FIGURE 4.

A single PTB binding protein binding site mediates splicing inhibition in vivo. IgM1–2, IgMΔE, and mutant derivatives of IgMΔE in which either or both of the PTB binding sites were mutated, were analyzed using an assay that monitored splicing of pre-mRNA substrates in transfected COS7 cells. (A) (Top) RNA was analyzed by RT-PCR using two sets of primer-pairs: IgM1–5′ and IgM1–2SJ, which overlaps the splicing junction between the first and second exons, were used to detect spliced products; IgM1–5′ and IgM1–3′ were used to detect both spliced and unspliced RNAs. As a control, a reaction was also performed in the absence of reverse transcriptase (RT–). (Bottom) PCR bands were quantitated, and the ratio of spliced product to the total RNA (spliced and unspliced) was calculated and normalized to the amount of spliced IgM1–2 product. (B) RNA was analyzed by RT-PCR using the IgM1–5′ /IgM2 primer-pair to investigate additional cryptic splicing products. Schematic diagrams of the IgM1–2 and IgME substrates and the location of the primers are shown.