From the
Splicing of precursors to messenger RNAs occurs via a two-step
mechanism. In the first step, the 5`-exon is released concomitant with
the production of a lariat intermediate, and in the second step, the
exons are joined, releasing the intron in the form of a lariat product.
Several gene products of the yeast Saccharomyces cerevisiae have been shown to be required exclusively for the second step.
Although mammalian proteins have been implicated in the second step of
splicing, none have been shown to act only at this step. We identify
here the first mammalian activity shown to be exclusively required for
the second step. The activity was shown to increase by 5-fold the rate
for this splicing step, whereas it had no effect on the rate of the
first step. The activity was not affected by treatment with micrococcal
nuclease, whereas it is sensitive to heating to 55 °C, suggesting
that it is not dependent on an RNA, but more likely is a protein. The
second step activity was separated from other factors required for the
first step and from PSF, a splicing factor thought to have a second
step activity. The activity does not require ATP hydrolysis, suggesting
that it acts at a late stage of the second step of splicing.
Introns are removed from precursors to messenger RNAs
(pre-mRNAs) in a precise process termed pre-mRNA splicing
(1) .
The mechanism for this removal has been conserved throughout evolution
of eukaryotes and is related to both group I and II self-splicing
mechanisms
(2, 3) . Both pre-mRNA and group II splicing
proceed via a two-step mechanism that involves the formation of a
2`,5`-phosphodiester bond. In the first step (also step I) of pre-mRNA
splicing, cleavage at the 5`-splice site yields a ``free''
5`-exon and a lariat intermediate (reviewed in Refs. 1 and 2). The
lariat intermediate has the 5`-position of the first nucleotide of the
intron, usually a G, involved in a 2`,5`-phosphodiester link with the
branch nucleotide in the intron. In the second step (also step II), the
5`-exon is ligated to the 3`-exon with displacement of the intron as a
lariat product. Both these steps are transesterification reactions and
involve no transfer of phosphates from ATP
(4) . Recent studies
have shown that the reaction mechanisms of both steps proceed via
S
Several gene products of the yeast Saccharomyces cerevisiae have been shown to be required only for the second step of
splicing (reviewed in Refs. 1 and 2). PRP16, a 120-kDa protein with a
DEAH box motif
(6) , is required for step
II
(7, 8, 9, 10) . PRP16 has
RNA-dependent NTPase and dNTPase activities, and its effect on step II
requires NTP binding and possibly hydrolysis
(11) . The broad
specificity range for these two activities argues that PRP16 may be the
last NTPase required in the splicing reaction. PRP16 associates with
the spliceosome after the completion of the first step of splicing and
mediates a conformational change in the spliceosome
(11) .
Another well studied factor required for the second step is PRP18
(12-15). This 28-kDa protein without identifiable motifs is
associated with U5 snRNP
Krainer and Maniatis
(18) described two activities required for the second step of
splicing: SF3 and SF4A. SF3 was described as a heat-sensitive activity
resistant to micrococcal nuclease treatment, and SF4A was not
characterized. More recently
In this report, we present the results of the study
of a mammalian activity required for the efficient catalysis of the
second step of splicing. Independent measurement of the rates of the
two steps of splicing was accomplished by determining initial rates of
these reactions. These measurements argue that this mammalian activity
is required exclusively for step II. Characterization of the activity
is described, and its relationship to other mammalian and yeast factors
is discussed. We show that the activity does not require ATP and is not
inhibited by the addition of an excess of nonhydrolyzable ATP
analogues. Finally, we show that spliceosomes formed in the absence of
this activity can be chased through step II.
Fractions were
assayed for the ability to catalyze the second step of splicing in two
ways, a simultaneous assay and a sequential assay. In the simultaneous
assay, DEAE II and the fraction being analyzed for second step activity
were combined prior to the splicing reaction. In the sequential assay,
DEAE II was added, and the splicing reaction was run for the indicated
length of time. At that point, the fraction to be analyzed for second
step activity was added to the original splicing reaction. The mixture
was then incubated at 30 °C for the indicated length of time.
In vitro splicing products and intermediates were analyzed as
described above.
The second
step activity in the DEAE I and CMFT fractions was heat-sensitive.
Treatment of DEAE I for 15 min at 37 °C did not affect second step
activity, whereas treatment at 55 °C for 15 min resulted in an
apparent defect in the second step (data not shown).
To evaluate the
likelihood for a role of RNAs in this activity, the CMFT fraction was
subjected to micrococcal nuclease treatment. Mock micrococcal nuclease
treatment of nuclear extract had no effect on either step of splicing
(Fig. 3, lane3). As expected, treatment with
micrococcal nuclease abolished splicing activity (lane4). The micrococcal nuclease was inactivated before
incubation with pre-mRNA as documented by lack of degradation of this
RNA. DEAE II was only capable of carrying out the first step of the
splicing reaction (lanes 5-8), but when supplemented
with the untreated CMFT fraction, the lariat intermediate converted to
the lariat product (lanes 9-11). The same was observed
when the CMFT fraction was either mock-treated (lanes
12-14) or micrococcal nuclease-treated (lanes
15-17). These data suggest, but do not prove, that the
second step activity does not depend on the integrity of an RNA.
In this report, we define an activity required for efficient
catalysis of the second step of splicing. This activity is not required
for the formation of splicing complexes or for the first
transesterification reaction. Several results suggested that the
activity is required at a step immediately preceding the second
transesterification reaction. First, the lack of a requirement for ATP
hydrolysis positions this activity late in the second step. ATP
hydrolysis is required at several stages of the splicing reaction,
including stages of the second step (Refs. 9, 12, 14, 18, 29, and 30;
see also review in Ref. 2). The specific stage after step I at which
ATP hydrolysis is required may be the PRP16-dependent conformational
change observed in yeast extracts
(11) . This change correlates
with the 3`-splice site becoming inaccessible to
oligonucleotide-directed cleavage by RNase H
(11) and is
probably linked to proofreading of the lariat intermediate and/or
branch point sequences before proceeding to step II
(31) .
Assuming evolutionary conservation of these stages from yeast to
humans, it is likely that our activity is required at a stage later
than PRP16. Horowitz and Abelson
(14) have proposed a similarly
late role for PRP18. Their data and ours argue that the second
transesterification reaction per se does not require ATP
hydrolysis.
The second argument in favor of a late role for our
activity is the ability to chase spliceosomes. Our interpretation of
the data was that spliceosomes that had undergone step I (complex C1)
were capable of functionally interacting with this activity and
catalyzing step II. A similar late entry for PRP18 has also been
suggested
(14) . This suggests the possibility that the catalytic
site for the second step is accessible to entry by factors acting late.
It is formally possible that in complete nuclear extract, the activity
may assemble early in spliceosome formation, but act late.
Of the
well characterized yeast gene products required for step II, PRP18 is
the best candidate as a homologue of our activity. Both share the lack
of a requirement for ATP and similar chromatographic properties. In
addition, we believe that our activity is required exclusively for step
II, as is PRP18. This requirement for the DEAE I activity may not be
absolute; the DEAE II fraction is capable of low levels of step II
activity. Horowitz and Abelson
(13, 14) showed that
PRP18-depleted extracts can still carry on the second step of splicing,
albeit at a much reduced rate. Moreover, a prp18 null mutant
is viable at low temperatures, suggesting that PRP18 is not absolutely
required for step II in vivo. Thus, PRP18 is a good candidate
homologue of the DEAE I activity. We tested this relationship in two
ways. Recombinant PRP18 did not substitute for the activity, and two
anti-PRP18 antisera did not inhibit the DEAE I activity (data not
shown). Neither of these two results permits a definitive conclusion,
and thus, PRP18 remains a viable homologue for the DEAE I activity.
At this time, we have no evidence to suggest that a single
polypeptide is responsible for this activity. In fact, based on the
results of the Sephacryl S-300 HR column, it is possible that the
activity is in a large complex. The only detectable U2, U4, U5 and U6
in the DEAE I fraction were observed in a large complex (data not
shown). This complex may be related to the pseudospliceosomes described
by Konarska and Sharp
(33) . It is possible that the step II
activity is associated with this complex; however, we do not favor a
requirement for a snRNP because of the insensitivity to micrococcal
nuclease.
Krainer and Maniatis
(18) described two activities
implicated in step II: SF3 and SF4A. SF3 was characterized as a
micrococcal nuclease-resistant activity that was destroyed by heating
at 45 °C for 10 min. Sawa et al.(30) showed that
SF3 activity required ATP and was inhibited by nonhydrolyzable
analogues of ATP. Thus, it is unlikely that our activity is SF3. SF4A
was distinguished from SF3 by chromatography on spermine-agarose, and
it is possible that our activity is the same as SF4A. Recently, Gozani
et al. (19) showed that PSF was found among a group of
proteins associated specifically with purified complex C1. Moreover,
PSF-depleted extracts were shown to have splicing defects that
suggested a role for PSF in step II
(19) . This factor was
originally described as a protein required for assembly of early
splicing complexes
(20) , and an early role for PSF has not been
ruled out. The DEAE I activity is not required for complex A formation,
which is clearly different than published for PSF. Also, we have shown
that PSF is not responsible for our step II activity by separating PSF
from the activity. Interestingly, anti-PRP18 antisera detect a human
54-kDa protein (p54
We thank members of the Garcia-Blanco laboratory for
helpful discussions. We are grateful to Drs. Adrian Krainer and David
Horowitz (Cold Spring Harbor Laboratory) for PRP18 cDNA and anti-PRP18
sera and to Dr. Jim Patton for PSF cDNA and anti-PSF serum. We thank
Sabina Sager for help in the preparation of this manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
2 displacements
(5) . This mechanism
suggests that the two steps of splicing probably occur in two distinct
catalytic sites in the spliceosome
(5) , which is consistent with
the existence of factors acting exclusively at one of the two sites.
(
)(13) . Extracts
depleted of PRP18 show an 80-fold slower rate for step II, whereas the
rate of step I is not significantly altered relative to wild-type
extract
(14) . The splicing intermediates formed in the depleted
extracts can be chased to mRNA and lariat product by the addition of
recombinant PRP18 in the absence of detectable levels of
ATP
(14) . Also required for step II are PRP17 (SLU4) and
SLU7
(16, 17) . Synthetic lethality experiments suggest
the existence of functional interactions between PRP16, PRP17, PRP18,
SLU7, and U5 snRNA
(17) .
15 proteins have been shown to be
associated with purified spliceosomal complex C1, in which splicing is
arrested after step I
(19) . It is reasonable to assume that some
of these will be required for the second step of splicing. PSF, a
protein previously described as a splicing factor required for
pre-spliceosome formation
(20) , was shown to be one of these
complex C1-associated proteins
(19) . Data both in the original
publication by Patton et al.(20) and in Gozani et
al.(19) suggest a possible involvement of PSF in the
second step of splicing. No mammalian activity has been subjected to
rigorous analysis in order to show a second step effect independent of
earlier effects such as has been accomplished for the yeast factors
PRP16 and PRP18.
Plasmids, RNAs, and Nuclear Extracts
pPIP7.A has
been described previously
(21) . pPIP7.A contains a 55-nucleotide
5`-exon, a 125-nucleotide intron, and a 56-nucleotide 3`-exon derived
from the adenovirus major late promoter. PIP7.A RNA was synthesized as
described previously
(22, 23) . Briefly, pPIP7.A was
linearized by digestion with HindIII. The linearized template
was transcribed with T7 RNA polymerase (New England Biolabs Inc.) in
the presence of [-
P]UTP at a final specific
activity of 300 Ci/mmol. Nuclear extract was prepared from HeLa cells
by the method of Dignam et al.(24) .
Nuclear Extract Fractionation
34 ml of HeLa
nuclear extract (in Buffer D (20 mM Hepes (pH 7.9), 20% (v/v)
glycerol, 0.2 mM EDTA, 0.5 mM dithiothreitol) with
100 mM KCl) containing 537 mg of protein were loaded onto a
DEAE-Sephacel column (cross-sectional area = 4.96
cm; height = 3.36 cm; volume = 16.5 ml) at a
rate of 2.5 ml/h/cm
. The DEAE-Sephacel column had been
equilibrated in Buffer D with 100 mM KCl. A total of 25 5-ml
fractions were collected. Fractions 4-8, containing 40% of the
protein, were found to have second step activity and were then pooled
and designated DEAE I. The DEAE-Sephacel column was then eluted with
Buffer D with 500 mM KCl at 2.5 ml/h/cm
, and 5-ml
fractions were collected. A 10-µl aliquot of each of the fractions
eluted from the column with Buffer D with 500 mM KCl was
analyzed for protein content, and fractions 2-15 were found to
contain the vast majority of protein (60% of protein loaded). These
fractions were pooled and dialyzed into Buffer D with 100 mM
KCl and termed DEAE II. 24 ml of the DEAE I fraction containing 211 mg
of protein were loaded onto a CM-Sepharose CL-6B column
(cross-sectional area = 1.77 cm
; height = 6.2
cm; volume = 11 ml) at a rate of 5 ml/h/cm
. The
column was washed with 15 ml of Buffer D with 100 mM KCl and
then step-eluted with 15 ml each of Buffer D with 250 mM KCl,
Buffer D with 500 mM KCl, and Buffer D with 1 M KCl.
5-ml fractions were collected and analyzed for second step activity.
Fractions 2-6, containing 75% of the protein, were pooled and
termed CM-Sepharose flow-through (CMFT). 2 ml of CMFT (12 mg) were
loaded onto a Sephacryl S-300 HR column (cross-sectional area =
2.0 cm
; height = 35 cm; volume = 70 ml) at a
rate of 2 ml/h/cm
. The Sephacryl column had been
standardized and then equilibrated in Buffer D with 100 mM
KCl. A total of 70 1-ml fractions were collected and assayed for step
II activity. Fractions 26-29 had activity. The overall -fold
purification can only be estimated roughly to be on the order of
10-fold. The activity is remarkably stable; little decay was observed
in crude fractions stored at -70 °C for over 1 year.
Splicing Reactions
Splicing reactions were run as
described previously
(25) . Splicing reactions were run at 30
°C for the indicated length of time with final concentrations of 64
mM KCl, 2 mM MgCl, 1 mM ATP, 5
mM creatine phosphate, and 12.8% glycerol (v/v). In vitro splicing products and intermediates were detected by extracting
the RNA and analyzing it on denaturing 15% polyacrylamide
gels
(25) . The splicing products and intermediates were
visualized by autoradiography using Hyperfilm (Amersham Corp.). For
splicing complex formation, reactions were incubated at 30 °C for
the indicated length of time, placed on ice, and supplemented with
heparin (Sigma H-3125) to a final concentration of 0.5 mg/ml. Splicing
complexes were analyzed by electrophoresis on nondenaturing 4%
polyacrylamide gels buffered with 50 mM Tris/glycine and run
at 200 V (12.5 V/cm) at room temperature for 3 h (26). The gels were
dried under vacuum at 80 °C for 1 h, and splicing complexes were
visualized by autoradiography using Hyperfilm.
Microccocal Nuclease Digestion
The CMFT fraction
was digested with micrococcal nuclease in the following manner. A
50-µl aliquot of CMFT was incubated at 30 °C for 30 min with
0.5 µl of 4 units/µl micrococcal nuclease in the presence of 1
mM CaCl. To stop the digestion, 6 µl of 100
mM EGTA were added, and the reaction mixture was incubated for
an additional 2 min at 30 °C. A second 50-µl aliquot of CMFT
was mock-treated by supplementing it with 1 mM CaCl
and incubating at 30 °C for 30 min. Aliquots of nuclear
extract were treated in the same manner.
Depletion of ATP
To deplete the reactions of ATP
after initial incubation with DEAE II, hexokinase (Sigma) and glucose
were added to final concentrations of 25 µg/ml and 12 mM,
respectively
(27) . This was allowed to incubate for 10 min at 30
°C. The reaction was chased with DEAE I for 90 min at 30 °C. To
follow ATP levels, the reactions were supplemented with
[-
P]ATP (12 µCi/ml). 1-µl aliquots
were taken for each condition before and after incubations and
incubated with 30 µl of 15% (w/v) chilled trichloroacetic acid for
15 min on ice. After spinning at 16,000
g for 15 min,
the supernatant fraction was collected and neutralized with an equal
volume of 1 N NaOH. 0.5 µl was spotted on
polyethyleneimine-cellulose thin-layer chromatography plates; the
plates were developed with 1 M LiCl; and products were
visualized by autoradiography
(28) .
Gradient Purification of Spliceosomes and
Chase
Procedures were the same as described previously
(22) with the following exceptions. The 100-µl splicing
reaction contained DEAE II rather than nuclear extract and was
incubated for 70 min at 30 °C. This was loaded onto a 1.9-ml
15-30% (w/v) glycerol gradient, and 10-200-µl fractions
were collected.
Preparation of PSF-depleted Nuclear Extract and Western
Blotting
PSF depletion was essentially as described previously
(20). Nuclear extract (8.5 ml) was adjusted to 500 mM KCl in
Buffer D and applied to a 4.5-ml poly(U)-agarose column and 1-ml
fractions were collected. Flow-through material and 500 mM KCl
were collected (fraction I). Step fractions were eluted with 1.0
M KCl (fraction II) and 1.0 M KCl and 2 M
guanidinium Cl (fraction III), and the resultant peaks were dialyzed in
Buffer D with 100 mM KCl. For Western blotting, protein was
run on a 12.5% SDS-polyacrylamide gel and then transferred onto
Immobilon affinity membranes (Millipore Corp.) in 20% methanol, 192
mM glycine, and 25 mM Tris. Following transfer, blots
were blocked in 5% nonfat dry milk with phosphate-buffered saline and
0.1% Tween 20. The PSF antibody (kindly provided by Dr. J. Patton,
Vanderbilt University) was diluted 1:5000 in the same buffer and
incubated at room temperature for 1 h, followed by incubation with a
peroxidase-conjugated secondary antibody (Amersham Corp.) for 1 h at
room temperature. Visualization was with the ECL reagents from Amersham
Corp. following their protocol.
A Mammalian Activity Required for the Second Step of
Splicing
HeLa nuclear extracts were fractionated over
DEAE-Sephacel to separate factors required for pre-mRNA splicing. Three
fractions were collected: a flow-through/wash 100 mM KCl
fraction (DEAE I), a fraction eluted with 500 mM KCl (DEAE
II), and a fraction eluted with 1 M KCl (DEAE III). The DEAE I
and II fractions were capable of reconstituting splicing with high
efficiency (Fig. 1A, lane5) relative
to the nuclear extract loaded onto the column (lanes1 and 2; note that lane1 is compressed
at the origin). The DEAE I fraction had no detectable splicing activity
(lane3). DEAE II was as active as the extract in
catalyzing step I: the formation of a lariat intermediate and a free
5`-exon (lane4). The conversion of these
intermediates to mature mRNA and lariat product of step II, however,
was very low compared with extract and with the reactions containing
both DEAE I and II fractions (lanes2 and
5). Therefore, DEAE II contained all the factors required to
complete the first step of splicing, but was missing at least one
activity required for the second step. DEAE I was capable of
complementing DEAE II for this missing activity. It was reasonable to
assume from this that the activity was not required for the first step
of splicing.
Figure 1:
An activity in the DEAE I fraction is
required for the second step of splicing. A, in vitro splicing using the simultaneous assay described under
``Experimental Procedures.'' Splicing reactions were
incubated in the absence of ATP with nuclear extract (NE;
lane1) or in the presence of 1 mM ATP and 5
mM creatine phosphate with nuclear extract (lane2), DEAE I (lane3), DEAE II (lane4), or DEAE I + II (lane5). Note
that lane1 is compressed near the origin and that
the top part of lane2 deviates to the left.
B, in vitro splicing using the sequential assay
described under ``Experimental Procedures.'' Splicing
reactions were incubated in the absence of ATP with nuclear extract
(lane1) or in the presence of 1 mM ATP and
5 mM creatine phosphate with nuclear extract (lane2), DEAE II (lane3), DEAE II followed
by chasing with DEAE I (lanes 4-6), or DEAE II followed
by a mock chase with Buffer D (lanes 7-9). Splicing
intermediates and products were purified and visualized as described
under ``Experimental Procedures.'' From top to bottom, icons
indicate the lariat intermediate, lariat product, pre-mRNA, spliced
product, and free 5`-exon.
A second assay was used to detect this activity in DEAE
I. Uniformly labeled pre-mRNA was incubated with DEAE II under splicing
conditions for 45 min. The results were similar to those obtained
before, and splicing was arrested after completion of step I
(Fig. 1B, lane3). The reactions were
divided into two, and one half was incubated further with Buffer D for
30, 60, or 90 min (lanes 7-9). This resulted in little
accumulation of products. The other half was incubated with DEAE I for
30, 60, or 90 min, and the result was a dramatic conversion of
intermediates into lariat products and mRNA (lanes 4-6).
This result was consistent with the data shown in
Fig. 1A, and it supported the idea that DEAE I had an
activity involved exclusively in the second step of splicing. Moreover,
the data suggest that the activity can interact with already assembled
spliceosomes.
The DEAE Activity Affects the Rate of the Second
Step
Experiments were performed to measure the initial rates of
both steps in the splicing reactions to confirm that the activity in
DEAE I was affecting the second step of splicing. DEAE I had absolutely
no activity on its own, and therefore, the rate of both steps was zero
(data not shown). The rate of the first step of splicing was measured
for splicing reactions containing DEAE II versus reactions
containing DEAE II + I. The assays were carried out as described
for Fig. 1A (see ``Experimental Procedures'').
The relative rates of accumulation of lariat intermediate and lariat
product for the reactions were derived from data shown in
Fig. 2
(A and B) and are plotted in
Fig. 2C. Quantification was based on scanning on a
PhosphorImager (Molecular Dynamics, Inc.). The level of the lariat
intermediate or lariat product was normalized to the total RNA level to
ensure that losses would not alter the results. The normalization did
not have much of an effect on the rate measurements given that the
different reactions had very similar levels of RNA. Measurements of
free 5`-exon and mRNA gave comparable results, but these were deemed
less reliable for quantification given the fact that in some cases the
mRNA appeared as multiple bands (data not shown). The initial rates of
the first step, calculated from the first three detectable levels of
the lariat intermediate, were similar for DEAE II and DEAE II + I.
The only effect of DEAE I on the first step was a shortening of the lag
phase of the reaction (Fig. 2C). The first detectable
levels of the lariat intermediate were observed at 60 min for DEAE II
and at 40 min for DEAE II + I.
Figure 2:
The activity in DEAE I affects the rate of
the second step, but not the rate of the first step. A, a
time course of in vitro splicing using the simultaneous assay
described under ``Experimental Procedures.'' Splicing
reactions were incubated for 20-180 min in the presence of 1
mM ATP and 5 mM creatine phosphate with DEAE II.
B, a time course of in vitro splicing using the
simultaneous assay described under ``Experimental
Procedures.'' Splicing reactions were incubated for 20-180
min in the presence of 1 mM ATP and 5 mM creatine
phosphate with DEAE I + II. Splicing intermediates and products
were purified and visualized as described under ``Experimental
Procedures.'' From top to bottom, icons indicate the lariat
intermediate, lariat product, pre-mRNA, spliced product, and free
5`-exon. C and D, the data in A and B were quantified using a Molecular Dynamics PhosphorImager. The
levels of the lariat intermediate, which was used to measure step I
(C), and of the lariat product, which was used to measure step
II (D), are plotted versus time.
The initial rate of the second
step of splicing, calculated from the first three detectable levels of
the lariat product, was markedly different for DEAE II versus DEAE II + I (Fig. 2D). The initial rate of the
second step for DEAE II + I was 6.6-fold higher than the initial
rate for DEAE II alone (Fig. 2D). There was also a
broadening of the effect on the lag, with the first detectable lariat
product appearing at 70 min for DEAE II versus 40 min for DEAE
II + I (Fig. 2D). The fact that the increased lag
was noted for both steps suggests that an early factor is present at
lower concentration in the DEAE II fraction relative to nuclear
extract. The results from two experiments are summarized in
. Direct comparison between rates of steps I and II is not
intended given that a conversion from signal strength (based on
counts/minute) to moles of lariat product and intermediate has not been
performed. Results from experiments using the sequential assay
indicated that the magnitude of the effect on step II may be even
greater than seen in (data not shown). These data
indicated the existence of factor required primarily, if not
exclusively, for the second step of splicing.
Characterization of the Second Step Activity
The
DEAE I fraction was loaded onto CM-Sepharose to further purify the
second step activity. Most, if not all, of the activity eluted in the
flow-through/wash fractions of the CM-Sepharose column (data not
shown). The properties of the activity for DEAE I and the CM-Sepharose
flow-through/wash (CMFT) fractions were found to be identical (data not
shown). The purification of this activity from nuclear extract was
5-fold. Other means of chromatographing this activity were tried,
including other ion exchangers, dyes, a hydrophobic resin, and
RNA-bound resins. In most of the cases, the activity was in the
flow-through fraction, thus resulting in very poor purification (data
not shown). The CMFT fraction was loaded onto Sephacryl S-300 HR to get
an estimate of the size. The activity eluted in the void volume,
indicating that it is very large, over 1.5 million Da.
Figure 3:
The second step activity is resistant to
micrococcal nuclease digestion. In vitro splicing was done
using the sequential assay described under ``Experimental
Procedures.'' Splicing reactions were incubated in the absence of
ATP with nuclear extract (NE; lane1) or in
the presence of 1 mM ATP and 5 mM creatine phosphate
with nuclear extract (lane2), mock-digested nuclear
extract (lane3), or nuclear extract digested with
micrococcal nuclease (MN, lane4). Splicing
reactions were incubated in the presence of 1 mM ATP and 5
mM creatine phosphate with DEAE II (lane5)
and chased for 30-60 min with Buffer D (lanes
6-8), CMFT (lanes 9-11), mock-digested CMFT
(lanes 12-14), or micrococcal nuclease-digested CMFT
(lanes 15-17). Splicing intermediates and products were
purified and visualized as described under ``Experimental
Procedures.'' From top to bottom, icons indicate the lariat
intermediate, lariat product, and pre-mRNA.
Northern analyses
(26) were performed to determine which
snRNPs were in the DEAE fractions. U2, U4, U6, and U5 snRNAs were found
in the DEAE II and I fractions, whereas U1 snRNA was exclusively found
in the DEAE II fraction (data not shown). Monomeric U2 snRNP, U5 snRNP,
and the U4/U6-U5 tri-snRNP complex were detected only in the DEAE II
fraction (data not shown). The U2, U4, U5, and U6 signals in DEAE I
were all in a very large complex (data not shown). Therefore, DEAE II
contained all of the required U snRNPs, making it unlikely that one of
these is the step II activity. These data are consistent with the fact
that DEAE II was competent to form spliceosomes (see below). It is
important to note, however, that these data do not exclude the
possibility that the activity is associated with U snRNPs, in
particular a novel subpopulation of snRNPs.
The Second Step Activity Does Not Require ATP
We
noted that the absence of exogenously added ATP had no effect on the
activity of the CMFT fraction (data not shown). Moreover, a 5000-fold
molar excess of the nonhydrolyzable ATP analogue AMP-PNP did not
inhibit this activity. We investigated further whether or not ATP was
required for the second step activity. To do this, we used the
sequential assay described above and modified such that any ATP present
during the first incubation was depleted before the chase (see
``Experimental Procedures''). The levels of ATP were followed
by the addition of [-
P]ATP to the reactions
and detection by autoradiography of polyethyleneimine-cellulose TLC
plates. High levels of ATP were detected after 70 min of incubation
with DEAE II, even if no creatine phosphate was added
(Fig. 4A, lanes1 and 2). In
contrast to what is observed in yeast extracts
(14) , glucose
alone did not completely deplete ATP (lane3).
Complete depletion of ATP required incubation with 25 µg/ml
hexokinase and 12 mM glucose for 10 min (lane4). All detectable ATP was shown to be converted into
ADP, AMP, and other products. Moreover, no ATP was regenerated during
the chase with the DEAE I fraction (data not shown). Mock-depleted
reactions and reactions incubated with 12 mM glucose alone,
both of which had high levels of ATP, were used as positive controls
for activity (Fig. 4B, lanes1,
2, 5, and 6). The depletion of ATP by
hexokinase and glucose did not significantly affect the conversion of
intermediates to products, which is most clearly seen for the lariat
product (lanes3 and 4).
Figure 4:
The second step activity does not require
ATP. A, thin-layer chromatographic separation of ATP in
splicing reactions was carried out as described under
``Experimental Procedures.'' Nucleotides were acid-extracted
and chromatographed on polyethyleneimine-cellulose TLC plates developed
with 1 M LiCl. Splicing reactions were incubated with DEAE II
for 0 min (lane1) or for 70 min (lane2), with DEAE II for 70 min followed by a 10-min
incubation with 12 mM glucose (lane3), or
with DEAE II for 70 min followed by a 10-min incubation with 25
µg/ml hexokinase and 12 mM glucose (lane4) or a 10-min mock depletion (lane5).
The chromatographic migration of ATP, ADP, and AMP is indicated.
B, in vitro splicing was carried out using the
sequential assay described under ``Experimental Procedures.''
Splicing reactions were incubated in the presence of 1 mM ATP
with DEAE II for 70 min followed by a 10-min mock incubation and
chasing with Buffer D (lane1) or DEAE I (lane2). Splicing reactions were incubated in the presence of
1 mM ATP with DEAE II for 70 min followed by a 10-min
incubation with 25 µg/ml hexokinase (Hex.) and 12
mM glucose (Gluc.) and chased with Buffer D (lane3) or DEAE I (lane4). Splicing
reactions were incubated in the presence of 1 mM ATP with DEAE
II for 70 min followed by a 10-min incubation with 12 mM
glucose and chased with Buffer D (lane5) or DEAE I
(lane6). Splicing intermediates and products were
purified and visualized as described under ``Experimental
Procedures.'' From top to bottom, icons indicate the lariat
intermediate, lariat product, pre-mRNA, spliced product, and free
5`-exon.
The Second Step Activity Can Complement Preassembled
Spliceosome
Based on the fact that DEAE II was capable of
catalyzing step I, we predicted that this fraction should be capable of
spliceosome formation. This was assayed using native gel
electrophoresis. Nuclear extract promoted the formation of two
ATP-dependent splicing specific complexes: the pre-spliceosome (complex
A) and the spliceosome (complex B) (Fig. 5A, lanes1 and 2). DEAE I was capable of forming complex
A, but not complex B (lane3). DEAE II, not
surprisingly, was capable of forming both (lane4),
and the addition of DEAE I did not seem to enhance this activity
(lane5). Thus, as expected, spliceosomes formed
efficiently in the absence of the second step activity.
Figure 5:
The
second step activity can chase preformed spliceosomes. A, the
second step activity was not required to form spliceosomes. Splicing
complexes were formed by splicing reactions incubated in the absence of
ATP with nuclear extract (NE; lane1) or in
the presence of 1 mM ATP and 5 mM creatine phosphate
with nuclear extract (lane2), DEAE I (lane3), DEAE II (lane4), or DEAE I +
II (lane5). Splicing complexes were separated and
visualized as described under ``Experimental Procedures.''
The splicing complexes spliceosome (B) and pre-spliceosome
(A), -ATP complex (*), and nonspecific heterogeneous
complexes (H) are indicated. B, a splicing reaction
was incubated in the presence of 1 mM ATP and 5 mM
creatine phosphate with DEAE II and subjected to velocity sedimentation
on a 15%-30% glycerol gradient. RNAs found in fractions 6-10 are
shown (lanes 1-5). Gradient fractions were chased with
DEAE I for 70 min at 30 °C, and the RNAs found after the chase are
shown (lanes 6-10). Splicing intermediates and products
were purified and visualized as described under ``Experimental
Procedures.'' From top to bottom, icons indicate the lariat
intermediate, lariat product, pre-mRNA, spliced product, and free
5`-exon.
Functional
spliceosomes were separated from pre-spliceosomes and commitment
complexes by glycerol gradient velocity sedimentation
(25) . On
15-30% glycerol (w/v) gradients, spliceosomes reproducibly
sedimented in fractions 8-10. Spliceosomes assembled by
incubation with DEAE II were sedimented on the same glycerol gradients.
Fractions 8-10 were shown to contain free 5`-exon and lariat
intermediate in addition to pre-mRNA, whereas all other fractions
contained only pre-mRNA (Fig. 5B, lanes
1-5; data not shown). Therefore, the DEAE II spliceosomes
could be separated after catalyzing the first step of splicing. The
addition of DEAE I to the fractions resulted in progression through
step II (lanes 6-10). These data suggest that the DEAE I
activity can interact with preassembled spliceosomes. It is unlikely
that all of the spliceosomes present in these fractions have undergone
step I, but the observed levels of the lariat intermediate and product
observed during the chase (lanes 6-10) argue that the
DEAE I fraction may interact with spliceosomes that have already
catalyzed the first step of splicing.
The Second Step Activity Can Be Purified Away from
PSF
Since PSF has been suggested to have a role in step
II
(21) , we tested whether or not PSF was responsible for the
second step activity in DEAE I. Nuclear extract was fractionated using
poly(U)-agarose as described previously
(20) . The
flow-through/wash fraction (fraction I), which is equivalent to the
PSF-depleted extract, had high levels of step II activity and no PSF
(Fig. 6, A and B). The 1 M KCl
fraction (fraction II) did not have any step II activity, but contained
PSF (Fig. 6, A and B). Fraction III, which
contains the splicing factor U2AF, had neither step II activity nor PSF
(Fig. 6, A and B). Moreover, we showed that
recombinant PSF did not have activity in our assay and that an
antiserum to PSF did not inhibit this activity (data not shown). All of
these data demonstrate that PSF is not a candidate for the novel step
II activity in DEAE I.
Figure 6:
A
PSF-depleted fraction has step II activity. A, the
poly(U)-agarose flow-through fraction (fraction I) has step II
activity. In vitro splicing was done using the sequential
assay described under ``Experimental Procedures.'' Splicing
reactions were incubated in the presence of DEAE II for 70 min and then
chased for 60 min with CMFT or fractions from nuclear extract
fractionation through poly(U)-agarose (fractions I-III). The data
were quantified using a Molecular Dynamics PhosphorImager. The step II
activity was defined as the counts/minute in the lariat product in the
tested fraction minus the counts/minute in the chase with Buffer D
reaction (background). The numbers have been normalized to the number
of counts/minute in each lane. B, Western analysis shows that
PSF is only detected in fraction II. 7 µg of nuclear extract
(NE) and fraction I and 3 µg of fractions II and III were
run on a 12.5% SDS gel, transferred, and probed with an anti-PSF
antiserum, which was detected by chemiluminescence as described under
``Experimental Procedures.''
) that, although seemingly
unrelated to PRP18, is highly homologous to PSF
(32) . PSF and
p54
may be members of a family of factors with
partially redundant activity analogous to SR
proteins
(34, 35, 36, 37) .
Table:
Relative initial rates for steps I and II
for DEAE fractions
,
-imidotriphosphate).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.