FIG. 7.

Effects of overexpression of Pnn protein subfragments or suppression of Pnn expression on mRNA export. (a) HeLa cells were transfected with vector as indicated at left. Thirty hours posttransfection, cells were immunostained with anti-myc antibody (A and C) or anti-β-Gal antibody (E, G, I, K, and M), followed by FISH with DIG-labeled oligo(dT) as probe (B, D, F, H, J, L, and N). Detection of oligo(dT) probe was made by staining with rhodamine-conjugated anti-DIG Fab fragment. Images were taken with a Bio-Rad MRC-1000 confocal microscope. Two samples are shown for cells expressing anti-Pnn RNA (G through J) and anti-TAP RNA (K through N). In all panels, positively transfected cells are indicated by an arrowhead. A diagram in the middle shows vector for coexpression of antisense RNA and β-Gal. (b) Similar FISH experiments were performed as in panel a, except that samples were collected 48 h posttransfection. The percentage of the cells with nuclear RNA accumulation over the positively transfected cells was measured. Note that ∼1,000 positively transfected cells were examined in each transfection and that ∼10% of anti-TAP RNA-expressing cells exhibited prominent nuclear accumulation of poly(A)⁺ RNA. The percentage obtained was then normalized against that of anti-TAP-expressing cells; averages and standard deviations were obtained from three to five independent experiments.