Title: Analysis and purification by ultracentrifugation of spliceosomes assembled in

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Title: Analysis and purification by ultracentrifugation of spliceosomes assembled in
vitro

Authors:
Klaus Hartmuth, Mari
a A.

van Santen, Reinhard Lührmann

Affiliation:

Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D
-
37077
Göttingen, Germa
ny

1. Abstract

Gradient centrifugation is a powerful purification step in the isolation of spliceosomes. It
involves the separation of spliceosomal complexes from nuclear extract under physiological
conditions. For the preparative isolation of spliceosomal

complexes, 10

30% (v/v) glycerol
gradients were found to be best. Here, we describe the purification procedure of spliceosomal
B complexes, which can easily be separated from earlier complexes by glycerol
-
gradient
centrifugation. Following gradient centri
fugation, spliceosomal complexes can be subjected
to
downstream applications
, such as affinity purification.


Keywords


2. Theoretical background

When a particle in solution is subjected to a gravitational field, it will respond by
sedimentation in the dir
ection of the applied field, whenever the particle is denser than the
solution. The rate of this sedimentation is a function of the particle's mass and shape, the
nature of the solute, and the strength of the gravitational field. In an ultracentrifuge, the

2


earth's gravitational field is replaced with a centrifugal field, which in present
-
day
instruments can attain up to 10
5

g. The sedimentation coefficient (
) is the particle
-
specific
parameter when measuring the rate of the sedimentation (
) of a particle as a function of the
rotor speed (
) and particle distance (
) from the centre using the following relation derived
from the well
-
known Svedberg equation,


where



the sedimentation coefficient

in seconds (usually expressed in Svedberg units
(

sec)




the distance between particle and the centre of rotation (cm)




the rotor speed (radians/sec)




the rate of movement of particle (cm/sec)

Thes
e relationships can be used to determine the S

value, the shape, the molecular weight, or
the buoyancy of a particular molecule or macromolecular complex in an analytical
ultracentrifuge
(Scott et al., 2006)
. The high reproducibility of the sedimentation properties of
macromolecules is due to the precise biophysical principles involved. This has made
ultracentrifugation the method of choice in

routine preparations of particles of a particular
size. To increase the effective separation range for particles within the limited range of radii
available in swinging
-
bucket rotors, the ultracentrifugation is performed in density gradients.
In these, th
e density increases with the distance from the centre of rotation, thereby retarding
the sedimentation of the fast
-
migrating particles. This is called the 'Rate Zonal Technique',
3


because differently sized particles will sediment thorough the gradient in se
parate zones,
where each zone contains particles with identical sedimentation coefficients. Clearly, the
intrinsic density of the particles must be higher than the highest density of the gradient, and
the run must be terminated before the zone with the par
ticle of interest reaches the bottom.
After centrifugation, the material in the gradient is harvested by fractionation, either manually
or by pumping from the bottom with a peristaltic pump. Depending on the type of experiment,
fractions can then be analys
ed for protein, RNA and/or radioactivity, and fractions containing
the complex of interest can then be used for downstream applications. In our laboratory,
centrifugation in glycerol
-
density gradients has been extensively used in the analysis and
preparati
on of spliceosomal complexes assembled
in vitro

(Behzadnia et al., 2007; Bessonov
et al., 2008; Deckert et al., 2006; Hartmuth et al., 2002)
.

3. Protocol

3.1 Preparation of the gradient

Gradients are preformed in th
e centrifuge tubes designed for the rotor to be used (see below).
Glycerol gradients are most versatile, because the stock solutions are easily prepared from
readily available materials. For the preparative isolation of spliceosomal complexes, 10

30%
(v/v
) glycerol gradients were found to be best. 10% and 30% (v/v) glycerol stock solutions
are prepared in standard gradient buffer (20 mM Hepes
-
KOH (pH 7.9), 150 mM NaCl (or
KCl), 1.5 mM MgCl
2
. As an alternative, sucrose gradients can be used. However, stock
solutions are more difficult to prepare owing to the solubility problems associated with high
concentrations of sucrose. In addition, contamination by RNase may pose a problem,
requiring the purchase of special RNase
-
free sucrose or cumbersome DEPC
-
treatme
nt of the
stock solutions.

4


3.1.1 Manual gradient formation.

When no mechanical devices such as pumps or gradient
formers are available, this is the method of choice. First, a discontinuous gradient is produced
in the centrifuge tube, either (i) by successi
vely overlaying less dense solutions on dense
solutions; or (ii) by successively underlaying less dense solutions under the denser ones. Best
results are obtained with three to four layers. A continuous gradient is then generated by
allowing the layers to
diffuse. This is best achieved by sealing the tube with Parafilm,
carefully rotating the tube into a horizontal position and allowing the layers to diffuse for 45

60 min. The tube is then returned to the vertical position and chilled to 4°C before use. The

gradients formed are highly reproducible, because of the purely biophysical principle
involved.

Alternatively, the gradient can be formed by using a two
-
chamber gradient maker. Two
identical chambers (one mixing chamber and one non
-
mixing chamber) are con
nected at the
bottom by a channel containing a tap. The mixing chamber has an outlet
with attached plastic
tubing to
let
the contents of the chamber pass
in
via a
peristaltic pump to the bottom of the
gradient tube. By using equal volumes of the two soluti
ons, first the heavy solution is placed
into the non
-
mixing chamber and the connection is flushed with this solution. Then the light
solution is placed in the mixing chamber. Identical magnetic stirring bars are put into the two
chambers and agitated. The
tap is then opened and, simultaneously, the peristaltic pump is
switched on. The gradient is formed from the bottom of the tube by underlaying a solution
that progressively becomes denser.

3.1.2

Automatic gradient formation with the Gradient Master.
This i
s the easiest and
most reproducible way to create
various
continuous gradients in tubes for a large diversity of
rotors, but it requires the Gradient Master instrument (see www.biocompinstruments.com).
This will produce the gradients according to predefine
d parameters. A large number of
5


programmes for gradients in different rotor tubes are included in the device, and with some
experimenting it is straightforward to adapt the parameters of a pre
-
existing program to a new
gradient
-
and
-
tube combination. The ro
tor tubes are
filled half
-
and
-
half with

the high (bottom)
and low (top) density solutions, plugged with a smooth rubber plug, and placed into the tube
holder of the instrument. Tubes are then rotated for a fixed time at a fixed angle (usually 80°).
A maxim
um of 6 gradients can be prepared simultaneously for a particular rotor, and
gradients are ready in about 2 min. Gradients are prepared at room temperature and then
equilibrated at the desired running temperature, usually 4°C, by placing them in a refriger
ator
or cold
-
room for a minimum of 1 hour.

3.2 Preparing the run

3.2.1 Loading the sample

The tube with the gradient is placed carefully in a rack, and the rubber plug is removed. With
an automatic pipette, an amount of liquid corresponding to the sample v
olume to be applied is
removed
from the top of the gradient. The sample is then slowly applied to the top by letting
it run down the wall of the tube, taking care not to disturb the gradient. It is usually not
necessary to balance the tubes before the actu
al centrifugation run, because all tubes will
have received identical volumes, so the above loading procedure should conserve the weight
of the tubes.

3.2.2 Sedimentation markers

To calibrate the gradients, particles of known sedimentation values are loade
d onto separate
gradients. For the spliceosomes fractionated on the 10

30% (v/v) gradients described here,
ribosomal subunits are ideal markers. The small and large subunits from bacterial ribosomes
sediment at 30S and 50S, respectively, and can be obtaine
d from any laboratory working on
6


mRNA translation. Alternatively, any other particles or molecules that sediment in the 20

50S range can be used, for example commercially available ribosomal RNA or other large
macromolecular complexes. Gradients are calibr
ated once for a particular set of solutions and
ultracentrifuge run parameter (see below).

3.3 The ultracentrifuge run

After the tubes have been carefully placed in the buckets, these are closed with their air
-
tight
seals and positioned in the rotor. The g
radients are then run for

the time and speed required to
achieve best resolution. Typical parameters for a number of rotors that we use to prepare
spliceosomes are listed in Table 1. While we use Sorvall Discovery 90 or 90SE centrifuges
with associated rot
ors, ultracentrifuges and rotors from Beckman are equivalent. For small
and precious amounts of sample
(Rhode et al., 2006)
, we run miniature gra
dients (1.4 mL) in
a Sorvall Discovery MC150 centrifuge with the S55
-
S rotor.

3.4 Harvesting the gradient

The method of choice for harvesting the gradients is manual pipetting. With an automatic
pipette, successive fractions are taken off from the top. The

tip of the pipette is immersed just
below the surface of the gradient, and the gradient solution is removed by slow suction while
the tip of the pipette follows the sinking meniscus of the gradient.

4. Example of an experiment

4.1 Purification of the spli
ceosomal B complex

To purify spliceosomal complexes, we make use of a pre
-
mRNA tagged with three MS2
RNA aptamers
at the 3' end
(
see
Chapter II.18
). This is pre
-
incubated with a fusion protein of
the MS2 coat protein and maltose
-
binding protein (MBP). Subs
equently, nuclear extract is
7


added and spliceosomes are allowed to form. Complexes are then fractionated by size on a
linear 10

30% (v/v) glycerol gradient. Gradient fractions containing spliceosomal complexes
of interest are then affinity
-
selected by usin
g amylose beads and subsequently eluted with
maltose. Here, we describe the gradient centrifugation step that is performed to purify
spliceosomal B complexes.

A 12
-
mL splicing reaction, containing 40% (v/v) HeLa nuclear extract in
dialysis buffer (
20
mM He
pes
-
KOH (pH 7.9), 0.1 M KCl, 1.5 mM MgCl
2
, 0.2 mM EDTA (pH 8.0), 10%
(v/v)
glycerol
),
supplemented with 25 mM KCl, 3 mM MgCl
2
, 20 mM creatine phosphate, 2 mM
ATP and 10 nM MINX
-
MS2 pre
-
mRNA, is incubated for 8 minutes at 30°C in small aliquots
in standard
1.5 ml tubes. The time point of 8 minutes was chosen, since after 8 minutes of
incubation, mainly A and B, but no activated complexes have formed. After incubation, the
tubes are placed on ice and then loaded onto 10

30% (v/v) glycerol gradients, prepared
in
standard gradient buffer.

In a Sorvall Tst 41.14 rotor, 6 gradients can be run in parallel. On each of the gradients, 2 ml
of splicing reaction was loaded as described above. To prevent reaction mixtures from
warming up, this is done at 4°C in the cold
-
room. Gradients are then centrifuged for 16 hours
at 25,000 rpm at 4
o
C and harvested manually by withdrawing 500

l fractions from the top.
Radioactivity, as a measure of the amount of pre
-
mRNA in each fraction, was then
determined by Cherenkov counting. An example of a resulting gradient profile is shown in
Figure 1. Fractions containing spliceosomal B complexes are

then subjected to affinity
chromatography (
see
Chapter II.18
). Subsequent RNA and protein analyses of the B complex
eluate are shown in Figure 2.


8


5. Trouble
-
shooting

1.

To make highly reproducible glycerol gradients with the Gradient Master, the

rotor
tubes

should be filled exactly
half
-
and
-
half

with the high (bottom) and low (top)
density solutions. The

boundary between 10% and 30% is indicated on the tube by
using a marker block supplied with the device.

2.

For most spliceosomal complexes, the standard gradi
ent buffer containing 150 mM
NaCl (or KCl) is best. However, for some less stable complexes, like the A complex,
salt concentrations may have to be reduced.

3.

If gradient samples are to be analysed directly by SDS
-
PAGE, NaCl should be used to
prevent precipi
tation of the insoluble salt potassium dodecyl sulphate.

4.

In order to load a sample without disturbing the gradient, the glycerol concentration of
the sample must be lower than that in the low
-
density solution on the top of the
gradient. If this is not the
case, the sample must be diluted with gradient buffer
containing no glycerol.

5.

The sample volume should not exceed 10% of the total volume of the gradient.

6.

For purification of native spliceosomes, it is important that all steps are carried out at
4°C.


Figu
re Legends

9


Figure 1: Separation of spliceosomal complexes on a glycerol gradients. A splicing reaction
was loaded on a 10

30% (v/v) glycerol gradient in standard gradient buffer. The distribution
of radioactively labelled pre
-
mRNA was determined by Cherenk
ov counting.

Figure 2: RNA and protein composition of a spliceosomal B complex were analysed by
electrophoresis, in an 8.3 M urea / 9.6% polyacrylamide gel and by SDS
-
PAGE (NuPAGE,
Invitrogen), respectively. RNA was visualised by silver
-
staining and autora
diography.
Proteins were visualised by Coomassie staining.

References


Behzadnia, N., Golas, M.M., Hartmuth, K., Sander, B., Kastner, B., Deckert, J., Dube, P.,
Will, C.L., Urlaub, H., Stark, H.
, et al.

(2007). Composition and three
-
dim
ensional EM
structure of double affinity
-
purified, human prespliceosomal A complexes. EMBO J

26
,
1737
-
1748.

Bessonov, S., Anokhina, M., Will, C.L., Urlaub, H., and Lührmann, R. (2008). Isolation of an
active step I spliceosome and composition of its RNP co
re.
Nature

452
, 846
-
850.

Deckert, J., Hartmuth, K., Boehringer, D., Behzadnia, N., Will, C.L., Kastner, B., Stark, H.,
Urlaub, H., and Lührmann, R. (2006).
Protein composition and electron microscopy structure
of affinity
-
purified human spliceosomal B comp
lexes isolated under physiological conditions.
Mol Cell Biol

26
, 5528
-
5543.

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Hartmuth, K., Urlaub, H., Vornlocher, H.P., Will, C.L., Gentzel, M., Wilm, M., and
Lührmann, R. (2002). Protein composition of human prespliceosomes isolated by a
tobramycin affini
ty
-
selection method. Proc Natl Acad Sci U S A

99
, 16719
-
16724.

Rhode, B.M., Hartmuth, K., Westhof, E., and Lührmann, R. (2006). Proximity of conserved
U6 and U2 snRNA elements to the 5' splice site region in activated spliceosomes. EMBO J

25
, 2475
-
2486.

Scott, D.J., Harding, S.E., and Rowe, A.J., eds. (2006). Analytical ultracentrifugation:
techniques and methods (London, RSC Publisher).



Abbreviations