FIG. 6.

Fox enhances splicing through tandem UGCAUG sequences in HeLa cells. Test enhancers were inserted into the second intron of pDUP4-1 β-globin splicing reporter. (A) An enhancer construct with three copies of UGCAUG element separated by 10-nt globin intron spacers. pDUP4-108 (100 ng) was cotransfected with empty expression vector (lane 1) or 100 ng or 900 ng of Fox-1 or Fox-2 expression plasmid. Lanes 2 and 3 have increasing Fox-1, and lanes 5 and 6 have increasing Fox-2. RNA was harvested after 48 h, and inclusion was assayed by RT-PCR. (B) Conditions similar to those for panel A but using the pDUP4-108Rev reporter plasmid, which has the spacer sequences reversed. (C) Conditions similar to those for panel A but with the pDUP4-108Mut reporter plasmid carrying three copies of UGACUG. (D) Knockdown of Fox-2 in HeLa cells results in loss of enhancer activity. The pDUP4-183 reporter plasmid is similar to that in panel A but carries the 5′ half of the c-src N1 exon, a known ASF/SF2-dependent exonic splicing enhancer (ESE) (41a). HeLa cells were cotransfected with 100 ng of pDUP4-183, 900 ng pUC carrier DNA, and the indicated siRNAs. RNA was harvested after 60 h, and exon inclusion was assayed by RT-PCR.