FIG. 6.

Functional analysis of Giardia eIF4E1. (A) Firefly luciferase in vitro transcripts with different cap structures were used to transfect Giardia trophozoites. The luciferase activity thus expressed was assayed 4 h after electroporation. Each value was derived from three independent electroporation experiments. (B) Total RNA was extracted from each of the knockdown cell lines described in the legend to Fig. 5 1 day after incubation in the presence of 4 mg/ml G418. These RNA samples were used as templates for semiquantitative RT-PCR to monitor the pre-mRNA and mature mRNA of 2Fe-2S ferredoxin in the eIF4E-depleted cells. (C) Giardia total RNA was incubated with the GST-eIF4E1 fusion protein and pulled down by glutathione-Sepharose 4B beads. Results from RT-PCR amplification indicate pulldown of snRNA D and snRNA H by GST-eIF4E1, whereas the uncapped snRNA J is not.