Figure 4.

Effects of EBNA5 on the processing of reporter gene pre-mRNA. (A) EBNA5 diminishes the fraction of reporter gene RNA exported from the nucleus. The pGL3-Control (20 µg) reporter plasmid was co-transfected with 1080 fmol either pCI or pCI-EBNA5 (designated pCI-E5 in the figure) into 10⁷ DG75 cells. Cytoplasmic and total RNA fractions were prepared and the level of luciferase RNA determined by RNase protection analysis using a riboprobe that protects the first 200 bp of luciferase RNA transcribed from the reporter gene. The RNA fragments were separated by PAGE under denaturing conditions. The length in bp of the DNA fragments used as size markers is indicated on the left side of the figure. Similar results were obtained in five independent experiments. (B) EBNA5 inhibits polyadenylation and nuclear export of reporter gene RNA. The RNA obtained from the transfections above were separated into poly(A)⁺ and poly(A)^– fractions with oligo(dT)-conjugated magnetic beads. Luciferase RNA was quantified by RNase protection analysis as described above. The relative level of polyadenylated luciferase RNA in total RNA in pCI-transfected cells was defined as 100% and the levels in the other RNA fractions were expressed as a percentage of this value. The means ± SEM of three independent experiments are presented.