Figure 5. A titratable trans-acting factor is required for the repression of deadenylation mediated by the 3′ UTR of the rabies virus G mRNA.

Panel A. Radiolabeled, capped and polyadenylated reporter RNA containing the 3′ UTR of the rabies virus G mRNA was incubated either in the absence (lane 0) or the presence of the indicated amount and type of competitor RNA for 10 minutes in the cell-free RNA deadenylation/decay assay using HeLa cytoplasmic extract. RNA products were isolated and analyzed on a 5% polyacrylamide gel containing urea. Panel B. Top: Competition assays using a capped and polyadenylated radiolabeled reporter RNA containing the 3′ UTR of the rabies virus G mRNA were performed as described in Panel A. Bottom: In parallel reactions, protein-RNA interactions with the radiolabeled RNA substrate in the presence of the indicated types and relative amounts of competitor RNA in the cell-free RNA deadenylation/decay system were assessed. EDTA was added to these reactions to block RNA degradation and afford a valid comparison between lanes. Samples were treated with short wave UV light, incubated with RNase One, and proteins covalently attached to short radioactive RNA oligomers were resolved on a 10% polyacrylamide gel containing SDS. Results were visualized by phosphorimaging. Panel C. Radiolabeled reporter RNA containing the 3′ UTR of the rabies virus G mRNA was incubated with HeLa cytoplasmic extract in the cell-free RNA deadenylation/decay system, UV cross linked and processed as described for Panel B, and analyzed by 10% SDS-PAGE either directly (input lane) or after immunoprecipitation with non-specific IgG (control lane) or with PCBP2-specific antibodies (PCBP2) lane. The positions of the 38 kDa and 85 kDa proteins are indicated on the left of the protein gels. Panel D: The sequence of the 72 base conserved region of the rabies virus G mRNA that was used in these experiments. A C-rich region, representing a possible PCBP2 binding site, is shown.