Protein analysis methods

For SDS-polyacrylamide gel electrophoresis (SDS-PAGE), protein samples were mixed with 4X SDS loading dye and denatured at 95°C for 10 min. Electrophoresis was carried out using 25 mA/gel in a discontinuous system with different composition and pH of the resolving and stacking gel buffers (Table 2.2). After the run, the gels were stained for 1 h with a Coomassie solution (0.1% Coomassie R-250, 10% acetic acid, 40% methanol) followed by destaining with a solution of 10% acetic acid and 20% methanol to enable direct protein visualization.

Alternatively, the samples were transferred onto a Hybond P 0.45 PVDF blotting membrane (GE Healthcare) in a wet-transfer system. The membrane was first activated in 100%

methanol for 2 min and the transfer was carried out for 75 min at 100 V in western blotting transfer buffer. For experiments involving detection of the G-patch protein SON, which has a molecular weight of more than 260 kDa, the buffer was supplemented with 0.05% SDS and the transfer was done for 16 h at 25 V. After transfer, the membrane was blocked for 1 h at room temperature in 5% milk in TBS with 0.1% Tween-20 (TBS-T), followed by incubation with primary antibodies (Table 2.8) overnight at 4°C. Next, the membrane was washed three times for 10 min each in TBS-T and incubated with HRP-coupled secondary antibodies (Table 2.9) for 1 h at room temperature. After removal of the secondary antibody, washing steps with TBS-T were carried out as before and detection was done by exposure to X-ray films using Immobilon Western Chemiluminescent HRP Substrate (Merck).

2.4.2 Immunoprecipitation (IP) of protein complexes

For each sample, approximately 7x10⁶ cells were plated in a 15 cm dish and the next day, expression of the FLAG-tagged proteins was induced with 1 µg/ml tetracycline. After 24 h, the cells were washed with PBS, harvested with 0.25% Trypsin-EDTA (Thermo Fisher) and centrifuged for 3 min at 1000 g. The cell pellet was resuspended in 1 ml IP buffer (20 mM HEPES-NaOH pH 7.5, 250 mM NaCl, 0.5% Triton X-100, 10% glycerol, 0.5 mM EDTA) supplemented with cOmplete Mini Protease Inhibitor Cocktail (Roche). Cells were lysed by

sonication (3 cycles of 15 sec with 0.3 sec on/0.7 off) using a Branson Digital Sonifier set to 20% amplitude, followed by centrifugation for 10 min at 20,000 g and 4°C to remove the insoluble material. The lysate was added to 30 µl slurry of pre-equilibrated anti-FLAG M2 Magnetic Beads (Sigma-Aldrich) and binding was carried out for 2 h at 4°C in the presence of 50 µg/ml RNase A (Applichem). Afterwards, the beads were washed five times with the IP buffer and bound complexes were eluted with 250 µg/ml FLAG Peptide (Sigma-Aldrich) diluted in IP buffer for 30 min at 4°C. The eluates were precipitated with 20% trichloroacetic acid for 20 min on ice and centrifuged for 20 min at 20,000 g and 4°C. The pellets were then washed with ice-cold acetone and air-dried briefly. For mass spectrometry analysis (2.4.4), the samples were resuspended in 1X NuPAGE LDS Sample Buffer (Thermo Fisher) supplemented with 50 mM DTT and denatured at 70°C for 10 min. For SDS-PAGE and western blotting analysis (2.4.1), the pellets were resuspended first in 100 mM Tris-HCl pH 8.4, mixed with 4X SDS loading dye and boiled at 95°C for 10 min.

To study the interactions between DHX15, NKRF and XRN2 (3.2.4), IPs using antibodies against the FLAG tag or against NKRF were performed as above with some modifications.

The buffer for cell lysis consisted of 20 mM HEPES pH 8.0, 150 mM KCl and 0.5 mM EDTA.

The cells were lysed by sonication and centrifuged to remove cell debris as described above. Afterwards, the lysates were supplemented with 10% glycerol, 1.5 mM MgCl₂ and 0.2% Triton X-100, and incubated with the pre-equilibrated beads for 2 h at 4°C. For IP with an anti-NKRF antibody, Protein G Sepharose beads (GE Healthcare) were prepared beforehand by an overnight incubation at 4°C with 5 µl antibody followed by washing with IP buffer (20 mM HEPES pH 8.0, 150 mM KCl, 10% glycerol, 1.5 mM MgCl₂ and 0.2% Triton X-100). After binding, the beads were washed with the IP buffer and in the case of the anti-NKRF IP, the co-precipitated proteins were eluted at 95°C with 4X SDS loading dye.

For the anti-FLAG IP, the bound proteins were eluted with 250 µg/ml FLAG Peptide (Sigma-Aldrich) and precipitated with 20% trichloroacetic acid as above. The samples were then analyzed by SDS-PAGE and western blotting (2.4.1). In the case of the IP experiments coupled to siRNA-mediated knockdowns, the cells were treated for 96 h with 50 nM siRNAs and expression of the FLAG-tagged proteins was induced 24 h prior to harvesting.

2.4.3 Purification of nucleoli and preparation of nucleolar lysates

HEK293 cell lines expressing FLAG-tagged DHX15 or the FLAG tag only were induced for 24 h with 1 µg/ml tetracycline and nucleoli were isolated based on Chamousset et al., 2010 with a few changes. Cells (4x15 cm plates) were harvested with trypsin, washed twice with ice-cold PBS and lysed for 10 min on ice in 5 ml lysis buffer containing 20 mM Tris-HCl pH 7.4, 10 mM KCl, 3 mM MgCl2, 0.1% NP-40 and 10% glycerol. The released nuclei were

pelleted by centrifugation at 1350 g for 10 min, resuspended in 3 ml of solution S1 (0.25 M sucrose, 10 mM MgCl₂) and layered on top of 3 ml solution S2 (0.35 M sucrose, 0.5 mM MgCl₂). After centrifugation at 1430 g for 5 min, the nuclear pellet was resuspended in 3 ml solution S2 and sonicated six times for 10 sec each at 20% amplitude using a Branson Digital Sonifier. The lysed sample containing intact nucleoli was layered on top of 3 ml solution S3 (0.88 M sucrose, 0.5 mM MgCl₂) and centrifuged at 3000 g for 10 min. The nucleolar pellet was resuspended in 500 µl solution S2 and centrifuged again at 1430 g for 5 min to remove contaminants. The isolated nucleoli were disrupted by incubation for 30 min on ice in 400 µl high-salt buffer (20 mM HEPES pH 8.0, 500 mM KCl, 0.5 mM EDTA, 0.8% Triton X-100, 0.4% CHAPS) supplemented with 16 U TURBO DNase (Thermo Fisher), followed by sonication as described for the IP protocol. The nucleolar lysate was diluted 1:4 with a buffer containing 20 mM HEPES pH 8.0, 2 mM MgCl2 and 13.3% glycerol, and immunoprecipitation with anti-FLAG M2 beads (Sigma-Aldrich) was performed as detailed above.

2.4.4 Liquid chromatography tandem mass spectrometry (LC-MS/MS)

Protein IP samples obtained as described in 2.4.2 were separated on 4-12% NuPAGE Bis-Tris gels (Thermo Fisher) and entire lanes were excised, divided into 12 fragments and digested with trypsin. The samples were analyzed on a mass spectrometer with two technical replicates for each. Proteins were identified with Mascot (Matrix Science) by searching against the UniProt human protein database. The results were further processed with Scaffold (Proteome Software) and proteins containing at least two detected peptides were identified at a false discovery rate of less than 1%. These steps were performed by the Proteomics Service Facility (University Medical Center Göttingen). For further data analysis, the proteins were ranked based on the total spectral counts and the fold change for each protein was calculated relative to the control sample. To enable calculations, a spectral count of 1 was added to proteins that had zero counts. The final results were expressed as the mean of two technical replicates or, when available, two biological replicates.

2.4.5 Separation of (pre)-ribosomal complexes on sucrose gradients

HeLa cells grown to 80% confluency in a 10 cm plate were detached with trypsin, centrifuged for 3 min at 1000 g and resuspended in 0.5 ml of lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 5 mM MgCl₂, 1 mM DTT). The cells were lysed by sonication as described for the IP method (2.4.2) and the lysates were cleared by centrifugation for 10 min at 20,000 g and 4°C. The obtained whole cell extracts (400 µl) were layered on top of 6 ml sucrose gradients (10-45%), which were prepared using a Gradient Master (BioComp). The separation of complexes was carried out for 16 h at 23,500 rpm and 4°C

in a SW40 Ti rotor (Beckman Coulter). After centrifugation, the gradient was fractionated into 530 µl samples that were precipitated with 20% trichloroacetic acid (2.4.2) and prepared for SDS-PAGE and western blotting analysis (2.4.1). When indicated, cells were treated with siRNAs for 96 h prior to the experiment.

2.5 RNA analysis methods