The tightly regulated production of distinct erythrocyte protein 4.1R isoforms involves differential splicing of 3 mutually exclusive first exons (1A, 1B, 1C) to the alternative 3′ splice sites (ss) of exon 2′/2. Here, we demonstrate that exon 1 and 2′/2 splicing diversity is regulated by a transcription-coupled splicing mechanism. We also implicate distinctive regulatory elements that promote the splicing of exon 1A to the distal 3′ ss and exon 1B to the proximal 3′ ss in murine erythroleukemia cells. A hybrid minigene driven by cytomegalovirus promoter mimicked 1B-promoter–driven splicing patterns but differed from 1A-promoter–driven splicing patterns, suggesting that promoter identity affects exon 2′/2 splicing. Furthermore, splicing factor SF2/ASF ultraviolet (UV) cross-linked to the exon 2′/2 junction CAGAGAA, a sequence that overlaps the distal U2AF35-binding 3′ ss. Consequently, depletion of SF2/ASF allowed exon 1B to splice to the distal 3′ ss but had no effect on exon 1A splicing. These findings identify for the first time that an SF2/ASF binding site also can serve as a 3′ ss in a transcript-dependent manner. Taken together, our results suggest that 4.1R gene expression involves transcriptional regulation coupled with a complex splicing regulatory network.
