FIG. 5.

The mRNA hyperadenylation phenotype is conserved in human cells depleted for NXF1/TAP. (A) Northern blotting analysis was done with total RNA isolated from HEK293T cells either mock-transfected (lane 1) or transfected with a plasmid expressing enhanced green fluorescent protein (EGFP) mRNA (lanes 2 and 3). In addition, cells were treated with either control (lanes 1 and 2) or NXF1/TAP (lane 3) siRNA. The blots were hybridized with probes directed against EGFP RNA or β-actin (control). Western blotting analysis used an antibody specific for NXF1/TAP or an antibody against hnRNP C as a loading control. Intervening lanes not relevant to this study were removed. (B) EGFP RNAs are hyperadenylated upon NXF1/TAP downregulation. RNase H/Northern blotting analysis of the 3′ ends of the EGFP transcripts from panel A. Positions of oligonucleotides used for the RNase H digestions (dT₁₈ and r-t) are indicated on top of the panel. Lanes 1 and 4, no oligonucleotide; lanes 2 and 5, oligo(dT)₁₈; lanes 3 and 6, oligonucleotide complementary to sequence downstream of the poly(A) site. (C) MS2 RNA FISH of U2OS cells stably expressing HIV-1 transcripts containing 24 MS2 sites. Cells were treated with either control siRNAs or siRNAs targeting TAP/NXF1. Cells were costained with DAPI as indicated. Arrows indicate nuclear dots.