Figure 5.

The role of RRM2 in alternative splicing. (A) A diagram of the E1A pre-mRNA. The alternative 5′ splice sites and splicing events that generate 13S, 12S and 9S mRNAs are shown schematically. (B) SF2/ASF or the indicated mutants, all N-terminally tagged with the T7 epitope, were overexpressed in HeLa cells to test the effect on the splicing pattern of transcripts encoded by the E1A minigene. After 48 h, the RNAs were extracted, subjected to RT–PCR with E1A specific oligos in the presence of [α-³²P]dCTP. The RT–PCR products were resolved through a 5% polyacrylamide gel and quantified with a PhosphorImager apparatus. The histogram shows the relative amount of 13S, 12S and 9S molecules in cells overexpressing the indicated mutants. The bars represent the average of at least three independent experiments. Δ2, SF2/ASF lacking RRM2. V, E1A minigene co-transfected with the empty pCGTHCF_FLT7 expression vector. Amino acid substitutions introduced in SF2/ASF are shown in Figure 2A.