opposite direction. For convenience, the protein-encoding sequences are not shown. (C) A thalassemic human
-globin
gene with a downstream SV40 enhancer (SV40E) was inserted in the positive orientation into the EcoRV site in the plasmids expressing Flag-tagged proteins or
Gal. The resulting constructs are generically termed "FusionProtein
-globinthal [+]." The thalassemic allele is mutated at first residue of intron 1 (G to A transition) (delta symbol). Splicing of exons 1 and 2 is achieved by using three cryptic
5
splice sites and the normal 3
splice site (see Caceres et al., 1995). The oligonucleotide used for RNase protection spans the 3
splice
site, but it is downstream of the cryptic 5
splice sites. Therefore, all three variably spliced transcripts register as 203 nucleotide RNAs in the RNase protection assay. For convenience, the protein-encoding sequences are not shown.
[View Larger Version of this Image (25K GIF file)]
). mAb M2 (Kodak) is an IgG that binds to the Flag® peptide, AspTyrLysAspAspAspAspLys. mAb anti-
Gal is an IgG that binds
to
Galactosidase (Promega). pAb anti-
Gal is a polyclonal antibody that
binds to
Galactosidase (Cappel, Malvern PA). mAb 138 is an IgG directed against ND55, a protein in N10 domains (Ascoli and Maul, 1991
).
Anti-coilin is a rabbit antiserum directed against p80 coilin (Andrade et al.,
1993
).
). For in vivo splicing assays, 106
HeLa cells were seeded in a 60-mm petri dish and transfected with each of
the plasmids (5 µg) using 45 µl of Lipofectamine®.
).
).
-globin gene, 5
-CAGGAGTGGACAGATCCC3
, was used for reverse transcription, followed by 10, 12, and 14 cycles of
PCR amplification using a forward oligo 5
-TCAAACAGACACCATGGTGCACCTGACT-3
which hybridizes to exon 1 of
-globin (167 nucleotides downstream from the HindIII site).
-globin was subcloned into the
corresponding sites of BlueScript-SKII (Strategene) and digested with
BbvII located within intron 1 to yield a linearized template. A complementary RNA probe was synthesized in vitro with T3 RNA polymerase (New
England Biolabs, Boston, MA) in the presence of 10 U of RNasin
(Promega), 0.5 mM of ATP, GTP, UTP, 3 µM of CTP, and 100 µCi [
-
32P]CTP, which yielded an internally radiolabeled 343-nt fragment covering exon 2 and the 3
half of intron 1. The probe was purified on a 4.5%
denaturing polyacrylamide gel and hybridized to total RNA prepared
from transfected HeLa cells at 45°C overnight. Hybridization mixtures
were digested with RNase A/T1, precipitated, solubilized with gel loading
buffer, and separated on a 4% denaturing polyacrylamide gel. The dried
gel was exposed to hyperfilm (Amersham Corp., Arlington Heights, IL)
overnight and scanned into a digital image using ScanJet (Hewlett Packard) and analyzed using NIH Image® software.
Results
) led us
to hypothesize that Pol IIo's association with pre-mRNA
splicing factors is mediated by the CTD. To test this hypothesis we first asked whether CTD-derived sequences,
which lack the catalytic and DNA-binding regions of the
Pol II LS, can target indicator proteins to speckle domains.
For this purpose, the Flag peptide (Flag) or
Galactosidase (
Gal) sequences were recombinantly added to the
NH2 terminus of the CTD-containing proteins. The resulting fusion proteins were transiently expressed and immunolocalized in CV1 cells. A similar approach has been used
to show that certain SR domains can target
Gal to speckle
domains (Li and Bingham, 1991
). Next, we asked whether
the CTD-derived fusion proteins interfere with splicing in
vivo. For this purpose, we co-expressed human
-globin
pre-mRNAs and CTD-derived proteins in HeLa cells, and
quantitated the efficiency of
-globin splicing in vivo. A similar approach has been used to assess the in vivo properties of splicing factors (Romac and Keane, 1995; Caceres,
1994).
Gal
(see below). During our investigations, we sought to determine the minimum number of heptapeptide repeats required to achieve certain biological effects (see below).
Therefore, the CTD-containing fusion proteins were unidirectionally truncated from the COOH terminus, giving
rise to a nested set of proteins containing heptapeptides 1-52 (F-CTD52 and
Gal-CTD52); 1-32 (F-CTD32 and
Gal-CTD32); 1-26 (F-CTD26 and
Gal-CTD26); 1-13 (F-CTD13); 1-6 (F-CTD6); 1-3 (F-CTD3), or only the first
heptapeptide (F-CTD1) (Fig. 1). Several control proteins
were used: (a) F-CTDless.1; (b) F-CTDless.2; (c) F-CTDless.3; (d)
Gal-CTDless and (e)
Gal (Materials and Methods).
Gal), and (b) an antibody directed at the
CTD portion of the fusion protein (mAb H5 or mAb H14).
). The above results indicate that
F-CTD52 accumulates in the nucleus, and suggest that
CTD heptapeptides on the F-CTD52 protein are phosphorylated in vivo. All of the CTD-derived and control proteins illustrated in Fig. 1 are expressed and enter the nucleus (see below).
Fig. 2.
Expression, in vivo
phosphorylation, and nuclear
localization of CTD-derived
fusion proteins. (A and B)
Immunoblotting. CV1 cells
were transfected with each of
the plasmids listed above the
panels (see Materials and
Methods). 2 d later, the cells
were lysed in SDS sample
buffer, subjected to 5-15%
gradient SDS-PAGE, and immunoblotted with the antibodies listed below each
panel. mAb M2 is directed
against the Flag® epitope,
anti-Gal is directed against
Galactosidase, and mAbs H5 and H14 are directed
against CTD phosphoepitopes (Kim et al., 1997
).
Numbers at the margins indicate apparent molecular
weights in kilodaltons. pSF,
control plasmid expressing a
Flag® tagged ~30-kD segment of human
-spectrin.
IIo, hyperphosphorylated largest subunit of Pol II. (C)
Immunofluorescence microscopy. CV1 cells were transfected with pF-CTD52. 2 d
later the cells were fixed and
double immunostained with anti-Flag® mAb M2 and anti-CTD mAb H14 (see Materials and Methods). Anti-Flag staining is visualized
by rhodamine (left panel) and mAb H14 staining is visualized by FITC (right panel). The cell at the top expresses the F-CTD52 protein,
and the cell at the bottom is an untransfected control. Note that mAb M2 labeling is almost exclusively intranuclear. In addition, the untransfected cell nucleus is weakly immunostained by mAb H14, whereas the transfected cell nucleus is intensely labeled. mAbs M2 and
H14 stain the diffuse nucleoplasm, but they also stain ~50 discrete "dots." Bar, 10 µM.
[View Larger Version of this Image (71K GIF file)]
; Kim et al.,
1997
; Blencowe et al., 1996
). Therefore, if the CTD-derived
proteins are expected to interact with splicing factors in
the nucleus, they probably need to be phosphorylated in
vivo. The immunolocalization studies described above suggest strongly that the CTD-derived fusion proteins are phosphorylated in vivo. To confirm this impression, and to
establish the electrophoretic mobility of each CTD-derived
protein, whole cell extracts were prepared from cells transfected with each plasmid in Fig. 1. The samples were subjected to 5-15% gradient SDS-PAGE and immunoblotted
with: (a) mAbs directed against CTD-specific phosphoepitopes (H5 or H14); or (b) mAbs directed at the indicator part of the protein (Flag or
Gal) (Fig. 2, A and B).
), these data indicate that the phosphorylation sites are within the CTD
portion of the fusion proteins.
Fig. 4.
Addition of heptapeptide
repeats to Flag-tagged fusion proteins potentiates their disruptive effect on B1C8 speckles. CV1 cells
were transfected with plasmids encoding fusion proteins listed at the
left margin. 2 d later, the cells were
fixed and double immunostained as
described in Fig. 3. The CTD-derived
fusion proteins were immunolocalized with anti-Flag® mAb M2 (A, D,
G, J, and M). A 160-kD SR-related
family splicing factor was immunolocalized with mAb B1C8 (B, E, H, K,
and N). Digital images were pseudocolored and merged as described in
Fig. 3 (C, F, I, L, and O). Red
pseudocolor indicates distribution of
CTD-derived fusion proteins. Green
pseudocolor indicates distribution of
SR splicing factor B1C8. Yellow
pseudocolor indicates overlap between red and green. White dots, nuclei expressing fusion proteins; thick
arrows, intact B1C8 speckles; thin arrows, Flag-tagged CTD-derived protein in discrete nuclear sites; arrowhead, B1C8 speckles immediately adjacent to F-CTD1 (B and C) and
FCTD6 (H and I). Bar, 10 µm.
[View Larger Version of this Image (58K GIF file)]
Fig. 3.
CTD52 disrupts
the speckled distribution of
SR splicing factor B1C8.
CV1 cells were transfected
with plasmids encoding fusion proteins listed at the left
margin. 2 d later, the cells
were fixed and double immunostained (see Materials and
Methods). CTD-derived fusion proteins and control
proteins were immunolocalized with anti-Flag® mAb M2
(A, D, and G) or anti-Gal (J
and M). A 160-kD SR-related family splicing factor
(Blencowe et al., 1995
) was
immunolocalized with mAb
B1C8 (B, E, K, and N).
ND55 was immunolocalized
with mAb 138 (Ascoli and
Maul, 1991
). Red pseudocolor indicates distribution of
Flag-tagged or
gal-linked
fusion proteins. Green
pseudocolor indicates distribution of endogenous nuclear proteins B1C8 or ND55. Red and green digital
images were merged (C, F, I,
L, and O), and areas of overlap between the distributions of transiently expressed
fusion proteins and endogenous proteins are pseudocolored yellow. White dots, nuclei expressing fusion
proteins; single arrows, B1C8
speckles; double arrows,
ND55 in N10/PML domains (H). Bars, 10 µm.
[View Larger Version of this Image (37K GIF file)]
Gal-linked CTD proteins
is presented in Fig. 2 B. mAb H14 immunoblots a ~240kD protein corresponding to endogenous Pol IIo (Fig. 2 B,
lanes 31-35). mAb H14 also immunoblots
GalCTD fusion proteins in cells transfected with p
Gal-CTD26, p
GalCTD32, and p
Gal-CTD52 (Fig. 2 B, lanes 33-36). Hyperphosphorylated
Gal-CTD52 comigrates with Pol IIo at
~240 kD; however, more rapidly migrating species are observed in some experiments (Fig. 2 B, lane 36). Finally, immunoblotting with an antibody directed at
Gal reveals
the expected stepwise increase in the PAGE mobility of
these proteins (Fig. 2 B, lanes 26-30).
Gal-CTD52 Induces the
SR-related Splicing Factor B1C8 to Redistribute from
Discrete Domains to a Diffuse Nucleoplasmic Pattern
, 1995).
Gal-CTD52, and the cells were
double immunostained with mAb B1C8 and anti-
Gal
(Fig. 3, J-L). Again, B1C8 has a speckled distribution in
control cells (Fig. 3 K, arrows), but it has a diffuse nuclear
distribution in cells expressing
Gal-CTD52 (Fig. 3 K).
B1C8 remains in a speckled distribution in nuclei expressing similar levels of a control protein,
Gal-CTDless (Fig.
3 N, arrows).
). N10 domains
are dynamic structures. For example, the number of N10
domains increases after growth factor stimulation, and
they disassemble following virus infection (Maul and
Everett, 1994
; Terris et al., 1995
). Several proteins in N10
domains have been identified, but none appear to have a role
in pre-mRNA splicing. Cells were transfected with pF-CTD52
and double immunostained with anti-ND55 mAb 138 (IgM) and anti-Flag mAb M2 (IgG). Our results indicate
that F-CTD52 does not alter the distribution of ND55, which remains exclusively in the N10 domains (Fig. 3, G-I).
). However, our experiment was complicated by the
fact that the B1C8 speckles, our intended landmarks, disperse in the presence of F-CTD52. Nevertheless, this outcome was gratifying, because CTD52 alters the distribution of an SR-related splicing factor that is colocalized
with native Pol IIo molecules in the speckles. These results
are consistent with the idea that the CTD interacts with
splicing factors in the speckles.
Gal (Du, L., and S.L. Warren, unpublished results). These data indicate that the speckled distribution of an SR splicing protein (B1C8) is incrementally
disrupted by the stepwise addition of heptapeptide repeats
to the fusion protein.
Fig. 5.
Relationship between CTD length and disruptive effect on B1C8
speckles. CV1 cells were
transfected with plasmids encoding the fusion proteins
listed above the histogram
bars. 2 d later, the cells were
fixed and double stained with
antibodies directed at the
Flag® epitope and B1C8 as
described in Fig. 4. The pattern of B1C8 staining in each
transfected cell nucleus was
scored as "intact" (20-50
prominent speckles) or "disrupted" (diffuse pattern or diminutive speckles). Data
were pooled from multiple
experiments performed on
different days. 150-250 nuclei were scored for each plasmid.
[View Larger Version of this Image (26K GIF file)]
). Other anti-SR mAbs, such
as 3C5 104 (Roth et al., 1991
), NM22, and NM4 (Blencowe
et al., 1995
) recognize multiple overlapping sets of SR
family proteins. To ascertain whether CTD-derived proteins alter the distribution of the SR proteins recognized
by these reagents, we repeated the experiment described
in Fig. 3, A-C, except mAb 3C5, mAb 104, mAb NM4, or
NM22 was substituted for mAb B1C8. Our results indicate that F-CTD52 disrupts the speckled staining pattern of all
four antibodies (Du, L., and S.L. Warren, unpublished results).
; Sharp, 1994
). The preceding study
showed that Pol IIo can be co-immunoprecipitated with
antibodies directed at Sm snRNPs (Kim et al., 1997
), so we
asked whether CTD-derived proteins induced Sm snRNP
antigens to become dispersed. The Sm snRNPs were localized with mAb Y12 (an IgG), so the anti-Flag mAb M2
could not be used for double staining. We addressed this
problem in two ways. In the first experiment, transfected
cell nuclei were distinguished from untransfected nuclei by
immunostaining with mAb H5 (IgM). This antibody recognizes phosphoepitopes on the CTD, and it stains nuclei
expressing phosphorylated CTD-derived proteins much
more intensely than control nuclei (Du, L., and S.L. Warren, unpublished results). In a second experiment, CV1
cells were transfected with p
Gal-CTD52, and double
stained with anti-
Gal (rabbit IgG) and mAb Y12.
Gal-linked CTD-52 protein has a more striking effect on
the Sm antigens. Immunostaining with anti-
Gal reveals a
brightly stained nucleus expressing the
Gal-CTD52 protein, and three faintly stained control cell nuclei (Fig. 6 D).
Examination of the same cells stained with mAb Y12 reveals that the Sm antigens are distributed more diffusely in
the transfected cell nucleus than in the untransfected cell
nuclei (Fig. 6 E, upper right).
Fig. 6.
CTD52 disrupts the speckled distribution of Sm snRNPs without altering the distribution of p80 coilin. Transfection and immunostaining were performed as described above. (A-
C) CV1 cells were transfected with pF-CTD52,
fixed and double immunostained with mY12 (directed at Sm snRNPs; Lerner et al., 1981), and
mAb H5 (directed at CTD phosphoepitopes;
Bregman et al., 1995
; Kim et al., 1997
). A nucleus expressing F-CTD52 is identified by the intense
immunostaining with mAb H5 (upper right corner). Three untransfected cell nuclei are identified by weaker mAb H5 immunostaining. (D-F)
CV1 cells were transfected with p
Gal-CTD52.
A nucleus expressing
Gal-CTD52 is identified
by intense immunostaining with mAb anti-
Gal,
and three untransfected cell nuclei are identified
by faint immunostaining with mAb anti-
Gal.
(G-I) CV1 cells were transfected with pF-CTD13. The distribution of F-CTD13 is revealed by red
pseudocolor in three transfected cell nuclei (G
and I). The distribution of p80 coilin is revealed
by green pseudocolor in the three cells expressing F-CTD13, and in one untransfected cell (H and I).
White dots, nuclei expressing CTD-derived fusion proteins; single arrows, speckles stained by
mAb Y12; double arrows, p80 coilin in coiled bodies (H and I). Bars, 10 µm.
[View Larger Version of this Image (40K GIF file)]
). Most cultured mammalian cells have 2-5 CBs,
which are easily visualized by immunostaining with antibodies directed at the p80 coilin autoantigen (Andrade et al.,
1993
). Speckles and CBs both contain certain splicing components, but their composition is otherwise very different: Pol IIo and SR splicing factors are present in
speckle domains, but they have not been reported in CBs.
Similarly, CBs contain p80 coilin, fibrillarin, and Nopp140,
which have not been reported in speckle domains. Finally,
transcriptional inhibitors and heat shock cause CBs to
shrink and speckle domains to enlarge, suggesting distinct
physiological roles for these two types of domains (Lamond and Carmo-Fonseca, 1993
).
-Globin
Transcripts In Vivo
), support the hypothesis that Pol IIo associates with SR splicing
factors and Sm snRNPs and via its CTD, but they do not
provide evidence indicating a functional relationship between the CTD and pre-mRNA splicing. If the processes
of transcription and splicing are linked via a CTD-mediated mechanism, then one might expect the CTD-derived proteins to interfere with transcription, splicing or both
processes. We approached this question by co-expressing
CTD-derived proteins and
-globin transcripts in the same
nucleus. A series of double expression plasmids was created by inserting intact
-globin genes into pF-CTD52, pFCTD13, pF-CTD6, and pF-CTD1 (Fig. 7 A). As controls, intact
-globin genes were inserted into pF-CTDless.1, pFCTDless.3, and p
Gal (Fig. 7 A). Each Flag-tagged CTD
coding sequence (or control sequence) is under the control
of a CMV promoter, whereas the
-globin gene is driven
by its own promoter. The
-globin genes were also inserted in the opposite orientation relative to the Flagtagged CTD coding sequences to control for possible cis
effects (Fig. 7 B).
globin [+], pF-CTD13
-globin [+], or pF-CTD52
-globin
[+] (Fig. 8 A). 24 h later, spliced and unspliced
-globin
transcripts were quantitated by RT-PCR. PCR primers
(P1 and P2) hybridize with sequences within exons 1 and 2, and therefore amplify a segment that includes intron 1 (Fig. 7 A). The PCR products corresponding to spliced
and unspliced
-globin transcripts are 170 and 300 nucleotides, respectively. The results of this experiment indicate
that co-expression of F-CTD52 reduces the amount of spliced
-globin transcript compared to the control, F-CTDless.1
(Fig. 8 A, S, lanes 2 and 4). In contrast, slightly more unspliced
-globin transcript accumulates in cells co-expressing F-CTD52 than in the control cells (Fig. 8 A, U, lanes 2 and 4). An intermediate effect is achieved by co-expressing F-CTD13 (Fig. 8 A, lane 3, U).
Fig. 8.
CTD-derived fusion proteins
block the accumulation of spliced, but
not unspliced, -globin transcripts in
vivo. (A) HeLa cells were transfected
with pF-CTDless.1
-globin [+], pFCTD13
-globin [+], or pF-CTD52
-globin [+]. 1 d later, total RNA was prepared from the cells as described in the
Materials and Methods. The RNA was
reverse transcribed and amplified by
PCR using primers P1 and P2 (see Fig. 7
A). The PCR products were separated
by agarose gel electrophoresis, stained
with ethidium bromide, and photographed. (B) HeLa cells were transfected
with pF-CTDless.1
-globin [+], pFCTD13
-globin [+], pF-CTD52
-globin
[+], pF-CTDless.1
-globin [
], pFCTD13
-globin [
], or pF-CTD52
-globin [
]. 1 d later, RNA was prepared
and subjected to the RNase protection
assay described in Fig. 7 and Materials
and Methods. Protected RNAs were
separated by electrophoresis and processed for autoradiography. (C) HeLa cells were transfected with pF-CTDless.1
-globinthal [+], pF-CTD13
-globinthal
[+], or pF-CTD52
-globinthal [+]. 1 d
later, RNase protection assays were performed as described in B. (D) HeLa cells
were transfected with pF-CTDless.3
globinthal [+], p
Gal
-globinthal [+], pFCTDless.1
-globinthal [+], pF-CTD1
-globinthal [+], pF-CTD6
-globinthal [+], pFCTD13
-globinthal [+], and pF-CTD52
-globinthal [+]. 1 d later, RNase protection assays were performed as described in B. In A-D, the expressed Flag-tagged proteins are indicated at the top of the panel. The
-globin transcripts, and their orientation relative to the CMV driven transcription unit, are indicated below the panels. U, unspliced; S, spliced; P, intact probe; C, control yeast
RNA; R, RNase added; M, molecular weight markers. MWs are indicated in base pairs at the left hand margin.
[View Larger Version of this Image (51K GIF file)]
-globin exon and 73 nucleotides at the 3
end of intron 1. Thus, unspliced
-globin transcripts protect 276 nucleotides, and spliced transcripts
protect 203 nucleotides of the radiolabeled probe (Fig. 7 A).
Similar amounts of spliced and unspliced
-globin transcripts
are present in cells expressing the control protein (Fig. 8 B,
lane 2); however, one observes no spliced
-globin RNA
in cells co-expressing the FCTD52 protein (Fig. 8 B, lane 4, S). Significantly, this reduction is accompanied by an increase of unspliced
-globin transcript (Fig. 8 B, lane 4, U).
Splicing is inhibited to a lesser degree by F-CTD13 than
F-CTD52 (Fig. 8 B, lane 3, U).
-globin
gene and CMV-Flag-CTD transcription unit, we reversed their
relative orientation on the plasmids. The resulting plasmid
constructs (pF-CTDless.1
-globin [
], pF-CTD13
-globin
[
], or pF-CTD52
-globin [
]) were transfected into
HeLa cells, and the RNase protection assay was performed.
Again, one observes a reduction of spliced
-globin RNA in
cells co-expressing the F-CTD52 protein (Fig. 8 A, lane 7,
U). A less severe inhibitory effect is achieved by co-
expressing F-CTD13 (Fig. 8 A, lane 6, U). More unspliced
-globin transcript accumulates in cells expressing CTDderived proteins than in control cells (Fig. 8, A and B).
This result indicates that CTD-derived proteins do not
block in transcription by RNA polymerase II. Indeed,
CTD-derived proteins selectively interfere with splicing.
-globin
gene that has a G to A transition at position 1 in intron 1 (see Caceres et al., 1994
). The thalassemic pre-mRNAs
are spliced at three cryptic 5
splice sites, each located upstream of the 343-nt RNA probe used in the RNase protection assay. All three cryptically spliced products should
protect 203 nucleotides of the radiolabeled probe, because
they all use the same 3
splice site. The thalassemic gene
was substituted for wild-type
-globin in pF-CTDless.1
globin [+], pF-CTD13
-globin [+], and pF-CTD52
globin [+] (Fig. 7 C), the resulting plasmids were transfected into HeLa cells, and RNase protection experiments
were performed as before. We have repeatedly found that
splicing of this thalassemic transcript is particularly sensitive to the inhibitory effects of the CTD-derived proteins (Fig. 8 C).
-globinthal
transcript and one of a nested set of CTD-derived proteins.
An RNase protection assay was performed as before (Fig.
8 D, lanes 2-8). The ratio of unspliced to spliced
-globinthal
transcripts is not significantly different in cells expressing F-CTD-less.3,
Gal, F-CTD-less.1, or F-CTD1 (Fig. 8 D,
lanes 2-5); however, this ratio increases progressively as
one adds 6, 13, and 52 heptapeptide repeats to the fusion
protein (Fig. 8 D, lanes 6-8). Indeed, a comparison of
spliced
-globinthal transcripts in cells expressing F-CTD1,
F-CTD6, F-CTD13, and F-CTD52 reveals a graded inhibition of splicing, which correlates with the number of heptapeptides added to the fusion protein.
Discussion
). However, we found that the
CTD-derived fusion proteins induce a striking disruption
of the speckle domains. The disruptive effect is global,
since multiple SR family proteins and Sm snRNPs redistribute from the speckles to a diffuse nucleoplasmic distribution. Moreover, the effect is specific for speckle domains, since the distribution of proteins in other types of
domains is unaffected.
-globin transcripts, but it does not block Pol II-
mediated transcription as indicated by the abundance of
unspliced
-globin transcripts (Fig. 8). The selective effect
of F-CTD52 on in vivo splicing provides the strongest evidence that one function of the CTD is related to premRNA splicing.
), while other transcripts are produced and spliced within, or at the periphery of, the speckle domains (Xing et al., 1993
, 1995
). The observation that CTDderived proteins disrupt speckle domains and interfere
with splicing argues that the CTD selectively affects (or interacts with) splicing components, but it does not help define where Pol II transcription and pre-mRNA splicing
take place relative to the SR protein-rich speckle domains.
). Four
proteins were identified, each containing repetitive SerArg
dipeptide (SR) motifs characteristic of the SR superfamily
of proteins; however, the SR domains in these proteins do
not bind to the CTD. One of these proteins, rA1, was
shown to bind yeast Pol II in overlay assays. These investigators also reported that wild-type, but not mutant CTD
peptides inhibit in vitro splicing reactions. Significantly,
rA1 is the only putative CTD-binding protein reported to
interact with Pol II molecules containing a hyperphosphorylated CTD. Independently, Pol IIo was also detected in
pre-mRNA splicing complexes assembled in vitro (Blencowe et al., 1996
).
).
Pol II transcripts are decorated with spliceosomal molecules in the lampbrush chromosomes (Gall, 1991
; and
references therein) and in polytene chromosomes (Matunis et al., 1993
; Barén and Wieslander, 1994
). Visualization of transcriptonally active chromatin by electron microscopy
strongly suggests that introns are excised cotranscriptionally (Beyer and Osheim, 1988
). Recently, co-transcriptional splicing has been directly demonstrated in polytene
chromosomes of C. tentans (Barén and Wieslander, 1994
).
Transcription and splicing are also closely associated in
mammalian cell nuclei. Fluorescent in situ hybridization experiments reveal that synthesis and splicing of specific
Pol II transcripts takes place in coincident foci in mammalian cell nuclei (Xing et al., 1993
; Zhang et al., 1994
; Xing
et al., 1995
). Additional evidence indicating that Pol II
transcription and splicing may be coordinated processes
comes from plasmid transfection and viral infection studies. All of the above studies indicate a close connection between Pol II transcription and splicing, but the mechanism
by which spliceosomes are recruited to Pol II transcripts remains poorly understood.
). According to this model, the phosphorylated
CTD helps recruit SR splicing factors to nascent Pol II
transcripts. Thus, in vivo spliceosome assembly would take
place processively on pre-mRNAs as they emerge from
the polymerase.
a few years ago. The preceding study showed that
splicing factors associate with Pol IIo without the direct involvement of RNA, and surprisingly, the association is
maintained at times when the polymerase is not engaged
in transcription (Kim et al., 1997
). The present study shows that CTD-derived proteins are phosphorylated in
vivo and accumulate in the nucleus, where they disrupt
splicing factor domains and interfere with pre-mRNA
splicing. In agreement with these in vivo results, CTD heptapeptides were shown to specifically inhibit in vitro splicing reactions (Yuryev et al., 1996
). Taken together, these
studies provide evidence for a functional interaction between Pol II's CTD and the splicing process, and they
strongly imply that transcription and pre-mRNA splicing
are coordinated by a mechanism involving a phosphorylated form of the CTD.
The current address of L. Du is Genome Therapeutics Corporation, Waltham, MA 02154.
Received for publication 1 June 1996 and in revised form 1 November 1996.
The work was supported by the Council for Tobacco Research (#3881) and National Institutes of Health (K08-CA01339) to S.L. Warren.We thank Euikyung Kim and David Bregman for helpful comments and
for critically reading the manuscript. We thank Michael Dahmus, Jeffery
Nickerson, Phil Cohen, Ed Chan, Gerd Maul, Xiang-Dong Fu, and Joan
Steitz for providing antibodies (see Materials and Methods). We thank
Adrian Krainer for providing -globin genes, and Jon S. Morrow for
pcDNA3AB and pSF. We thank David Ward for the use of his imaging facilities, and we are particularly grateful for the excellent technical assistance of Xun Sun.
CBs, coiled bodies; CTD, carboxy-terminal domain of RNA polymerase II; MIG, mitotic interchromatin granule cluster; nt, nucleotide; Pol IIa, unphosphorylated largest RNA polymerase II subunit; Pol II LS, largest subunit of RNA polymerase II; Pol IIo, hyperphosphorylated largest RNA polymerase II subunit; RT-PCR, reverse transcription-polymerase chain reaction; Sm sn RNP, Smith antigen-containing small nuclear ribonucleoprotein; SR, serine-arginine dipeptide repeat motif.