The splice regulatory function of the C-rich tract adjacent to the intron 1 splice donor is independent of the C-rich element within the 3′ UTR. The indicated set of intron 1 mutations (as in Figure 3) was assayed by in vitro splicing of a full-length WT hα-globin transcript with an intact 3′ UTR C-rich tract or with the C-rich tract substituted with a previously reported neutral (neut) sequence segment of the same size (green box).^7,8  Products of the splicing reaction were assessed by RT-PCR using the indicated amplimer set (horizontal arrows). The increase in cryptic RNA (cryptic donor) and reciprocal decrease in normally spliced α-globin RNA (cognate donor) in all substrates containing mutation 1 (Mut 1) confirm the regulatory activity of the C-rich tract 3′ to the intron 1 splice donor (Mut 1) on splice donor utilization. The lack of a similar impact by Muts 2, 3, or 23 or Neut further demonstrated that this activity of the donor site splice regulatory element is independent of the C-rich elements at the intron 1 splice acceptor and within the 3′ UTR. The 25-bp ladder size marker is indicated on the left.