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Molecular and Cellular Biology, December 1998, p. 7510-7520, Vol. 18, No. 12
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The Pre-mRNA 5' Cap Determines Whether U6 Small
Nuclear RNA Succeeds U1 Small Nuclear Ribonucleoprotein Particle at
5' Splice Sites
Laura
O'Mullane and
Ian C.
Eperon*
Department of Biochemistry, University of
Leicester, Leicester LE1 7RH, United Kingdom
Received 4 June 1998/Returned for modification 10 August
1998/Accepted 31 August 1998
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ABSTRACT |
Efficient splicing of the 5'-most intron of pre-mRNA requires a 5'
m7G(5')ppp(5')N cap, which has been implicated in U1 snRNP
binding to 5' splice sites. We demonstrate that the cap alters the
kinetic profile of U1 snRNP binding, but its major effect is on U6
snRNA binding. With two alternative wild-type splice sites in an
adenovirus pre-mRNA, the cap selectively alters U1 snRNA binding at the
site to which cap-independent U1 snRNP binding is stronger and that is
used predominantly in splicing; with two consensus sites, the cap acts
on both, even though one is substantially preferred for splicing.
However, the most striking quantitative effect of the 5' cap is neither
on U1 snRNP binding nor on the assembly of large complexes but on the
replacement of U1 snRNP by U6 snRNA at the 5' splice site. Inhibition
of splicing by a cap analogue is correlated with the loss of U6
interactions at the 5' splice site and not with any loss of U1 snRNP binding.
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INTRODUCTION |
All precursor mRNAs transcribed by
RNA polymerase II have a 5' cap (m7G[5']ppp[5']N) added
cotranscriptionally (63, 68) by enzymes associated with RNA
polymerase II (8, 45). The 5' cap protects pre-mRNA from 5'
exoribonucleases (17, 20, 70) and has been shown to have an
important role in mRNA translation (62, 69). The cap also
has roles in pre-mRNA processing; it is implicated in both
polyadenylation and export from the nucleus, even if it is not
essential for these processes (12, 15, 21, 28, 43, 51, 77,
78), and it has an important role in splicing.
A role for the 5' cap structure in pre-mRNA splicing was shown by early
studies in extracts. In HeLa whole-cell extracts, substrate pre-mRNA
was spliced efficiently only when capped (32), and splicing
reactions could be inhibited by adding low levels of
m7GpppG or m7GTP cap analogues to the extract.
In HeLa nuclear extracts, splicing was reduced, but only by one-third,
in the absence of a cap (37, 52). Subsequent work showed
that cap dependence was enhanced if nuclear extracts were preincubated
in the presence of magnesium before the splicing reaction
(13). The splicing of introns other than the cap-proximal
intron is not affected much by the 5' cap (25, 55) because
of the presence of a polypyrimidine tract in an upstream intron
(42). This is consistent with the suggestion that the cap
functions in a manner analogous to that of an upstream polypyrimidine
tract in the definition of the adjacent exon (1, 23, 24, 42,
58).
The effects of the cap on pre-mRNA processing and export are mediated
by a cap-binding complex (CBC). In mammals, this comprises two proteins
of 80 and 20 kDa (26, 30, 54). Immunodepletion of the CBC
led to a loss of spliceosome assembly (26), including the
loss of most U1 snRNP base pairing with the 5' splice site (42). In Saccharomyces cerevisiae, loss of either
capping enzyme activity or a CBC component affected splicing efficiency
in vivo (9, 16, 66); in vitro, depletion of CBC reduced
commitment complex formation and splicing (9, 41), but the
absence of a 5' cap on the pre-mRNA did not affect the efficiency of
splicing (9, 66).
The finding that the CBC was required for efficient interactions of U1
snRNA with the 5' splice site seemed to be inconsistent with other
evidence. Not only are there reports that uncapped RNA can be spliced,
but in some cases uncapped RNA has been used intentionally to detect
complexes at 5' splice sites (52, 53, 61). Furthermore,
there are numerous reports of unaided interactions between pure U1
snRNPs and 5' splice sites (5, 22, 27, 31, 49, 60, 75).
These findings could be reconciled if the cap-CBC interaction was not
required constitutively but only at specific sites where U1 snRNP
binding is hindered by, for example, weak 5' splice sites or
sequestration of sites by proteins or RNA secondary structure. If this
is true, then the exact mechanism of the cap effect on U1 snRNP binding
would be expected to affect the selection of alternative 5' splice
sites in a 5' exon. Thus, if binding of U1 snRNPs was enhanced by the
cap, and the cap-proximal site was favored, then splicing would favor
that site. If the cap enhanced binding at all sites, then weak sites
would behave like strong sites and the presence of the cap would lead
to a shift in splicing preferences towards the cap-distal (downstream) site, according to the model described in reference
14. If the cap enhanced binding only at specific
sequestered sites, then the presence of the cap would lead to a shift
towards those sites at the expense of the site favored in the absence
of the cap. These schemes all suggest that the presence of the cap or
the concentration of cap-binding proteins may have important effects on
5' splice site selection. Previous studies have shown that the cap
cannot just promote cap-proximal U1 snRNP binding with alternative
strong 5' splice sites, because initial U1 snRNP binding did not appear
to depend on the proximity of the sites to the cap and the cap-distal
site was spliced (14, 50), but discriminatory effects of the
cap cannot be discounted for weaker sites, where the initial binding of
U1 snRNPs is less well characterized (5, 74) and the
cap-proximal site is sometimes preferred (57). The
uncertainties about the generality of the cap requirement for U1 snRNP
binding have such important implications for splice site selection that
we sought to determine whether the cap is required for binding and
splicing at all alternative splice sites, including strong consensus
splice sites.
We investigated U1 snRNP binding to alternative 5' splice sites on
pre-mRNA capped with m7GpppG or ApppG. Splicing at all the
sites was cap dependent. These experiments revealed that there were two
kinetic modes for U1 snRNP binding, a rapid binding mode that depended
on the 5' cap and a slower mode that was seen in the absence of the
correct cap or at sites that were not used for splicing. The cap itself did not appear to determine which site was bound rapidly and spliced. Strikingly, the m7GpppG cap was required for efficient interactions of
U6 snRNA at active 5' splice sites.
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MATERIALS AND METHODS |
Preparation of pre-mRNA.
Ad1 DNA constructs were derived
from pBSAd1 (34). The Ad1 template used was mutated to
introduce either a wild-type 5' splice site (GGG/GTGAGT), a
consensus 5' splice site (CAG/GTAAGT), or a nonfunctional
mutant sequence (GGCGAATTC) in place of either the natural
5' splice site or a cryptic 5' splice site in the intron. Mutagenesis
of only the cryptic splice site produced templates named Ad1WW (wild
type-wild type) and Ad1WM (wild type-mutant); mutations at both sites
produced Ad1MW (mutant-wild type), Ad1CC (consensus-consensus), Ad1CM
(consensus-mutant), Ad1MC (mutant-consensus), and Ad1MM
(mutant-mutant). The templates were linearized with SauIIIa
and transcribed with T3 RNA polymerase. The human 
-globin IVS-1
substrate pSP64 5'
16 (37) was linearized with
BamHI and transcribed with Sp6 RNA polymerase. Transcription
reaction mixtures contained 0.5 µg of DNA template; 500 µM (each)
ATP, CTP, and UTP; 100 µM GTP; 1 mM m7GpppG or ApppG cap
analogues (New England Biolabs); 20 µCi [
-32P]GTP
(NEN); and either T3 or SP6 RNA polymerase (Promega).
Splicing reactions.
HeLa nuclear extracts were obtained from
the Computer Cell Culture Centre (Mons, Belgium). The extracts depleted
of CBC were kindly given by J. Lewis and I. W. Mattaj (EMBL).
Extracts were preincubated at 30°C for 15 min to deplete ATP.
Splicing reactions were started by the addition of the pre-mRNA and
splicing mix and then were incubated at 30°C for a maximum of 90 min.
Reaction mixtures contained 40% (vol/vol) extract, 
0.5 ng (10 to
20,000 cpm) of substrate per µl, and 1.6 mM MgCl2
(13, 26). Reactions were initiated such that different
incubation times terminated together. The spliced products were
resolved on 6% denaturing polyacrylamide gels.
Native gel analysis.
Splicing reactions were assembled as
described previously. After incubation, 30 µl of buffer D
supplemented with 0.4 mg of heparin per ml and an additional 150 mM KCl
was added to each 7.5-µl reaction mixture, and the reaction mixtures
were incubated at 30°C for an additional 10 min. Samples were then
filtered through cellulose acetate filters (Costar) and loaded directly
onto 0.5% agarose-3.0% polyacrylamide gels.
RNase H digestion.
Protection of consensus 5'splice sites in
extracts was assayed by the addition of 100 pmol of the appropriate
oligonucleotide in 2 µl of buffer D plus 2 mM MgCl2 and 4 U of RNase H (Pharmacia) to 10-µl splicing reactions either with the
pre-mRNA for zero time points or 25 min later. Digestion was for 5 min.
The oligonucleotides were 14-mers complementary to the consensus 5'
splice sites. For RNase H digestion of Ad1 pre-mRNA cross-linked to
snRNA, splicing reaction products or gel-purified RNA was precipitated
with ethanol and dissolved in Tris-EDTA; portions were incubated with
50 pmol of oligonucleotide in 3 µl at 80°C for 15 s and
digested in 5 µl by the addition of buffer D, MgCl2 (to
3.2 mM), Nonidet P-40, (to 0.05% [vol/vol]), and RNase H. The
products were analyzed on denaturing gels. Oligonucleotides used to
cleave the snRNA were complementary to U1 snRNA nucleotides (nt) 123 to
142, U2 snRNA nt 1 to 15, U5 snRNA nt 68 to 88, or U6 snRNA nt 69 to
88. Oligonucleotides used to map the positions of the snRNA cross-links were complementary to the Ad1 pre-mRNA sequences centered at nt 40, 80, 120, 170, 210, 250, 290, and 330. The 5' splice sites are at nt 92 and
93 and 185 and 186; the 3' splice site is at position 326.
Psoralen cross-linking.
Splicing reaction mixtures were as
normal but included 0.8% (vol/vol) of a
4'-aminomethyl-4,5',8-trimethyl (AMT)-psoralen solution (2 mg of
dimethylsulfoxide [DMSO]; HRI Associates). Reactions (2.5 µl each)
were incubated in open wells of Thermowell C strips (Costar) in a
30°C water bath. Each strip was removed after a fixed time of
incubation. When samples were to be irradiated, the strip was placed in
a rigid holder that aligned the wands of a SpotCure (UVP) with the
wells, and samples were irradiated in pairs with two wands that
produced UV light of identical intensities. Irradiation was for 1 min
at room temperature with long-wave UV filtered via a thin glass plate.
Electrophoresis of the RNA was on 5% polyacrylamide gels containing 7 M urea and 30% (vol/vol) formamide. Unirradiated but parallel and
otherwise identical reactions were run on the same gels and also on 8%
gels. Dried gels were analyzed by PhosphorImager. The cross-links were
quantified by the use of profiles derived from the summation of signals
across the whole width of each lane; the intensity of each cross-linked band is given as a percentage of the sum of the intensities of the
pre-mRNA, the major internal cross-links, and the identifiable snRNA
cross-links. Where the cross-linked bands were very close (as in
upstream U1, b, and U2), the baseline was set to the background outside
the group and peaks were defined by vertical lines from the lowest
point between the peaks. In some cases, the long exposures required for
accurate measurement of faint signals led to saturation of the
precursor, and the level of pre-mRNA was calculated from a scale factor
derived from shorter exposures. The reactions shown in Fig. 3 differed
in that the volumes were 5 µl, the psoralen was added at 1.6%
(vol/vol), and where present, MgCl2 was at 2.7 rather than
1.6 mM.
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RESULTS |
Dependence of alternative 5' splice sites on the 5' cap.
Adenovirus 1 and human -globin IVS1 pre-mRNA transcripts primed with
either m7GpppG or ApppG caps were spliced in vitro in HeLa
nuclear extracts to confirm that splicing depended on the correct cap
under the conditions used in this study (Fig.
1). The ApppG cap structure is unable to
bind the CBC which mediates the effect of the m7GpppG
(26) and therefore was used as a control. The ApppG-capped pre-mRNAs have been shown to behave as uncapped substrates in splicing, both in extracts and in microinjected Xenopus
oocytes (25, 55). The substrates used all had alternative 5'
splice sites; the Ad1 template was mutated to create either an
additional wild-type 5' splice site in place of a cryptic 5' splice
site in the intron (Ad1WW) or a consensus sequence at both sites
(Ad1CC).
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FIG. 1.
Pre-mRNA substrates containing alternative 5' splice
sites require the m7GpppG 5' cap structure for splicing to
both sites. (A) Splicing of ApppG- and m7GpppG-capped human
-globin IVS-1 substrate psp64 5'16, which contains alternative 5'
splice sites. ApppG-capped RNA is in lanes 1 to 10 and 21 to 23;
m7GpppG-capped RNA is in lanes 11 to 20 and 24 to 26. Splicing reaction mixtures were incubated for the times indicated (in
minutes) above each lane. Lanes 8 to 10 and 18 to 26 were incubated for
120 min. Lanes A, B, and C contain 0, 100, and 1 mM m7GpppG
cap analogue as a competitor. Lanes D, E, and F contain increasing
amounts of SR proteins: 0.75, 1.5, and 3 µg. Cap analogues and SR
proteins were preincubated with the extract before the addition of the
pre-mRNA and splicing mix. Lane 27 (M) contains size markers. Spliced
products and intermediates were resolved on a 6% denaturing
polyacrylamide gel. (B) Splicing of m7GpppG- and
ApppG-capped adenovirus pre-mRNA with a duplicated wild-type 5' splice
site (Ad1WW). (C) Splicing of adenovirus pre-mRNA with duplicated
consensus 5' splice sites (Ad1CC). Splicing reactions were for 40 min
(lanes 1 and 3) or 1.5 h (lanes 2 and 4).
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Splicing to all 5' splice sites was severely reduced with
ApppG-capped substrates (Fig.
1A, lanes 1 to 7; 1B, lane 2; 1C,
lanes 3 and 4). The inhibition of splicing of the
-globin substrate
by exogenous m
7GpppG (Fig.
1A, lanes 19 and 20) and the
ability of SR proteins
to compensate for an ApppG cap (Fig.
1A, lanes
21 to 23) show
that the
-globin substrate behaves like the Ad1
pre-mRNA studied
previously (
42). We conclude that (i) the
m
7GpppG 5' cap structure enhances splicing at all three 5'
splice
site sequences (
-globin, Ad1, and consensus) and that (ii)
both
cap-proximal (
-globin and Ad1WW) and cap-distal (Ad1CC) sites
are affected. The absence of the cap did not noticeably alter
preferences in the residual splicing reactions. These results
suggest
that the role of the cap is not restricted to enhancing
U1 snRNP
binding at sites where it is hindered but that the cap
affects a
constitutive step in the
reaction.
Protection of consensus 5' splice sites in either
m7GpppG- or ApppG-capped pre-mRNAs.
The effect of the
5' cap on the interactions of U1 snRNPs was examined in extracts either
depleted of CBC or mock depleted. The efficiency of the depleted
extract in splicing was reduced compared to that of the mock-depleted
extract by 50 to 90% in several trials (data not shown). U1 snRNP
binding was measured by an incubation-dependent increase in the
protection of consensus 5' splice sites against ribonuclease H when RNA
was added to extracts lacking MgCl2, ATP, and creatine
phosphate (14). Under these conditions, splicing complex
assembly proceeds only as far as the E complex, which contains U1 snRNP
bound to the 5' splice site (47, 48, 71, 79). The substrate
used was a capped -globin IVS-2 pre-mRNA (C174C) containing two
consensus sites. When an oligonucleotide complementary to the upstream
5' splice site was added to the reaction mixture before the pre-mRNA,
incubation with RNase H produced almost complete cleavage (i.e., most
of the pre-mRNA was cleaved into two fragments in Fig. 2, lanes 4, 8, 12, and 16), but if the oligonucleotide
was added 25 min after the RNA, the site was almost completely
protected (Fig. 2, lanes 5, 9, 13, and 17). A smaller proportion of the
pre-mRNA was cleaved initially at the downstream site, probably because
of interfering RNA secondary structure (Fig. 2, lanes 6, 8, 14, and
16), but this, too, became completely protected after 25 min (Fig. 2,
lanes 7, 9, 15, and 17). If the reduction in splicing of 50% or more was caused by a corresponding loss of U1 snRNP binding in the depleted
extract, then at least 50% of the RNase H product fragments in lanes
12, 14, and 16 should have remained in lanes 13, 15, and 17. In fact,
lanes 13, 15, and 17 were identical to lanes 5, 7, and 9, from which we
conclude that there was equivalent U1 snRNP association in the two
extracts. This was also found to be the case for Ad1 substrates that
contained duplicated consensus 5' splice sites and for C174C capped
with m7GpppG or ApppG (data not shown). Thus, the 5' cap
was required for splicing but not for U1 snRNP-dependent complex
assembly at 5' splice sites. This suggested that the cap, via CBC and
interacting factors, may have a critical role in splicing beyond
permitting U1 snRNP binding.
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FIG. 2.
RNase H protection of consensus 5' splice sites. The
substrate pre-mRNA was m7GpppG-capped C174C, derived from
-globin IVS-2, which contained two alternative consensus 5' splice
sites. Pre-mRNA was incubated in HeLa nuclear extract that had been
depleted of CBC or had been mock depleted. Oligonucleotides that direct
RNase H cleavage to the consensus 5' splice sites were added either
just before the pre-mRNA (0) or 25 min after the pre-mRNA
(25). No MgCl2 or ATP was added to the reaction
mixtures. No oligonucleotide was added to the reaction mixtures in
lanes 2, 3, 10, and 11; an oligonucleotide directed against the
upstream site was added in lanes 4, 5, 12, and 13; an oligonucleotide
directed against the downstream site was added in lanes 6, 7, 14, and
15; both were added to the reaction mixtures in lanes 8, 9, 16, and 17. The reaction mixtures were resolved on a 6% polyacrylamide gel. The
products of RNase H cleavage are indicated with bars above the diagrams
of the pre-mRNA on the left-hand side.
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Analysis of snRNA binding by cross-linking.
The lack of a
requirement for the cap to mediate U1 snRNP binding appeared to be
inconsistent with previous findings (42), but the
inconsistency could be attributed to differences in the substrate or
method of detection; Ad1 pre-mRNA and psoralen-mediated UV
cross-linking were used in the earlier study (42). The
effect of differences in the 5' splice site sequence could not be
tested by RNase H cleavage, which does not detect initial interactions at nonconsensus 5' splice sites efficiently (14). Thus, we
used psoralen-mediated UV cross-linking to examine Ad1 pre-mRNA. The presence of the psoralen did not affect splicing (see below). The Ad1
substrates were modified at the 5' splice site and at a cryptic site 93 nt downstream such that they contained two wild-type sites (WW), two
consensus sites (CC), two nonfunctional sites (MM), or mixtures (WM and
MW). The sites of cross-linking by psoralen to Ad1 wild-type substrate
(equivalent to Ad1WM) have been characterized previously
(79).
A number of UV-dependent bands were detected by electrophoresis that
moved with lower mobility than the pre-mRNA (Fig.
3).
These were characterized by
oligonucleotide-directed RNase H cleavage
of purified cross-linked
molecules. A preparative cross-linking
reaction was carried out with
Ad1WW, and RNA was extracted from
each band after electrophoresis. The
RNA was treated with RNase
H and oligonucleotides complementary to
either U1, U2, U5, or
U6 snRNA (Fig.
4).
The results showed that there were two major
cross-links to U1 snRNA
and one to each of the U2 and U6 snRNAs.
No cross-links to U5 snRNA
were detected. The assignments of bands
to the upstream and downstream
5' splice sites were confirmed
by separate RNase H digestions of each
purified cross-linked product
at eight sites in the pre-mRNA spaced
approximately 40 nt apart
(data not shown). The bands visible in Fig.
3
between the two
major U1 snRNA-pre-mRNA cross-linked products (labelled
band a)
and between the upstream U1 cross-link and the U2-pre-mRNA
cross-linked
products (labelled band b) were also isolated and shown to
comprise,
respectively, wholly or largely U1 snRNA cross-linked to the
downstream
and upstream 5' splice sites; their intensities changed in
proportion
to changes in the intensities of the corresponding major U1
snRNA
cross-links. Double bands resulting from U1 snRNA cross-linking
to the Ad1 5' splice site have been observed previously (
79,
80).
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FIG. 3.
Psoralen cross-linking of U1 snRNA to
m7GpppG- and ApppG-capped Ad1 substrates. The substrates
used had two alternative 5' splice sites, each of which was either
consensus (C), wild-type (W), or mutant (M), as indicated above each
lane. (A) Pre-mRNA capped with m7GpppG (m7G) or
ApppG (A) was incubated for 20 min in splicing mixes containing
AMT-psoralen in the presence (lanes 1 to 8) or absence (lanes 9 to 16)
of magnesium, ATP, and phosphocreatine. Cross-linked products were
resolved on a 5% polyacrylamide gel. The positions of the snRNA
cross-links are shown diagramatically. Bands a and b are minor forms of
the two U1 cross-links.
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FIG. 4.
Identification of snRNA cross-linked to pre-mRNA. (A)
Splicing reaction mixtures containing m7GpppG-capped Ad1WW
were irradiated (as shown in Fig. 3, lane 3) and resolved on a 5%
preparative polyacrylamide gel. (B) The cross-linked products shown in
panel A were digested with RNase H and oligonucleotides complementary
to either U1, U2, U5, or U6 snRNA and then fractionated onto a 5%
polyacrylamide gel. The diagram above each gel indicates the identity
of the snRNA shown to be cross-linked to the pre-mRNA.
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The assignments made in Fig.
4 were shown to be correct for all the
substrates shown in Fig.
3 by cleavage of parallel portions
of each
m
7G-capped RNA reaction mixture with oligonucleotides
complementary
to U1 or U2 snRNA (data not shown). This showed that
comigrating
bands produced with different substrates contained the same
snRNAs.
In addition, the assignments fit the interactions expected with
different substrates; Ad1WM has only the upstream U1 cross-links
(Fig.
3, lanes 5 and 6), Ad1MM has no snRNA cross-links (Fig.
3, lanes 7 and
8), and neither U2 nor U6 cross-links are seen
in reaction mixtures
lacking ATP (Fig.
3, lanes 9 to
16).
With Ad1CC pre-mRNA, the cross-linking experiments confirmed the
findings from RNase H protection (Fig.
2) that U1 snRNP interacted
with
both consensus 5' splice sites and was not dependent on the
m
7GpppG or ApppG cap structure (Fig.
3, lanes 1 to 2 and 9 to 10).
Similarly, U1 snRNA was cross-linked to wild-type 5' splice
sites
whether the pre-mRNA had been primed with m
7GpppG or
ApppG (Fig.
3, lanes 3 to 4 and 11 to 12). This was true
even when only
E complex could be formed, in experiments with
splicing reactions
lacking magnesium and ATP (Fig.
3, lanes 11
to 14). Quantification
showed that the absence of a m
7GpppG cap caused no
proportional reduction in the level of U1
cross-links in the latter
experiments. In some other experiments,
the level of U1 cross-linking
in reactions lacking magnesium and
ATP was reduced by one-third or a
half with ApppG-capped RNA,
but the extent was variable. We conclude
that the association
of U1 snRNPs with the pre-mRNA is not
substantially dependent
on the 5'
cap.
Effects of the 5' cap on splicing complex assembly.
Comparisons on native gels of splicing complexes formed with
m7GpppG- and ApppG-primed pre-mRNA indicated that both
substrates can form A and B complexes (Fig. 5A, lanes 1 to 8;
quantification in Fig. 5B). The A complex
is the first ATP-dependent step in spliceosome assembly in which U2
snRNP enters the complex (2, 19, 33, 34, 47), and the
ability of the ApppG-capped Ad1WW pre-mRNA to form this complex was
expected from the psoralen cross-links to U2 snRNA described above.
However, complex formation was less than might have been expected from
the cross-linking. Thus, even though U1 snRNP binding to the substrate
appeared to be normal, the ApppG cap appears to affect either the rate
of the subsequent steps in complex assembly or the stability of the
complexes on native gel electrophoresis (56). Furthermore,
the B complexes formed do not splice in proportion to their abundance;
even after prolonged incubation, the splicing efficiency of
ApppG-capped RNA was usually extremely low (see below).
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FIG. 5.
(A) Native gel analysis of splicing complexes formed
with m7GpppG and ApppG primed Ad1 pre-mRNA. The incubation
times for the splicing reactions are shown. The presumed identities of
the bands are shown as complexes B, A, and H plus E. (B) Quantitative
analysis of the data in panel A. Numbers correspond to the lanes in
panel A.
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Appearance of cap-dependent RNA cross-links.
The native gels
suggested that the 5' cap affects both the rate of formation of
complexes and steps after the assembly of complex B but prior to step
1. These effects were examined by psoralen cross-linking. Splicing
reactions with the Ad1 substrate WW were incubated in the presence of
psoralen at 30°C for the times shown. Samples were then irradiated in
pairs at ambient temperature for 1 min each. Representative results are
shown in Fig. 6A; Fig.
7A shows the results of quantitative
analysis. Samples were removed also at 40 min and 1.5 h,
without cross-linking, for analysis of splicing. The splicing
efficiencies (calculated as mRNA/[mRNA + pre-mRNA]) after
1.5 h were 44 and 4% (for mRNA resulting from upstream and
downstream 5' splice site use, respectively) for
m7GpppG-Ad1WW and 1.8 and 0% for the corresponding
products from ApppG-Ad1WW. Thus, the difference in splicing efficiency
was over 20-fold.
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FIG. 6.
Effects of the 5' cap and splice site sequence on snRNA
interactions with pre-mRNA during time courses of splicing reactions.
(A) Ad1WW pre-mRNA, capped with m7GpppG or ApppG, was
incubated under splicing conditions in the presence of psoralen at
30°C. After the times shown above the lanes, reaction mixtures were
removed and irradiated for 1 min each at ambient temperature. The
results were analyzed by gel electrophoresis. The bands at the foot of
the panel are intramolecular cross-links. Parallel reactions included
(i) duplicates that contained an exogenous protein but showed the same
features on quantitative analysis, (ii) reactions in the absence of
MgCl2 and ATP, and (iii) reactions that were incubated for
40 and 90 min but not irradiated to allow splicing efficiency to be
measured. (B) Ad1WW, Ad1WM, and Ad1MW pre-mRNAs were incubated under
splicing conditions as in panel A, but for longer periods; irradiated;
and analyzed as in panel A. The unlabelled lanes contained RNA from
parallel reactions for 2.7 h that had not been irradiated so that
the efficiency of splicing could be measured. The band shown as
comprising U6 snRNA cross-linked to the downstream site, which is most
obvious in the reaction of Ad1MW (lanes 21 to 24), has not been
demonstrated directly to be this molecule, but its substrate
specificity, kinetics, and mobility are consistent with this
identification. (C) Identical to panel A in all respects, including the
parallel reactions, but with Ad1CC pre-mRNA.
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FIG. 7.
Quantitative analysis of the cross-links shown in Fig.
6A, B, and C refers to the three panels of Fig. 6, with the
corresponding substrate designations for panel B. The y axis
in every case shows the percentage of the cross-linked RNA molecule in
the reaction mixture (see Materials and Methods); the x axis
shows the time of incubation under splicing conditions (in minutes).
The reactions of RNA capped with m7GpppG and ApppG are
shown on the same graphs. The three graphs in each row show results for
U1, U2 and U6 cross-links, respectively, with the identities of the
lines shown below. u/s, upstream; d/s, downstream.
|
|
The cross-linking results quantified in Fig.
7A showed five main
features: (i) the m
7GpppG cap did not increase the final
level of U1 binding to the
upstream 5' splice site, but it increased
the rate of both the
initial binding and subsequent dissociation; (ii)
there was no
significant effect of the cap on U1 snRNA cross-linking to
the
downstream 5' splice site; (iii) U1 snRNA cross-linking to the
upstream 5' splice site predominated over the downstream 5' splice
site, regardless of the cap; (iv) the cap made little reproducible
difference to the level of U2 snRNA cross-linking; (v) U6 snRNA
cross-linking was virtually undetectable in the absence of the
correct
cap.
The steady increase in U6 cross-linking appeared to be the major
quantitative difference between the reaction mixtures containing
Ad1WW
RNA capped with m
7GpppG or ApppG. The sharp rise and fall
in U1 cross-linking to
the upstream 5' splice site also appeared to
depend on the cap,
but it was not clear whether this property was
associated with
use of a site for splicing or with the upstream site in
particular.
Both of these aspects were followed during longer time
courses
(Fig.
6B; quantitative results shown in Fig.
7B). The results
from the comparison of Ad1WW caps were very much the same as before,
but it was noticeable that, whereas U6 snRNA cross-linking to
the
upstream 5' splice site of m
7GpppG pre-mRNA continued to
accumulate, its cross-linking to the
RNA capped with ApppG did not
accelerate with time, indicating
that this step was slower rather than
delayed in its onset. The
sharp rise and fall in U1 snRNA
cross-linking, noted at the predominant
(upstream) 5' splice site of
Ad1WW, was seen at the upstream site
of Ad1WM and at the downstream
site of Ad1MW. Thus, this pattern
was seen with both splice sites
when they were used significantly
for splicing but neither at the
less-used site in m
7GpppG-capped Ad1WWn or in the absence
of the m
7GpppG cap. The samples incubated for
2.7 h without irradiation
were also run on an 8% polyacrylamide
gel. The percentages of
RNA at that time that had undergone at least
step 1 of splicing
were as follows: Ad1WW G cap, 24 and 6.3% (upstream
and downstream,
respectively); Ad1WW A cap, 3.3 and 0.5%; Ad1 WM G
cap, 49%; Ad1
MW G cap, 55%.
The cross-linking results with wild-type splice sites are consistent
not with a role for the cap in determining whether U1
snRNPs will bind
but, rather, in enabling binding to be productive.
One possible
mechanism might involve the recruitment of U1 snRNPs
such that they
bind rapidly to the site closest to the cap and
permit subsequent
interactions there. This possibility was tested
with Ad1CC RNA which,
as predicted for a pre-mRNA with two consensus
5' splice sites
(
14), splices preferentially to the downstream
(cap-distal)
site. A time course of incubation, followed by cross-linking,
is shown
in Fig.
6C; quantitative data are given in Fig.
7C. After
1.5 h,
5% of the RNA had undergone at least step 1 at the upstream
site and
22% at the downstream site (where total RNA at 1.5 h
is defined
as follows: pre-mRNA + both 5' exon intermediates,
corrected + both mRNA products, corrected). No splicing was detected
with the
ApppG-capped RNA. If the 5' cap is responsible for determining
the site
of splicing, the cap effect on U1 snRNA cross-linking
should be the
converse of the findings with Ad1WW, i.e., the effect
at the upstream
site should be greatly reduced or absent. As with
Ad1WW, U1 snRNP
binding to ApppG-capped RNA peaked at 15 min,
but the levels of binding
to both sites were approximately equal.
However, although the
m
7GpppG-capped RNA did show more rapid U1 snRNP binding,
peaking
at 5 min, binding was enhanced equally at both sites (Fig.
7C,
U1). The subsequent decline in U1 snRNA cross-linking was more
marked
at the site used predominantly for splicing. We conclude
that the 5'
cap is required for the productive binding of U1 snRNPs
at 5' splice
sites but that it does not determine the site at
which this happens.
The twofold difference in U6 snRNA cross-linking
between the two RNA
samples is comparatively small, but the intensities
were very low, so
the measurements are not
reliable.
Inhibition of the U6 but not U1 snRNA cross-links by the addition
of m7GpppG cap analogue as a competitor.
The cap
dependence of splicing and cross-linking reported here could be
attributed to inhibition by the ApppG cap rather than to dependence on
an m7GpppG cap. This was tested by incubation of
m7GpppG-capped Ad1WW RNA with increasing concentrations of
free cap analogues; a requirement for recognition of the correct cap would be reflected in inhibition by m7GpppG but not by
ApppG. The results showed that the U6 snRNA cross-link was reduced by
50 and 100 µM m7GpppG but that it was unaffected by
equivalent concentrations of ApppG (Fig.
8). The efficiency of splicing was also
reduced at these concentrations. In contrast, U1 snRNA cross-linking
was not reduced. The drop in U6 cross-linking and splicing at 50 and 100 µM m7GpppG is highly reproducible. The apparent
increase in U6 snRNA cross-linking at lower concentrations was also
seen in other experiments, but it has not been investigated further. We
conclude that m7GpppG is an effective competitor and that
recognition of the m7GpppG 5' cap is required for the
replacement of U1 snRNA at a 5' splice site by U6 snRNA.
View larger version (65K):
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|
FIG. 8.
Inhibition of U6 snRNA interactions with
m7GpppG-capped Ad1WW pre-mRNA by the m7GpppG
cap analogue but not the ApppG cap analogue. HeLa nuclear extract was
preincubated with the m7GpppG or the ApppG cap analogue for
15 min prior to the addition of the pre-mRNA. The final concentrations
of m7GpppG and ApppG were 0, 5, 10, 20, 50, and 100 µM,
as marked above the gel. The cross-linked RNA was resolved on a 5%
polyacrylamide gel. The bar charts aligned below the image show the
relative intensities of specific products and the pre-mRNA (expressed
as percentages) for each lane. The products measured are U1
cross-linked to the upstream 5' splice site (the major site of
splicing), U6 cross-linked, and the upstream (u/s) mRNA, as indicated
by the y axis labels.
|
|
| |
DISCUSSION |
The purpose of these experiments was to investigate further the
implications of previous evidence that the binding of U1 snRNPs to an
adenovirus 5' splice site depended on an interaction between the
m7GpppG cap and the CBC and that this caused the observed
dependence of splicing on the cap. Strikingly, our first results showed
that U1 snRNP binding did not require the correct cap, even though splicing was all but eliminated in the absence of the cap. Thus, even
though the low concentrations of pre-mRNA used may have precluded the
detection of changes in the overall affinity of the U1 complex for the
5' splice site in the presence of different caps, the fact that
splicing was inhibited in these reactions allows us to conclude that
the dependence of splicing on a 5' cap is not a consequence of any
effect of the cap on the overall level of U1 binding. Subsequent
quantitative analysis of psoralen cross-linking during time courses of
splicing showed that there are two different kinetic pathways for U1
snRNA to cross-link to the 5' splice sites: wild-type splice sites that
were not to undergo splicing (either because of their location or
because of the lack of a cap) bound U1 snRNA more slowly, although the
final level might be substantial, but sites that were to be spliced
bound U1 snRNPs much more rapidly. The 5' cap was required for splicing
and thus for the productive mode of binding; binding to an unused
alternative site was unaffected by the 5' cap. Substrates that lacked
the correct cap did form large complexes, albeit less efficiently, but
these did not lead to proportional levels of splicing. Instead, there
was a very marked effect of the cap on U6 snRNA interactions at the
wild-type 5' splice site.
Our finding that the correct cap was not required for U1 snRNP binding
as such is consistent with early studies on complex formation (52,
61) and with the properties of purified U1 snRNPs (see the
introduction) but in clear disagreement with the first report that the
cap and CBC are required in extracts (42). However, most of
the evidence in that report was based on psoralen cross-linking and gel
filtration with a 5' fragment of the Ad1 pre-mRNA, and it is possible
that U1 snRNP binding to this portion is unstable in nuclear extracts
in the absence of the cap. Thus, the kinetic effect on U1 snRNP binding
that we have detected on intact pre-mRNA may reflect interactions that,
in the absence of the 3' splice site region, stabilize binding.
Does the 5' cap act directly or via U1 to promote U6 snRNP
interactions at the 5' splice site?
The rapid rise and fall in U1
snRNP binding to active 5' splice sites was seen in earlier studies
with psoralen and site-specific 4-thiouridine cross-linking (71,
79, 80). The fall in U1 snRNP binding is likely to be associated
with displacement by U6 snRNA, as it in turn base pairs with the 5'
splice site (24, 29, 35, 36, 40, 44, 64, 65, 73, 79). The
effects of the cap on these two processes might be contingent or
separate. Thus, it is possible that the slower binding of U1 snRNPs to
ApppG-capped pre-mRNA results in a conformation that permits the
association of other components to form large complexes (albeit at a
slower rate) but that is not compatible with the interactions required to displace U1 snRNA and introduce U6 snRNA. It is notable that U1
snRNP binding to short uncapped 5' splice site RNA oligonucleotides prevented the assembly of U2-U5-U6 complexes (36). The
alternative possibility is that the CBC acts directly at a later stage
in the spliceosome to facilitate U6 snRNA interactions.
Some support for the contingent effect on U6 snRNA comes from
experiments in which m
7GpppG was added as a competitor at
various times before or after
the addition of
m
7GpppG-capped Ad1WW RNA; splicing was inhibited if the
competitor
was added before or with the substrate but not if it was
added
even as early as 5 min afterwards (data not shown). The ability
of exogenous SR proteins (members of the serine/arginine family
of
proteins) to circumvent cap dependence (reference
42
and
Fig.
1) might also suggest that the determining effect of the
cap
is on U1 snRNP binding to 5' splice sites, because some SR
proteins
enhance this (
14,
27,
31,
42,
74,
81), but
there is evidence
that they affect splicing reactions at later
stages as well (
3,
46,
59,
74). A separate and direct
effect of the cap on the
replacement of U1 snRNP by U6 snRNA at
the 5' splice site might require
the presence of the CBC in mature
spliceosomes. The CBC appears to
remain associated throughout
splicing (
42). Thus, neither
mechanism can be ruled out yet.
It should be possible to determine
whether the effect on U6 snRNA
cross-linking is an indirect consequence
of the cap effect on
U1 snRNP binding by testing pre-mRNA sequences
that can splice
in the absence of U1 snRNPs and without additional SR
proteins
(
10).
The 5' cap acts on U1 snRNPs that bind to the preferred 5' splice
sites.
The effects of the cap on the binding of U1 snRNPs to the
wild-type splice sites in Ad1WW might be consistent with several possible functions. Given that Ad1WW spliced almost exclusively from
the upstream 5' splice sites, it might be argued that the rapid rise
and fall in U1 cross-linking was a unique property of that site.
However, when that site was deleted (Ad1MW), the downstream site of the
cap-dependent substrate (data not shown) showed a similar profile (Fig.
6B and 7B). Thus, the cap seems to induce the profile associated with
productive binding at whichever site is used. The function of the 5'
cap, therefore, might be to determine the site at which U1 snRNP
binding would be rapid, or it might simply be required for rapid
binding at sites specified previously. The first possibility can be
excluded by the results with Ad1CC (Fig. 6B and 7B), which showed that
the cap promoted rapid binding of U1 snRNPs at both alternative sites,
even though the use of the upstream site was as small as that of the
downstream site in Ad1WW. Instead, it appears that the stronger U1
snRNP binding to consensus sites is sufficient for rapid binding in the
presence of the cap, whereas cap-dependent productive binding to Ad1WW
was restricted to the upstream site. We conclude that the cap is
required for rapid binding of U1 snRNPs but that this activity itself
is not selective. We cannot exclude an additional earlier role in other
substrates for the cap in the process that determines which of the two
wild-type sites is to be used.
Potentially productive U1 snRNP binding at both consensus 5' splice
sites is associated with use of the downstream site.
The role of
U1 snRNP binding in 5' splice site selection is still unclear (reviewed
in reference 4). Despite genetic evidence showing
that U1 snRNA base pairing can affect preferences between alternative
sites (67, 82), at least when they are well separated (24), and that this is in turn can determine U6 recruitment (24), the molecular mechanisms have not been established. We have shown previously that both of two alternative consensus 5' splice
sites can be occupied initially simultaneously by U1 snRNPs (14), and we proposed that this would be normal with strong sites; the marked preference among tandem strong 5' splice sites for
the downstream site, both in vitro and in vivo (11, 14), could be accounted for by interactions of 3' components with the nearest occupied site (14). Interestingly, the same
preference for a downstream site was elicited upon transfection of U1
and U6 snRNA genes that complemented tandem weak 5' splice sites
perfectly, suggesting that interactions with these two components were
sufficient for a site to behave as a strong site (24). The
cross-linking results shown here (Fig. 7C) with Ad1CC are also
consistent with this proposal; the cap-dependent rapid binding to both
sites, followed by splicing primarily to the downstream site, suggests that potentially productive or competent U1 snRNP binding has taken
place on both sites simultaneously and that selection of the downstream
site depends neither on the 5' cap nor on any selectivity in the U1
snRNP interaction. Genetic evidence (24) suggests that U6
snRNA is likely to mediate the selection.
Mechanisms that restrict the cap effect on U1 snRNP binding to one
alternative wild-type site.
For weaker sites, we suggested
previously (14) that the probability of simultaneous binding
was lower and that the outcome would depend on the relative levels of
U1 snRNP binding; if the use of a site depended on its being occupied
at some critical point in the reaction, the relative probabilities of
the sites being occupied at that point would determine the ratio of
use. The psoralen cross-linking data from Ad1WW show that both sites can be bound by U1 snRNPs, but the levels are quite unequal. In the
absence of the m7GpppG cap, the level of binding to the
downstream site is considerably lower, especially at early times. Thus,
cap proximity does not appear to determine the preferences. Instead,
the exclusive effect of the cap on binding to the upstream site may
merely reflect the existence of a limited period of time, up to the
critical point described above, during which the cap accelerates
binding; afterwards, when one U1 snRNP has occupied 100% of the
upstream sites and is interacting with 3' components, the 5' cap may
not be able to facilitate U1 snRNP binding elsewhere (it might, for example, be sequestered within the nascent spliceosome). This interpretation is consistent with the evidence from Ad1CC, in which the
levels of U1 snRNP binding to the two sites in ApppG-capped RNA are
more equal and the m7GpppG 5' cap accelerates both.
The identities of the proteins that mediate the effects of the cap on
U1 snRNA interactions at the 5' splice site are not
known. Yeast
two-hybrid screens have shown that CBP80 can interact
with several
proteins (
30), including hnRNP F (
18), but none
of these has been shown to affect U1 snRNP binding directly. Some
of
the factors that might influence cap-independent U1 snRNP binding
rates
can be surmised. In the present case, for example, the sequences
of the
two 5' splice sites to which U1 snRNA could base pair in
Ad1WW are
identical, but the rates of U1 snRNP binding might be
determined by
either RNA secondary structure or the proximity
of proteins that impede
or hasten binding. U1 snRNP binding is
enhanced by several SR proteins
(
14,
27,
31,
81), which
in Ad1 pre-mRNA are known to
cross-link in the E complex to sequences
in the 5' exon 26 to 31 nt 5'
of the 5' splice site (
6); at
limiting concentrations, these
may favor U1 snRNP binding at the
closest 5' splice site. Cross-linking
does not support the proposal
(
83) that SR proteins bind to
or select the 5' splice site directly.
Several other unidentified
proteins have been shown by cross-linking
to contact the 5' splice site
with or before U1 snRNA (
80).
These might affect U1 snRNP
preferences for the upstream 5' splice
site, or they might mediate the
effects of the 5'
cap.
The 5' cap had relatively little effect on the interaction of U2 snRNA
at the branch site. Given that U2-containing complexes
can assemble in
the absence of a functional 5' splice site (
7,
33,
36,
38,
39,
48,
61), this result suggests that
the primary effect of the cap is
at the 5' splice site and that
it does not have a more general role in
facilitating the assembly
of
complexes.
Correlations between the 5' cap, splicing efficiency, and U6
cross-links.
The effect of the cap on U6 snRNA interactions
at the 5' splice site was substantial. Interestingly, the rate of
the linear increase in U6 snRNA cross-linking to the upstream 5'
splice site between 5 and 45 min for three substrates
(m7GpppG-capped Ad1WW, m7GpppG-Ad1WM,
and ApppG-Ad1WW) in Fig. 6B was proportional to the level of splicing
(step 1) after 2.7 h (data not shown), perhaps implying that no
subsequent step is affected grossly by the cap. In contrast, the
m7GpppG-capped substrates Ad1WM and Ad1MW spliced
with similar efficiencies, even though the level of cross-linking to
U6 snRNA was quite different (Fig. 6B and 7B). This result might
imply that U6 snRNA interactions are not rate limiting at the
downstream splice site because of its distance from the 5' cap or for
other reasons, but it is also possible that the site itself affects the
cross-linking efficiency and that comparisons should not be made
between sites.
A candidate protein for mediating the effects of the cap directly on
the replacement of U1 snRNP at the 5' splice site by
U6 snRNP is the U5
100-kDa component of the U5 and [U4-U6-U5]
tri-snRNPs, which contains
an N-terminal RS domain and a C-terminal
domain that has sequences
suggestive of RNA unwinding activity
(
76). The C-terminal
domain is similar in sequence to the yeast
Prp28p, which has been
proposed to facilitate the U1-to-U6 transition
(
72). It
would be most interesting to determine whether these
proteins interact
with the
CBC.
Whether the 5' cap participates directly in snRNA rearrangements in the
spliceosome or whether it only ensures that U1 snRNP
is bound in a
manner that is sterically or kinetically compatible
with such
rearrangements, its role seems to be more subtle and
more complex than
the mere recruitment of U1 snRNPs to the 5'
splice
site.
| |
ACKNOWLEDGMENTS |
We are grateful to J. Lewis and I. W. Mattaj for
hospitality, advice, and materials during a visit by L.O. to EMBL.
This work was supported by a Human Capital and Mobility grant from the
Commission of the European Communities and by the Medical Research
Council, with an equipment grant from the Wellcome Trust.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, University of Leicester, Leicester LE1 7RH, United
Kingdom. Phone: (116) 2523482. Fax: (116) 2523369. E-mail:
eci{at}le.ac.uk.
| |
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Molecular and Cellular Biology, December 1998, p. 7510-7520, Vol. 18, No. 12
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
