(Received for publication, February 6, 1997, and in revised form, April 16, 1997)
From the Department of Cell Biology, University of New Mexico Health Sciences Center, Cancer Research and Treatment Center, Albuquerque, New Mexico 87131
U1 small nuclear ribonucleoprotein (snRNP) may
function during several steps of spliceosome assembly. Most spliceosome
assembly assays, however, fail to detect the U1 snRNP. Here, I used a
new native gel electrophoretic assay to find the yeast U1 snRNP in three pre-splicing complexes (,
1,
2) formed in vitro. The order of complex
formation is deduced to be
1
2
1
2, the active
spliceosome. The
complex is formed when U1 snRNP binds to
pre-mRNA in the absence of ATP. There are two forms of
: a major
one,
un, unstable to competitor RNA; and a minor one,
commit, committed to the splicing pathway. The other
complexes are formed in the presence of ATP and contain the following
snRNPs:
1, the pre-spliceosome, has both U1 and U2;
2 has all five, however, U1 is reduced compared with the
others; and
1 and
2 have U2, U5, and U6.
Prior work by others suggests that U1 is "handing off" the 5
splice site region to the U5 and U6 snRNPs before splicing begins. The
reduced levels of U1 snRNP in the
2 complex suggests
that the handoff occurs during formation of this complex.
The U1 small nuclear ribonucleoprotein (snRNP)1 is one of several components including the U2, U4, U5, and U6 snRNPs and several non-snRNP proteins that form the spliceosome on pre-mRNA (for review, see Refs. 1-7). U1 is the first snRNP to bind to pre-mRNA during spliceosome assembly as studied by in vitro assays. Subsequently the U2 snRNP binds and the pre-spliceosome is formed. Next the U4/U5/U6 tri-snRNP binds, and the spliceosome is created. Finally, the spliceosome is activated for splicing by at least one conformational change in which the U4 and U6 snRNAs dissociate from one another.
Although the order of binding of snRNPs and some non-snRNP proteins
during spliceosome assembly is known, the presence and function of U1
snRNP in several of the spliceosome assembly intermediates are
controversial. The U1 snRNP has been thought to function in recognizing
the intron primarily during the early steps of spliceosome assembly. As
the first snRNP to bind to the pre-mRNA, U1 has an important,
hierarchic role in spliceosome assembly and in recognition of the
splice sites in the yeast Saccharomyces cerevisiae. The nucleotides 3-8 at the 5 end of U1 snRNA base pair with the
pre-mRNA 5
splice site region (8). This interaction probably
occurs when U1 binds to pre-mRNA because mutations in the
pre-mRNA 5
splice site decrease U1 snRNP binding and inhibit
subsequent steps in spliceosome assembly (9, 10). RNase H degradation
(9) or depletion (11) of the U1 snRNP prevents the other snRNPs from
binding to the pre-mRNA as well.
In addition to base pairing with the 5 splice site, U1 snRNP interacts
directly or indirectly with several other spliceosomal components. A
protein bridge may span the 5
splice site and branchpoint region (12,
13) and contribute to U1 snRNP binding to the pre-mRNA (9, 14). U1
snRNP may also associate directly with the U2 snRNP as a U1/U2 snRNP
complex can be induced in Hela cell extracts by an RNA complementary to
the 5
end of U1 snRNA (15). Finally, U1 snRNP may interact with
additional spliceosomal components during the late steps of spliceosome
assembly as suggested by the finding that a 2
-O-methyl
oligoribonucleotide complementary to U5 snRNA can induce the formation
of a U1/U4/U5 snRNP complex in Hela cell extracts (16). Most of these
interactions are probably important for spliceosome assembly but not
for splicing catalysis because once a yeast spliceosome is formed on
the pre-mRNA, it can lose U1 snRNA and still catalyze splicing
(17).
These interactions suggest that the U1 snRNP is present in several
intermediates in spliceosome assembly. At least in HeLa cell splicing
extracts, this is the case. When pre-mRNA is added to HeLa cell
extracts, the pre-splicing complexes E, A, and B form in that order as
precursors to the active spliceosome, complex C (18-22). The U1 snRNP
can be detected in all four complexes when purified by affinity
chromatography. However, it is found predominantly in E, and its
concentration decreases relative to the U2 and U4/U5/U6 snRNPs as these
snRNPs bind during formation of the A and B complexes, respectively
(20). These and other studies (23, 24) have proposed that the
association of the U1 snRNP with the spliceosome changes sometime
before splicing is catalyzed. In fact, U1 is supplanted by the U5 and
U6 snRNAs at the pre-mRNA 5 splice site before catalysis (for
review, see Refs. 5 and 6). Recently, it has been suggested that the
base pairing between U1 and pre-mRNA is disrupted even earlier,
before U2 snRNP binds (25, 26).
Although the U1 snRNP has been thought to participate in several steps of spliceosome formation in yeast, its presence in most of the yeast pre-splicing complexes formed in vitro has not been shown. Pre-splicing complexes III, I, and II, as detected initially by one-gel electrophoretic assay (27, 28), and complexes B, A2-1, and A2-2, as identified by a different gel assay (29), form in that order and contain the U2, U2/U4/U5/U6, and U2/U5/U6 snRNPs, respectively. U1 snRNP binding to the pre-mRNA during the first step of yeast spliceosome assembly was discovered subsequently by affinity chromatography (9) and by gel-electrophoresis (11). All gel electrophoretic assays, however, have failed to find U1 snRNA in pre-splicing complexes formed after this first assembly step (11, 18, 27, 29), although the U1 snRNP-specific 70K protein was found in pre-splicing complexes formed in HeLa extracts and resolved by gel electrophoresis (30).
To detect the U1 snRNP during yeast spliceosome assembly in
vitro, I developed a new native gel electrophoretic assay. I found U1 snRNA in three pre-splicing complexes (,
1, and
2). One of these complexes,
1 (the
pre-spliceosome), contains both U1 and U2 snRNAs as had been proposed
by previous studies (9, 11). The
2 complex contains the
U2, U4, U5, and U6 snRNAs, as well as reduced amounts of U1. This
reduction in the amount of U1 suggests that the association of U1 with
pre-mRNA or other spliceosomal components that makes this snRNP
stable in the pre-spliceosome is disrupted about the time the
2 complex is formed. This change in U1 may indicate the
"handing off" of the 5
splice site region from the U1 to the U5
and U6 snRNPs.
Plasmid for in vitro synthesis of Sp6-Wt (31) actin pre-mRNA was as described. The plasmid was cut with HpaII restriction endonuclease (New England Biolabs Inc.) for synthesizing the transcripts used in the native gel electrophoretic assays. Uncapped, radiolabeled pre-mRNAs for splicing and spliceosome assembly assays were synthesized in vitro with SP6 polymerase (Promega) and [32P]UTP (Amersham Corp.) as described previously (31). For Northern blot analyses, the pre-mRNA was synthesized with very low or no specific activity as described previously (29).
Whole cell splicing extract was made from strain EJ101 as described previously (31). A typical splicing reaction contained 0.4 nM radiolabeled pre-mRNA, 60 mM KPO4 (pH 7.4), 3 mM MgCl2, 2 mM ATP, 3% PEG8000 and 40% extract in 20 mM Hepes-K+ (pH 7.8 at 0 °C), 0.2 mM EDTA, 50 mM KCl, 0.5 mM dithiothreitol, and 20% glycerol. A 10-µl splicing reaction with radiolabeled pre-mRNA was incubated at 23 °C and then quenched by adding it to 10 µl of ice-cold 50 mM Hepes-K+ (pH 7.4), 2 mM (CH3COO)2Mg (R buffer; see Ref. 11) with 2 µg/µl carrier RNA. Carrier RNA was prepared from mouse intestine as described (32). The sample was incubated on ice for 10 min, and then 5 µl of loading buffer (200 mM Tris-phosphate (pH 8.0 at 0 °C), 50% (v/v) glycerol, 0.1% xylene cyanol, and 0.1% bromphenol blue) were added. The samples were fractionated on a 3.2% polyacrylamide gel (50:1, acrylamide:bisacrylamide) in TPM8 buffer (48 mM Tris-phosphate (pH 8.0 at 0 °C), 1.5 mM (CH3COO)2Mg)) at 4 °C for 16-20 h at 5.5-6.7 V/cm. The gel was placed on 3MM Whatman paper, and the complexes were visualized by autoradiography with film or a Molecular Dynamics PhosphorImager.
The previously described prp6-1 mutant (33) was used for
mutant extract. A UV-induced, temperature-resistant revertant of this
prp6-1 mutant (prp6-1R1), was isolated and used
for wild-type extract, as shown in Fig. 5.
Northern Blot Analyses
Ten-µl splicing reactions containing 4 nM pre-mRNA were run on native gels as described above. After electrophoresis, the gel was placed on 3MM Whatman paper and soaked in two changes of a 5-fold volume of 1 × TBE (8 mM Tris-borate (pH 8), 2 mM EDTA) with 8 M urea for 15 min each; this denaturation is essential for efficient transfer of the snRNAs in the next step. The RNAs in the gel were electrophoretically transferred to Gene Screen (New England Nuclear) in 0.25 × TBE at 120 V for 1 h at 4 °C in an apparatus described by Church and Gilbert (34). The wet membrane was then irradiated with three germicidal bulbs (General Electric, G15T8) at a distance of 35 cm for 15 min. The membrane was next simmered for 10 min in about 500 ml of boiling 0.1 × SSCP (120 mM NaCl, 15 mM Na citrate, and 20 mM NaPO4 (pH 7.0)) with 0.1% SDS in the microwave and then shaken for 10 min at room temperature. After prehybridization in hybridization buffer (50% formamide, 5 × SSCP, 0.1% SDS, 3 × Denhardt's solution, and 100 µg/ml sonicated, denatured salmon sperm DNA) at 42 °C for at least 30 min, the radiolabeled probe was added in 15-20 mls of fresh hybridization buffer and incubated with the membrane overnight. The blot was washed three times at 23 °C for 10 min each in 3 × SSCP with 0.1% SDS, once for 10 min at 55 °C, and once in 0.1 × SSCP with 0.1% SDS for 10 min at 55 °C. The hybridization was detected by autoradiography with either film or a Molecular Dynamics PhosphorImager.
For radiolabeled snRNA probes, the DNA fragments encoding the snRNAs on plasmids pT7-U1 and pT7-U2 (from D. McPheeters, Case Western Reserve University, Cleveland, OH), pT7-U4 and pT7-U6 (from P. Fabrizio, Phillips University Marburg, Marburg, Germany), and pT7-U5 (from L. Krinke) encoding the snRNAs were amplified by the polymerase chain reaction (35). The sizes of amplified fragments are the following: U1, 575 bp; U2, ~1200 bp; U4, 170 bp; U5, 180 bp; and U6, 115 bp. The amplified DNAs were then radiolabeled by random oligodeoxynucleotide-primed extension (36).
Deoxyoligonucleotide-directed RNase H Degradation of snRNAs in Splicing ExtractsDeoxyoligonucleotides for targeted degradation
of the U1 (oSR19), U2 (oSR20-also called SRU2), and U6
(d1) were as described (9, 37). For the experiment shown in
Fig. 7, A and B, the deoxyoligonucleotide oSR20
was added to a concentration of 1.3 µM in splicing
extract with 1.6 mM MgCl2 and 2 mM
dithiothreitol. After incubating the extract for 30 min at 30 °C,
water was added to bring the extract to 40% of the volume, and
KPO4 (pH 7.6), MgCl2, ATP, and
PEG8000 were added to final concentrations of 60, 3, and 2 mM and 3%, respectively. This brought the calculated concentration of the deoxoligonucleotide oSR20 to 600 nM
for the splicing reactions incubated in Step 1. After addition of an
equal volume of splicing reaction with active extract in Step 3, the final calculated concentration of oSR20 was 300 nM. The
conditions for inactivating the U1 and U6 snRNAs in extracts were as
described (9, 37).
Commitment and stability of the complex.
A, a flow scheme for determining if pre-mRNA in the
complex can be chased into the spliceosome. The scheme corresponds to
the reactions shown in lanes 6, 7, and 8 in panel B.
Step 1, radiolabeled pre-mRNA was incubated for 10 min
at 23 °C in a splicing reaction with ATP and with the U2 snRNA
inactivated by deoxyoligonucleotide-directed RNase H degradation.
Step 2, a 25-fold excess of cold pre-mRNA was added to
the reaction that was then incubated for 1 min at 23 °C. Step
3, an equal volume of a splicing reaction made with active whole
cell extract and ATP was added to the reaction. After 15 and 30 min,
samples were removed and assayed by native gel electrophoresis.
B, autoradiogram of the native gel electrophoretic assay.
The samples are from the following reactions: lane 1,
radiolabeled pre-mRNA was incubated in Step1 only; lane
2, radiolabeled pre-mRNA was incubated in Step1 and Step3 but
with no ATP in Step 3; lane 3, radiolabeled pre-mRNA was
incubated in Step1 and Step 3; lane 4, 25-fold molar excess
cold pre-mRNA was incubated in the splicing reaction in Step 1 and
radiolabeled pre-mRNA and active splicing extract and ATP were
added in Step 3; lane 5, radiolabeled pre-mRNA was added
in Step 1, excess cold pre-mRNA was added in Step 2, and carrier
RNA was added before active extract to quench the reaction; lanes
6, 7 and 8 are as diagrammed in panel A;
lanes 9 and 10, radiolabeled pre-mRNA was
added in Step 1, and active extract and ATP were added in Step 3;
lane 11, "0" time point, radiolabeled pre-mRNA was
added to the splicing reaction after the reaction had been stopped for
native gel electrophoresis; and lanes 12 and 13,
radiolabeled pre-mRNA was added to active splicing extract with ATP
(Step 3 only), and samples were removed at 15 and 30 min, respectively.
C, samples designated pre show splicing extract
with ATP and the U2 snRNA inactivated by deoxyoligonucleotide-directed RNase H degradation were incubated first for 10 min with 0, 1-, 5-, or 10-fold
molar amounts of cold pre-mRNA, and then for 10 min with
radiolabeled pre-mRNA, after which the samples were assayed by
native gel electrophoresis and visualized by autoradiography. Samples
designated post show radiolabeled pre-mRNA was incubated in extract first for 10 min and then for 10 min after the indicated amounts of cold pre-mRNA were added.
As the U1
snRNA is not found in most pre-splicing complexes resolved by native
gel electrophoresis, I specifically sought electrophoretic conditions
that would retain the U1 snRNP in pre-splicing complexes. Among the gel
buffer components tested, magnesium ion had the most significant effect
on U1 snRNP retention (Fig. 1). U1 snRNA, as detected by
Northern blot hybridization, is present with pre-mRNA in two bands,
and
1, in a gel run with or without magnesium
acetate, but there is an average of 16-fold more U1 in the
band
(n = 4, standard deviation = 1.9) with magnesium ion than without it. Additional assays revealed that the amount of U1
in the
band is proportional to the magnesium ion concentration in
the buffer up to 1.5 mM (data not shown). In a gel with or without magnesium ion, "free" U1 snRNP not bound to the added pre-mRNA migrates as two diffuse bands (designated by asterisks in
Fig. 1). There is less free U1 snRNP with than without magnesium, and
yet equivalent amounts of free U1 snRNP are detected in splicing reactions without added pre-mRNA. This suggests that most U1 snRNP bound to added pre-mRNA dissociates during electrophoresis without magnesium. The
1 band migrates near the slower form of
free U1 snRNP; however, it is distinct from free U1 snRNP as it is
sharp and its formation depends on the presence of added pre-mRNA.
That there is more
band and less free U1 snRNP in gels with
magnesium ion than without it suggests that magnesium ion stabilizes U1 snRNP bound to pre-mRNA. I included 1.5 mM magnesium
acetate in all subsequent assays.
To begin to determine the order of formation of the pre-splicing
complexes, I assayed their kinetics of formation in splicing reactions
at the normal 23 or at 15 °C, which slows the splicing reaction
(Fig. 2). Radiolabeled pre-mRNA was added to
splicing reactions with or without added ATP, and at various times
thereafter, samples were removed and analyzed by native gel
electrophoresis. In the absence of added ATP at either temperature,
there are two major bands, a rapidly migrating band that forms
nonspecifically on any RNA and the band. The
band forms quickly
at 23 °C with the maximum amount appearing by 2-5 min. Later, there
are low amounts of a slowly migrating smear that consists of two nearly unresolved bands (
and
); their formation without added ATP is
due to the low levels of ATP endogenous to the splicing extract (see
below; also see Ref. 38). In the presence of added ATP at 23 °C, the
band accumulates first with maximal amounts reached at 2-5 min and
then declines after 10 min. The
-
band reaches maximal amounts at
about 10 min. The reactions incubated at 15 °C form most of the
bands more slowly than at 23 °C, allowing additional kinetic
differentiation. In particular, the
band now appears first in the
presence of added ATP, followed by the more slowly migrating
band.
Little
band accumulates at these early times. The simplest
interpretation of the temporal relationship of the formation of these
bands is that one or more complexes in the
band is the precursor of
the complexes in the
and
bands. The
and
bands migrate
close to one another and do not resolve well when formed on the amount,
specific activity, and type of radiolabeled actin pre-mRNA used
here. Subsequent experiments described below show that these two bands
can be resolved.
If the band represents the first pre-splicing complex to form, then
its formation should depend on the U1 but not the U2 snRNP (9, 11). I
therefore analyzed its formation with radiolabeled pre-mRNA in
splicing extracts in which either the U1 or U2 snRNA was inactivated by
deoxyoligonucleotide-directed RNase H degradation (Fig.
3). The reaction with inactivated U1 snRNA forms no detectable pre-mRNA-specific bands (Fig. 3A). The reaction with
inactivated U2 snRNA forms the
band in the presence or absence of
ATP but is unable to form additional complexes. Additionally, extracts pre-incubated with the control deoxyoligonucleotide do not form the
and
bands unless ATP is added subsequently for the assay as the
pre-incubation step depletes the endogenous levels of ATP (39). U2
snRNA in the gel was detected by Northern blot analysis with a
U2-specific probe after sufficient time had elapsed for the radiolabel
in the pre-mRNA to decay. As previously reported for the conditions
used in this study (9, 40), greater than 97% of U2 snRNA was destroyed
when targeted for degradation (Fig. 3B, lanes
9-12). This destruction, however, has no effect on the formation
or migration of the
band even though free U2 snRNP normally
migrates close to the
complex. Furthermore, the
band has the
same mobility when formed in extracts with either wild-type U2 snRNP or
a smaller, but functional, U2 snRNP (41) despite the fact that the
small U2 snRNP migrates nearly twice as far as wild-type U2 snRNP in
the gel (data not shown). Thus, the formation and migration of the
band requires U1 but not U2 snRNP while the formation of the
and
bands requires ATP and both U1 and U2 snRNPs. The properties of the
band (that it contains U1 snRNA, forms in the absence of ATP and U2
snRNP, and forms first in the presence of ATP), as well as additional
properties described below, indicate that the
band contains a
pre-splicing complex that I have called the
complex.
To identify additional pre-splicing complexes that contain U1 snRNP, a
splicing reaction was incubated either with or without non-radiolabeled
pre-mRNA, fractionated by native gel electrophoresis, and analyzed
by Northern blot hybridization with the snRNA-specific probes
(Fig. 4). This experiment resolved three slowly
migrating complexes in addition to the complex. They are
distinguished by their mobilities, kinetics of formation, and snRNA
content, and are called
1,
, and
2.
The
and
complexes as detected with the U1 probe appear early in
the splicing reaction. Of note is that both U1 and U2 snRNAs are
present in the
complex at the early times (Fig. 4, lanes
1 and 6). At 30 min, however, U2, U5, and U6 snRNAs
accrue, and U1 snRNA remains the same (lane 3) or decreases
(data not shown). Furthermore, only pre-mRNA can be isolated from
the
complex at early times in splicing reactions, whereas
pre-mRNA and intermediates can be isolated from the
complex at
late times (data not shown). These results suggest that there are two
co-migrating
complexes that are kinetically distinguishable:
1, which forms early in the splicing reaction and
contains U1 and U2 snRNPs, and
2, which forms later,
contains U2, U5 and U6 snRNPs, and is the active spliceosome.
A third band formed on the pre-mRNA in the presence of ATP is also
apparent in this assay (Fig. 4). The band, as seen most clearly
with the U4 snRNA probe, is identified by having 1) a slower migration
than the
complexes, 2) a slower rate of formation than the
1 complex, and 3) the U4 as well as the U2, U5, and U6
snRNAs. A very small amount of U1 snRNA may also be present in this
band. The
band is more readily identified by Northern blot
hybridizations in part because the splicing reactions usually have
10-fold more pre-mRNA than those with radiolabeled pre-mRNA. Subsequent assays described below identified two complexes in this
band.
To confirm the nature and composition of the 1 complex,
I assayed its formation in a splicing extract in which the Prp6 protein was inactivated. The Prp6 protein is required for the U4/U5/U6 tri-snRNP to form and to be incorporated into the developing
spliceosome (42). Active splicing extract made from a
temperature-sensitive prp6-1 mutant grown at the permissive
temperature has been shown to be temperature sensitive in
vitro due to the mutant prp6 protein (33). The prp6-1
mutant extract is active for splicing in vitro at or below
26 °C but inactive at 30 °C, whereas the wild-type extract is
active at all the temperatures tested (data not shown). When splicing
reactions with mutant extract and radiolabeled pre-mRNA are
analyzed by native gel electrophoresis, however, the reactions incubated at 23 °C look similar to those at 30 °C in that most of
the radiolabeled pre-mRNA is in the
band (Fig.
5A). However, a defect in complex formation due to
inactivation of the mutant prp6 protein at 30 °C is detected by
Northern blot analyses (Fig. 5B). A wild-type reaction
incubated at 30 °C forms all three bands (
,
, and
) with
the U2, U4, U5, and U6 snRNAs accumulating in the
band by 20 min.
Again, a small amount of U1 snRNA may be present in the
band. The
U2, U5, and U6 snRNAs also accumulate in the
band at 20 min. In
contrast, a splicing reaction with mutant prp6 extract
incubated at 30 °C accumulates U1 and U2 but not U5 and U6 snRNAs in
the
band. Furthermore, little or no U2, U4, U5, or U6 is present in
the
band at 20 min. Additionally, little or no tri-snRNP can be
seen in the 30 °C reactions with mutant prp6 extract even
in the absence of added pre-mRNA (Fig. 5B, lane
7, in U4, U5, and U6 panels),
whereas this extract can form the tri-snRNP at 23 °C (data not
shown). These results indicate that the reactions with the mutant
prp6 extract at 30 °C form no tri-snRNP,
band, or
2 complex. They do, however, accumulate the U1 and U2
snRNAs in the
band, consistent with the properties of the
1 complex. Additionally, the results show that
1 is formed before the
and
2
complexes.
Inactivation of U6 snRNA by deoxyoligonucleotide-directed RNase H
degradation has also been shown to prevent the U4, U5, and U6 snRNPs
from forming the tri-snRNP and binding to the pre-spliceosome (37). I
found that both control and U6-inactivated extracts form and
1 complexes with
1 containing both U1 and
U2 snRNAs. The
and
2 complexes form only when U6
snRNA is intact (data not shown).
Given these data on the kinetics of formation, snRNA and pre-mRNA
content, and formation of the pre-splicing complexes in inactivated
extracts, I deduced the order of formation and composition of the ,
1, and
2 complexes to be as follows;
first
(with U1 snRNA), then
1 (with U1 and U2
snRNAs), and eventually
2 (with U2, U5, and U6 snRNAs).
The identities of complexes in the
band and their relationships to
the other complexes were revealed in the experiments described
below.
I next asked if U1 snRNA could be detected in any additional pre-splicing complexes. Two different approaches were used to increase the amounts of late-forming complexes.
In one approach, EDTA was added to the splicing reactions. Previously,
Cheng and Abelson (29) added EDTA to splicing reactions to identify and
distinguish in their native gel electrophoretic assay two complexes (A1
and A2-1) that form late in the assembly pathway. They showed that 5 mM EDTA in the yeast splicing reaction induces accumulation
of A1 complex in particular and almost completely inhibits splicing.
Complex A2-1 contains the U2, U4, U5, and U6 snRNAs and is most likely
the precursor to complex A1, which contains the U2, U5, and U6 snRNAs.
The A1 complex is the immediate precursor of the active spliceosome,
designated A2-2 in the gel system of Cheng and Abelson (29). I assayed
the effects of 5 mM EDTA on pre-splicing complex formation
by my native gel electrophoretic assay and Northern blot hybridizations
(Fig. 6).
Six complexes are distinguishable in the reaction with 5 mM
EDTA: , three
complexes (
1, and
1*, and
2), and two
complexes (
1 and
2). Three complexes that hybridize with the U1 probe form within 2 min after addition of pre-mRNA,
,
1, and
1*.
The
1 complex is so designated in this reaction because
it forms early, contains the U1 and U2 snRNAs, and migrates in the same
position as the
1 complex in reactions with no EDTA. A
novel
1* complex also contains U1 and U2
snRNAs and appears early but only in the presence of EDTA, and it
migrates more slowly than
1. The
2
complex, as most clearly seen with the U4 probe, contains U2, U4, U5,
and U6 snRNAs like the A2-1 complex of Cheng and Abelson (29).
Notably, a distinguishable, but small amount of U1 snRNA compared with
the other snRNAs is also present in the
2 complex. Finally, two complexes containing the U2, U5, and U6 snRNAs accumulate late in the splicing reaction. The most abundant of these two probably
corresponds to the A1 complex of Cheng and Abelson (29) as it
accumulates in the presence of 5 mM EDTA and contains the U2, U5, and U6 snRNAs, and hence, I have called it
1.
The second, more rapidly migrating and less abundant complex migrates
the same as the
2 complex in the reaction without EDTA
and is most likely the active spliceosome. Neither U1 or U4 snRNA is
detected in the spliceosome. Consistent with the low levels of
2 in this reaction, splicing was greatly inhibited by 5 mM EDTA (data not shown). When compared for their kinetics
of formation, snRNA content, and response to EDTA, the
2
and
1 complexes are analogous to the A2-1 and A1
complexes, respectively, of Cheng and Abelson (29). This comparison, as
well as the snRNA composition of the complexes, suggests that the order
of formation of the late pre-splicing complexes detected in this gel
assay is
1
2
1
2.
An endogenous U1-U5 bi-snRNP complex can also be observed in the presence or absence of EDTA in the splicing reaction (Fig. 6). The presence of this complex varies among extract preparations.
Because the amount of complex is dependent on magnesium ion in this
gel assay, the addition of EDTA to the splicing reaction might
destabilize U1 snRNP in the complexes. I therefore used a second
approach to determine if U1 is in the
1 complex.
Previously Cheng and Abelson (29) showed that inactivation of a
temperature-sensitive, mutant prp2 protein in vitro results
in accumulation of the A1 complex. I found that inactivation of the
prp2 protein caused the U2, U5, and U6 snRNAs, but not the U1 and U4
snRNAs to accumulate in the
band, and prevented
2
complex formation (data not shown). These results again indicate that
U1 snRNA is not in the
1 complex. They also suggest that
the
2 complex is normally short lived as this complex,
like the A2-1 complex (43), is not readily detected.
The complex has
several of the properties of the commitment complex (CC) (11, 44): 1)
it contains U1 snRNA and 2) forms on pre-mRNA in the absence of ATP
and the U2, U4, U5, and U6 snRNPs. CC was originally defined in yeast
splicing extracts as a complex that is formed in the absence of ATP
and, once formed, is stable in the presence of excess competitor
pre-mRNA and can be chased into an active spliceosome in the
presence of competitor and ATP (44). To test if the
complex is the
functional equivalent of CC, I determined if the
complex could be
chased into an active spliceosome in the presence of excess competitor
pre-mRNA. Radiolabeled pre-mRNA was incubated in extract in the
absence of ATP and an intact U2 snRNP (Fig.
7A, Step 1). After 10 min of incubation, a
25-fold molar excess of cold pre-mRNA was added (Fig.
7A, Step 2). One min later, additional extract
and ATP were added and incubated for an additional 15 or 30 min (Fig.
7A, Step 3). Samples were removed at the various steps and
assayed by native gel electrophoresis. When a 25-fold excess cold
competitor pre-mRNA is added at Step 2, only a small fraction of
the pre-mRNA in the
complex is subsequently chased into the
other complexes during the incubation in Step 3 (Fig. 7B,
lanes 6-8). That these complexes are formed from the Step 1
complex is indicated by the datum that the 25-fold excess of cold
competitor RNA is sufficient to block any pre-splicing complex
formation if it is added in Step 1 before the radiolabeled pre-mRNA
is added (lane 4). In contrast, the
complex formed in
Step 1 is stable in the extract in the absence of ATP and competitor RNA during the Step 3 incubation (Fig. 7B, compare lanes
6-8 with lanes 1-2). When ATP and complete extract
are subsequently added in Step 3, the pre-mRNA can efficiently form
additional complexes (lane 3). Thus, like the commitment
complexes described by Rosbash and coworkers (10, 11, 44), some of the
pre-mRNA in the
complex is committed to the splicing pathway.
Unlike the CC, however, a significant amount of pre-mRNA in the
complex cannot be chased into the spliceosome by this assay.
One possible explanation for the fraction of unchaseable pre-mRNA
in the complex is that some of the
complex is not stable in the
presence of excess cold competitor pre-mRNA. I, therefore, assayed
the stability of the
complex in the presence of competitor pre-mRNA. To measure the levels pre-mRNA in the
complex
only, I used splicing extract in which the U2 snRNP had been
inactivated by deoxyoligonucleotide-directed RNase H degradation. If
the
complex is formed first on the radiolabeled pre-mRNA,
subsequent incubation with even an equal molar amount of cold
pre-mRNA is sufficient to reduce the amount of radiolabeled
pre-mRNA in the
complex, with increasing reductions occurring
with increasing amounts of cold competitor pre-mRNA (Fig.
7C). In contrast, at least a 10-fold excess of cold
competitor pre-mRNA is required to block formation of the
complex on radiolabeled pre-mRNA added after the cold competitor.
Although this is the same extract used in the experiments in Fig. 7,
B and C, I have found the same results with other
extracts. Thus most of the radiolabeled pre-mRNA that cannot be
chased into the spliceosome can be accounted for by being in a
complex that is unstable in the presence of the excess cold
competitor.
Although it has been proposed that the U1 snRNP has
several functions in spliceosome assembly and in splice site
recognition and selection, most spliceosome assembly assays have failed
to detect the U1 snRNP. Here I used a new native gel electrophoretic assay to detect the yeast U1 snRNA in three pre-splicing complexes (,
1,
2) formed in an in
vitro splicing reaction. I determined the most probable order of
formation of the complexes to be
1
2
1
2, where
2 is the active spliceosome. Unlike all previous
electrophoretic gel assays, this new assay has detected the U1 snRNA
together with other snRNAs in two complexes: 1) with U2 snRNA in the
1 complex and 2) with U2, U4, U5, and U6 snRNAs in the
2 complex. Although U1 is in the
2
complex, little of it is present compared with the other four snRNAs in
the complex and to the levels of both U1 and U2 in
1.
This reduction of U1 snRNA in
2 precedes the complete
loss of U1 and U4 that occurs subsequently in the transition from
2 to
1. Both
1 and the active spliceosome as isolated by this gel electrophoretic assay contain the U2, U5, and U6 snRNAs.
As U1 snRNA is found in three pre-splicing complexes, the gel conditions used in this study most likely preserve several U1 interactions with other spliceosomal components. This gel assay will be useful for studying factors that affect these interactions during spliceosome assembly.
There Are Two Forms ofA small fraction of the complex is
probably the functional equivalent of the previously identified yeast
commitment (11) and mammalian E (45) complexes. Like the CC and E
complexes, the
complex is formed in the absence of ATP. It is the
first splicing-specific complex formed in the presence of ATP (Fig. 2).
Its formation depends on the 5
end of U1 snRNP but not on other snRNPs
(Figs. 3 and 5). Also, the
complex (33) like CC (13, 46) forms in
the absence of active Prp9 or Prp11 protein, each of which is required
for pre-spliceosome assembly. Additionally, the effects of some
mutations in both actin and RP51 pre-mRNAs on
complex
formation2 are similar to those previously
reported for CC formation (11) and for U1 snRNP binding to pre-mRNA
(9), 5
splice site mutations reduce the total amount of
complex as
well as inhibit formation of other pre-splicing complexes. Finally,
some, but not all of the pre-mRNA in the
complex is refractory
to excess competitor pre-mRNA and can be chased into succeeding
assembly intermediates (Fig. 7).
A substantial fraction of complex represents a new complex that
contains U1 snRNP and pre-mRNA but is not committed to splicing (Fig. 7). The failure to chase most of the pre-mRNA in the
complex into subsequent steps in the splicing pathway is probably due to the instability of this fraction in the presence of excess competitor pre-mRNA (Fig. 7C). Thus, there are two forms
of
complex: 1) one scarce form,
commit, which is
stable to exogenous competitor pre-mRNA and can be chased into
subsequent steps of the splicing pathway; and 2) another, abundant
form,
un ("un" for unstable and uncommitted), which
apparently dissociates in the presence of competitor.
There are several possibilities to consider regarding the relationship
between un and
commit. The 5
pre-mRNA cap and the nuclear cap binding complex may be pertinent
to this relationship because the cap and complex help to stabilize
interactions between the U1 snRNP and pre-mRNA (47, 48).
Furthermore, an uncapped pre-mRNA of the RP51 gene is
not as efficiently chased as the capped form from the commitment
complex to the spliceosome in vitro in the presence of
competitor RNA (49). However, the uncapped actin pre-mRNA used here
in this study is spliced with equal efficiency as the capped form
in vitro (31, 50). Thus,
un may be a
precursor of the pre-spliceosome or a precursor to
commit. Alternatively,
un may not be
involved in splicing. Finally, I cannot exclude the possibility that
there are more than two complexes in the
band. Two forms of
commitment complex formed on RP51 pre-mRNA migrate as two distinct
bands, CC1 and CC2, in another gel assay system (11). Clearly,
additional experiments are necessary to understand the parameters
affecting the formation and stabilities of
un and
commit, the significance of these two complexes, and their relationship to each other and to the previously characterized commitment complexes, CC1 and CC2.
The yeast pre-spliceosome
has been thought to contain the U1 as well as the U2 snRNP; however,
previous gel electrophoretic assays have not found U1 snRNA in the B or
III complex analogue of the pre-spliceosome (11, 27, 29).
1, the second complex formed during spliceosome
formation in this study, contains both the U1 and U2 snRNAs and most
probably represents the pre-spliceosome. Like the mammalian A and yeast
B and III complexes,
1 arises early during an in
vitro splicing reaction and requires ATP and the U1 and U2 snRNPs
for its formation (Figs. 1, 2, 3, 4). It does not require a functional
U4/U5/U6 tri-snRNP (Fig. 5). Furthermore, anti-Prp4 antibody that
inhibits the U4/U5/U6 in yeast splicing extracts still allows formation
of
1 (33) and B complexes (51). Finally, the Prp5, Prp9,
and Prp11 proteins are necessary for
1 formation (33).
The mammalian homologs of the Prp9 and Prp11 proteins are also required
for pre-spliceosome formation in HeLa cell splicing extracts (52-54).
Therefore, although I have not shown that pre-mRNA in the
1 complex can be chased into an active spliceosome, the
kinetics and other properties of its formation strongly suggest that
the
1 complex is the pre-spliceosome.
Recently, it has been proposed that disruption of base pairing between
the U1 snRNA and 5 splice site of the pre-mRNA occurs after U1
snRNP binds to the pre-mRNA and before U2 snRNP binds during
pre-spliceosome formation (25, 26). In support of this idea are the
observations that less U1 than U2 snRNA is present in the mammalian
pre-spliceosome (complex A) (20) and that U2 snRNP can bind to one
mutant pre-mRNA without U1 snRNP and ATP in yeast splicing
reactions (25). I have not observed any changes at this step of
spliceosome assembly as evidence of this proposed alteration; in
the absence of competitor pre-mRNA, the
complex is stable.
Furthermore, there are only low levels of U1 snRNA in the
complex
run in a gel without magnesium ion but higher levels of U1 snRNA in the
pre-spliceosome (
1 complex) (Fig. 1). This suggests that
the U1 and U2 snRNPs stabilize each other in the pre-spliceosome.
I have also detected a second complex, 1*,
that, like
1, contains pre-mRNA, and the
U1 and U2 snRNAs.
1* appears early in
splicing reactions with added EDTA and does not accumulate at later
times. As its mobility is different than the
1 complex
formed in either the presence or absence of added EDTA,
1* is most likely a novel complex.
1* may be an assembly intermediate that, in
the absence of added EDTA, co-migrates with
1 or is
short-lived. If it is an intermediate, then its existence may mean that
additional non-snRNP factors bind to the pre-spliceosome or the
conformation of the pre-spliceosome changes in preparation for the
binding of the U4/U5/U6 tri-snRNP. Alternatively, it may be an aberrant
complex formed as a result of the EDTA.
In the native gel electrophoretic assay described
here, little band is present in splicing reactions with
radiolabeled actin pre-mRNA, and the
band migrates close to the
complexes. The
band becomes apparent when more pre-mRNA is
used in the reaction and it is assayed by Northern blot analyses. This
band may have two complexes: 1) the
2 complex that
contains U2, U4, U5, and U6 snRNAs as well as a small amount of U1
snRNA (Fig. 4-6); and 2) the
1 complex that contains
the U2, U5, and U6 snRNAs. Both of these complexes require a functional
U4/U5/U6 tri-snRNP to form. Furthermore, splicing reactions blocked
with anti-prp4 antibody that prevents the tri-snRNP from binding to
pre-mRNA can only form the
and
1 complex (33).
These two complexes are distinguished by their behavior in reactions
with 5 mM EDTA (Fig. 6). In the presence of 5 mM EDTA, the
1 complex migrates slightly
faster than
2 and slower than
2. They can
also be differentiated under other experimental conditions. In the
presence of low concentrations of added ATP,
2
accumulates and little
1 forms; and in heat-inactivated mutant prp2 extracts, mostly
1
accumulates.2 Given the known order of snRNP binding to the
pre-mRNA during spliceosome assembly (for reviews, see Refs. 1, 2,
7, and 55), the simplest interpretation of the data is that
2 is formed from the pre-spliceosome and that
1 is formed from
2.
The comparison of 2 and
1 with the
previously described A2-1 and A1 complexes (29, 38) is also consistent
with this interpretation. A2-1 and
2 are formed after
the pre-spliceosome, contain the U2, U4, U5, and U6 snRNAs, and appear
to be short lived but accumulate in the presence of low ATP. A1 and
1 contain the U2, U5, and U6 snRNAS and accumulate in
reactions with 5 mM EDTA or inactivated Prp2 protein. The
similarities of the
2 and
1 complexes to
the A2-1 and A1 complexes suggests that
2 and
1 are intermediates in the spliceosome assembly pathway.
However, that they are intermediates and equivalent to the A2-1 and A1 complexes remains to be shown.
The presence of the U1 snRNP with the other snRNPs in the the
2 complex is supported by recent evidence that U1 and U5
snRNPs associate with each other in a complex. Recently, Ast and Weiner induced the formation of a U1/U4/U5 complex in HeLa extracts (16) as
well as found that the U1 and U5 snRNAs can be cross-linked in a
splicing related complex (56). Here I have observed that the U1 and U5
snRNPs co-migrate in a complex in the absence of added pre-mRNA
although the function, signficance, and origin of this complex has yet
to be determined.
The amount of U1 snRNA is reduced relative to the other snRNAs in the
2 complex. This reduction suggests that the U1 snRNP dissociates from the
2 complex during gel
electrophoresis or during spliceosome assembly about the time
2 is formed. A decrease in the levels of U1 relative to
U2 in the transition from the pre-spliceosome to complex B in HeLa
extracts has also been observed (57). Because studies in HeLa cell
extracts have detected some U1 snRNP in late assembly intermediates
(20, 23, 30, 57) as well as the formation of a U1/U4/U5 snRNP complex
(16), I favor the first explanation that the loss of U1 is due to its instability in the
2 complex during gel electrophoresis.
This instability may be due to a disruption of the base pairing between U1 snRNA and pre-mRNA or of the U1 snRNP interactions with other spliceosomal components that make this snRNP stable to electrophoresis in the pre-spliceosome.
Genetic experiments in yeast have predicted a loss of base pairing
between the pre-mRNA and U1 snRNA and the formation of base pairs
between the 5 splice site and the U5 and U6 snRNAs sometime in the
splicing pathway (for reviews, see Refs. 3 and 5). Subsequently,
in vitro assays revealed that U5 and U6 snRNAs can be
cross-linked to the 5
splice site region in a spliceosome formed in
either yeast or HeLa cell extracts (for review, see Ref. 6)
Furthermore, the U1 and U5 interact with the 5
splice site region
during spliceosome assembly, whereas U5 and U6 associate with this
region during splicing catalysis. These data support the notion that
the U1 snRNP is "handing off" the 5
splice site region to U5 and
U6 snRNPs. The low level of U1 snRNA in the
2 complex
that I observe suggests that the handoff occurs when the U4/U5/U6
tri-snRNP binds the pre-spliceosome or shortly thereafter.
A working model of yeast
spliceosome assembly showing the U1 snRNP in several pre-splicing
complexes is shown in Fig. 8. Although numerous
non-snRNP proteins are required at several steps in the assembly
pathway (for reviews, see Refs. 1, 55, 58, and 59), these are not
included here.
The U1 snRNP binds to the pre-mRNA in the absence of ATP to form
the complex. The folding of the pre-mRNA designates the dependence of U1 snRNP binding to the pre-mRNA on both the 5
splice site and branch point regions of the pre-mRNA (9, 14). Some
of this complex
commit is committed to subsequent steps in the splicing pathway. The uncommitted complex,
un,
may be a precursor of either
commit or the
pre-spliceosome, or not in the pathway.
The U2 snRNP binds to commit in a reaction requiring
ATP, and the pre-spliceosome with the U1 and U2 snRNPs is formed. The U4/U5/U6 tri-snRNP binds to the pre-spliceosome, and the
2 complex with all five snRNPs is created. The relative
positions of the snRNPs are arbitrarily drawn. During this time or
shortly thereafter, the association of the U1 snRNP with the developing
spliceosome changes, depicted as a change in shading and positioning of
the U1 snRNP. In the transition from the
2 to the
1 complex, the association of U1 and U4 with the complex
becomes tenuous as these snRNPs are not detected in the
1 complex. These two snRNPs may dissociate from the
complex during assembly or electrophoresis. Finally, the active
spliceosome containing the U2, U5, and U6 snRNAs is formed in the
transition from the
1 to the
2
complex.
I thank J. Banroques, P. Fabrizio, L. Krinke, R-J. Lin, D. McPheeters, M. Robinson, U. Vijayraghavan, and T. Wilkie for antibodies, plasmids, prp2 splicing extracts, and carrier RNA samples. I thank M. Ares, J. Staley, J. Summers, and J. Warner for comments on the manuscript. I thank J. Abelson for support during the initial development of the native gel assay and J. Sampson for suggestions.