FIG. 3.

Specific binding of chicken NPM to the 3′-100/50 element in the chicken ccn2 mRNA 3′-UTR. (A) Schematic representation of the radiolabeled probes utilized in this study. The 3′ half of the ccn2 mRNA, including the open reading frame (ORF) and the 3′-UTR, is illustrated at the top. The sense-strand RNA fragments of the chicken ccn2 mRNA 3′-UTR (3′-100/50 and 3′-50) were transcribed in vitro in the presence of [α-³²P]UTP. (B) REMSA of the RNA fragments of ccn2 mRNA 3′-UTR. Two radiolabeled and folded RNA probes (3′-100/50 and 3′-50) corresponding to the ones shown in panel A were incubated with or without (−) 50 to 1,000 ng of recombinant chicken NPM or BSA. After RNase digestion, the mixtures were analyzed by electrophoresis through a 6% native polyacrylamide gel. (C) UV cross-linking assay. The radiolabeled and folded RNA probes were incubated with the protein and digested with RNase, as was performed in REMSA. Then, the mixtures were irradiated by UV on ice and analyzed by SDS-PAGE using a 12.5% gel. The positions of molecular mass standards (in kilodaltons) are shown at the left side of the panel. The arrow (labeled NPM) indicates the position corresponding to the molecular mass of the recombinant chicken NPM. (D) RNA immunoprecipitation analysis to confirm the specific binding of endogenous NPM in the nucleus to ccn2 mRNA. Total CEF RNA was mixed with CEF nuclear extract and was immunoprecipitated with the antibody indicated, and the presence of ccn2 or actin (control) mRNA in the immunocomplex was examined by RT-PCR after RNA extraction (Nuclear Extract). NPM, His, and Pre in the figure denote anti-chicken NPM antibody, antipolyhistidine antibody, and a preimmune IgG, respectively. Experiments were also repeated with recombinant NPM only, instead of CEF nuclear extract, which detected the interaction of NPM and ccn2 mRNA through the polyhistidine tag in the recombinant NPM as well. The RT-PCR analysis of input RNA is shown in lane Total. (−), negative-control antibody.