FIGURE 4.

RNPC1 can directly bind to MIC-1 transcript at its 3′-UTR. A, RKO cell lysates were immunoprecipitated with RNPC1 antibody or control IgG followed by RT-PCR to measure transcript levels of MIC-1, p21, and GAPDH within RNPC1 or IgG immunocomplexes. B, schematic diagram of MIC-1 transcript includes the MIC-1 coding region, 5′-UTR, 3′-UTR, and the ARE. MIC-1 3′-UTR containing an ARE was used as an RNA probe and designated as ARE. The mutant probe was designated as UtC. C, RNA-EMSA was performed by mixing ³²P-labeled MIC-1 3′-UTR with either GST alone or GST-fused RNPC1a. Unlabeled wild-type MIC-1 3′-UTR was used as cold probe for competition. The bracket labeled RPC represents an RNA-protein complex. D, the experiment was performed as in C except p21 cold probe was used for competition. E, the experiment was performed as in C except that ³²P-labeled wild-type and mutant MIC-1 probes were used for RNA-EMSA. F, schematic diagram details RNPC1a and ΔRNP2 and ΔRNP1 mutants. RRM, RNA recognition motif region in RNPC1. G, the experiment was performed as in C except that RNA-EMSA was performed with GST alone, GST-RNPC1a, GST-ΔRNP2, or GST-ΔRNP1.