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Vol. 13, No. 10, pp. 1234-1239, May 15, 1999
Department of Biological Sciences, Columbia University, New York, New York 10027 USA
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Abstract |
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 Top
 Abstract
 Introduction
Results and Discussion
Materials and methods
References
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RNA polymerase II (RNAP II) is responsible for transcription of mRNA precursors in eukaryotic cells. Recent studies, however, have suggested that RNAP II also participates in subsequent RNA processing reactions through interactions between the carboxy-terminal domain (CTD) of the RNAP II largest subunit and processing factors. Using reconstituted in vitro splicing assays, we show that RNAP II functions directly in pre-mRNA splicing by influencing very early steps in assembly of the spliceosome. We demonstrate that the phosphorylation status of the CTD dramatically affects activity: Hyperphosphorylated RNAP IIO strongly activates splicing, whereas hypophosphorylated RNAP IIA can inhibit the reaction.
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Introduction |
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Top
Abstract
Introduction
Results and Discussion
Materials and methods
References
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Splicing of mammalian pre-mRNA is a nuclear process in which
introns are removed from primary transcripts synthesized by RNA polymerase II (RNAP II). Splicing takes place in a
large macromolecular complex called the spliceosome, which is composed
of small nuclear ribonucleoprotein (snRNP) particles and non-snRNP
proteins including members of the serine/arginine-rich
(SR) protein family (for review, see Moore et al. 1993
; Kramer 1996).
Although cytological studies have suggested that splicing can occur
cotranscriptionally (e.g., Beyer et al. 1988; Bauren and Wieslander
1994) and factors required for splicing are found localized at sites of
active transcription (e.g., Zhang et al. 1994), functional coupling
between transcription and splicing does not seem obligatory because
splicing can be reconstituted in vitro with pretranscribed RNA and
splicing-competent cell extracts.
Recent studies, however, have provided evidence indicating that the
carboxy-terminal domain (CTD) of the largest subunit of RNAP II links
transcription with pre-mRNA processing (for reviews, see Corden and
Patturajan 1997; Neugebauer and Roth 1997; Steinmetz 1997). The CTD is
comprised of multiple repeats of the consensus sequence YSPTSPS, which
is highly conserved throughout evolution (for review, see Corden 1990)
and subject to reversible phosphorylation during the transcription
cycle (for review, see Dahmus 1996). RNAP II with a hypophosphorylated
CTD (RNAP IIA) is preferentially included in the preinitiation complex
at the promoter, whereas RNAP II with a hyperphosphorylated CTD (RNAP
IIO) is associated with elongation complexes.
Biochemical studies have shown that RNAP II, via the CTD, can
physically interact with capping enzymes (Cho et al. 1997; McCracken et
al. 1997a; Yue et al. 1997), polyadenylation factors (McCracken et al.
1997b), and splicing factors, including both snRNPs and SR-like
proteins (Chabot et al. 1995; Yuryev et al. 1996; Mortillaro et al.
1996; Kim et al. 1997). Notably, only RNAP IIO has been found to
associate with capping and splicing factors, and this isoform has also
been detected in active spliceosomes (Mortillaro et al. 1996). In
addition, in vivo studies using mammalian cultured cells have
demonstrated that RNAs transcribed by RNAP II with a shortened CTD
undergo inefficient capping, splicing, and polyadenylation (McCracken
et al. 1997a,b) and that overexpression of phosphorylated CTD peptides
inhibits splicing (Du and Warren 1997). Antibodies directed against the
CTD and CTD peptides have also been shown to inhibit splicing in vitro
(Yuryev et al. 1996). These observations have supported the view that
the phosphorylated CTD of elongating RNAP II may serve as a platform on
which processing factors bind, thus helping to promote efficient
splicing by recruiting necessary factors to the vicinity of the nascent
transcript. But an important, unaddressed issue is whether RNAP II
plays a direct, active role in the splicing reaction. Perhaps the
simplest view has been that RNAP II functions only as an `escort,'
helping to deliver factors to sites of processing, but does not
participate in the actual reaction. However, our recent finding that
RNAP II, and specifically the CTD, is necessary for polyadenylation in
vitro in the absence of transcription (Hirose and Manley 1998) led us
to consider the possibility that RNAP II might also function directly
in the more complex pre-mRNA splicing reaction.
Here, using in vitro splicing assays, we show that RNAP II participates directly in splicing in the absence of transcription. Purified RNAP IIO was found to strongly activate splicing of several different premRNAs, whereas RNAP IIA was capable of inhibiting the reaction. RNAP IIO functions very early to accelerate one of the first steps in spliceosome assembly, working in a manner distinct from, but complementary to, that of SR proteins. We discuss these results with respect to the coupling of mRNA transcription and processing.
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Results and Discussion |
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Top
Abstract
Introduction
Results and Discussion
Materials and methods
References
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That RNAP II plays a direct role in polyadenylation (Hirose and
Manley 1998) led us to consider the possibility that the enzyme might
also function in splicing. Arguing against this, S100 extracts of HeLa
cells lack RNAP II (Weil et al. 1979), yet can be activated for
splicing of many pre-mRNAs by addition of SR protein splicing factors
(for review, see Fu 1995; Manley and Tacke 1996; Valcárcel and
Green 1996). In the case of polyadenylation, the requirement for RNAP
II had escaped attention because high concentrations of another factor,
the small molecule creatine phosphate (CP), can substitute for it
(Hirose and Manley 1998). Perhaps CP, or some other factor, has masked
an RNAP II requirement.
To examine the possibility that RNAP II functions directly in splicing,
we first purified RNAP II from HeLa cells, following the procedure of
Lu et al. (1991). Given that some studies suggested that only the RNAP
IIO isoform is able to interact with splicing factors, this procedure
was especially appropriate because it allowed separation of RNAP IIO
from RNAP IIA. Aliquots of the two purified preparations were resolved
by SDS-PAGE and visualized by silver staining (Fig.
1a), which revealed that both were essentially homogeneous. Western blotting with an antibody that recognizes the CTD
(Besse et al. 1995) confirmed that neither isoform was detectably
contaminated with the other (Fig. 1b). Given the known ability of SR
proteins to activate splicing, we wished to provide additional evidence
that the RNAP preparations were not contaminated with SR proteins.
Figure 1c presents a Western blot utilizing an antibody that recognizes
an epitope shared in all SR proteins (Roth et al. 1990). SR proteins
were not detectable in either RNAP preparation (lanes 5,6) nor in an
S100 extract used in the experiments described below (lane 2). For
comparison, nuclear extract (NE; lane 1) and a preparation of purified
SR proteins (lane 4) are also shown. The SR proteins and RNAP II
preparations were then tested in standard S100 complementation splicing
assays, using a 
-globin-derived pre-mRNA (Fig. 1d). As expected,
the SR proteins strongly activated splicing (lanes 2,3), and RNAP IIA
and IIO were both inactive (lanes 4-7). Addition of either RNAP to
reaction mixtures containing SR proteins but lacking CP failed to
activate splicing (results not shown), indicating that the roles of
RNAP II in splicing, if any, and of CP are distinct from their function
in polyadenylation.
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Next we set out to determine whether RNAP II might be capable of
activating splicing under conditions where the efficiency of the
reaction was reduced. In Figure 2a, splicing of the -globin pre-mRNA was again assayed in S100 extract plus SR proteins, but the
efficiency was lowered (lane 2) simply by a slight alteration in the
concentrations of monovalent salts (see Materials and
Methods). Strikingly, addition of RNAP IIO to
reaction mixtures resulted in strong concentration-dependent activation
of splicing, ~15-fold at the highest amount tested (120 ng; lane 5).
In sharp contrast, identical amounts of RNAP IIA not only failed to
activate splicing but actually inhibited it, such that splicing became
undetectable as the concentration of RNAP IIA was increased (lanes
6-8). Equivalent amounts of control proteins, for example, glutathione
S-transferase (GST; lanes 9-11), BSA, and heat-inactived RNAP
II (data not shown) were without effect. To corroborate these results,
we repeated the experiment except substituting a single recombinant SR
protein, ASF/SF2 for the total SR protein preparation.
The results (Fig. 2a, lanes 12-20) were essentially identical: RNAP
IIO strongly stimulated splicing and RNAP IIA inhibited it.
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We also wished to determine whether the RNAP II isoforms might be
capable of influencing splicing when added to NE, which contains both
RNAP II and SR proteins. In this case, splicing efficiency was reduced
by using a lower amount of NE, a strategy employed previously to show
that splicing could be activated by certain SR proteins (Yeakley et al.
1996) or by the splicing regulator Tra2 (Tacke et al. 1998). The
results (Fig. 2b) show that the two RNAP II isoforms had the same
effects on splicing of the -globin pre-mRNA in NE as in S100: RNAP
IIA inhibited splicing (lanes 2-4) and RNAP IIO activated it
(lanes 5-7). Therefore, under these conditions RNAP IIO can be a
limiting factor for splicing, whereas the IIA isoform can have a
dominant-negative effect.
How does RNAP II actually function to influence splicing? One
possibility is that the enzyme, specifically the CTD, simply mimics SR
proteins. To test this we compared the effect of adding additional SR
proteins with that of adding RNAP IIO. The results in Figure 3a show
that splicing reactions reconstituted with S100 and 250 ng of SR
proteins (as in Fig. 2a) were not affected significantly by an
additional 120 ng of SR proteins, whereas the same mass of RNAP IIA or
IIO again had large effects. (These amounts
constitute nearly a 20-fold molar excess of additional SR proteins
relative to the added RNAP II.) Essentially identical results (not
shown) were obtained with a range of SR protein concentrations and with another pre-mRNA (IgM; see below). Given the difference in the behavior
of the two RNAP II isoforms, it seemed possible that the CTD alone
might be necessary and/or sufficient for the effects observed. Figure 3b, lanes 1-8, shows, however, that the CTD was not
sufficient, as neither phosphorylated nor unphosphorylated recombinant
GST-CTD (both of which could activate polyadenylation; results not
shown) had a significant, reproducible effect on splicing. This
distinguishes the effect of RNAP IIO on splicing from its previously
described function in 3'-end formation, where the CTD was shown to
be sufficient to activate 3' cleavage (Hirose and Manley 1998).
Not, unexpectedly, however the CTD was necessary, as RNAP IIB, a
proteolytic form lacking the CTD, was without significant effect,
positive or negative, on splicing (Fig. 3b, lanes 9-15). We also
examined a time course of the RNAP IIO-supplemented S100 splicing
reaction to determine when activation of splicing could first be
detected. As shown in Figure 3c, RNAP IIO enhanced splicing very early
in the reaction, at or before the first catalytic step, as judged by
the significant increase in reaction intermediates (5' exon and
lariat-3' exon) observed as early as 10 min and clearly by the
20-min time point.
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We also tested whether effects of RNAP IIO and IIA on splicing could be
observed with additional pre-mRNAs. Splicing of the IgM M1 and M2 exons
has been well studied, known, for example, to require an SR
protein-dependent exonic splicing enhancer (e.g., Watakabe et al.
1993). Figure 4a shows that this pre-mRNA responded to addition of both
RNAP IIO and IIA almost exactly as did the -globin pre-mRNA. RNAP
IIO strongly activated IgM splicing (~10-fold) in both S100 and NE,
whereas RNAP IIA nearly abolished it. We also
examined splicing of HIV tat pre-mRNA, which behaves distinctively from
the above pre-mRNAs. tat RNA is spliced very poorly in NE, except when
supplemented with additional ASF/SF2 (Krainer et al. 1990), and it can also be committed to splicing specifically by ASF/SF2 (Fu 1993). In vitro splicing of the tat pre-mRNA
was performed with S100 plus SR proteins or with NE, in the presence of
increasing amounts of both isoforms of RNAP II (Fig. 4b). RNAP IIO
again activated splicing in a concentration-dependent manner in the S100-reconstituted system (lanes 6-8). However, unlike with the other
pre-mRNAs, RNAP IIA did not significantly affect tat splicing (lanes
3-5). As expected, the tat pre-mRNA was not spliced efficiently in NE
(lane 10). Strikingly, RNAP IIO (lanes 13,14), but not IIA (lanes
11,12), was able to activate splicing, without supplementation with exogenous ASF/SF2. Together, these results suggest
that RNAP IIO can function as a general activator of splicing, whereas
the function of RNAP IIA as a splicing inhibitor may be more substrate specific.
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Pre-mRNA splicing requires the assembly of a series of spliceosomal
complexes on the substrate RNA preceding the chemical steps of the
reaction (for review, see Moore et al. 1993). Upon incubation of
pre-mRNA in splicing extract, three specific complexes, which can be
resolved on nondenaturing polyacrylamide gels, are formed:
prespliceosomal complex A, early spliceosomal complex B, and
catalytically active late spliceosomal complex C. To investigate whether either form of RNAP II affects spliceosome assembly, we performed nondenaturing gel analysis of spliceosomes formed on -globin pre-mRNA with S100 plus SR proteins and RNAP II. Figure 5
shows a time course of spliceosome assembly in the presence of buffer
(lanes 1-6), 80 ng of RNAP IIO (lanes 7-12), or 80 ng of RNAP IIA
(lanes 13-18). Addition of RNAP IIO resulted in
substantial increases in all three complexes. Most notable were the
significant increases in A complex at very early times of the reaction.
Complex formation was markedly enhanced even at the earliest time point tested (2 min; cf. lanes 2 and 8), indicating that RNAP IIO accelerates the rate of one of the first steps in spliceosome assembly. In contrast, RNAP IIA inhibited appearance of all complexes at all time
points, with one significant exception: There was no reduction in A
complex formation at the 2-min time point (cf. lanes 2 and 14). This
finding leads to the conclusions that the step(s) enhanced by RNAP IIO
is distinct from the step(s) inhibited by RNAP IIA and that RNAP IIA
can intervene in spliceosome assembly subsequent to initial recognition
of the pre-mRNA by splicing factors.
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We have presented evidence that RNAP II participates directly in
pre-mRNA splicing and that the phosphorylation state of the CTD
strongly affects its activity. These findings extend previous in vitro
and in vivo studies that suggested functional interactions between RNAP
II and splicing factors, that is, inhibition of splicing by antibodies
directed against the CTD (Chabot et al. 1995; Yuryev et al. 1996), by
peptides corresponding to CTD heptapeptide repeats (Yuryev et al. 1996;
Du and Warren 1997) and by truncation of the CTD of actively
transcribing RNAP II (McCracken et al. 1997b). The CTD has been shown
to interact directly with SR-like proteins (Yuryev et al. 1996), and
RNAP IIO can be detected associated with known splicing factors,
including snRNPs (Chabot et al. 1995; Mortillaro et al. 1996). Because
RNAP IIO appears to be the elongating form of the enzyme (Dahmus 1996)
and functions very early in spliceosome assembly, an attractive model
is that the enzyme, via the CTD and likely associated proteins (e.g.,
SCAFs; Patturajan et al. 1998b), facilitates binding of U1
and/or U2 snRNP particles to the pre-mRNA 5' splice
site and/or branch site, respectively. These RNAP
II-snRNP interactions are likely transient and serve to help commit
the nascent RNA to splicing and assure proper pairing of splice sites.
By this view, the final steps in spliceosome assembly, and certainly
splicing catalysis, could come after RNAP IIO has dissociated from the
splicing complex, perhaps already functioning to nucleate complex
assembly at downstream splicing signals. This is consistent with
electron micrographic visualization of actively transcribed genes,
which indicates the presence of possible splicing complexes (i.e.,
snRNPs) on putative splice sites of nascent RNAs (Beyer et al. 1988).
RNAP IIA, on the other hand, can inhibit splicing, apparently by
disrupting early pre-splicing complexes. This effect may be related to
findings of Yuryev et al. (1996), who reported that an unphosphorylated
CTD peptide containing eight heptapeptide repeats strongly inhibited
splicing in vitro, whereas mutant derivatives did not. However,
inhibition required at least a 103-fold higher molar
concentration of peptide relative to the levels of RNAP IIA used here,
suggesting either a different mechanism or that intact RNAP IIA
functions to inhibit splicing much more efficiently than does a CTD
peptide. Considerable evidence suggests that RNAP IIA assembles in the
preinitiation complex but is rapidly phosphorylated upon initiation of
transcription (Dahmus 1996). How then could this isoform play a role in
splicing? Although RNAP IIO is the principal elongating form, IIA has
been implicated in elongation on a small number of genes. Remarkably,
where identified, these tend to be intronless genes, such as those
encoding histones or heat shock proteins (Weeks et al. 1993; O'Brien
et al. 1994). RNAP IIA's negative effect on splicing in vitro may
reflect a proofreading mechanism that prevents inaccurate splicing by
disrupting inappropriate prespliceosomal complexes. In any case,
especially given that RNAP II appears to exist in multiple partly
phosphorylated forms (e.g., Patturajan et al. 1998a), our results not
only indicate that RNAP II is a direct participant in splicing but also
suggest that differential CTD phosphorylation has the potential to play an important role in splicing regulation.
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Materials and methods |
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Top
Abstract
Introduction
Results and Discussion
Materials and methods
References
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Proteins
NE and cytoplasmic S100 for splicing assays were prepared from
HeLa cells essentially as described (Tacke and Manley 1995; Tacke et
al. 1998). SR proteins were purified from HeLa cells by the method of
Zahler et al. (1992). Recombinant baculovirus-encoded ASF/SF2 was expressed in SF9 cells and purified as
described (Tacke and Manley 1995). Purification and separation of the
RNAP IIA and IIO from HeLa cell nuclear extract pellets was performed
as described (Lu et al. 1991; Hirose and Manley 1998). Recombinant GST-CTD was purified from Escherichia coli, phosphorylated or mock phosphorylated, and repurified as described (Hirose and Manley 1998). Purified calf thymus RNAP IIB was a gift of X. Sun and D. Reinberg (Rutgers University) (see also Hirose and Manley 1998). Western blots were performed as described previously (Tacke and Manley
1995).
In vitro splicing
32P-Labeled pre-mRNA substrates were prepared as
described (Tacke et al. 1998). Unless stated otherwise in the figure
legends, splicing reactions were carried out at 30°C for 80 min in
15 µl containing 4 µl of NE or S100 supplemented with the
amounts of purified SR proteins, recombinant ASF/SF2,
GST-CTD, and/or RNAP IIO, IIA, or IIB indicated in the
figure legends. The final concentrations of buffer components in all
splicing reactions were 13 mM HEPES (pH 7.9), 0.13 mM EDTA, 13% glycerol, 1 mM DTT, 2 mM
MgCl2, 1 mM ATP, 10 mM CP (disodium
salt), 0.16 units of RNasin (Promega), 1-2 ng of labeled substrate
RNA, and 2% (wt/vol) polyvinyl alcohol. With respect to
monovalent salts, in the experiment in Figure 1d, reaction mixtures
also contained 60 mM KCl and 8 mM
(NH4)2SO4, whereas in Figures 2-5, they
contained 27 mM KCl and 20 mM
(NH4)2SO4. The former concentrations were
optimal for SR protein-dependent splicing in S100, whereas the latter
reproducibly allowed significant response to RNAP II. We do not know if
this was due to the slight reduction in monovalent cations (76 vs. 67 mM) or the changed ratio of KCl to
(NH4)2SO4. Splicing products were
deproteinized and analyzed on 6% polyacrylamide gels containing 8 M urea. Splicing efficiencies were determined by
PhosphorImager (Molecular Dynamics).
Spliceosome formation assays
Reaction mixtures were exactly as above, except that 2%
polyvinyl alcohol was replaced with 7% glycerol. -Globin pre-mRNA was incubated in S100 extract plus 250 ng of purified SR proteins in
the absence or presence of 80 ng of RNAP IIA or IIO at 30°C for the
times indicated in Figure 5. Heparin (0.5 mg/ml) was then added, and reaction mixtures were incubated for an additional 5 min at 30°C. Five microliters of each reaction mixture was resolved on a nondenaturing 4% polyacrylamide gel.
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Acknowledgments |
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We thank C. Kedinger and M. Vigneron for mAb 7G5; X. Sun and D. Reinberg for purified RNAP IIB; S.H. Xiao and J. Prasad for plasmids; and X.H. Shi and Y. Sun for assistance in preparation of HeLa cell extracts. We are grateful to S.H. Xiao and J. Prasad for advice and discussions. This work was supported by grant R37GM 48259 from the National Institutes of Health. Y.H. was partly supported by the Japanese Ministry of Education, Science, and Culture.
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked `advertisement' in accordance with 18 USC section 1734 solely to indicate this fact.
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Footnotes |
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[Key Words: RNAP II isoforms; RNA processing; in vitro slicing; phosphorylation]
Received March 3, 1999; revised version accepted April 6, 1999.
1 Present address: Department of Biophysics, Cancer Research Institute, Kanazawa University, Kanazawa, Ishikawa 920, Japan.
2 Corresponding author.
E-MAIL jlm2{at}columbia.edu; FAX (212) 865-8246.
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References |
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Abstract
Introduction
Results and Discussion
Materials and methods
References
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