Figure 6.

Expression of Zeb2 NAT in epithelial cells prevents splicing of the 5′-UTR intron. (A) The different constructions used in this figure are shown, indicating also the relative position of the NAT. (B) Epithelial RWP-1 cells were transiently transfected with pcDNA3-Zeb2 NAT or pcDNA3 as control. RNA was collected and analyzed by RT–PCR with oligonucleotides specific for NAT, Zeb2 5′-UTR intron, or HPRT as control. (C) RWP-1 or SW-480 cells were cotransfected with pcDNA3 NAT, pGL3 (−842/+2998), or pGL3 (−842/+475) and Renilla Luciferase expression plasmid. Firefly Luciferase was determined and normalized according to the value of Renilla Luciferase. The values correspond to the average ± SD of stimulation observed in three experiments performed in these two cell lines. (D) Levels of transcript from transfected cells from C were analyzed by RT–PCR with two oligonuclotides corresponding to 5′-UTR exon. (E,F) pGL3 (−842/+2998) plasmid, either wild type or with a deletion comprising nucleotides 1920–2845, was transfected to RWP-1 control or Snail1-expressing cells. (E) Levels of NAT were determined as above by RT–PCR. At this number of cycles, RWP-1 Snail1 cells not transfected with pGL3 plasmids presented levels of NAT similar to those detected for pGL3 (−842/+2998)(Δ1920–2845). (F) Analysis of the splicing of the 5′-UTR intron in the exogenous RNA was carried out as above by RT–PCR with two oligonucleotides flanking this intron. When indicated, pcDNA3-Zeb2 NAT or pcDNA3 as control were cotransfected. In D and F, no amplified fragments corresponding to exon or intron sequences were detected at this number of cycles in mock-transfected cells.