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From: Methods in Molecular Biology, Vol. 118: RNA-Protein Interaction ProtocolsEdited by: S. Haynes © Humana Press Inc., Totowa, NJ

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Preparation of Hela Cell Nuclear and Cytosolic S100Extracts for In Vitro Splicing

Akila Mayeda and Adrian R. Krainer

1. IntroductionFollowing the initial discovery of split genes in 1977, it took several years

before in vitro systems were successfully developed to study the biochemistryof pre-mRNA splicing. The first systems relied on coupling of transcriptionand splicing in whole-cell extracts and were fairly inefficient, because of thedifferent optima for these two reactions (1,2). It was later shown that thesereactions could be uncoupled (3,4), but obtaining discrete pre-mRNAs in use-ful amounts remained an obstacle until in vitro transcription with bacterioph-age RNA polymerases (3) was adopted for this purpose. Another usefuldevelopment was a nuclear extract preparation procedure that was initiallydeveloped for in vitro transcription studies (5). No splicing was detected in thisstudy. However, the same extract preparation procedure, in conjunction withpre-mRNAs transcribed from cloned genes by SP6 RNA polymerase, was usedto define optimal conditions for in vitro splicing (6). This system results inrelatively efficient and accurate splicing, and is now in wide use, with slightvariations from laboratory to laboratory. Variations in extract preparationinclude primarily the use of slightly different buffers and salts for nuclearextraction or dialysis.

HeLa cells are used most frequently, but the same procedure has been usedto prepare splicing-competent extracts from other cells that grow in spinnerculture. Many cell lines fail to yield active extracts, and this is sometimes dueto the presence of ribonucleases, proteases, or other nonspecific inhibitors. Thisproblem appears to be exacerbated when animal tissues are used as the source.Studies of in vitro transcription have been much more successful in this regard,and the reason may be that the splicing apparatus includes highly sensitive,

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multisubunit snRNP components. In addition, owing to the very large number ofcomponents required to assemble an active spliceosome, it appears that limiting com-ponents are lost when attempts are made to obtain more purified nuclei, e.g., fromanimal tissues. A very similar extract preparation procedure using HeLa cells hasbeen used to carry out accurate polyadenylation (8; see Chapter 32), and coupling ofsplicing and polyadenylation can be studied in this system (9). The extract prepara-tion procedure described here is also useful to study splicing of the recently discov-ered nonconventional (AT–AC) introns, in addition to the major splicing pathway(10,11). A similar procedure has been used to prepare splicing extracts from Droso-phila cell lines (12). An efficient system for yeast pre-mRNA splicing has been widelyused, and is based on whole-cell extracts from S. cerevisiae (13; see Chapter 26). Incontrast, pre-mRNA splicing in plant extracts has not yet been reported.

Here we provide a detailed protocol for the preparation of pre-mRNA splic-ing-competent extracts from HeLa cells. It is based on the method for prepara-tion of transcription-competent extracts (5,6) with slight variations to improvesplicing efficiency (7), and has been used in our laboratory for many yearswith reproducible results. The same preparation yields two kinds of extract: anuclear salt wash and a cytosolic S100. The nuclear extract is fully competentfor splicing of numerous pre-mRNAs. The S100 extract contains many splic-ing factors, but is not competent for splicing because it has limiting amounts ofSR proteins, which are required for splicing. However, this extract can becomplemented by one or more SR proteins to give efficient splicing (14). Thesetwo systems (nuclear extract and S100 plus SR proteins) differ in interestingand useful ways, in part because of the somewhat different ratios of varioussplicing factors (15–17). Both systems have been widely used to study consti-tutive splicing and/or alternative splicing of different pre-mRNAs.

The protocol described here is for extract preparation on a medium scale. The proce-dure can be easily scaled up using appropriate glassware and rotors, although cell lysisis more difficult with larger Dounce homogenizers. A procedure for nuclear extractpreparation on a very small scale has been described, which should be useful for sur-veying many different cell lines or cells grown under many different conditions (18).

2. MaterialsAll reagents should be prepared with high quality autoclaved water, e.g.,

Milli Q (Millipore, Bedford, MA, USA) or double-distilled water. Sterilizationof all components used for cell growth is carried out by autoclaving or, in thecase of thermolabile materials, by filtration through 0.22-μm filters (Millipore).

2.1. HeLa Cell Suspension Culture

1. Medium: Joklik’s modification of Eagle’s minimal essential medium for suspen-sion culture, or equivalent (ICN Pharmaceuticals, Costa Mesa, CA, USA; cat. no.

Mammalian Extracts for In Vitro Splicing 311

10-323-24). This powdered medium already contains all required additions except

serum. A 5× stock is prepared by dissolving the powder in Milli Q water and issterilized by filtration. The working growth medium is prepared by dilution with

filter sterilized water and addition of sterile calf serum (Gibco-BRL, Grand

Island, NY, USA) to 50 mL/L. The pH should be approx 7.0.2. HeLa cells: Use a strain adapted to growth in suspension culture, e.g., the S-3

strain (available from ATCC, Manassas, VA, USA).

2.2. Preparation of Extracts

Reagents 1 and 2 are stock solutions that should be stored at –20°C andadded to reagents 3–6 immediately prior to use.

1. 1 M Dithiothreitol (DTT).2. 20 mg/mL (115 mM) Phenylmethanesulfonyl fluoride (PMSF) dissolved in ethanol.3. Phosphate-buffered saline (PBS) solution: 137 mM NaCl, 2.7 mM KCl, 8 mM

Na2HPO4, 1.5 mM KH2PO4, 0.5 mM MgCl2.4. Buffer A: 10 mM HEPES-KOH, pH 8.0, 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT.5. Buffer B: 0.3 M HEPES-KOH, pH 8.0, 1.4 M KCl, 30 mM MgCl2.6. Buffer C: 20 mM HEPES-KOH, pH 8.0, 0.6 M KCl, 1.5 mM MgCl2, 0.2 mM

EDTA, 25% (v/v) glycerol, 0.5 mM PMSF, 1 mM DTT.7. Buffer D: 20 mM HEPES-KOH, pH 8.0, 100 mM KCl, 0.2 mM EDTA, 20% (v/v)

glycerol, 0.5 mM PMSF, 1 mM DTT.8. 40-mL Dounce glass homogenizer with loose-fitting pestle (Kontes, Vineland,

NJ, USA; 0.003–0.006 in. clearance).9. Dialysis tubing with 12–14,000 nominal mol wt cutoff (Spectrum, Houston, TX, USA).

3. Methods3.1. Suspension Culture of HeLa Cells

The cells are grown in spinner flasks at 37°C. The cell density should bemaintained daily between 2 × 105 and 5 × 105. The doubling time is approx 24h (see Note 1). It is important to use magnetic stirrers that do not generate heatat the surface, or to avoid direct contact between the stirring plate and the bot-tom of the flask. The cell density can be checked with a hemocytometer orCoulter counter.

3.2. Preparation of HeLa Cell Nuclear Extract1. Harvest the cells at the logarithmic growth stage (4–6 × 105 cells/mL, do not

exceed 1 × 106 cells/mL) by low-speed centrifugation at ~1800g (e.g., Sorvall H-6000A rotor, 2500 rpm, 10 min).

2. Wash the cells by gently resuspending the pellet in ice-cold PBS, centrifuge again,and measure the packed cell volume (PCV). All subsequent steps should be carriedout on ice or in the cold room, and all centrifugation steps are carried out at 4°C.

312 Mayeda and Krainer

3. Gently resuspend the cell pellet in 5× PCVs of buffer A and keep on ice for 10min to swell the cells in the hypotonic buffer.

4. Pellet the cells by centrifugation at ~1,800g (e.g., Sorvall H6000-A, 2500 rpm,10 min) and resuspend in 2× PCVs of buffer A. The cell pellet approximatelydoubles in volume due to swelling; use the original PCV in the calculation.

5. Homogenize with approx 10 strokes in a Dounce glass homogenizer. The num-ber of strokes may vary depending on the homogenizer but is fairly consistent ifthe same set is used (see Note 2). The extent of cell lysis can be measured on ahemocytometer using trypan blue staining. Aim for ~90% lysis, but do not usemore strokes than necessary. Note that the HeLa nuclei are very large and notmuch smaller than the intact cells.

6. Transfer the lysate into high-speed centrifuge tubes and centrifuge first at lowspeed, using appropriate adapters (~1200g, e.g., Sorvall H6000-A, 2000 rpm, 10min); remove the supernatant carefully into a graduated cylinder with a pipet. (Thesupernatant is further processed to prepare the S100 extract, see Subheading 3.3.)

7. Centrifuge the pellet at higher speed in the same tubes (~33,000g, e.g., SorvallSS-34, 16,500 rpm, 20 min); remove the supernatant carefully with a pipet anddiscard it.

8. The packed nuclei volume (PNV) is usually 9–15 mL for a 12-L culture of HeLacells. Add 2 mL of buffer C and dislodge the pellet by swirling, but do not dis-perse the pellet. Transfer the pellet and liquid into a graduated 50-mL disposabletube (Corning Labware, Corning, NY, USA). Estimate the PNV by subtracting 2mL from the measured volume, and add more buffer C (which contains 0.6 MKCl) to obtain a final KCl concentration of 0.24 M. The acceptable range is 0.20–0.25 M KCl (see Note 3).

9. Transfer the pellet and buffer (do not resuspend yet, or the solution will becomesticky and difficult to transfer without losses) into a clean 40-mL glass Dounce.Suspend by several strokes with a loose-fitting pestle. A total of 10 strokes shouldbe sufficient to make a homogeneous suspension.

10. Transfer the homogenate into one or more screw-capped high-speed centrifugetubes and mix gently by rocking for 30–45 min.

11. Pellet the salt-washed nuclei by high-speed centrifugation (~33,000g, e.g.,Sorvall SS-34, 16,500 rpm, 30 min) and carefully transfer the supernatant into adisposable tube, avoiding contamination with material from the pellet.

12. Dialyze the supernatant twice against buffer D (usually 1–2 L). One of the dialy-sis steps is done overnight and the other for at least 4 h.

13. Remove the cloudy precipitate formed during dialysis by high-speed centrifuga-tion (~33,000g, e.g., Sorvall SS-34, 16,500 rpm, 20 min) and carefully withdrawthe supernatant, which is the nuclear extract, into a disposable tube.

14. Aliquot the extract into microcentrifuge tubes, in 0.1–1-mL portions, as desired.Quick freeze by dropping the tubes into liquid nitrogen, and store at –70°C orlower. The usual yield of nuclear extract, starting from a 12-L spinner culture, is8–10 mL with a total protein concentration of 10–15 mg/mL. The extract remainsactive for several years.

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3.3. Preparation of HeLa Cell Cytosolic S100 Extract

While waiting for one of the centrifugation steps in Subheading 3.2.,the supernatant obtained in Subheading 3.2.6. can be processed further toobtain an S100 extract, which contains cytosolic components as well asmany, but not all, nuclear components, which are extracted in the hypo-tonic buffer A.

1. Record the volume of supernatant, add 0.11 vol of buffer B, and mix gently.2. Spin in an ultracentrifuge at ~100,000g (38,000 rpm, Beckman 60Ti rotor or

36,500 rpm, Beckman 45Ti rotor) for 1 h.3. Transfer the supernatant into a disposable tube.4. Dialyze twice against 2 L of buffer D, once overnight and once for at least 4 h.

The volume of extract is reduced considerably during dialysis because of theglycerol in buffer D, thus resulting in a concentrated extract.

5. Remove insoluble material by high-speed centrifugation (~33,000g, e.g., SorvallSS-34 rotor, 16,500 rpm) for 20 min.

6. Aliquot the extract into microcentrifuge tubes, in 0.1–1-mL portions, as desired.Quick freeze and store as described in Subheading 3.2., step 14. The usual yieldof cytosolic S100 extract, starting from a 12-L spinner culture, is 40–55 mL witha total protein concentration of 10–15 mg/mL. The extract remains active forseveral years (see Note 4).

4. Notes

1. The use of healthy cells under optimal growth conditions is essential. Slowlydividing cells (longer than 24 h doubling time) or overgrown cells should not beused. Cultures that show a lot of dead cells and debris also fail to yield goodextracts, and reflect problems with the medium, serum batch, and/or mechanicalinjury due to the spinner flask setup.

2. The cell lysis process is also critical. Mild homogenization keeps the isolatednuclei intact and prevents excessive leakage of splicing factors. Avoid using avery tight-fitting pestle and limit the number of strokes to prevent excessive dam-age to nuclei.

3. The salt extraction of nuclei in buffer C is also a critical step. The final KClconcentration should be 0.20–0.25 M, and the buffer should be mixed rapidly toprevent transient localized exposure to higher salt concentration. Lower salt con-centrations fail to extract splicing factors efficiently, whereas higher salt concen-trations disrupt chromatin, resulting in a substantial increase in viscosity, and inextraction of inhibitory components. The final salt concentration can be checkedby measuring the conductivity, if desired.

4. The activity of S100 extracts is not as reproducible as that of nuclear extracts,and some batches may fail to be complemented efficiently by SR proteins. It maybe necessary to prepare and test more than one batch of S100 extract for particu-lar purposes.

314 Mayeda and Krainer

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