In vitro pull-downs on M2-L3 pre-mRNA

The pull-downs were performed in pull-down buffer containing 20 mM HEPES-NaOH (pH 8.0), 50 mM NaCl, 0.5 mM TCEP, and 2 mM Mg(OAc)₂. First, 520-nt MS-L3 pre-mRNA was incubated with MBP-tagged MS2 protein at molar ratio 1:3 for 45 min on ice. Then, 3 µM RBBP6, 1 µM CPSF, or 3 µM RBBP6 + 1 µM CPSF was added and incubated for 1.5 h. The mixture containing RBBP6/CPSF/RBBP6 + CPSF and MBP-MS2-bound L3 pre-mRNA was mixed with amylose beads (NEB E8021) equilibrated in pull-down buffer and incubated rotating for 1.5 h at 4°C. The beads were washed with pull-down buffer. Protein–RNA complexes were eluted in pull-down buffer supplemented with 20 mM maltose (Merck 63418). The eluates were loaded onto a NuPAGE 4%–12% Bis-Tris 1.0-mm mini protein gel. The proteins were transferred onto a nitrocellulose membrane using Trans-Blot Turbo transfer system (Bio-Rad 1704158). StrepII-tagged proteins (RBBP6, symplekin, and WDR33) were detected using streptavidin-HRP conjugate (Merck Millipore 18152) and Amershan ECL detection reagents (Cytiva RPN2106). The blots were visualized using a ChemiDoc XRS+ (Bio-Rad).