FIGURE 3.

Mutation of nucleotides in pRL-XIAP-FL reduces apparent IRES activity and affects RNA splicing in transfected cells. (A) Schematic depicting the pRL-XIAP-FL DNA construct. Gray solid lines indicate splicing of chimeric introns. Dashed lines indicate splicing associated with XIAP sequences. Mutations to pRL-XIAP-FL are indicted by arrowheads and identified in parentheses. Splice-donor and -acceptor locations are denoted by forward and backward slash marks, respectively. (B) Indicated DNA constructs were transfected into 293T cells and lysates generated 24 h post-transfection. FL and RL activities were determined and FL/RL plotted for each construct. (C) RNA was harvested from cells in one well for each of the transfections shown in B and subjected to RT-PCR (as indicated in Materials and Methods). Products were separated in a 1% (w/v) agarose gel and visualized by EtBr staining/UV. PCR products from the HCV and XIAP DNA constructs were included as size controls (DNA). RT was included (+) or excluded (−) from each reaction as indicated.