PCBPs enforce utilization of the cognate intron 1 splice donor. HeLa cell nuclear extracts were used for in vitro splicing studies after depletion of PCBPs (polyC) or mock depletion. The in vitro splicing of the full-length WT α-globin transcript was carried out in each of the 2 extracts (duplicate assays indicated as lanes 1 and 2). Comparison of the splicing products in the mock vs PCBP-depleted extract reveals the role of PCBPs in donor site selection and in enforcing functional splicing from the cognate splice donor. M indicates 25-bp ladder size marker.