Rabies virus infected 293T cells were treated with formaldehyde to stabilize RNA-protein complexes and cell lysates were immunoprecipitated using either control IgG or a PCBP2-specific antisera. Co-precipitating rabies viral mRNAs were analyzed by RT-PCR after reversal of the formaldehyde cross links and products run on a 1% agarose gel and visualized with ethidium bromide. The 10% input lane represents total RNA present in the lysate prior to immunoprecipitation. The numbers below the lanes represent quantification and standard deviation from three independent experiments.

Total RNA was isolated from rabies virus infected 293T cells at the times indicated post infection. qRT-PCR assays were performed for each of the viral mRNAs and Ct values converted to numbers of RNA molecules per µg of total cellular RNA using a standard curve that was developed for each set of primers.

A radiolabeled, capped and polyadenylated reporter RNA derived from pGem-A60 (reporter only), or a variant containing a 72 base segment from the 3′ UTR of the rabies virus G mRNA (reporter+G 3′ UTR), were electroporated into BHK cells. Total RNA was isolated from cells at the times indicated, analyzed on a 5% polyacrylamide gel containing urea, and visualized by phosphorimaging. A graphical representation of the results obtained independent representative experiment is shown. The standard deviation shown in the calculation of half-life differences was determined from 3 independent experiments.

Panel A: Radiolabeled capped and polyadenylated RNAs containing either reporter only (Gem A60) or the reporter plus the 3′ UTR of the rabies virus P, (P 3′ UTR), M, (M 3′ UTR) or G (G 3′ UTR) mRNAs were incubated with HeLa cytoplasmic extract in a cell-free deadenylation/decay assay. Aliquots were taken at the times indicated and reaction products were analyzed on a 5% polyacrylamide gel containing urea. The position of the polyadenylated (A₆₀) input RNA is indicated in the ‘0’ lane and a deadenylated marker RNA is run in the ‘A₀’ lane. Panel B: Graphical representation of three independent experiments as described in Panel A. The error bars represent standard deviations.

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.

Panel A. Radiolabeled, capped and polyadenylated reporter RNA containing the 3′ UTR of the rabies virus G mRNA was incubated in the cell-free RNA deadenylation/decay system using HeLa cytoplasmic extract for 10 minutes in the presence of the indicated amount of poly(C) competitor RNA. Reaction products were analyzed on a 5% polyacrylamide gel containing urea. The positions of the input polyadenylated (A₆₀) and deadenylated product (A₀) are indicated at the left of the gel. The lane marked input indicates the RNA in a reaction that was set up but not incubated. Panel B: Same as panel A. 2.5 µgs of the indicated competitor RNA were added in the lanes marked poly C and poly G. The lane marked ‘0’ indicates a control deadenylation reaction to which no competitor RNA was added. Reactions were incubated for 10 minutes. Panel C: HeLa cytoplasmic extracts were immunodepleted using the indicated antisera. In the left panel, the extent of immunodepletion was examined by western blot using the indicated antisera. GAPDH was used as a normalization control. In the right panel, extracts immunodepleted using either control IgG or PCBP2-specific antibodies were used in our cell-free deadenylation assays with a reporter RNA containing the 3′ UTR of the G mRNA. Samples were taken at the times indicated and analyzed on a 5% polyacrylamide gel containing urea. The positions of the input polyadenylated (A₆₀) and deadenylated product (A₀) are indicated at the left of the RNA gel.