Rapid experiments and controls: To identify CFB2 specifically interacting with VSG2 mRNA, we performed captures of lambda-GFP-SBP proteins in cells expressing boxB-tagged VSG2 mRNA and lambdaN-GFP-SBP. Cells lacking either component were used as controls.

Cells at mid-log phase were collected, resuspended in PBS, and then cross-linked with 0.01% formaldehyde at room temperature for 10 min with slow shaking. The cross-linking reaction was terminated by adding 1 M glycine-NaOH buffer (pH 8.0) to a final concentration of 0.125 M and additional shaking for 2 min. The cells were then washed once with ice-cold PBS buffer and the pellet was flash-frozen in liquid nitrogen, and stored at −80°C. Cell pellets were thawed by addition of ice-cold lysis buffer (25 mM Tris-HCl, pH 7.5, 175 mM KCl, 0.5% NP40, 1 mM DTT, and 120 U RNAse inhibitor) (New England Biolabs) supplemented with EDTA-free protease inhibitors (Roche). Samples were then subjected to three cycles of sonication (10 pulses of 0.5 s) followed by 1 min rest between cycles at 4°C (Branson Ultrasonics Sonifier S-250). The extract was then supplemented with 1x DNAse salt solution and 100 U DNAse I (Roche) and incubated for 30 min at 4°C on rotator. Samples were returned to ice and the reaction was immediately terminated by the addition of 10 mM EDTA and 10 mM EGTA. The extract was finally clarified by centrifugation for 20 min at 15,000 × g, the supernatant removed to a new microcentrifuge tube and protein concentration determined using the Bradford assay (Bio-Rad).

RNA-binding protein purification and identification (RaPID) was performed essentially as described by Slobodin and Gerst, 2010 with a few modifications. In order to block endogenous biotinylated moieties, the protein aliquot taken for pull-down was incubated with 10 mg of free avidin (Sigma) per 1 mg of protein input at 4°C for 1 hr with constant rotation. In parallel, streptavidin magnetic beads (New England Biolabs) were washed five times with 1 ml of lysis buffer supplemented with EDTA 5 mM. Pull-down was then performed by adding the indicated amount (see figure legends) of avidin-blocked total cell lysate to the beads, followed by incubation at 4°C for 3 hr with constant rotation. Beads with captured mRNPs were washed three times with lysis buffer and three times with washing buffer (25 mM Tris-HCl, pH 7.5, 300 mM KCl, 0.5% NP40, 1 mM DTT, 5 mM EDTA, and 40 U/ml RNAse inhibitor) (New England Biolabs) at 4°C for 5 min to remove non-specifically associated proteins. For elution of the cross-linked RNP complexes, 250 µl of washing buffer supplemented with 6 mM free biotin (Sigma) was added to the beads, followed by 1 hr of incubation at 4°C with rotation. To reverse the cross-link for RNA analysis, samples were incubated at 70°C for 1 hr with cross-link reversal buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 10 mM DTT, and 1% SDS).

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