Purification of Spliceosomal C and C* Complexes

HeLa S3 cells were obtained from the Helmholtz Zentrum für Infektionsforschung, Braunschweig, and tested negative for mycoplasma. HeLa nuclear extracts were prepared according to Dignam et al. (46 (link)) and dialyzed twice for 2.5 hours against 50 volumes of Roeder D buffer [20 mM Hepes-KOH (pH 7.9), 0.2 mM EDTA (pH 8.0), 1.5 mM MgCl₂, 100 mM KCl, 10% (v/v) glycerol, 0.5 mM dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonyl fluoride]. For purification of spliceosomal C complexes assembled on MINX or PM5 pre-mRNA, dialyzed nuclear extracts were preincubated for 10 min at 30°C with 1 μM of dominant-negative mutant of PRP16 protein (dnPRP16) (8 (link)). For both C and C* purifications, m7G(5′)ppp(5′)G-capped PM5, MINX, or MINX GG pre-mRNAs (5 nM) were preincubated with 20 nM MS2-MBP fusion protein for 30 min on ice before addition to the splicing reaction. Splicing reactions were carried out at 30°C with 40% (v/v) nuclear extract in splicing buffer [3 mM MgCl₂, 65 mM KCl, 20 mM Hepes-KOH (pH 7.9), 2 mM ATP, and 20 mM creatine phosphate]. Splicing was carried out for 1.5 hours for purification of C complexes assembled on MINX and PM5 pre-mRNA, 3 hours for C* complexes assembled on PM5, and 1 hour for C* complexes assembled on MINX GG pre-mRNA. Splicing reactions were then chilled on ice, centrifuged for 30 min at 12,000 rpm to remove aggregates, and loaded onto an MBP Trap HP column (GE Healthcare) after the addition of 100 mM NaCl. The column was washed with G-150 buffer [20 mM Hepes-KOH (pH 7.9), 1.5 mM MgCl₂, and 150 mM NaCl], and the complexes were eluted with G-150 buffer containing 1 mM maltose. Eluted complexes were loaded onto a 36-ml linear 5 to 20% (w/v) sucrose gradient prepared in G-150 buffer and centrifuged at 27,200 rpm for 9 hours at 4°C in a Surespin 630 (Thermo Fisher Scientific) rotor, and fractions were harvested from the bottom of the gradient. RNA from peak gradient fractions was separated on denaturing 4 to 12% NuPAGE gels (Life Technologies) and visualized by staining with SYBER Gold (Thermo Fischer Scientific).