The C-rich motif 3′ to the intron 1 splice donor enforces functional splicing of the hα-globin transcript. (A) Mutations introduced in intron 1. The hα-globin gene diagram and the C-rich segments are as described in Figure 2A. Mutations that interrupt the C-rich elements within intron 1 are indicated in green font. The combinations of mutations listed below the sequence were assayed for impact on splicing in vitro. (B) In vitro splicing analysis reveals that the C-rich tract adjacent to the cognate splice donor enforces functional splicing of exon 1. Each ³²P internally labeled RNA substrate was incubated with HeLa nuclear extract. The products of each in vitro splicing reaction were assessed by denaturing polyacrylamide gel electrophoresis. The results reveal that mutations in the C-rich tract adjacent to the cognate splice donor (mutation 1 [Mut 1]) accentuates splicing from the cryptic site with a reciprocal decrease in normally spliced RNA. Additional mutations within the C-rich tracts adjacent to the splice acceptor fail to alter the activation of the cryptic donor by Mut 1 (also described in “Results”). The differential usages of the 2 donor sites (shown below the gel) are calculated as percentages of the individual band intensities (cognate or cryptic site splicing) over the total RNA (unspliced plus cognate site splicing plus cryptic site splicing). WT input substrate alone (not incubated with HeLa cell extract) is shown. M indicates 25-bp size marker ladder.