FIG. 4.

Effects of PTB1 and PTB4 on translation directed by the p27 IRES element. (A) Schematic diagram of dicistronic mRNAs used in monitoring IRES activities in vitro. Dicistronic mRNAs Rp27F, REF, and RHF contained the p27 IRES, EMCV IRES, and HCV IRES, respectively. The dicistronic mRNAs were synthesized by T7 RNA polymerase in the presence of 7methyl-GpppG for addition of cap structures at the 5′ ends. (B, i) Translation reactions were carried out as described in Materials and Methods. Various amounts of PTB1, PTB4, and La (a control RNA-binding protein) were generated by in vitro translation of effector mRNAs encoding PTB1, PTB4, and La, respectively. Dicistronic mRNAs were preincubated with RRL containing the effector proteins, and then translation reactions were carried out by addition of fresh RRL and amino acids. After translation, firefly and Renilla luciferase activities were measured, and the relative IRES activities were calculated. The value obtained by the addition of effector mRNA-free RRL was set to 1 for each mRNA (bars 1, 5, and 11). The numbers in parentheses indicate the volume (in microliters) of effector-containing RRL added. (ii) The amounts of effector proteins newly synthesized in RRL were monitored by labeling newly synthesized proteins with [³⁵S]Met. The labeled proteins were analyzed by SDS-12% PAGE.