Special methods

3.2.3.1 Cell culture

HeLa S3 cells (Computer Cell Culture Center, Belgium) were grown in suspension in S-MEM media supplemented with 5% (v/v) newborn calf serum, 50 μg/ml penicillin and 100 μg/ml streptomycin to a density of 2.5-5x 10⁵ cells/ml. Cultivation and harvesting of the cells was essentially performed as previously described in (Kastner 1998).

38 3.2.3.2 Preparation of splicing active HeLa nuclear extract

Splicing active nuclear extract was prepared from HeLa cells as described in (Dignam, Lebovitz et al.

1983). Six to eight liters of HeLa cells were grown to a density of 2.5-5 x 10⁵ cells/ml (Kastner 1998).

The cells were pelleted by centrifugation in a Megafuge 1.0R (Heraeus) for 10 min at 2000 rpm. The supernatant was discarded and the cells were washed three times with ice-cold 1x PBS, pH 7.4. Then, the cell pellet was resuspended in 1.25 volumes of 1x MC buffer supplemented with two complete EDTA-free protease inhibitor cocktail tablets per 50 ml of buffer. After an incubation of 5 min on ice, the cells were lysed with 18 strokes of a Dounce homogenizer at 4 °C. The mixture was transferred to 50 ml tubes and the nuclei were pelleted by centrifugation in a FiberLite F14-14 × 50 rotor for 5 min at 13000 g. Afterwards the supernatant was discarded and nuclei were treated in one of the following ways:

 According to the general procedure, the nuclei were resuspended in 1.3 volumes of 1x Roeder C buffer containing 0.5 mM DTE and 0.5 mM PMSF. For lysis of the nuclei the suspension was again subjected to 20 strokes of a Dounce homogenizer at 4 °C. The lysate was stirred for 40 min at 4 °C, followed by centrifugation in a FiberLite F14-14 × 50 rotor at 16000 rpm for 30 min to pellet particulate material. The supernatant was recovered and dialyzed against 50 volumes of 1x Roeder D buffer at 4 °C for 5 h with one change of buffer after 2.5 h. The dialyzed nuclear extract was recovered and centrifuged in a FiberLite F14-14 × 50 rotor at 10000 rpm for 10 min at 4 °C. The supernatant was aliquoted, frozen in liquid nitrogen and stored at -80 °C.

 According to the optimized for cryo-EM procedure, the nuclei were resuspended in 1.3 volumes of 1x Roeder D or 1x Roeder C buffer and homogenized with 20 strokes of a Dounce homogenizer at 4 °C. The lysate was stirred for 40 min at 4 °C, followed by centrifugation in a FiberLite F14-14 × 50 rotor at 16000 rpm for 30 min to pellet particulate material, if not stated otherwise. The supernatant was immediately used in an in vitro splicing reaction.

3.2.3.3 In vitro splicing reactions

A typical splicing reaction was carried out in the presence of 10 nM pre-mRNA and 40 % (v/v) HeLa nuclear extract in a buffer containing 20 mM HEPES-KOH, pH 7.9, 3 mM MgCl2, 65 mM KCl, 2 mM ATP and 20 mM creatine phosphate. Alternatively, splicing was carried out in the presence of 5 nM pre-mRNA and 20% (v/v) HeLa nuclear extract (optimized procedure) in a splicing mixture containing 20 mM HEPES-KOH, pH 7.9, 3 mM MgCl2, 65 mM KCl or 50 mM NaCl, 2 mM ATP and 20 mM creatine phosphate. Spliceosomal complexes were allowed to form by incubating at 30 °C. B^act complexes were assembled on PM5-10 pre-mRNA by incubating at 30 °C for 0-3 h. A 30-fold molar excess of DNA oligonucleotides complementray to nucleotides -6 to -18 and -12 to -24 relative to the 5’ss (M6 and M12 oligos, respectively) was added and the reaction was incubated at 30°C for an additional 20 min. After splicing, the reactions were chilled on ice.

39 3.2.3.4 Analysis of splicing complexes by native agarose gel-electrophoresis

Spliceosomal complexes A, B and C are formed in sequentially and show decreased mobility on a native agarose gel (Lamond, Konarska et al. 1987, Kent and MacMillan 2002). To resolve spliceosomal complexes, 20 µl splicing reactions were incubated at 30 °C for different time points. Heparin was added to 18 μl aliquots of the splicing reaction to a final concentration of 0.125 mg/ml and the mixtures were incubated for another 1 min at 30°C before addition of 2 μl of 4X agarose loading buffer. The samples were loaded onto a native agarose gel (26 cm x 15 cm) containing 1.5% (w/v) of low melting point agarose (Invitrogen) in 0.5 x TBE buffer. Electrophoresis was performed by applying 100 V for 5 h at room temperature. The gels were dried at 60 °C for 5 h and bands were visualized by exposing to a phosphoimager screen.

3.2.3.5 MS2 affinity-selection of splicing complexes

Spliceosomal complexes were isolated by MS2 affinity-selection as previously described (Bessonov, Anokhina et al. 2010). Pre-mRNA was incubated with a 20-fold molar excess of purified MS2-MBP fusion protein for 30 min at 4 °C prior to splicing. After performing in vitro splicing the reaction was equilibrated with HEPES-KOH, pH 7.9 and then loaded onto a MBPTrap HP column (GE Healthcare) equilibrated with 1x G-150 buffer (20 mM HEPES-KOH, pH 7.9, 1.5 mM MgCl2, 150 mM NaCl). The column was washed with 40 volumes of 1x G-150 buffer and complexes were eluted with 1x G-150 buffer containing 1 mM maltose. Eluted complexes were loaded onto a 36 ml linear 10-30% (v/v) glycerol gradient containing G-150 buffer (20 mM HEPES- KOH, pH 7.9, 1.5 mM MgCl2, 150 mM NaCl), centrifuged at 23000 rpm for 15 h at 4 °C in a Surespin 630 (Thermo Scientific) rotor and gradient fractions were harvested from the bottom. The distribution of ³²P-labeled pre-mRNA across the gradient was determined by Cherenkov counting. Fractions were analysed on denaturing 4-12%

NuPAGE gels (Invitrogen), followed by autoradiography or fixation and staining (3.2.1.9 and 3.2.2.3).

Peak fractions containing the activated spliceosomal complexes were pooled, concentrated by centrifugation with an Amicon Ultra concentration unit (Merck), diluted to decrease the glycerol concentration and reloaded on a second gradient with or without glutaraldehyde as fixative (for EM analysis and for biochemical sample validation, respectively). The RNA and protein compositions of purified complexes were analysed on denaturing 4-12% NuPAGE gels (Invitrogen).

3.2.3.6 ProteoPlex

A ProteoPlex screen, to identify the optimal buffer conditions for spliceosomal complex stability, was performed in 20 μl reactions in a 96-well plate (Bio-Rad) (Chari, Haselbach et al. 2015). Each individual 20 μl reaction was composed of 16 μl of the isolated complex at a concentration of 0.1 - 1 μM, 2 μl of SYPRO orange (Life Technologies) at a final concentration of 1x and 2 μL of the buffer/chemical under investigation. Buffer substance screens at various pH values were performed with a custom made buffer screen, where individual stock solutions were 1 M (Table 2). Each plate

40 contained control wells, which contained the purification buffer of the spliceosome complex and wells without complex. Unfolding transitions were recorded in a CFX connect real-time PCR machine (Bio-Rad). A reader program was utilized, where the entire plate was equilibrated to 30°C for 2 min followed by fluorescence measurement. Subsequently, the samples were incrementally heated in 1°C steps, equilibrated for 30 s and fluorescence was measured. Finally, the sample was held at 95°C for 2 min, followed by a final fluorescence measurement.

Table 2: Composition of custom made buffer screen

All individual stock solutions were 1 M. Column 12 is intentionally left blank for controls

1 2 3 4 5 6 7 8 9 10 11 12 1) 1M SPG Buffer contains: 0.125M Succinic Acid, 0.5M NaH2PO4, 0.375M Glycine adjusted to the appropriate pH with 10M NaOH.

2) 1M MMT Buffer contains: 0.2M DL-Malic Acid, 0.4M MES monohydrate, 0.4M Tris adjusted to the appropriate pH with either 10M HCl or 10M NaOH 3) 1M PCB Buffer contains: 0.4M Sodium Propionate, 0.2M Sodium Cacodylate trihydrate, 0.4M Bis-Tris Propane adjusted to the appropriate pH with 10M HCl 4) MES(2-(N-morpholino)ethanesulfonicacid)monohydrate

The structure of isolated spliceosomal complexes was analyzed by electron microscopy (EM). Cryo EM experiments were performed in collaboration with Dr. David Haselbach (Department of Structural Dynamics, MPI-BPC). Carbon films and grids for cryo EM were kindly prepared by Frank Würriehausen (Department of Structural Dynamics, MPI-BPC).

3.2.4.1 Preparation of continuous carbon film and holey carbon grids

For continuous carbon films, mica (Plano G250- 1, 25x75 mm) was used as the coating carrier. To obtain a smooth surface that does not have beforehand any direct air contact, the mica was freshly split. The fresh side was indirectly exposed to carbon in an Edwards E12E vacuum coating unit (BOC Edwards, Kirchheim) by conducting electric current through two carbon rods (Ringsdorff Werke GmbH, Bonn).

Low-cost grids with irregular holes were made to evaluate sample quality. Copper grids were covered with self-made triafol film containing holes. Then carbon was directly sputtered onto the triafol film.