RNA binding of UPF2 variants tested by electromobility shift assays. (A) Schematic showing UPF2L (residues 121–1227) and UPF2 MIF4G domain 3 (D3, 761–1054) constructs. (B) Hexachlorofluorescein (HEX)-labeled hairpin RNA (hp-RNA) construct used in this study (prediction based on RNAfold web server) (Lorenz et al. 2011). (C,D) Electromobility shift assays (EMSAs) using HEX_hp-RNA (250 nM) and increasing concentrations of MIF4G-D3 and UPF2L. Remodeled RNA bands appearing in the presence of UPF2 variants are highlighted (red). (E) UPF2 constructs used for EMSAs shown in F. (F) EMSAs using no RNA (−), HEX-labeled ss-RNA (ss), and HEX_hp-RNA (hp) (250 nM each) and UPF2 constructs. Corresponding Coomassie-stained gel sections are shown in Supplemental Figure S1C. Remodeled RNA-containing bands are highlighted with asterisks.
