Identifying the in situ sites of IRP-1 interaction with the TFRC mRNA. (A) The RNA-CLIP strategy used to identify sites of IRP-1 interaction. The immunoprecipitation step enriched for IRP-1 and associated crosslinked RNAs relative to unrelated RNA binding proteins (RBP). (B) The anti-IRP-1 polyclonal antibody exploited for the RNA-CLIP immunoprecipitates IRP-1–IRE complexes but not IRP-2–IRE complexes. One picomol of each recombinant IRP was UV crosslinked to a radiolabeled 43-nt IRE transcript for direct loading on the gel (pre) or incubation with either anti-IRP-1 polyclonal or control rabbit IgG antibody that had been bound to magnetic beads. The nonbound complexes in the supernatant (sup) and the complexes eluted from the beads, by heating in gel-loading buffer after two washes in lysis buffer, are indicated. Gels are representative of three sets of reactions. (C) IRP-1 interacting sites identified by the RNA-CLIP. Upper case letters represent the most abundant CLIP sequences and lower case the longest 3′ and 5′ extensions present within the nine libraries. (D) The TFRC mRNA level in HUVECs is iron-responsive. Cells were treated with either DFO or FAC for 14 h prior to the isolation of total RNA. Both RPL4 and POLR2A were used as qRT-PCR reference amplicons.
