Figure 4

Verification of the predicted dBP of PTB exon 11. (a) Output for PTB exon 11 from our prototype dataset. 'AGEZ' gives the size of the AGEZ; 'AG' gives the positions of three AGs upstream of the 3' splice site and two downstream. -2 is the 3' splice site. 'PPT' and 'U2BP' give the positions of predicted PPT and BPS, with bit scores in square brackets for BPS. 'SEQ1' is the sequence from the third upstream AG to the 3' splice site, whereas 'SEQ2' is the exon sequence to the second downstream AG. Predicted PPTs are in capitals. See Materials and methods for more detailed explanation of terms. Potential BPS that were mutagenized are indicated in red and blue. (b) PTB exon 11 and flanking intron sequences were cloned in an EGFP exon trapping vector [21]. Mutants ΔBP-351 and -51 contained the indicated mutations in potential branch points. Constructs were transfected into HeLa cells, and RNA analyzed by reverse transcriptase polymerase chain reaction. Splicing of exon 11 was abolished in ΔBP-351. AGEZ, AG exclusion zone; BPS, branch point sequence; dBP, distant branch point; PPT, polypyrimidine tract.