FIG 1.

TIA1 facilitates exon 16 inclusion through two pyrimidine-rich regions located between the branch point and the 3′ ss. (A) Schematic representation of the exon 16 minigene construct. Primers used in RT-PCR are indicated by arrows. (B) RT-PCR primers are minigene specific. MEL and HeLa cells were transiently transfected with the exon 16 minigene for 40 h; RNA isolated from transfected (+) and untransfected (−) cells was analyzed by RT-PCR. (C) TIA1 activates exon 16 splicing on both endogenous 4.1R and minigene pre-mRNA. The WT minigene was cotransfected with a vector (−) or HA-TIA1 (+) in MEL cells and analyzed for endogenous and exogenous exon 16 expression. E16 inclusion was calculated as the percentage of total RNA products containing exon 16. Averages and SDs were obtained for three independent experiments (n = 6), and results are presented at the bottom of each lane and as a bar graph. Anti-HA antibody detected HA-TIA1. β-Actin served as a loading control. (D) TIA1 effect depends on the U-rich (U-ISE) and C-rich (C-ISE) region upstream of exon 16. The top panel shows mutated minigene constructs with the replaced nucleotides indicated. The middle panel shows the effect of each mutation on exon 16 inclusion in the presence of vector (V), TIA1, or TIAR. Anti-HA antibody detected HA-TIA1 or HA-TIAR. As shown in the bottom panel, the C-rich region is critical for exon 16 inclusion even in the presence of a consensus 5′ ss. Minigenes with a consensus 5′ ss in the presence of the wild-type (AA) or mutated C-rich region (Xm/AA) were analyzed for exon 16 inclusion in response to TIA1. IB, immunoblotting.