3 Materials and methods
3.1 Materials .1 Chemicals
3.2.3 Cell culture, nuclear extract, purification of spliceosomal complexes
3.2.3.1 Metabolic Labeling of HeLa S3 cells (SILAC labeling)
HeLa S3 cells were grown in custom made DMEM medium containing normal (light) or stable isotope labeled (medium and heavy) L-arginine and L-lysine (see table 3.2). Cells were grown for at least six passages at smaller volumes and then expanded to 2.0 l in spinner
flasks (0.5-1.0 × 106 cells/ml). The cells were transferred to a 2.5 l fermenter and grown under standard conditions (2.5-5.0 × 106 cells/ml).
SILAC medium:
500 ml DMEM w/o Arginine, w/o Lysine 50 ml dialyzed FBS
5ml 100 × Penicillin/Streptomycin 50 mg/l L-Arginine
50 mg/l L-Lysine
Table 3.2: Isotope composition of amino acids used for different SILAC media. Due to the number of incorporated 13C, 15N or 2D atoms a mass difference between the light amino acid and the stable isotope labeled amino acid is obtained.
L-Arginine ∆m L-Lysine ∆m
duplex SILAC light - 0 - 0
heavy 13C6,15N4 +10 Da 13C6 +6 Da
triple SILAC
light - 0 - 0
medium 13C6 +6 Da 2D4 +4 Da
heavy 13C6,15N4 +10 Da 13C6,15N2 +8 Da
3.2.3.2 Preparation of splicing-active HeLa cell nuclear extract
Cells from a fermenter were used to prepare nuclear extract according to Dignam et al., 1983. Briefly, cells were harvested by centrifugation for 5 min at 2000 rpm (Cryofuge 6000i, Hereaus) and washed with ice-cold PBS. The cells were resuspended in 1.25 vol. MC buffer supplemented with 1/500 vol. 0.25 M DTE and 1/100 vol. EDTA free protease inhibitor cocktail, incubated on ice for 5 min and lyzed in a Dounce homogenizer (18 strokes) at 4 °C.
Nuclei were pelleted by centrifugation for 5 min at 18,000 × g (Sorvall SS34 rotor) and dounced (20 strokes) at 4 °C in 1.3 vol. of Roeder C buffer supplemented with 1/500 vol.
0.25 M DTE and 1/200 vol. 0.1 M PMSF. The mixture was stirred for 40 min at 4 °C followed by centrifugation for 30 min at 16,000 rpm (Sorvall SS34 rotor). The supernatant was dialyzed three times for 2 h against 50 vol. of Roeder D buffer. The dialysate was centrifuged for 2 min at 9000 × g (Sorvall SA600 rotor) and aliquots of the supernatant were frozen in liquid nitrogen, stored at -80 °C and tested for splicing activity.
3.2.3.3 In vitro splicing
In vitro splicing was performed using m7G(5’)ppp(5’)G-capped, 32P-labeled and MS2-tagged pre-mRNA. Splicing reactions contained 50 % (v/v) HeLa nuclear extracts, 65 mM KCl, 3 mM
MgCl2, 2 mM ATP, 20 mM creatine phoshphate and 30 nM pre-mRNA and were incubated for different time intervals at 30 °C. Assembled spliceosomal complexes were analyzed by native gel electrophoresis. RNA was recovered by PCI extraction and analyzed by denaturing gel electrophoresis.
3.2.3.4 Purification of human Prp19/CDC5L complex
hPrp19/CDC5L complex was isolated from AD-002-FLAG/HA-tagged HeLa nuclear extract.
Briefly, HeLa S3 cell lines stably expressing FLAG/HA-tagged human AD-002 were generated according to general protocols. Nuclear extract from these cells was prepared according to Dignam et al., 1983. The hPrp19/CDC5L complex was affinity-purified from nuclear extract by using anti-FLAG-M2-Agarose beads and FLAG peptide. Isolated complexes were purified on a 5–20 % glycerol gradient. Protein tagging, stable cell line construction and hPrp19/CDC5L complex purification were performed by M. Grote (Dept. for Cellular Biochemistry; Grote et al., 2010).
3.2.3.5 Purification of human tri-snRNP (U4/U6.U5)
Total snRNPs were isolated from HeLa cell nuclear extract by immunoaffinity chromatography using monoclonal anti-m3G-specific H20-antibody (Bach et al., 1990;
Bringmann et al., 1983). Total snRNPs were separated on 10-30 % glycerol gradients. 1.5 ml fractions were collected yielding the single snRNP components (12S U1 snRNP, 17S U2 snRNP, 20S U5 snRNP and 25S U4/U6.U5 snRNP) in different fractions. This was always verified by analyzing RNA and protein composition after PCI extraction by gel electrophoresis. All fractions were routinely frozen in liquid nitrogen and peak fractions containing only the U4/U6.U5 snRNP were used for the experiments described herein.
Isolation of total snRNPs was performed on a routine basis by P. Kempkes and H. Kohansal (Dept. for Cellular Biochemistry) and purification of U4/U6.U5 snRNP was performed by M.
Raabe (Bioanalytical Mass Spectrometry Group).
3.2.3.6 Overexpression of MS2-MBP fusion protein
The MS2-MBP fusion protein was used to affinity purify spliceosomal complexes assembled on MS2-tagged pre-mRNA. To overexpress the MS2-MBP protein, 50 µl chemical competent BL21 cells were transfected with 50 ng MS2-MBP fusion protein plasmid DNA by
electroporation and subsequently transferred to LB medium containing ampicillin and chloramphenicol. After incubation at 37 °C overnight, cells were diluted with LB medium, grown at 37 °C until they reached an OD of 0.7 and induced with IPTG. When the cells reached an OD of 1.6 they were harvested by centrifugation for 30 min at 5000 rpm (SL6000). Cells were washed with PBS and the cell pellet was resuspended in Tris-HCl pH 7.5 supplemented with 200 mM NaCl and protease inhibitor cocktail. The cells were solubilized by sonication and centrifuged for 15 min at 4000 rpm (Megafuge) at 4 °C. The supernatant was incubated overnight with amylose beads and the MS2-MBP fusion protein was eluted from the beads with 15 mM maltose in Na-P buffer. The protein was further purified on Heparin beads using 20 mM Hepes pH 7.9, 100 mM KCl, 15 % (v/v) glycerol, 0.5 M DTT and 0.2 M PMSF as elution buffer. The protein concentration of the collected fractions was determined by the Bradford assay (section 3.2.2.1) and the fractions containing MS2-MBP fusion protein were identified by PAGE.
3.2.3.7 Purification of human B and C complexes
Spliceosomal B and C complexes were purified as described before (Bessonov et al., 2008;
Deckert et al., 2006). Briefly, 32P-labeled pre-mRNA was incubated with a 20-fold molar excess of MS2-MBP fusion protein and used without further purification in a standard splicing reaction. This contained 10 nM (B complex) or 30 nM (C complex) pre-mRNA and was incubated at 30 °C for 6 min (B) or 180 min (C). A 30-fold molar excess of M6 and M12 DNA oligonucleotides was added and the reaction was incubated for further 2 min (B) or 20 min (C). Assembled complexes were separated on 10-30 % glycerol gradients and 40-45 S gradient fractions were affinity purified on amylose beads. Spliceosomal B and C complexes were purified in collaboration with S. Bessonov and J. Deckert (Dept. for Cellular Biochemistry).
3.2.3.8 Investigating the protein composition during the spliceosomal assembly
To investigate the time dependent protein assembly of spliceosomal proteins, two different approaches were followed: (i) The time dependent assembly of proteins on different mRNAs, and (ii) the direct comparison of the time dependent protein assembly on PM5 pre-mRNA and on a splicing-inactive pre-pre-mRNA.
To analyze the time dependent assembly of proteins on the tagged pre-mRNA during 30 min,
32P-labeled pre-mRNA was first incubated with a 20-fold molar excess of MS2-MBP fusion protein. Several standard splicing reactions, each containing 20 pmol pre-mRNA, were then
assembled and incubated at 30 °C for different time intervals using triple (i.e. light, medium and heavy) SILAC extracts (as outlined in detail in Table 3.3). Assembled complexes were affinity purified on amylose beads and the concentration of samples from different time points to be compared (Table 3.3) was determined according to the radioactivity of the used pre-mRNA. Samples from different time points to be compared were pooled in equal molar amounts and proteins were precipitated with ethanol.
To directly compare the protein assembly on PM5 mRNA and on a splicing-inactive pre-mRNA, 32P-labeled pre-mRNA was incubated with a 20-fold molar excess of MS2-MBP fusion protein. Standard splicing reactions were incubated at 30 °C for different time intervals during 30 min using duplex SILAC extracts (Table 3.4). Assembled complexes from different time points were affinity purified on amylose beads and the concentration was determined according to the radioactivity of the pre-mRNAs. Samples from the same time point but assembled on different pre-mRNAs were pooled in equal molar amounts and proteins were subsequently precipitated with ethanol.
Precipitated proteins were separated by PAGE, hydrolyzed with trypsin as described in section 3.2.4.1 except that 100 mM ammonium bicarbonate buffer was used instead of 50 mM TEAB and analyzed by LC-MS/MS.
Table 3.3: Used SILAC nuclear extracts for investigation of the protein assembly on pre-mRNAs during 30 min. The concentration of samples from different time points to be compared was determined according to the radioactivity contained within the used pre-mRNAs and samples were then pooled in equal molar amounts (Pool 1, Pool 2, Pool 3).
time 0’ 2’ 5’ 0’ 10’ 15’ 0’ 20’ 30’
NE light medium heavy light medium heavy light medium heavy
Pool 1 Pool 2 Pool 2
Table 3.4: Used SILAC nuclear extracts for direct comparison of the protein assembly on PM5 pre-mRNA and on a splicing-inactive pre-mRNA during 30 min. The concentration of samples from different time points to be compared was determined according to the radioactivity contained within the used pre-mRNAs and samples were then pooled in equal molar amounts (Pools 1 - 8).
time PM5 pre-mRNA Splicing-inactive pre-mRNA
0’ heavy NE light NE → Pool 1
2’ heavy NE light NE → Pool 2
5’ heavy NE light NE → Pool 3
10’ heavy NE light NE → Pool 4
15’ heavy NE light NE → Pool 5
20’ heavy NE light NE → Pool 6
25’ heavy NE light NE → Pool 7
30’ heavy NE light NE → Pool 8
3.2.4 Mass spectrometry methods