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| Section: Cell Biology Methods » Organelles and Cellular Structures » Isolation: Plasma Membrane, Organelles & Cellular Structures
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Purification of Intermediate-Containing Spliceosomes
|Purification of Intermediate-
The spliceosome is the dynamic macromolecular
machine responsible for removing intervening
sequences (introns) from pre-mRNA transcripts. In
vitro studies have been used to define a pathway of a
spliceosome assembly consisting of several intermediate
subcomplexes (Moore et al.
, 1993). Isolating any one
of these splicing complexes for subsequent characterization,
and structural analysis requires a means to
accumulate that specific complex (Jurica and Moore,
2002). A number of methods for purifying complexes
along the spliceosome assembly pathway have been
described (Das et al.
, 2000; Hartmuth et al.
, 2002; Jurica et al.
, 2002; Makarov et al.
, 2002; Zhou et al.
These protocols use similar approaches of combining
size fractionation and affinity purification to isolate in
vitro-assembled splicing complexes under native conditions.
Presented here is a procedure developed for
isolating intact splicing complexes arrested between
the first and the second chemical steps of splicing
(Jurica et al.
Spliceosomes are assembled on an in vitro
splicing substrate from components
present in the nuclear extract of HeLa cells. Using a
substrate with a mutation at the 3' splice site, complex
assembly is arrested by a block at the second step of
splicing. The substrate also contains binding sites for
an RNA-binding protein that serves as an affinity tag.
Purification of arrested spliceosomes is carried out in
three steps: (1) RNase H digestion of excess pre-mRNA
substrate, (2) size-exclusion chromatography, and (3)
affinity selection via the RNA-binding adapter protein
(Fig. 1). Digestion serves to improve the ratio of fully
assembled complexes to earlier species by targeting substrate molecules not fully assembled into spliceosomes.
Cleavage is mediated by endogenous RNase H
present in nuclear extracts and DNA oligonucleotides
targeted to the last 30 nucleotides of the 5' exon in the
pre-mRNA substrate, a region protected from digestion
in arrested complexes, but accessible in earlier
ones (Reichert et al.
, 2002). Size exclusion employs a
small sizing column to quickly separate a splicing reaction
into basically two fractions: larger molecules,
including the spliceosomes, that are excluded by the
sizing resin and elute in the void volume, and smaller
molecules that can enter the pores of the resin and are
retained on the column, such as the bulk of nuclear
extract proteins, degraded RNA, and unbound affinity
adapter protein. Affinity selection is mediated by
binding sites for the bateriophage MS2 coat protein in
the pre-mRNA substrate. The substrate can then be
bound to a fusion of MS2 and maltose-binding protein
(MBP). The MS2:MBP serves as an affinity adapter so
that spliceosomes assembled on the tagged substrate
can be captured by amylose resin and eluted under
native conditions with maltose. The main objectives in
developing this protocol were to isolate homogeneous
splicing complexes in the quantities (~100µl) and
) useful for both biochemical
characterization and electron microscopy analysis.
|FIGURE 1 Purification scheme.
II. MATERIALS AND
HEPES (Cat. No. H-3375), EDTA (Cat. No. E-5134),
isopropyl-β-D-thiogalactoside (IPTG, Cat. No. 1-5502),
maltose (Cat. No. M-5895), phenylmethylsulfonyl
flouoride (PMSF, Cat. No. P-7626), L
-glutamic acid, monopotassium salt (Cat. No. G-1501), Nonidet P-40
(Cat. No. N-6507), heparin (Cat. No. H-3900), phosphocreatine
(Cat. No. P-7936), yeast tRNA (Cat. No. R-
9001), Trizma base (Cat. No. T-1503), and formamide
(Cat. No. 47671) are from Sigma. Tris-HCl (Cat. No.
BP153), glycerol (Cat. No. G33), magnesium acetate
(Cat. No. M13), and potassium chloride (Cat. No. P217)
are from Fisher. Adenosine triphosphate (Cat. No. 27-
2056-01), HiTrap heparin column (Cat. No. 17-0407-
01), and S-400 Sephacryl (Cat. No. 217-0609-10) are
from Amersham Biosciences. RNasin (Cat. No. N2515)
is from Promega, and amylose resin (Cat. No. E8021)
is from New England Biolabs. Acrylamide (Cat. No.
EC-852) is from National Diagnostics, and urea (Cat.
No. 821527) is from ICN. The splicing substrate is generated
by in vitro
runoff transcription under standard
conditions (Moore and Query, 1998) from construct
MJM273. MJM273 is derived from an AdML gene
construct containing a single intron with an extended
polypyrimidine tract, an AG->GG 3' splice site mutation
(Anderson and Moore, 1997; Gozani et al.
and contains a tag of three MS2-binding sites located
in the intron between the 5' splice site and the branch
point (Jurica et al.
, 2002). The substrate is transcribed
with T7 RNA polymerase, radiolabeled uniformly
P]UTP and capped with G(5')ppp(5')G. The
RNA should be gel purified and quantified by comparison
with the specific activity of the transcription
reaction. Nuclear extract is derived from HeLa cells as outlined in Dignam et al.
(1983) and stored in 400-µl
aliquots at -80°C. DNA oligonucleotides for RNase
H-mediated digestion were synthesized with the
following sequences: oligol (5'-AGCTGGCCCTCG-3'),
oligo2 (5'-CAGACAGCGATG-3') and stored in 100 µM
aliquots of 100 µl at -20°C.
The FPLC (Cat. No. 18-1118-67) is from Amersham
Biosciences, and the Centricon-10 (Cat. No. 4205) and
Kontes Flex-column (Cat. No. K420401-1010) are from
Fisher. Mobicols (Cat. No. M1002) and filter (Cat. No.
M2135) are obtained from Mo Bi Tec, LLC, and lowadhesion
microcentrifuge tubes (Cat. No. 1415-2600)
are from USA Scientific. Gel plates, combs, spacers,
and electrophoresis rigs are homemade. Power supplies
are from Thermo EC (EC3000) or similar, and the
phosphorimager is from Molecular Dynamics.
A. Purification of Spliceosomes
- Potassium glutamate: 1M, pH 7.5. To make 50ml,
dissolve 9.26 g of L-glutamic acid, monopotassium salt
in 35ml of H2O, and adjust the pH to 7.5 with 10N potassium hydroxide. Complete to 50 ml with H2O and
filter sterilize. Store at 4°C.
- Magnesium acetate: 1M pH 7.5. To make 50ml,
dissolve 10.72 g of magnesium acetate in 35 ml of H2O
and adjust pH to 7.5 with sodium hydroxide. Complete
to 50ml with H2O and filter sterilize. Store at
- Creatine phosphate: 250mM. To make 5ml, dissolve
319mg phosphocreatine disodium salt in 5ml of
H2O. Store at -20°C in 1-ml aliquots. Keep a 100-µl
working aliquot at -20°C.
- Yeast tRNA: 5 mg/ml. Add 5 ml of H2O to a 500
unit bottle of Sigma yeast tRNA and store at -20°C. Keep a 100-µl working aliquot at -20°C.
- Heparin: 10mg/ml. Make a 1-ml stock by dissolving
10mg heparin in 1 ml H2O and filter sterilize.
Store at 4°C.
- Maltose: 0.5M. To make 50ml, dissolve 9g of
maltose in 40ml of H2O and complete to 50ml. Filter
sterilize and store at room temperature.
- 5x SB: 0.75M KCl, 25 mM EDTA, 100mM Tris, pH
7.9. Make 50ml with 18.75ml of 2M KCl stock solution,
2.5 ml of 0.5M of EDTA stock solution, 5 ml of
1M Tris, pH 7.9, stock solution, and 23.75 ml of H2O.
Store at room temperature.
- SB-N: 150mM KCl, 20mM Tris, pH 7.9, 5mM EDTA, 1 mM dithiothreitol (DTT), and 0.5% NP-40. Make 50ml fresh for each preparation with 10ml of 5
× SB, 50 µl of 1M DTT stock solution, 250 µl of 10% NP-
40 stock solution, and 39.7ml of H2O. Keep at room
- SB: 150mM KCl, 20mM Tris, pH 7.9, 5mM EDTA, and 1 mM DTT. Make 15ml fresh for each
preparation with 3 ml of 5 × SB, 15 µl of 1M DTT stock
solution, and 12ml of H2O. Keep on ice.
- SB-M: 150mM KCl, 20mM Tris, pH 7.9, 5mM EDTA, 1 mM DTT, and 10 mM maltose. Make 1 ml fresh
for each preparation with 200µl of 5 × SB, 1 µl of 1M DTT stock solution, 20µl of 0.5M maltose, and 779 µl
of H2O. Keep on ice.
- At room temperature, pour 5 ml of S-400 resin
equilibrated and resuspended in an equal volume of
SB-N into a 1.0 × 10-cm glass column. Allow the resin
to settle by gravity flow until the buffer is near the top
of the column bed. This sizing column can be used
multiple times by washing with 10ml of SB-N before
each use (Fig. 2A).
- Set up a 1-ml splicing reaction as described in
steps 3 and 4 to contain final concentrations of the following:
10nM splicing substrate (MJM273), 500nM MS2-MBP, 80mM potassium glutamate, pH 7.5, 2mM magnesium acetate, pH 7.5, 2 mM ATP, 5 mMcreatine
phosphate, 0.05mg/ml yeast tRNA, 1% RNasin, and
40% nuclear extract.
- First, place 50 µl substrate (0.2 pmol/µl) in a lowadhesion
microcentrifuge tube. Heat at 95°C for 1 min and then place on ice to cool. Add 10µl MS2-MBP
(50µM) and mix by flicking. Incubate on ice for
- In a second tube, add the following in order:
381 µl H2O, 80µl potassium glutamate (1M, pH 7.5), 2
µl magnesium acetate (1M, pH 7.5), 20µl ATP (100
mM), 20 µl creatine phosphate (250 mM), 10 µl tRNA
(5mg/ml, yeast), 10µl RNasin, contents of the first
tube (substrate plus MS2-MBP), and 400µl nuclear
extract. Mix by flicking the tube gently. (Place a 5-µl aliquot of the reaction on ice as a 0' time point for RNA
denaturing gel analysis.)
- Aliquot 200µl into five tubes. Incubate tubes at
30°C for 60min to assemble spliceosomes. (Place a 5-
µl aliquot of the reaction on ice as a 60' time point for
RNA denaturing gel analysis.)
- During this incubation, assemble the MoBiCol
column by placing a small filter in the bottom of the
column. The column can be supported in a microcentrifuge
tube on ice. Add 200µl of amylose resin equilibrated
and resuspended in an equal volume of SB-N.
Allow the buffer to drain by gravity flow (Fig. 3A).
- After the 60' incubation add 2µl each of oligo 1
and oligo 2 (100 µM) to each tube of the splicing reaction.
Mix by flicking the tube gently. Incubate tubes at
30°C for 20min to induce RNase H digestion. (Place a
5-µl aliquot of the reaction on ice as an 80' time point
for RNA denaturing gel analysis.)
- Add 10µl of heparin (10mg/ml) to each tube.
Mix by flicking the tubes gently. Incubate tubes at 30°C for 5 min.
- Run the sizing column by gravity flow until the
buffer head just reaches the top of the resin bed. Pool
the splicing reactions from the five tubes and carefully
load onto the sizing column. Run the column until the
resin bed is exposed and load 500 µl SB-N to wash the
column wall. Again run the column until the resin bed
- Run 10 ml of SB-N through the column, taking
500-µl fractions in tubes on ice. Gravity flow should be
about ~0.4 ml/min. Collect 18-20 fractions total.
- Count the fractions for the radioactively labeled
the splicing substrate with a Geiger counter. There
should be two peaks of radioactivity, with the first
smaller peak (usually within the first 11 fractions) containing
splicing intermediates (spliceosomes) and the
larger second peak containing degraded splicing
substrate (Fig. 2B). (This can be checked by taking
10-µl aliquots of the fractions for RNA denaturing gel
- Pool the first peak (usually ~2ml) and flow this
through the amylose column by gravity. Collect the
column flow-through in tubes on ice and reapply to the
amylose column. To wash the column, attach a 10-ml
syringe barrel to the top of the column using a luer
adaptor cap and place in a 15-ml Falcon tube. Fill the
syringe with 10ml of cold SB to wash the column by
gravity flow at 4°C (Fig. 3B).
- Elute the splicing complexes by pipetting 50-µl
aliquots of SB-M onto the amylose resin and taking
fractions dropwise into tubes on ice. Count the fractions
for the radioactively labeled splicing substrate
with a Geiger counter. The complexes should be contained
within the first 100 µl of elution.
|FIGURE 2 (A) Size-exclusion column. (B) Elution profile of size-exclusion column measured with a Gieger
B. Purifying Affinity-Tag Adapter Protein,
|FIGURE 3 (A) Amylose affinity resin in a small MoBiCol column.
(B) Setup for washing the amylose column.
The affinity-tag adapter protein is a recombinant
MS2-MBP fusion expressed in Escherichia coli
a gift from Josep Vilardell). This fusion places
MBP N-terminal to MS2. The MS2 portion carries
a double mutation (V75Q and A81G) that prevents
oligomerization (LeCuyer et al.
, 1995). Single-step
purification of MS2-MBP over an amylose column
yields a single band on a Coomassie-stained gel, but
ratio (<1) reveals that a significant amount
of bound nucleic acid remains as a contaminant.
Heparin chromatography as a second purification step
eliminates this contaminant.
- AB1: 20mM HEPES, pH 7.9, 200mM KCl, and
1 mM EDTA. Make 500ml by combining 10ml of 1M HEPES, pH 7.9, stock solution, 50ml of 2M KCl stock
solution, 1 ml of 0.5 M EDTA stock solution, and 439 ml
- AB2: 20mM HEPES, pH 7.9, 5mM KCl, and
1 mM EDTA. Make 500 ml by combining 10 ml of 1M HEPES, pH 7.9, stock solution, 1.25ml of 2M KCl
stock solution, 1 ml of 0.5 M EDTA stock solution, and
487.75 ml of H2O.
- ABE: 20mM HEPES, pH 7.9, 5mM KCl, 1 mM EDTA, and 10 mM maltose. Make 100 ml by combining
2ml of 1M HEPES, pH 7.9, stock solution, 0.25 ml of
2M KCl stock solution, 200µl of 0.5M EDTA stock
solution, 2 ml of 0.5 M maltose, and 474.75 ml of H2O.
- PMSF: 100mM. To make 5ml, dissolve 87.1mg
PMSF in 5 ml of ethanol. Store at 4°C.
- IPTG: 1M. To make 10ml, dissolve 1.19g of IPTG
in 5 ml of H2O. Store in 1-ml aliquots at -20°C.
- HB1: 20mM HEPES, pH 7.9, and 1 mM EDTA.
Make 500ml by combining 10ml of 1M HEPES, pH
7.9, stock solution, 1 ml of 0.5 M EDTA stock solution,
and 489ml of H2O.
- HB2: 20mM HEPES, pH 7.9, 1M KCl, and 1 mM EDTA. Make 500 ml by combining 10 ml of 1M HEPES,
pH 7.9, stock solution, 250ml of 2M KCl stock
solution, 1 ml of 0.5 M EDTA stock solution, and 239ml
C. Denaturing Gel Analysis and
- Inoculate a 5-ml culture of Luria broth (LB) with
single bacterial colony of DH5oc cells transformed with
a plasmid expressing MS2-MBP and grow overnight to
saturation at 37°C with shaking. The next morning,
inoculate 1 liter of LB plus 2% glucose with the 5-ml
culture. Grow the cells at 37°C with shaking to an
OD600 of ~0.5 and then induce expression of the protein by adding 1 ml of 1M IPTG. Continue to grow the cells
for 2-3 h and harvest by centrifugation at 6000rpm for
10min. Pour off the supernatant, freeze the cell pellet,
and store at -20°C.
- Thaw and resuspend ~1 g cells in 10ml cold AB1
plus 200 µl PMSE Break open the cells by sonication on
ice. Centrifuge for 30min at 15,000rpm at 4°C.
- Perform all the following steps of purification at
4°C. Load the supernatant on a -5-ml amylose column
equilibrated with AB1, running the column at 0.3 ml/
min. Wash the column with 40ml of AB1, followed by
10 ml of AB2 to lower the salt concentration in preparation
for heparin chromatography.
- Elute the protein with 20ml of ABE, taking 1-ml
fractions. Check the OD280 of the fractions and pool the
peak fractions. (The column can be cleaned with 5 ml
of 0.1% SDS and reequilibrated with AB1 for future
- Concentrate the pooled peak fraction to -1 ml in
a Centricon -30.
- On an FPLC, equilibrate a 1-ml heparin column
with a mixture of HB1 and HB2 to 5 mM KCl. Load the
concentrate on the column and wash with 5 ml at 5 mM KCl.
- Run a gradient from 5 to 400mM KCl over
10 column volumes. The MS2-MBP protein elutes at
~60mM KCl. Pool peak fractions and concentrate to
~500µl in a Centricon-30. Add glycerol to 10% and
freeze at -20°C in 100-µl aliquots.
- Determine the protein concentration for MS2-
MBP. An OD280 of 1 corresponds to 16.5µM or
- Splicing dilution buffer: 100mM Tris, pH 7.5,
10mM EDTA, 1% SDS, 150mM NaCl, and 0.3 M NaAc,
pH 5.2. To make 50 ml, combine stock solutions of 5 ml
of 1M Tris, pH 7.5, 1 ml of 0.5M EDTA, 5 ml of 10%
SDS, 3.75 ml of 2M NaCl, 5 ml of 3M NaAc, pH 5.2,
and 30.25 ml of H2O. Store at room temperature.
- Acrylamide solution: 15% acrylamide (29:1 acrylamide:
bis-acrylamide), 8M urea, and 1 × TBE. To
make 500ml, dissolve 240.24g of urea in 100ml of
stock solution of 5 × TBE and 187.5ml of 40% acrylamide
solution with stirring over low heat. Complete
to 500 ml with H2O and filter. Store in the dark at room
- FEB: 94% formamide, 20mM EDTA, 0.01% cyan
blue, and 0.01% bromphenol blue. To make 10ml,
combine 9.4ml of formamide, 0.4ml of 0.5M EDTA, 0.1 ml of 1% cyan blue, and 0.1 ml of 1% bromphenol
blue stock solutions. Store 1-ml aliquots at -20°C.
- To prepare the time point aliquots taken during
the spliceosome purification for electrophoresis,
remove the tubes from ice and add 95µl of splicing
dilution buffer. Then add 100 µl of phenol:chloroform:
isoamyl alcohol (25:24:1, pH 4.5) stock solution. Vortex
well and spin at 14,000rpm in a microcentrifuge for
10min at room temperature. (If taken, aliquots
from the sizing column elution should be treated
- Take the top 90µl, avoiding the interface, and
place in a new tube. To this add 3 volumes ethanol and
invert to mix. Place at -70°C for 30min. Discard the
- Spin tubes in a microcentrifuge at 14,000 rpm for
30 min at 4°C. Remove the ethanol with a pipettor and
wash the pellet with 100µl of 70% ethanol. Dry the
- Resuspend the pellet in 5 µl of FEB. Load 2.5µl
on the denaturing acrylamide gel.
- To prepare elution samples, add 4µl of FEB to
1 µl of elution aliquots. Load 2.5 µl on the gel.
- To prepare standard, add 39µl of FEB to 1 µl of
10nM splicing substrate used during the purification.
Load 1 µl on the gel.
- Assemble a gel mold using 20 × 25-cm glass
plates with 0.1mm spacers and comb. Pour the gel
using 25 ml acrylamide solution and polymerize with
75 µl of 20% APS and 25 µl of TEMED stock solutions.
Allow the gel to set for at least 30min.
- Assemble the gel on a gel rig with TBE buffer
stock. Prerun at 30 W for 30min.
- Prior to loading on the gel, incubate the samples
at 95°C for 1 min and then place on ice.
- After rinsing out the wells, load samples and
standard. Run the gel at 30W for 2h. The cyan blue
should migrate to near the bottom of the gel.
- Take down the gel onto a piece of exposed X-ray
film and cover with plastic wrap. Expose to a phosphorimager
- To quantify the concentrations of the purified
spliceosomes, the specific activity of the labeled splicing
intermediates should be compared to the standard.
|FIGURE 4 Example of denaturing gel (15% polyacrylamide)
analysis of substrate RNA during purification. Time points taken
during the splicing reaction (0 and 60min) and after RNase H digestion
(80min), size-exclusion peak fraction (S), affinity column elutions
(1-4), and standard used for quantification (St) are shown. The
positions of MS2-tagged pre-mRNA, lariat intermediate, 5' exon,
and RNase H digestions products are indicated..
- Starting with 10 pmol of pre-mRNA substrate,
this protocol generally yields 0.3-0.5 pmol of purified complexes. The yield is highly dependent on the efficiency
of the first step of splicing in the reactions.
Nuclear extracts can differ in their splicing efficiencies,
and therefore titrations of potassium glutamate and
magnesium acetate concentrations should be performed
to maximize splicing efficiency for a given
nuclear extract preparation.
- The final buffer conditions for the purification
were chosen with electron microscopy studies in mind.
We found that the splicing complexes tended to aggregate
when 2mM MgCl2 was present in the buffer
instead of the 5 mM EDTA. Although some spliceosome
proteins are disassociated in the presence of
EDTA, the core splicing components remain intact
(Jurica et al., 2002).
- We have yet to find a method to concentrate
spliceosomes after purification, as the complexes
adhere to the membranes of all concentration devices
tested. By using the very small amount of affinity resin
with a column geometry, the spliceosomes can be
eluted at a high enough concentration for structural
and biochemical analyses without further concentration. Typically peak elution fractions of 15-50µl containing
5 to 15 nM splicing intermediates are obtained.
- Contamination of reagents, tubes, and so on by
RNases is always a concern when handling RNA. Care
should be taken to wear clean gloves during the purification
and, if possible, to designate reagents, tips,
tubes, and so on as being for RNA use only.
Anderson, K., and Moore, M. J. (1997). Bimolecular exon ligation by
the human spliceosome. Science 276
Das, R., Zhou, Z., and Reed, R. (2000). Functional association of U2
snRNP with the ATP-independent spliceosomal complex E. Mol.
Dignam, J. D., Lebovitz, R. M., and Roeder, R. D. (1983). Accurate
transcription initiation by RNA polymerase II in a soluble extract
from isolated mammalian nuclei. Nucleic Acids Res
Gozani, O., Patton, J. G., and Reed, R. (1994). A novel set of spliceosome-
associated proteins and the essential splicing factor PSF
bind stably to pre-mRNA prior to catalytic step II of the splicing
reaction. EMBO J
Hartmuth, K., Urlaub, H., Vornlocher, H. P., Will, C. L., Gentzel, M.,
Wilm, M., and Luhrmann, R. (2002). Protein composition of
human prespliceosomes isolated by a tobramycin affinityselection
method. Proc. Natl. Acad. Sci. USA 99
Jurica, M., Licklider, L., Gygi, S., Grigorieff, N., and Moore, M.
(2002). Purification and characterization of native spliceosomes
suitable for three-dimensional structural analysis. RNA 8
Jurica, M. S., and Moore, M. J. (2002). Capturing splicing complexes
to study structure and mechanism. Methods 28
LeCuyer, K. A., Gehlen, L. S., and Uhlenbeck, O. C. (1995). Mutants
of the bacteriophage MS2 coat protein that alter its cooperative
binding to RNA. Biochemistry 34
Makarov, E. M., Makarova, O. V., Urlaub, H., Gentzel, M., Will, C.
L., Wilm, M., and Luhrmann, R. (2002). Small nuclear ribonucleoprotein
remodeling during catalytic activation of the spliceosome. Science 298
Moore, M. J., and Query, C. C. (1998). Uses of site-specifically
modified RNAs constructed by RNA ligation. In "RNA-Protein
Interactions: A Practical Approach"
(C. W. J. Smith, ed.), pp. 75-108.
IRL Press, Oxford.
Moore, M. J., Query, C. C., and Sharp, P. A. (1993). Splicing of precursors
to mRNA by the spliceosome. In "The RNA World"
Gesteland and J. Atkins, eds.), pp. 303-357. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY.
Reichert, V., Le Hir, H., Jurica, M. S., and Moore, M. J. (2002). 5' exon
interactions within the human spliceosome establish a framework
for exon junction complex structure and assembly. Genes
Zhou, Z., Sim, J., Griffith, J., and Reed, R. (2002). Purification and
electron microscopic visualization of functional human spliceosomes. Proc. Natl. Acad. Sci. USA 99