mRNA Decay Factor AUF1 Binds the Internal Ribosomal Entry Site of Enterovirus 71 and Inhibits Virus Replication
Figure 2
AUF1 interacts with the EV71 5′UTR.
(A) RNA-protein pull-down experiments were performed to examine the interaction between AUF1 and the EV71 5′UTR. The EV71 5′UTR was synthesized in vitro in the presence of biotin-UTP. Biotinylated RNA was incubated with SF268 cell lysate (200 µg proteins) for 15 min at 30°C. Unlabeled RNA was added to parallel reactions as a negative control. Streptavidin-linked beads were used to pull-down biotin-labeled EV71 5′UTR and its associated cellular proteins. The beads were washed and then resuspended in SDS-PAGE loading buffer to dissociate proteins from RNA. Samples were boiled and analyzed by Western blot using anti-AUF1 antibody. (B) Secondary structures within the 5′UTR were predicted by MFold. Numbers below each stem-loop indicate 5′UTR nucleotides encompassing the respective stem-loop. RNA substrates used for protein-RNA pull-down experiments contained the indicated stem-loop and the flanking region immediately 5′ to it. (C) Identification of AUF1 interaction site(s) within the EV71 5′UTR. Biotinylated RNAs containing the indicated stem-loops were synthesized; control RNAs lacked biotin. SL-VI extends to nt 745, so it contains linker sequence between the IRES and the AUG. RNA-protein pull-downs and Western blot analysis were carried out as described for panel (A). The blot was stripped and re-probed with anti-FBP1 antibody as a positive control for RNA-binding activity.