Rddx5

The RNA pull down assay was carried out using Dynabeads™ MyOne™ Streptavidin C1 (65,001, Thermo-Fisher) blocked with 2% UltraPure™ BSA (Ambion). The following reagents: 2 nmol of 5′-biotynylated and 3′-Cyanine 5 labelled RNA (Sigma Aldrich), 100 nM rMBNL1 (H00004154-P02, Abnova) and 400 nM rDDX5 (TP300371, OriGene) or 400 nM BSA were incubated in PBST buffer (137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, and 2 mM KH₂PO₄, 0.02% Tween-20, pH 8.0) with 10U RNasin^® (Promega) in a total volume of 20 μL for 15 min at 37 °C. The reaction was combined with the beads and incubated for 15 min at RT followed by a collection of fraction #2 of unbound proteins and three rounds of washing steps with PBST. The beads were transferred to a new tube and elution of RNA–protein complexes (fraction #1) was performed by adding 4 × Bolt™ LDS sample buffer and 10 × reducing agent (Thermo Fisher). All samples were heated to 95 °C for 5 min and separated on a gradient Bolt™ 4–12% Bis–Tris Plus gel according to the manufacturer’s protocol. Immunoblotting was performed as above mentioned for MBNL1 antibody.

The electrophoretic mobility shift assay (EMSA) was carried out by incubation of 15 nM of 5′-biotynylated and 3′-Cyanine 5 labelled RNAs (Sigma Aldrich) with the indicated concentrations of recombinant rMBNL1 (H00004154-P02, Abnova) and rDDX5 (TP300371, OriGene). The reactions were performed in a volume of 10 µl in buffer A (50 mM NaCl, 50 mM KCl, 50 mM Tris–HCl, 1 mM MgCl₂) at 37 °C for 15 min. The samples were mixed with loading buffer (30% glycerol) and run on a non-denaturing 6% PA gel. Gels were scanned using Amersham Typhoon RGB Biomolecular Imager. Cyanine 5 fluorescence was excited by 635 nm and detected using a Cy5 filter. RNA-DDX5 interaction was calculated based on the signal of free RNA which was extrapolated into RNA–protein complexes. A dissociation constant, K_d, was calculated using the equation: one site specific binding Y = B_max*X/(K_d + X) with B_max = 100, in Graph Pad program.