FIGURE 5.

UCUU elements in the polypyrimidine tract are important for PTB binding and splicing repression. (A) Sequence of NM-SM PCR fragment 3. Nucleotide substitutions in the ΔUCUU and ΔCUG mutants are indicated. (B) UV cross-linking of HeLa nuclear extract proteins to [³²P]-labeled WT and mutant transcripts after incubation in nuclear extract for 25 min. Lane 1, fragment 3; lane 2, fragment 3ΔUCUU; lane 3, fragment 3ΔCUG; lane 4, NM-^∇-SM WT; and lane 5, NM-^∇-SMΔUCUU. All five lanes are derived from the same gel. (C) NM-^∇-SM transcripts were spliced in HeLa nuclear extract for 0.5, 1, 2, or 3 h. RNA species were analyzed by 12% PAGE. T indicates transcript alone. Transcripts were WT NM-^∇-SM or had the ΔUCUU point mutations in fragment 3. Quantitation of splicing of these two transcripts is shown in Figure 6D.