RNA structural changes mediated by UPF2L. (A) A schematic illustrating potential UPF2-mediated RNA annealing or RNA unfolding activities. (B) EMSAs using HEX-labeled ss-RNA oligonucleotides of 13 and and 24 nt, HEX_ds-RNA, and HEX_hp-RNA as controls. Incubation of HEX_hp-RNA with UPF2L or MIF4G-D3 produces remodeled RNA bands running at similar height as HEX-labeled 13 nt ss-RNA and 24 nt ss-RNA/ds-RNA, respectively (red arrows). (C) Molecular beacon hp-RNA (HEX_hp-RNA_BHQ) construct labeled 5′ with HEX dye and 3′ with a blackhole quencher 1 (BHQ-1). (D) EMSAs using same controls as in B, 250 nM molecular beacon, and MIF4G-D3 incubated with HEX_hp-RNA or molecular beacon. The remodeled RNA band is highlighted (red arrow). (E) Normalized fluorescence signal of HEX_hp-RNA (light gray bars) and molecular beacon (dark-gray bars) in the presence of UPF2L compared to RNA only (no UPF2L). Experiments were performed in duplicate. (F) Changes in hp-RNA and ss-RNA tertiary structure upon addition of 10 μM UPF2L monitored by CD spectroscopy. (Black dashed line) hp-RNA, (blue dashed line) ss-RNA, (black line) hp-RNA spectrum subtracted by UPF2L spectrum, (blue line) ss-RNA spectrum subtracted by UPF2L spectrum. Peak wavelengths are indicated. For reference: ss poly-uridine peaks at 272 nm.
