Figure 3.

Characterization of vigilin binding to vitellogenin mRNA 3′-UTR and albumin mRNAs. (A) EMSA analysis was performed on 500 fmol of a 160-nt uniformly labeled albumin RNA containing the mapped PMR-1 cleavage sites (16) (Left) and vitellogenin 3′-UTR (Right). Lanes marked “No protein” contain input RNA. RNAs were incubated with either 5 μg of protein extract from liver polysomes of control animals (− E extract), 5 μg of extract from polysomes of estrogen-treated animals (+ E extract), or 750 ng of recombinant vigilin (Vigilin). The vigilin–RNA complex is indicated by the open arrow. The probes used had equal specific activity, and the albumin RNA EMSA gel was exposed for 20 vs. 4 h for vitellogenin RNA gel. (B) Complexes assembled on ice as in A were crosslinked for 5 min at 0.12 joules/cm² at a distance of 5 cm. After digestion with RNase A to remove unbound probe, the protein samples were separated by SDS/PAGE on a 10% polyacrylamide gel and cross-linked peptides were visualized by PhosphorImager. The mobility of a 158-kDa maltose-binding protein-β-galactosidase fusion protein size marker is indicated. (C) Samples (10 μg) of extract from liver polysomes of control (− E) and estrogen-treated (+ E) frogs were analyzed by Western blot by using a polyclonal antibody to epitope-tagged hnRNP K homology domains 3–5 of human vigilin, which was expressed in Escherichia coli and then purified.