FIGURE 4.

Identification of the ∼50-kDa protein binding to MT-1 3′UTR. (A–C) Data from an RNA-affinity experiment in which proteins from a CHO S100 extract (1 mg) were incubated with biotinylated MT-1 3′UTR RNA anchored to SA-PMP (see Materials and Methods) and eluted proteins separated by SDS-PAGE. (A) A gel stained with Coomassie Brilliant Blue in which lane 1 shows the eluate from no-RNA negative control beads, and lane 2 contains proteins eluted from beads conjugated to MT-1 3′UTR RNA. The black arrowhead points to the major ∼50-kDa band and white arrowheads indicate bands of 35–40 and 60–65 kDa in the MT-1 3′UTR eluate that are reduced or absent in the negative eluate. In B, 100 μM CaCl₂ was added to the protein binding reaction, and a 15-min postincubation in which heparin was added to a final concentration of 10 mg/mL was performed. Lane 2 shows the eluate from beads conjugated to MT-1 3′UTR, compared to that from β-globin 3′UTR RNA (lane 1). The black arrowhead indicates the ∼50-kDa band present in the MT-1 3′UTR eluate. In C, eluates from beads conjugated to either MT-1 3′UTR RNA (lane 2) or β-globin 3′UTR RNA (lane 1) were subjected to Western blotting using 0.5 μg/mL anti-eEF1α antibody and chemiluminescence detection. In D, binding reactions were carried out using [α-³²P]CTP-labeled MT-1 3′UTR RNA (12 fmol) and 2 μg S100 protein (from CHO cells, prepared as described in Materials and Methods). For supershift reactions protein extract was preincubated with 8 μg IgG. Complexes were separated by native PAGE. Lane 1 contains labeled MT-1 3′UTR transcripts alone (free probe); lanes 2–7 transcripts are incubated with S100 extract either in the absence of added antibodies (lanes 2,3), or in the presence of 0.5 μg anti-eEF1α antibodies (lanes 4,5) or anti-biotin antibodies (lanes 6,7). Formation of an RNP complex with S100 extract is indicated by the black arrowhead (lanes 2,3), and the formation of a larger complex (supershift) with anti-eEF1α antibodies (lanes 4,5) is indicated by the white arrowhead. There was no apparent supershift by antibiotin antibodies (lanes 6,7).