Figure 5

Skipping of exon P3A is promoted by impairing the formation of exon-defined E (EDE) complex in the wild-type pre-mRNA.

(a) Time-course data obtained from in vitro splicing of the ³²P-labeled pre-mRNAs from E3P3A (wt and mut) and P3AE4 (wt and mut) minigenes. The splicing products are shown schematically on the right. The spliced mRNA (asterisk) is increased in E3P3A-mut compared to E3P3A-wt. Although intron lariats are apparently increased in E3P3A-wt, the intron lariat and high molecular weight RNAs are not clearly resolved for E3P3A and the increase of the intron lariats cannot be precisely estimated. Poor resolution of splicing products of E3P3A compared to P3AE4 is likely due to binding of a splicing repressing PTB. (b) Time-course analysis of early exon-defined spliceosome (EDE complex) that assembles across the P3A exon of ³²P-labeled substrates (iP3Ai-wt and iP3A-mut) in the absence of ATP. Native agarose gel electrophoresis resolves the indicated nonspecific complex H (H) and the exon-defined E complex (EDE). (c) Schematic structures of MS2-attached wild-type (wt) and mutant (mut) substrates used for isolation of EDE complex. Immunoblotting (IB) and RT-PCR analyses of purified E complex assembled on indicated substrates. PTB was likely bound to a CUCUCUCU sequence in intron 1 of β-globin-MS2 pre-mRNA.