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

Analysis and purification of spliceosomes assembled in vitro by ultracentrifugation

In the overview picture: what are the question that you can address: determine to composition of spliceosomes associated with a substrate? Prepare spliceosomes for affinity purification?

Authors: Klaus Hartmuth, Maria A. van Santen, Reinhard Lührmann

Affiliation: Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany

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 centrifugation, 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 direction 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 earth's gravitational field is replaced with a centrifugal field, which in present-day instruments can attain up to 105 g, ie. the 100,000 fold of the earth’s gravity (g is the gravitational constant 6.674 x 10 –11 N (m/kg)2 I have this from Wikipdia, could you check the number(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)

These relationships can be used to determine the Svalue, 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, the 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', because differently sized particles will sediment thorough the gradient in separate 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 particle 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 analysed 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 preparation of spliceosomal complexes assembled in vitro(Behzadnia et al., 2007; Bessonov et al., 2008; Deckert et al., 2006; Hartmuth et al., 2002).

Can you give a textbook description of the ultracentrifuge property?, especially the connection between S and shape. I learnt this from the cantor schimmel book, which is non intuitive, A good short intro is given in PW atkins, Physical Chemistry, page 611 in my 3rd edition, this includes some solved problems about geometry etc

3. Protocol

3.1 Preparation of the gradient

Gradients are preformed in the 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 MgCl2. 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-treatment of the stock solutions. Out of my own curiosity: is normal “sigma-glycerol RNAse free”?

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 successively 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 ( what kind of volumes and concentrations would you use for a 10 to 30% gradient?, 1 ml 30%, 1 ml 25%..etc, we always use a gradient mixer). 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 connected 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 solutions, 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. Can you give the type of gradient mixer, ie. sigma number xxx, and maybe make a little cartoon.

3.1.2Automatic gradient formation with the Gradient Master. This is 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 This will produce the gradients according to predefined parameters. A large number of 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 rotor 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 maximum 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 refrigerator or cold-room for a minimum of 1 hour. (can you let them sit over night?)

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 volume 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 actual centrifugation run, because all tubes will have received identical volumes, so the above loading procedure should conserve the weight of the tubes. (I would say something: beginner should balance the tubes to avoid damage of the ultracentrifuge, otherwise someone might sue, only half kidding)

3.2.2 Sedimentation markers

To calibrate the gradients, particles of known sedimentation values are loaded 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 obtained from any laboratory working on mRNA translation (this is a bit vague, is there an easy way to get this markers, does someone sells them?, for example, can you put a reticulate lysate into it?). 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 Can you give a vendor here?. Gradients are calibrated 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 gradients 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 rotors, ultracentrifuges and rotors from Beckman are equivalent. For small and precious amounts of sample (Rhode et al., 2006), we run miniature gradients (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. Can you make a diagram here? From our own experience: we used a pump attached to a glass capillary to ‘suck’ the gradient of the bottom. Do you rather recomment pipetting it out and why?

4. Example of an experiment

4.1 Purification of the spliceosomal 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, not clear to stefan which chapter this refers to, authors?. Pre-mRNA is transcribed in vitro, as described in chapter mayeda 26)This The pre-mRNA is pre-incubated with a fusion protein of the MS2 coat protein and maltose-binding protein (MBP). Subsequently, nuclear extract is added and spliceosomes are allowed to form. How much total protein and RNA (cpm or ug do you load) Complexes are then fractionated by size on a linear 10–30% (v/v) glycerol gradient. (what is the gradient volume, is there a relationship ug nuclear extract per ml gradient?) Gradient fractions containing spliceosomal complexes of interest are then affinity-selected by using amylose beads (vendor) and subsequently eluted with maltose. Here, we describe the gradient centrifugation step that is performed to purify spliceosomal B complexes.

A 12mL splicing reaction, containing 40% (v/v) HeLa nuclear extract in dialysis buffer (20 mM Hepes-KOH (pH 7.9), 0.1 M KCl, 1.5 mM MgCl2, 0.2 mM EDTA (pH 8.0), 10% (v/v) glycerol), supplemented with 25 mM KCl, 3 mM MgCl2, 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. ( is this 10 ug/ul nuclear extract, this would be 120 mg NE?, can you give the amount of minx rna in ug and or cpm) 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 (what is the volume of each tube?).

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 4oC 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, not clear to stefan which chapter this refers to, authors?.). Subsequent RNA and protein analyses of the B complex eluate are shown in Figure 2.

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 gradient 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 precipitation 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.

Figure Legends

Klaus can you add here a schematic figure and/or photo showing the making of the gradient and the harvesting

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 Cherenkov 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 are these premade gels? (NuPAGE, Invitrogen), respectively. RNA was visualised by silver-staining (reference for RNA silver staining?, it there a reason you don’t use an RNA dye acridin or more modern versions?) and autoradiography. Proteins were visualised by Coomassie staining. Only the input pre-mRNA is radioactively labelled and gives a signal upon autoradiography. The U1-U6 RNAs are from the nuclear extract. Can you also give the sizes of the RNAs either in the Figure legend or the Figure. Why is the MINX RNA running as a doublet? Does this reflect processing or an in vitro transcription stop?

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-dimensional 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 core. 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 complexes isolated under physiological conditions. Mol Cell Biol 26, 5528-5543.

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 affinity-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